WO1994028133A1 - Recombinant neu differentiation factors - Google Patents
Recombinant neu differentiation factors Download PDFInfo
- Publication number
- WO1994028133A1 WO1994028133A1 PCT/US1994/005769 US9405769W WO9428133A1 WO 1994028133 A1 WO1994028133 A1 WO 1994028133A1 US 9405769 W US9405769 W US 9405769W WO 9428133 A1 WO9428133 A1 WO 9428133A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- ser
- ndf
- glu
- thr
- lys
- Prior art date
Links
- 230000004069 differentiation Effects 0.000 title description 23
- 210000004027 cell Anatomy 0.000 claims abstract description 309
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 108
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 91
- 229920001184 polypeptide Polymers 0.000 claims abstract description 80
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 claims abstract description 67
- 238000000034 method Methods 0.000 claims abstract description 57
- 108020004414 DNA Proteins 0.000 claims abstract description 56
- 101710100968 Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 claims abstract description 55
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 46
- 108020004511 Recombinant DNA Proteins 0.000 claims abstract description 12
- 238000004519 manufacturing process Methods 0.000 claims abstract description 11
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 4
- 108090000556 Neuregulin-1 Proteins 0.000 claims description 347
- 108020004635 Complementary DNA Proteins 0.000 claims description 208
- 239000002299 complementary DNA Substances 0.000 claims description 186
- 238000010804 cDNA synthesis Methods 0.000 claims description 185
- 230000014509 gene expression Effects 0.000 claims description 79
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 67
- 241000588724 Escherichia coli Species 0.000 claims description 57
- 239000013612 plasmid Substances 0.000 claims description 49
- 239000002773 nucleotide Substances 0.000 claims description 46
- 125000003729 nucleotide group Chemical group 0.000 claims description 46
- 239000012634 fragment Substances 0.000 claims description 42
- 210000001519 tissue Anatomy 0.000 claims description 42
- 206010028980 Neoplasm Diseases 0.000 claims description 38
- 108020004705 Codon Proteins 0.000 claims description 30
- 230000001086 cytosolic effect Effects 0.000 claims description 24
- 210000004978 chinese hamster ovary cell Anatomy 0.000 claims description 19
- 238000003556 assay Methods 0.000 claims description 16
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 15
- 230000004936 stimulating effect Effects 0.000 claims description 15
- 210000000981 epithelium Anatomy 0.000 claims description 14
- 239000003085 diluting agent Substances 0.000 claims description 12
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 claims description 12
- 239000000203 mixture Substances 0.000 claims description 12
- 239000013598 vector Substances 0.000 claims description 11
- 210000001072 colon Anatomy 0.000 claims description 10
- 210000003734 kidney Anatomy 0.000 claims description 10
- 210000000481 breast Anatomy 0.000 claims description 9
- 230000004663 cell proliferation Effects 0.000 claims description 9
- 210000004072 lung Anatomy 0.000 claims description 9
- 230000024245 cell differentiation Effects 0.000 claims description 8
- 230000000295 complement effect Effects 0.000 claims description 7
- 230000002500 effect on skin Effects 0.000 claims description 7
- 210000001035 gastrointestinal tract Anatomy 0.000 claims description 7
- 210000004962 mammalian cell Anatomy 0.000 claims description 7
- 102400000058 Neuregulin-1 Human genes 0.000 claims description 6
- 150000007523 nucleic acids Chemical group 0.000 claims description 6
- 230000037309 reepithelialization Effects 0.000 claims description 6
- 210000002784 stomach Anatomy 0.000 claims description 6
- 108091029865 Exogenous DNA Proteins 0.000 claims description 5
- 230000001580 bacterial effect Effects 0.000 claims description 5
- 238000006243 chemical reaction Methods 0.000 claims description 5
- 208000023514 Barrett esophagus Diseases 0.000 claims description 4
- 208000023665 Barrett oesophagus Diseases 0.000 claims description 4
- 239000002202 Polyethylene glycol Substances 0.000 claims description 4
- 239000002671 adjuvant Substances 0.000 claims description 4
- 239000002246 antineoplastic agent Substances 0.000 claims description 4
- 230000002496 gastric effect Effects 0.000 claims description 4
- 229920001223 polyethylene glycol Polymers 0.000 claims description 4
- 229920001451 polypropylene glycol Polymers 0.000 claims description 4
- 230000008439 repair process Effects 0.000 claims description 4
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 3
- 208000018522 Gastrointestinal disease Diseases 0.000 claims description 3
- 239000000427 antigen Substances 0.000 claims description 3
- 230000002068 genetic effect Effects 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 3
- 210000003932 urinary bladder Anatomy 0.000 claims description 3
- 229920003169 water-soluble polymer Polymers 0.000 claims description 3
- 210000005253 yeast cell Anatomy 0.000 claims description 3
- 208000022559 Inflammatory bowel disease Diseases 0.000 claims description 2
- 230000002708 enhancing effect Effects 0.000 claims description 2
- 230000003053 immunization Effects 0.000 claims description 2
- 210000000496 pancreas Anatomy 0.000 claims description 2
- 229920005606 polypropylene copolymer Polymers 0.000 claims description 2
- 210000002307 prostate Anatomy 0.000 claims description 2
- 230000008929 regeneration Effects 0.000 claims description 2
- 238000011069 regeneration method Methods 0.000 claims description 2
- 210000005000 reproductive tract Anatomy 0.000 claims description 2
- 230000000241 respiratory effect Effects 0.000 claims description 2
- 210000002345 respiratory system Anatomy 0.000 claims description 2
- 210000001635 urinary tract Anatomy 0.000 claims description 2
- 102000053602 DNA Human genes 0.000 claims 17
- 239000013060 biological fluid Substances 0.000 claims 2
- 108010043921 rat pro-neuregulin-alpha2C Proteins 0.000 claims 2
- 208000026372 Congenital cystic kidney disease Diseases 0.000 claims 1
- 208000026292 Cystic Kidney disease Diseases 0.000 claims 1
- 108020005202 Viral DNA Proteins 0.000 claims 1
- 230000001093 anti-cancer Effects 0.000 claims 1
- 208000020832 chronic kidney disease Diseases 0.000 claims 1
- 208000010643 digestive system disease Diseases 0.000 claims 1
- 208000028208 end stage renal disease Diseases 0.000 claims 1
- 201000000523 end stage renal failure Diseases 0.000 claims 1
- 208000018685 gastrointestinal system disease Diseases 0.000 claims 1
- 238000002649 immunization Methods 0.000 claims 1
- 235000015097 nutrients Nutrition 0.000 claims 1
- 230000001737 promoting effect Effects 0.000 claims 1
- 230000001850 reproductive effect Effects 0.000 claims 1
- 230000026731 phosphorylation Effects 0.000 abstract description 18
- 238000006366 phosphorylation reaction Methods 0.000 abstract description 18
- 239000013604 expression vector Substances 0.000 abstract description 15
- 210000005260 human cell Anatomy 0.000 abstract description 3
- 239000012620 biological material Substances 0.000 abstract description 2
- 102000048238 Neuregulin-1 Human genes 0.000 description 343
- 241000700159 Rattus Species 0.000 description 160
- 108090000623 proteins and genes Proteins 0.000 description 107
- 102000004169 proteins and genes Human genes 0.000 description 94
- 235000018102 proteins Nutrition 0.000 description 91
- 229940024606 amino acid Drugs 0.000 description 81
- 235000001014 amino acid Nutrition 0.000 description 76
- 150000001413 amino acids Chemical class 0.000 description 73
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 69
- 239000002953 phosphate buffered saline Substances 0.000 description 69
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 60
- RLMISHABBKUNFO-WHFBIAKZSA-N Ala-Ala-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O RLMISHABBKUNFO-WHFBIAKZSA-N 0.000 description 57
- 239000000523 sample Substances 0.000 description 56
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 52
- 239000013615 primer Substances 0.000 description 42
- 239000003636 conditioned culture medium Substances 0.000 description 41
- 101000996935 Homo sapiens Putative oxidoreductase GLYR1 Proteins 0.000 description 37
- 102000057482 human GLYR1 Human genes 0.000 description 37
- 108020004999 messenger RNA Proteins 0.000 description 37
- 229910000162 sodium phosphate Inorganic materials 0.000 description 37
- 238000004458 analytical method Methods 0.000 description 36
- 108010029485 Protein Isoforms Proteins 0.000 description 35
- 102000001708 Protein Isoforms Human genes 0.000 description 35
- 239000001488 sodium phosphate Substances 0.000 description 35
- 229960003339 sodium phosphate Drugs 0.000 description 35
- 235000011008 sodium phosphates Nutrition 0.000 description 35
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 35
- 230000000694 effects Effects 0.000 description 34
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 33
- 229920002684 Sepharose Polymers 0.000 description 32
- 238000010367 cloning Methods 0.000 description 31
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 30
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 29
- 239000002243 precursor Substances 0.000 description 28
- 208000027418 Wounds and injury Diseases 0.000 description 27
- 239000012091 fetal bovine serum Substances 0.000 description 26
- 239000011780 sodium chloride Substances 0.000 description 26
- 206010052428 Wound Diseases 0.000 description 25
- 238000011282 treatment Methods 0.000 description 25
- 239000013592 cell lysate Substances 0.000 description 24
- 239000000047 product Substances 0.000 description 24
- 239000000243 solution Substances 0.000 description 24
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 22
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 22
- 235000011130 ammonium sulphate Nutrition 0.000 description 22
- 201000011510 cancer Diseases 0.000 description 22
- 150000002632 lipids Chemical class 0.000 description 21
- 230000036961 partial effect Effects 0.000 description 21
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 19
- 102000005962 receptors Human genes 0.000 description 19
- 108020003175 receptors Proteins 0.000 description 19
- 230000002829 reductive effect Effects 0.000 description 19
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 19
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 19
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 18
- 239000000872 buffer Substances 0.000 description 18
- 239000002609 medium Substances 0.000 description 18
- 108010022366 Carcinoembryonic Antigen Proteins 0.000 description 17
- 102100025475 Carcinoembryonic antigen-related cell adhesion molecule 5 Human genes 0.000 description 17
- 206010009944 Colon cancer Diseases 0.000 description 17
- 108010031372 Tissue Inhibitor of Metalloproteinase-2 Proteins 0.000 description 17
- 230000014759 maintenance of location Effects 0.000 description 17
- 239000008188 pellet Substances 0.000 description 17
- 102100026262 Metalloproteinase inhibitor 2 Human genes 0.000 description 16
- 239000013613 expression plasmid Substances 0.000 description 16
- 230000012010 growth Effects 0.000 description 16
- 230000002209 hydrophobic effect Effects 0.000 description 16
- 239000000463 material Substances 0.000 description 16
- 238000000746 purification Methods 0.000 description 16
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 15
- 102000012545 EGF-like domains Human genes 0.000 description 14
- 108050002150 EGF-like domains Proteins 0.000 description 14
- 101800003838 Epidermal growth factor Proteins 0.000 description 14
- 102400001368 Epidermal growth factor Human genes 0.000 description 14
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 14
- 230000015572 biosynthetic process Effects 0.000 description 14
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 14
- 229940116977 epidermal growth factor Drugs 0.000 description 14
- 210000002919 epithelial cell Anatomy 0.000 description 14
- JYPCXBJRLBHWME-UHFFFAOYSA-N glycyl-L-prolyl-L-arginine Natural products NCC(=O)N1CCCC1C(=O)NC(CCCN=C(N)N)C(O)=O JYPCXBJRLBHWME-UHFFFAOYSA-N 0.000 description 14
- 230000035755 proliferation Effects 0.000 description 14
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 13
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 13
- 239000000499 gel Substances 0.000 description 13
- 238000009396 hybridization Methods 0.000 description 13
- 238000000338 in vitro Methods 0.000 description 13
- 238000011068 loading method Methods 0.000 description 13
- 210000004881 tumor cell Anatomy 0.000 description 13
- 101001012157 Homo sapiens Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 description 12
- 108091034117 Oligonucleotide Proteins 0.000 description 12
- 238000007792 addition Methods 0.000 description 12
- 125000000539 amino acid group Chemical group 0.000 description 12
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 12
- 229960004198 guanidine Drugs 0.000 description 12
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 12
- 229930182817 methionine Natural products 0.000 description 12
- 239000006228 supernatant Substances 0.000 description 12
- 238000002560 therapeutic procedure Methods 0.000 description 12
- 206010006187 Breast cancer Diseases 0.000 description 11
- 230000001143 conditioned effect Effects 0.000 description 11
- 230000000875 corresponding effect Effects 0.000 description 11
- 239000006167 equilibration buffer Substances 0.000 description 11
- 238000011534 incubation Methods 0.000 description 11
- 230000007935 neutral effect Effects 0.000 description 11
- 238000002360 preparation method Methods 0.000 description 11
- 238000003786 synthesis reaction Methods 0.000 description 11
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 10
- 208000026310 Breast neoplasm Diseases 0.000 description 10
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 10
- 241000283973 Oryctolagus cuniculus Species 0.000 description 10
- 102000006747 Transforming Growth Factor alpha Human genes 0.000 description 10
- 101800004564 Transforming growth factor alpha Proteins 0.000 description 10
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 10
- 238000003277 amino acid sequence analysis Methods 0.000 description 10
- 230000000692 anti-sense effect Effects 0.000 description 10
- 238000005119 centrifugation Methods 0.000 description 10
- 238000012512 characterization method Methods 0.000 description 10
- 108010015792 glycyllysine Proteins 0.000 description 10
- 238000004007 reversed phase HPLC Methods 0.000 description 10
- 230000014616 translation Effects 0.000 description 10
- 201000009030 Carcinoma Diseases 0.000 description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 9
- 239000004471 Glycine Substances 0.000 description 9
- 238000002835 absorbance Methods 0.000 description 9
- 201000008275 breast carcinoma Diseases 0.000 description 9
- 239000002131 composite material Substances 0.000 description 9
- 238000002474 experimental method Methods 0.000 description 9
- 108010051307 glycyl-glycyl-proline Proteins 0.000 description 9
- 230000001965 increasing effect Effects 0.000 description 9
- 239000000178 monomer Substances 0.000 description 9
- 230000028327 secretion Effects 0.000 description 9
- 238000012163 sequencing technique Methods 0.000 description 9
- 238000013519 translation Methods 0.000 description 9
- 108091026890 Coding region Proteins 0.000 description 8
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 8
- 108010024636 Glutathione Proteins 0.000 description 8
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 8
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 description 8
- 108010076504 Protein Sorting Signals Proteins 0.000 description 8
- 108091034057 RNA (poly(A)) Proteins 0.000 description 8
- COYHRQWNJDJCNA-NUJDXYNKSA-N Thr-Thr-Thr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O COYHRQWNJDJCNA-NUJDXYNKSA-N 0.000 description 8
- 238000010521 absorption reaction Methods 0.000 description 8
- 238000009825 accumulation Methods 0.000 description 8
- 108010024078 alanyl-glycyl-serine Proteins 0.000 description 8
- 230000008033 biological extinction Effects 0.000 description 8
- 238000005341 cation exchange Methods 0.000 description 8
- 238000004440 column chromatography Methods 0.000 description 8
- 239000012467 final product Substances 0.000 description 8
- 230000006870 function Effects 0.000 description 8
- 239000008103 glucose Substances 0.000 description 8
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 8
- 238000001727 in vivo Methods 0.000 description 8
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 8
- 239000012071 phase Substances 0.000 description 8
- 239000013600 plasmid vector Substances 0.000 description 8
- 108010026333 seryl-proline Proteins 0.000 description 8
- 230000000638 stimulation Effects 0.000 description 8
- 230000001225 therapeutic effect Effects 0.000 description 8
- 108010061238 threonyl-glycine Proteins 0.000 description 8
- 108010076441 Ala-His-His Proteins 0.000 description 7
- YBKKLDBBPFIXBQ-MBLNEYKQSA-N Ile-Thr-Gly Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)O)N YBKKLDBBPFIXBQ-MBLNEYKQSA-N 0.000 description 7
- 108060003951 Immunoglobulin Proteins 0.000 description 7
- 229930182816 L-glutamine Natural products 0.000 description 7
- YKBSXQFZWFXFIB-VOAKCMCISA-N Lys-Thr-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](CCCCN)C(O)=O YKBSXQFZWFXFIB-VOAKCMCISA-N 0.000 description 7
- 102000005741 Metalloproteases Human genes 0.000 description 7
- 108010006035 Metalloproteases Proteins 0.000 description 7
- 229920002274 Nalgene Polymers 0.000 description 7
- 108700026244 Open Reading Frames Proteins 0.000 description 7
- 239000012980 RPMI-1640 medium Substances 0.000 description 7
- 238000012300 Sequence Analysis Methods 0.000 description 7
- 108010077245 asparaginyl-proline Proteins 0.000 description 7
- 210000004556 brain Anatomy 0.000 description 7
- 239000004202 carbamide Substances 0.000 description 7
- 230000010261 cell growth Effects 0.000 description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 7
- 210000002950 fibroblast Anatomy 0.000 description 7
- 239000003102 growth factor Substances 0.000 description 7
- 102000018358 immunoglobulin Human genes 0.000 description 7
- 230000003993 interaction Effects 0.000 description 7
- 108010054155 lysyllysine Proteins 0.000 description 7
- 238000005259 measurement Methods 0.000 description 7
- VOFUROIFQGPCGE-UHFFFAOYSA-N nile red Chemical compound C1=CC=C2C3=NC4=CC=C(N(CC)CC)C=C4OC3=CC(=O)C2=C1 VOFUROIFQGPCGE-UHFFFAOYSA-N 0.000 description 7
- 238000012545 processing Methods 0.000 description 7
- 230000001105 regulatory effect Effects 0.000 description 7
- 210000000278 spinal cord Anatomy 0.000 description 7
- 238000001262 western blot Methods 0.000 description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- KLKARCOHVHLAJP-UWJYBYFXSA-N Ala-Tyr-Cys Chemical compound C[C@H](N)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](CS)C(O)=O KLKARCOHVHLAJP-UWJYBYFXSA-N 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 6
- NHHKSOGJYNQENP-SRVKXCTJSA-N Leu-Cys-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCCN)C(=O)O)N NHHKSOGJYNQENP-SRVKXCTJSA-N 0.000 description 6
- YBAFDPFAUTYYRW-UHFFFAOYSA-N N-L-alpha-glutamyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CCC(O)=O YBAFDPFAUTYYRW-UHFFFAOYSA-N 0.000 description 6
- 230000004988 N-glycosylation Effects 0.000 description 6
- 238000000636 Northern blotting Methods 0.000 description 6
- WXUBSIDKNMFAGS-IHRRRGAJSA-N Ser-Arg-Tyr Chemical compound NC(N)=NCCC[C@H](NC(=O)[C@H](CO)N)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 WXUBSIDKNMFAGS-IHRRRGAJSA-N 0.000 description 6
- 108010005233 alanylglutamic acid Proteins 0.000 description 6
- 108010062796 arginyllysine Proteins 0.000 description 6
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 6
- 229940127089 cytotoxic agent Drugs 0.000 description 6
- 238000001962 electrophoresis Methods 0.000 description 6
- 238000010828 elution Methods 0.000 description 6
- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 description 6
- 230000003834 intracellular effect Effects 0.000 description 6
- 230000001394 metastastic effect Effects 0.000 description 6
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 6
- 108010031719 prolyl-serine Proteins 0.000 description 6
- 108010048818 seryl-histidine Proteins 0.000 description 6
- 238000003756 stirring Methods 0.000 description 6
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 6
- GHWWTICYPDKPTE-NGZCFLSTSA-N Asn-Val-Pro Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC(=O)N)N GHWWTICYPDKPTE-NGZCFLSTSA-N 0.000 description 5
- 102000011632 Caseins Human genes 0.000 description 5
- 108010076119 Caseins Proteins 0.000 description 5
- 238000011537 Coomassie blue staining Methods 0.000 description 5
- PORWNQWEEIOIRH-XHNCKOQMSA-N Cys-Gln-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CS)N)C(=O)O PORWNQWEEIOIRH-XHNCKOQMSA-N 0.000 description 5
- 238000001712 DNA sequencing Methods 0.000 description 5
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 5
- TWHDOEYLXXQYOZ-FXQIFTODSA-N Gln-Asn-Gln Chemical compound C(CC(=O)N)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N TWHDOEYLXXQYOZ-FXQIFTODSA-N 0.000 description 5
- PCBBLFVHTYNQGG-LAEOZQHASA-N Glu-Asn-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCC(=O)O)N PCBBLFVHTYNQGG-LAEOZQHASA-N 0.000 description 5
- MXJYXYDREQWUMS-XKBZYTNZSA-N Glu-Thr-Ser Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O MXJYXYDREQWUMS-XKBZYTNZSA-N 0.000 description 5
- PUUYVMYCMIWHFE-BQBZGAKWSA-N Gly-Ala-Arg Chemical compound NCC(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N PUUYVMYCMIWHFE-BQBZGAKWSA-N 0.000 description 5
- BUEFQXUHTUZXHR-LURJTMIESA-N Gly-Gly-Pro zwitterion Chemical compound NCC(=O)NCC(=O)N1CCC[C@H]1C(O)=O BUEFQXUHTUZXHR-LURJTMIESA-N 0.000 description 5
- WNZOCXUOGVYYBJ-CDMKHQONSA-N Gly-Phe-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)CN)O WNZOCXUOGVYYBJ-CDMKHQONSA-N 0.000 description 5
- JYPCXBJRLBHWME-IUCAKERBSA-N Gly-Pro-Arg Chemical compound NCC(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(O)=O JYPCXBJRLBHWME-IUCAKERBSA-N 0.000 description 5
- YWCJXQKATPNPOE-UKJIMTQDSA-N Ile-Val-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N YWCJXQKATPNPOE-UKJIMTQDSA-N 0.000 description 5
- IWWMPCPLFXFBAF-SRVKXCTJSA-N Lys-Asp-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O IWWMPCPLFXFBAF-SRVKXCTJSA-N 0.000 description 5
- WTZUSCUIVPVCRH-SRVKXCTJSA-N Lys-Gln-Arg Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N WTZUSCUIVPVCRH-SRVKXCTJSA-N 0.000 description 5
- WTHGNAAQXISJHP-AVGNSLFASA-N Met-Lys-Val Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O WTHGNAAQXISJHP-AVGNSLFASA-N 0.000 description 5
- 206010027476 Metastases Diseases 0.000 description 5
- 230000004989 O-glycosylation Effects 0.000 description 5
- 108700020796 Oncogene Proteins 0.000 description 5
- 240000007711 Peperomia pellucida Species 0.000 description 5
- 102000035195 Peptidases Human genes 0.000 description 5
- 108091005804 Peptidases Proteins 0.000 description 5
- OKQQWSNUSQURLI-JYJNAYRXSA-N Phe-Met-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CCSC)NC(=O)[C@H](CC1=CC=CC=C1)N OKQQWSNUSQURLI-JYJNAYRXSA-N 0.000 description 5
- 239000012564 Q sepharose fast flow resin Substances 0.000 description 5
- DKKGAAJTDKHWOD-BIIVOSGPSA-N Ser-Asn-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)N)NC(=O)[C@H](CO)N)C(=O)O DKKGAAJTDKHWOD-BIIVOSGPSA-N 0.000 description 5
- PVDTYLHUWAEYGY-CIUDSAMLSA-N Ser-Glu-Arg Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O PVDTYLHUWAEYGY-CIUDSAMLSA-N 0.000 description 5
- CAOYHZOWXFFAIR-CIUDSAMLSA-N Ser-His-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CO)C(O)=O CAOYHZOWXFFAIR-CIUDSAMLSA-N 0.000 description 5
- QYSFWUIXDFJUDW-DCAQKATOSA-N Ser-Leu-Arg Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O QYSFWUIXDFJUDW-DCAQKATOSA-N 0.000 description 5
- SYSWVVCYSXBVJG-RHYQMDGZSA-N Val-Leu-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C(C)C)N)O SYSWVVCYSXBVJG-RHYQMDGZSA-N 0.000 description 5
- 230000002159 abnormal effect Effects 0.000 description 5
- 238000005349 anion exchange Methods 0.000 description 5
- 238000000211 autoradiogram Methods 0.000 description 5
- 238000004166 bioassay Methods 0.000 description 5
- 235000018417 cysteine Nutrition 0.000 description 5
- 238000010790 dilution Methods 0.000 description 5
- 239000012895 dilution Substances 0.000 description 5
- 108010025801 glycyl-prolyl-arginine Proteins 0.000 description 5
- 210000003000 inclusion body Anatomy 0.000 description 5
- 238000002955 isolation Methods 0.000 description 5
- 239000003446 ligand Substances 0.000 description 5
- 210000004185 liver Anatomy 0.000 description 5
- 230000007246 mechanism Effects 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- 230000009401 metastasis Effects 0.000 description 5
- 206010061289 metastatic neoplasm Diseases 0.000 description 5
- 239000013642 negative control Substances 0.000 description 5
- 108020004707 nucleic acids Proteins 0.000 description 5
- 102000039446 nucleic acids Human genes 0.000 description 5
- 230000003287 optical effect Effects 0.000 description 5
- 108010029020 prolylglycine Proteins 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 238000010186 staining Methods 0.000 description 5
- 238000001890 transfection Methods 0.000 description 5
- 108010073969 valyllysine Proteins 0.000 description 5
- WOVKYSAHUYNSMH-RRKCRQDMSA-N 5-bromodeoxyuridine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(Br)=C1 WOVKYSAHUYNSMH-RRKCRQDMSA-N 0.000 description 4
- 101100295756 Acinetobacter baumannii (strain ATCC 19606 / DSM 30007 / JCM 6841 / CCUG 19606 / CIP 70.34 / NBRC 109757 / NCIMB 12457 / NCTC 12156 / 81) omp38 gene Proteins 0.000 description 4
- SVBXIUDNTRTKHE-CIUDSAMLSA-N Ala-Arg-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O SVBXIUDNTRTKHE-CIUDSAMLSA-N 0.000 description 4
- NBTGEURICRTMGL-WHFBIAKZSA-N Ala-Gly-Ser Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O NBTGEURICRTMGL-WHFBIAKZSA-N 0.000 description 4
- VWVPYNGMOCSSGK-GUBZILKMSA-N Arg-Arg-Asn Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(O)=O VWVPYNGMOCSSGK-GUBZILKMSA-N 0.000 description 4
- FRMQITGHXMUNDF-GMOBBJLQSA-N Arg-Ile-Asn Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N FRMQITGHXMUNDF-GMOBBJLQSA-N 0.000 description 4
- CVXXSWQORBZAAA-SRVKXCTJSA-N Arg-Lys-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CCCN=C(N)N CVXXSWQORBZAAA-SRVKXCTJSA-N 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 241000972773 Aulopiformes Species 0.000 description 4
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 4
- GRNOCLDFUNCIDW-ACZMJKKPSA-N Cys-Ala-Glu Chemical compound C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CS)N GRNOCLDFUNCIDW-ACZMJKKPSA-N 0.000 description 4
- JLZCAZJGWNRXCI-XKBZYTNZSA-N Cys-Thr-Glu Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(O)=O JLZCAZJGWNRXCI-XKBZYTNZSA-N 0.000 description 4
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 4
- 102000001301 EGF receptor Human genes 0.000 description 4
- 108060006698 EGF receptor Proteins 0.000 description 4
- LKDIBBOKUAASNP-FXQIFTODSA-N Glu-Ala-Glu Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(O)=O LKDIBBOKUAASNP-FXQIFTODSA-N 0.000 description 4
- BPLNJYHNAJVLRT-ACZMJKKPSA-N Glu-Ser-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O BPLNJYHNAJVLRT-ACZMJKKPSA-N 0.000 description 4
- 108010053070 Glutathione Disulfide Proteins 0.000 description 4
- RJIVPOXLQFJRTG-LURJTMIESA-N Gly-Arg-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)CN)CCCN=C(N)N RJIVPOXLQFJRTG-LURJTMIESA-N 0.000 description 4
- BIRKKBCSAIHDDF-WDSKDSINSA-N Gly-Glu-Cys Chemical compound NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CS)C(O)=O BIRKKBCSAIHDDF-WDSKDSINSA-N 0.000 description 4
- 102000003886 Glycoproteins Human genes 0.000 description 4
- 108090000288 Glycoproteins Proteins 0.000 description 4
- 101100118545 Holotrichia diomphalia EGF-like gene Proteins 0.000 description 4
- 108090001061 Insulin Proteins 0.000 description 4
- 102000004877 Insulin Human genes 0.000 description 4
- 241000880493 Leptailurus serval Species 0.000 description 4
- FJUKMPUELVROGK-IHRRRGAJSA-N Leu-Arg-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N FJUKMPUELVROGK-IHRRRGAJSA-N 0.000 description 4
- UWKNTTJNVSYXPC-CIUDSAMLSA-N Lys-Ala-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCCN UWKNTTJNVSYXPC-CIUDSAMLSA-N 0.000 description 4
- IMAKMJCBYCSMHM-AVGNSLFASA-N Lys-Glu-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CCCCN IMAKMJCBYCSMHM-AVGNSLFASA-N 0.000 description 4
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- RMLLCGYYVZKKRT-CIUDSAMLSA-N Met-Ser-Glu Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(O)=O RMLLCGYYVZKKRT-CIUDSAMLSA-N 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 4
- 229910019142 PO4 Inorganic materials 0.000 description 4
- XQLBWXHVZVBNJM-FXQIFTODSA-N Pro-Ala-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1 XQLBWXHVZVBNJM-FXQIFTODSA-N 0.000 description 4
- JUJGNDZIKKQMDJ-IHRRRGAJSA-N Pro-His-His Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CNC=N1)C(O)=O JUJGNDZIKKQMDJ-IHRRRGAJSA-N 0.000 description 4
- 239000004365 Protease Substances 0.000 description 4
- ZUGXSSFMTXKHJS-ZLUOBGJFSA-N Ser-Ala-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O ZUGXSSFMTXKHJS-ZLUOBGJFSA-N 0.000 description 4
- DINQYZRMXGWWTG-GUBZILKMSA-N Ser-Pro-Pro Chemical compound OC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 DINQYZRMXGWWTG-GUBZILKMSA-N 0.000 description 4
- GYDFRTRSSXOZCR-ACZMJKKPSA-N Ser-Ser-Glu Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(O)=O GYDFRTRSSXOZCR-ACZMJKKPSA-N 0.000 description 4
- XQJCEKXQUJQNNK-ZLUOBGJFSA-N Ser-Ser-Ser Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O XQJCEKXQUJQNNK-ZLUOBGJFSA-N 0.000 description 4
- SNXUIBACCONSOH-BWBBJGPYSA-N Ser-Thr-Ser Chemical compound OC[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](CO)C(O)=O SNXUIBACCONSOH-BWBBJGPYSA-N 0.000 description 4
- VLMIUSLQONKLDV-HEIBUPTGSA-N Ser-Thr-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O VLMIUSLQONKLDV-HEIBUPTGSA-N 0.000 description 4
- 108091081024 Start codon Proteins 0.000 description 4
- SHOMROOOQBDGRL-JHEQGTHGSA-N Thr-Glu-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O SHOMROOOQBDGRL-JHEQGTHGSA-N 0.000 description 4
- XPNSAQMEAVSQRD-FBCQKBJTSA-N Thr-Gly-Gly Chemical compound C[C@@H](O)[C@H](N)C(=O)NCC(=O)NCC(O)=O XPNSAQMEAVSQRD-FBCQKBJTSA-N 0.000 description 4
- UGFSAPWZBROURT-IXOXFDKPSA-N Thr-Phe-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CS)C(=O)O)N)O UGFSAPWZBROURT-IXOXFDKPSA-N 0.000 description 4
- NDXSOKGYKCGYKT-VEVYYDQMSA-N Thr-Pro-Asp Chemical compound C[C@@H](O)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O NDXSOKGYKCGYKT-VEVYYDQMSA-N 0.000 description 4
- KERCOYANYUPLHJ-XGEHTFHBSA-N Thr-Pro-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O KERCOYANYUPLHJ-XGEHTFHBSA-N 0.000 description 4
- BCYUHPXBHCUYBA-CUJWVEQBSA-N Thr-Ser-His Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](Cc1cnc[nH]1)C(O)=O BCYUHPXBHCUYBA-CUJWVEQBSA-N 0.000 description 4
- RVMNUBQWPVOUKH-HEIBUPTGSA-N Thr-Ser-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O RVMNUBQWPVOUKH-HEIBUPTGSA-N 0.000 description 4
- 102000004142 Trypsin Human genes 0.000 description 4
- 108090000631 Trypsin Proteins 0.000 description 4
- UDNYEPLJTRDMEJ-RCOVLWMOSA-N Val-Asn-Gly Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)NCC(=O)O)N UDNYEPLJTRDMEJ-RCOVLWMOSA-N 0.000 description 4
- PZTZYZUTCPZWJH-FXQIFTODSA-N Val-Ser-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)O)N PZTZYZUTCPZWJH-FXQIFTODSA-N 0.000 description 4
- KOSRFJWDECSPRO-UHFFFAOYSA-N alpha-L-glutamyl-L-glutamic acid Natural products OC(=O)CCC(N)C(=O)NC(CCC(O)=O)C(O)=O KOSRFJWDECSPRO-UHFFFAOYSA-N 0.000 description 4
- 101150042295 arfA gene Proteins 0.000 description 4
- 108010013835 arginine glutamate Proteins 0.000 description 4
- 108010069926 arginyl-glycyl-serine Proteins 0.000 description 4
- 230000004071 biological effect Effects 0.000 description 4
- 239000005018 casein Substances 0.000 description 4
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 4
- 235000021240 caseins Nutrition 0.000 description 4
- 230000015556 catabolic process Effects 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 238000002512 chemotherapy Methods 0.000 description 4
- 208000029742 colonic neoplasm Diseases 0.000 description 4
- 201000010989 colorectal carcinoma Diseases 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 4
- 238000012217 deletion Methods 0.000 description 4
- 230000037430 deletion Effects 0.000 description 4
- 230000001419 dependent effect Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 238000004520 electroporation Methods 0.000 description 4
- 239000011521 glass Substances 0.000 description 4
- YPZRWBKMTBYPTK-BJDJZHNGSA-N glutathione disulfide Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@H](C(=O)NCC(O)=O)CSSC[C@@H](C(=O)NCC(O)=O)NC(=O)CC[C@H](N)C(O)=O YPZRWBKMTBYPTK-BJDJZHNGSA-N 0.000 description 4
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 4
- 239000010931 gold Substances 0.000 description 4
- 229910052737 gold Inorganic materials 0.000 description 4
- 108010018006 histidylserine Proteins 0.000 description 4
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 4
- 230000002055 immunohistochemical effect Effects 0.000 description 4
- 230000006698 induction Effects 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 229940125396 insulin Drugs 0.000 description 4
- 210000002510 keratinocyte Anatomy 0.000 description 4
- 108010073472 leucyl-prolyl-proline Proteins 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 108010017391 lysylvaline Proteins 0.000 description 4
- 210000004379 membrane Anatomy 0.000 description 4
- 239000002751 oligonucleotide probe Substances 0.000 description 4
- 101150087557 omcB gene Proteins 0.000 description 4
- 101150115693 ompA gene Proteins 0.000 description 4
- YPZRWBKMTBYPTK-UHFFFAOYSA-N oxidized gamma-L-glutamyl-L-cysteinylglycine Natural products OC(=O)C(N)CCC(=O)NC(C(=O)NCC(O)=O)CSSCC(C(=O)NCC(O)=O)NC(=O)CCC(N)C(O)=O YPZRWBKMTBYPTK-UHFFFAOYSA-N 0.000 description 4
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 4
- 239000010452 phosphate Substances 0.000 description 4
- 238000003566 phosphorylation assay Methods 0.000 description 4
- DCWXELXMIBXGTH-UHFFFAOYSA-N phosphotyrosine Chemical compound OC(=O)C(N)CC1=CC=C(OP(O)(O)=O)C=C1 DCWXELXMIBXGTH-UHFFFAOYSA-N 0.000 description 4
- 230000008488 polyadenylation Effects 0.000 description 4
- 230000002062 proliferating effect Effects 0.000 description 4
- 230000017854 proteolysis Effects 0.000 description 4
- 230000002797 proteolythic effect Effects 0.000 description 4
- 230000004044 response Effects 0.000 description 4
- 235000019515 salmon Nutrition 0.000 description 4
- 108010048397 seryl-lysyl-leucine Proteins 0.000 description 4
- 210000003491 skin Anatomy 0.000 description 4
- FQENQNTWSFEDLI-UHFFFAOYSA-J sodium diphosphate Chemical compound [Na+].[Na+].[Na+].[Na+].[O-]P([O-])(=O)OP([O-])([O-])=O FQENQNTWSFEDLI-UHFFFAOYSA-J 0.000 description 4
- 229940048086 sodium pyrophosphate Drugs 0.000 description 4
- 108010005652 splenotritin Proteins 0.000 description 4
- 210000000130 stem cell Anatomy 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 235000019818 tetrasodium diphosphate Nutrition 0.000 description 4
- 239000001577 tetrasodium phosphonato phosphate Substances 0.000 description 4
- 239000012588 trypsin Substances 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- HXNNRBHASOSVPG-GUBZILKMSA-N Ala-Glu-Leu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O HXNNRBHASOSVPG-GUBZILKMSA-N 0.000 description 3
- HKRXJBBCQBAGIM-FXQIFTODSA-N Arg-Asp-Ser Chemical compound C(C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CO)C(=O)O)N)CN=C(N)N HKRXJBBCQBAGIM-FXQIFTODSA-N 0.000 description 3
- DGFGDPVSDQPANQ-XGEHTFHBSA-N Arg-Cys-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CCCN=C(N)N)N)O DGFGDPVSDQPANQ-XGEHTFHBSA-N 0.000 description 3
- HPKSHFSEXICTLI-CIUDSAMLSA-N Arg-Glu-Ala Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O HPKSHFSEXICTLI-CIUDSAMLSA-N 0.000 description 3
- NXDXECQFKHXHAM-HJGDQZAQSA-N Arg-Glu-Thr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O NXDXECQFKHXHAM-HJGDQZAQSA-N 0.000 description 3
- SLNCSSWAIDUUGF-LSJOCFKGSA-N Arg-His-Ala Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](C)C(O)=O SLNCSSWAIDUUGF-LSJOCFKGSA-N 0.000 description 3
- CRCCTGPNZUCAHE-DCAQKATOSA-N Arg-His-Ser Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CO)C(O)=O)CC1=CN=CN1 CRCCTGPNZUCAHE-DCAQKATOSA-N 0.000 description 3
- KWQPAXYXVMHJJR-AVGNSLFASA-N Asn-Gln-Tyr Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 KWQPAXYXVMHJJR-AVGNSLFASA-N 0.000 description 3
- KOWWUKUFQYDZID-SRVKXCTJSA-N Asn-Gly-Pro-His Chemical compound C1C[C@H](N(C1)C(=O)CNC(=O)[C@H](CC(=O)N)N)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O KOWWUKUFQYDZID-SRVKXCTJSA-N 0.000 description 3
- RAQMSGVCGSJKCL-FOHZUACHSA-N Asn-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC(N)=O RAQMSGVCGSJKCL-FOHZUACHSA-N 0.000 description 3
- NKLRWRRVYGQNIH-GHCJXIJMSA-N Asn-Ile-Ala Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O NKLRWRRVYGQNIH-GHCJXIJMSA-N 0.000 description 3
- BZWRLDPIWKOVKB-ZPFDUUQYSA-N Asn-Leu-Ile Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O BZWRLDPIWKOVKB-ZPFDUUQYSA-N 0.000 description 3
- NNDSLVWAQAUPPP-GUBZILKMSA-N Asn-Met-Met Chemical compound CSCC[C@@H](C(=O)N[C@@H](CCSC)C(=O)O)NC(=O)[C@H](CC(=O)N)N NNDSLVWAQAUPPP-GUBZILKMSA-N 0.000 description 3
- JNCRAQVYJZGIOW-QSFUFRPTSA-N Asn-Val-Ile Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O JNCRAQVYJZGIOW-QSFUFRPTSA-N 0.000 description 3
- WVDDGKGOMKODPV-UHFFFAOYSA-N Benzyl alcohol Chemical compound OCC1=CC=CC=C1 WVDDGKGOMKODPV-UHFFFAOYSA-N 0.000 description 3
- 102000004266 Collagen Type IV Human genes 0.000 description 3
- 108010042086 Collagen Type IV Proteins 0.000 description 3
- 239000003155 DNA primer Substances 0.000 description 3
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 3
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- IOFDDSNZJDIGPB-GVXVVHGQSA-N Gln-Leu-Val Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O IOFDDSNZJDIGPB-GVXVVHGQSA-N 0.000 description 3
- GURIQZQSTBBHRV-SRVKXCTJSA-N Gln-Lys-Arg Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O GURIQZQSTBBHRV-SRVKXCTJSA-N 0.000 description 3
- PBEQPAZRHDVJQI-SRVKXCTJSA-N Glu-Arg-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCC(=O)O)N PBEQPAZRHDVJQI-SRVKXCTJSA-N 0.000 description 3
- RDDSZZJOKDVPAE-ACZMJKKPSA-N Glu-Asn-Ser Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O RDDSZZJOKDVPAE-ACZMJKKPSA-N 0.000 description 3
- KLJMRPIBBLTDGE-ACZMJKKPSA-N Glu-Cys-Asn Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(O)=O KLJMRPIBBLTDGE-ACZMJKKPSA-N 0.000 description 3
- MUSGDMDGNGXULI-DCAQKATOSA-N Glu-Glu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CCC(O)=O MUSGDMDGNGXULI-DCAQKATOSA-N 0.000 description 3
- OQXDUSZKISQQSS-GUBZILKMSA-N Glu-Lys-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O OQXDUSZKISQQSS-GUBZILKMSA-N 0.000 description 3
- SYAYROHMAIHWFB-KBIXCLLPSA-N Glu-Ser-Ile Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O SYAYROHMAIHWFB-KBIXCLLPSA-N 0.000 description 3
- OVSKVOOUFAKODB-UWVGGRQHSA-N Gly-Arg-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CCCN=C(N)N OVSKVOOUFAKODB-UWVGGRQHSA-N 0.000 description 3
- QSVMIMFAAZPCAQ-PMVVWTBXSA-N Gly-His-Thr Chemical compound [H]NCC(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O QSVMIMFAAZPCAQ-PMVVWTBXSA-N 0.000 description 3
- LYSMQLXUCAKELQ-DCAQKATOSA-N His-Asp-Arg Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N LYSMQLXUCAKELQ-DCAQKATOSA-N 0.000 description 3
- BZKDJRSZWLPJNI-SRVKXCTJSA-N His-His-Ser Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CO)C(O)=O BZKDJRSZWLPJNI-SRVKXCTJSA-N 0.000 description 3
- GNBHSMFBUNEWCJ-DCAQKATOSA-N His-Pro-Asn Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(O)=O GNBHSMFBUNEWCJ-DCAQKATOSA-N 0.000 description 3
- JGFWUKYIQAEYAH-DCAQKATOSA-N His-Ser-Val Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O JGFWUKYIQAEYAH-DCAQKATOSA-N 0.000 description 3
- CSTDQOOBZBAJKE-BWAGICSOSA-N His-Tyr-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H](CC2=CN=CN2)N)O CSTDQOOBZBAJKE-BWAGICSOSA-N 0.000 description 3
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 3
- WTOAPTKSZJJWKK-HTFCKZLJSA-N Ile-Cys-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H]([C@@H](C)CC)C(=O)O)N WTOAPTKSZJJWKK-HTFCKZLJSA-N 0.000 description 3
- VUPHVQCDULLACF-NAKRPEOUSA-N Ile-Met-Cys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CS)C(=O)O)N VUPHVQCDULLACF-NAKRPEOUSA-N 0.000 description 3
- ZSESFIFAYQEKRD-CYDGBPFRSA-N Ile-Val-Met Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCSC)C(=O)O)N ZSESFIFAYQEKRD-CYDGBPFRSA-N 0.000 description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 3
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 3
- 229930064664 L-arginine Natural products 0.000 description 3
- 235000014852 L-arginine Nutrition 0.000 description 3
- RCFDOSNHHZGBOY-UHFFFAOYSA-N L-isoleucyl-L-alanine Natural products CCC(C)C(N)C(=O)NC(C)C(O)=O RCFDOSNHHZGBOY-UHFFFAOYSA-N 0.000 description 3
- GRZSCTXVCDUIPO-SRVKXCTJSA-N Leu-Arg-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(O)=O GRZSCTXVCDUIPO-SRVKXCTJSA-N 0.000 description 3
- JIHDFWWRYHSAQB-GUBZILKMSA-N Leu-Ser-Glu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(O)=O JIHDFWWRYHSAQB-GUBZILKMSA-N 0.000 description 3
- 239000006137 Luria-Bertani broth Substances 0.000 description 3
- MLLKLNYPZRDIQG-GUBZILKMSA-N Lys-Cys-Gln Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N MLLKLNYPZRDIQG-GUBZILKMSA-N 0.000 description 3
- HVAUKHLDSDDROB-KKUMJFAQSA-N Lys-Lys-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O HVAUKHLDSDDROB-KKUMJFAQSA-N 0.000 description 3
- YKWHHKDMBZBMLG-GUBZILKMSA-N Met-Cys-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CCSC)N YKWHHKDMBZBMLG-GUBZILKMSA-N 0.000 description 3
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 3
- 108010069196 Neural Cell Adhesion Molecules Proteins 0.000 description 3
- 241001494479 Pecora Species 0.000 description 3
- 229930182555 Penicillin Natural products 0.000 description 3
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 3
- IAOZOFPONWDXNT-IXOXFDKPSA-N Phe-Ser-Thr Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O IAOZOFPONWDXNT-IXOXFDKPSA-N 0.000 description 3
- ZCXQTRXYZOSGJR-FXQIFTODSA-N Pro-Asp-Ser Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O ZCXQTRXYZOSGJR-FXQIFTODSA-N 0.000 description 3
- XQSREVQDGCPFRJ-STQMWFEESA-N Pro-Gly-Phe Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O XQSREVQDGCPFRJ-STQMWFEESA-N 0.000 description 3
- AQSMZTIEJMZQEC-DCAQKATOSA-N Pro-His-Ser Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CC2=CN=CN2)C(=O)N[C@@H](CO)C(=O)O AQSMZTIEJMZQEC-DCAQKATOSA-N 0.000 description 3
- SBVPYBFMIGDIDX-SRVKXCTJSA-N Pro-Pro-Pro Chemical compound OC(=O)[C@@H]1CCCN1C(=O)[C@H]1N(C(=O)[C@H]2NCCC2)CCC1 SBVPYBFMIGDIDX-SRVKXCTJSA-N 0.000 description 3
- DCHQYSOGURGJST-FJXKBIBVSA-N Pro-Thr-Gly Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O DCHQYSOGURGJST-FJXKBIBVSA-N 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 3
- UICKAKRRRBTILH-GUBZILKMSA-N Ser-Glu-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CO)N UICKAKRRRBTILH-GUBZILKMSA-N 0.000 description 3
- UPLYXVPQLJVWMM-KKUMJFAQSA-N Ser-Phe-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(O)=O UPLYXVPQLJVWMM-KKUMJFAQSA-N 0.000 description 3
- VGQVAVQWKJLIRM-FXQIFTODSA-N Ser-Ser-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O VGQVAVQWKJLIRM-FXQIFTODSA-N 0.000 description 3
- XJDMUQCLVSCRSJ-VZFHVOOUSA-N Ser-Thr-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O XJDMUQCLVSCRSJ-VZFHVOOUSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 208000007107 Stomach Ulcer Diseases 0.000 description 3
- KGKWKSSSQGGYAU-SUSMZKCASA-N Thr-Gln-Thr Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N)O KGKWKSSSQGGYAU-SUSMZKCASA-N 0.000 description 3
- SLUWOCTZVGMURC-BFHQHQDPSA-N Thr-Gly-Ala Chemical compound C[C@@H](O)[C@H](N)C(=O)NCC(=O)N[C@@H](C)C(O)=O SLUWOCTZVGMURC-BFHQHQDPSA-N 0.000 description 3
- LECUEEHKUFYOOV-ZJDVBMNYSA-N Thr-Thr-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@@H](N)[C@@H](C)O LECUEEHKUFYOOV-ZJDVBMNYSA-N 0.000 description 3
- KBKTUNYBNJWFRL-UBHSHLNASA-N Trp-Ser-Asn Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O)=CNC2=C1 KBKTUNYBNJWFRL-UBHSHLNASA-N 0.000 description 3
- AVIQBBOOTZENLH-KKUMJFAQSA-N Tyr-Leu-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)N AVIQBBOOTZENLH-KKUMJFAQSA-N 0.000 description 3
- PMDOQZFYGWZSTK-LSJOCFKGSA-N Val-Gly-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)C(C)C PMDOQZFYGWZSTK-LSJOCFKGSA-N 0.000 description 3
- QTPQHINADBYBNA-DCAQKATOSA-N Val-Ser-Lys Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCCN QTPQHINADBYBNA-DCAQKATOSA-N 0.000 description 3
- AEFJNECXZCODJM-UWVGGRQHSA-N Val-Val-Gly Chemical compound CC(C)[C@H]([NH3+])C(=O)N[C@@H](C(C)C)C(=O)NCC([O-])=O AEFJNECXZCODJM-UWVGGRQHSA-N 0.000 description 3
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 3
- 238000011481 absorbance measurement Methods 0.000 description 3
- 208000009956 adenocarcinoma Diseases 0.000 description 3
- 238000000376 autoradiography Methods 0.000 description 3
- 239000011324 bead Substances 0.000 description 3
- 230000033228 biological regulation Effects 0.000 description 3
- 229940098773 bovine serum albumin Drugs 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 3
- 229960004316 cisplatin Drugs 0.000 description 3
- 230000000112 colonic effect Effects 0.000 description 3
- 239000002254 cytotoxic agent Substances 0.000 description 3
- 231100000599 cytotoxic agent Toxicity 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 208000035475 disorder Diseases 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 108700020302 erbB-2 Genes Proteins 0.000 description 3
- 239000003797 essential amino acid Substances 0.000 description 3
- 235000020776 essential amino acid Nutrition 0.000 description 3
- 239000011536 extraction buffer Substances 0.000 description 3
- 239000012737 fresh medium Substances 0.000 description 3
- 230000013595 glycosylation Effects 0.000 description 3
- 238000006206 glycosylation reaction Methods 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 230000035876 healing Effects 0.000 description 3
- 108010028295 histidylhistidine Proteins 0.000 description 3
- 229960000890 hydrocortisone Drugs 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 239000003112 inhibitor Substances 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 229960000310 isoleucine Drugs 0.000 description 3
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 3
- 108010027338 isoleucylcysteine Proteins 0.000 description 3
- 210000002429 large intestine Anatomy 0.000 description 3
- 230000003902 lesion Effects 0.000 description 3
- 239000012160 loading buffer Substances 0.000 description 3
- 239000006166 lysate Substances 0.000 description 3
- 230000003211 malignant effect Effects 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 235000013336 milk Nutrition 0.000 description 3
- 239000008267 milk Substances 0.000 description 3
- 210000004080 milk Anatomy 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- PJUIMOJAAPLTRJ-UHFFFAOYSA-N monothioglycerol Chemical compound OCC(O)CS PJUIMOJAAPLTRJ-UHFFFAOYSA-N 0.000 description 3
- 210000002220 organoid Anatomy 0.000 description 3
- 210000001672 ovary Anatomy 0.000 description 3
- 230000002018 overexpression Effects 0.000 description 3
- 229940049954 penicillin Drugs 0.000 description 3
- 230000001817 pituitary effect Effects 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 108700042226 ras Genes Proteins 0.000 description 3
- 102000027426 receptor tyrosine kinases Human genes 0.000 description 3
- 108091008598 receptor tyrosine kinases Proteins 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 230000002441 reversible effect Effects 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 229910052708 sodium Inorganic materials 0.000 description 3
- 239000012064 sodium phosphate buffer Substances 0.000 description 3
- 125000006850 spacer group Chemical group 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 210000000952 spleen Anatomy 0.000 description 3
- 238000010561 standard procedure Methods 0.000 description 3
- 229960005322 streptomycin Drugs 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 238000001356 surgical procedure Methods 0.000 description 3
- 238000013518 transcription Methods 0.000 description 3
- 230000035897 transcription Effects 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- 230000010474 transient expression Effects 0.000 description 3
- 230000029663 wound healing Effects 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- NMWKYTGJWUAZPZ-WWHBDHEGSA-N (4S)-4-[[(4R,7S,10S,16S,19S,25S,28S,31R)-31-[[(2S)-2-[[(1R,6R,9S,12S,18S,21S,24S,27S,30S,33S,36S,39S,42R,47R,53S,56S,59S,62S,65S,68S,71S,76S,79S,85S)-47-[[(2S)-2-[[(2S)-4-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-amino-3-methylbutanoyl]amino]-3-methylbutanoyl]amino]-3-hydroxypropanoyl]amino]-3-(1H-imidazol-4-yl)propanoyl]amino]-3-phenylpropanoyl]amino]-4-oxobutanoyl]amino]-3-carboxypropanoyl]amino]-18-(4-aminobutyl)-27,68-bis(3-amino-3-oxopropyl)-36,71,76-tribenzyl-39-(3-carbamimidamidopropyl)-24-(2-carboxyethyl)-21,56-bis(carboxymethyl)-65,85-bis[(1R)-1-hydroxyethyl]-59-(hydroxymethyl)-62,79-bis(1H-imidazol-4-ylmethyl)-9-methyl-33-(2-methylpropyl)-8,11,17,20,23,26,29,32,35,38,41,48,54,57,60,63,66,69,72,74,77,80,83,86-tetracosaoxo-30-propan-2-yl-3,4,44,45-tetrathia-7,10,16,19,22,25,28,31,34,37,40,49,55,58,61,64,67,70,73,75,78,81,84,87-tetracosazatetracyclo[40.31.14.012,16.049,53]heptaoctacontane-6-carbonyl]amino]-3-methylbutanoyl]amino]-7-(3-carbamimidamidopropyl)-25-(hydroxymethyl)-19-[(4-hydroxyphenyl)methyl]-28-(1H-imidazol-4-ylmethyl)-10-methyl-6,9,12,15,18,21,24,27,30-nonaoxo-16-propan-2-yl-1,2-dithia-5,8,11,14,17,20,23,26,29-nonazacyclodotriacontane-4-carbonyl]amino]-5-[[(2S)-1-[[(2S)-1-[[(2S)-3-carboxy-1-[[(2S)-1-[[(2S)-1-[[(1S)-1-carboxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-1-oxopropan-2-yl]amino]-1-oxopropan-2-yl]amino]-3-(1H-imidazol-4-yl)-1-oxopropan-2-yl]amino]-5-oxopentanoic acid Chemical compound CC(C)C[C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](Cc1c[nH]cn1)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H]1CSSC[C@H](NC(=O)[C@@H](NC(=O)[C@@H]2CSSC[C@@H]3NC(=O)[C@H](Cc4ccccc4)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](Cc4c[nH]cn4)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H]4CCCN4C(=O)[C@H](CSSC[C@H](NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](Cc4c[nH]cn4)NC(=O)[C@H](Cc4ccccc4)NC3=O)[C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](Cc3ccccc3)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N3CCC[C@H]3C(=O)N[C@@H](C)C(=O)N2)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](Cc2ccccc2)NC(=O)[C@H](Cc2c[nH]cn2)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)C(C)C)[C@@H](C)O)C(C)C)C(=O)N[C@@H](Cc2c[nH]cn2)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](Cc2ccc(O)cc2)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N1)C(=O)N[C@@H](C)C(O)=O NMWKYTGJWUAZPZ-WWHBDHEGSA-N 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 2
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 2
- IMIZPWSVYADSCN-UHFFFAOYSA-N 4-methyl-2-[[4-methyl-2-[[4-methyl-2-(pyrrolidine-2-carbonylamino)pentanoyl]amino]pentanoyl]amino]pentanoic acid Chemical compound CC(C)CC(C(O)=O)NC(=O)C(CC(C)C)NC(=O)C(CC(C)C)NC(=O)C1CCCN1 IMIZPWSVYADSCN-UHFFFAOYSA-N 0.000 description 2
- 208000003200 Adenoma Diseases 0.000 description 2
- 206010001233 Adenoma benign Diseases 0.000 description 2
- CVGNCMIULZNYES-WHFBIAKZSA-N Ala-Asn-Gly Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O CVGNCMIULZNYES-WHFBIAKZSA-N 0.000 description 2
- STACJSVFHSEZJV-GHCJXIJMSA-N Ala-Asn-Ile Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O STACJSVFHSEZJV-GHCJXIJMSA-N 0.000 description 2
- GORKKVHIBWAQHM-GCJQMDKQSA-N Ala-Asn-Thr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O GORKKVHIBWAQHM-GCJQMDKQSA-N 0.000 description 2
- BUDNAJYVCUHLSV-ZLUOBGJFSA-N Ala-Asp-Ser Chemical compound C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O BUDNAJYVCUHLSV-ZLUOBGJFSA-N 0.000 description 2
- WKOBSJOZRJJVRZ-FXQIFTODSA-N Ala-Glu-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O WKOBSJOZRJJVRZ-FXQIFTODSA-N 0.000 description 2
- XYTNPQNAZREREP-XQXXSGGOSA-N Ala-Glu-Thr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O XYTNPQNAZREREP-XQXXSGGOSA-N 0.000 description 2
- ATAKEVCGTRZKLI-UWJYBYFXSA-N Ala-His-His Chemical compound C([C@H](NC(=O)[C@@H](N)C)C(=O)N[C@@H](CC=1NC=NC=1)C(O)=O)C1=CN=CN1 ATAKEVCGTRZKLI-UWJYBYFXSA-N 0.000 description 2
- OYJCVIGKMXUVKB-GARJFASQSA-N Ala-Leu-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@@H]1C(=O)O)N OYJCVIGKMXUVKB-GARJFASQSA-N 0.000 description 2
- YEBZNKPPOHFZJM-BPNCWPANSA-N Ala-Tyr-Val Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C(C)C)C(O)=O YEBZNKPPOHFZJM-BPNCWPANSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 2
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 2
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 description 2
- 108010039627 Aprotinin Proteins 0.000 description 2
- USNSOPDIZILSJP-FXQIFTODSA-N Arg-Asn-Asn Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O USNSOPDIZILSJP-FXQIFTODSA-N 0.000 description 2
- GDVDRMUYICMNFJ-CIUDSAMLSA-N Arg-Cys-Glu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(O)=O)C(O)=O GDVDRMUYICMNFJ-CIUDSAMLSA-N 0.000 description 2
- BEXGZLUHRXTZCC-CIUDSAMLSA-N Arg-Gln-Ser Chemical compound C(C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CO)C(=O)O)N)CN=C(N)N BEXGZLUHRXTZCC-CIUDSAMLSA-N 0.000 description 2
- NYZGVTGOMPHSJW-CIUDSAMLSA-N Arg-Glu-Cys Chemical compound C(C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CS)C(=O)O)N)CN=C(N)N NYZGVTGOMPHSJW-CIUDSAMLSA-N 0.000 description 2
- HQIZDMIGUJOSNI-IUCAKERBSA-N Arg-Gly-Arg Chemical compound N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(O)=O HQIZDMIGUJOSNI-IUCAKERBSA-N 0.000 description 2
- GXXWTNKNFFKTJB-NAKRPEOUSA-N Arg-Ile-Ser Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O GXXWTNKNFFKTJB-NAKRPEOUSA-N 0.000 description 2
- LVMUGODRNHFGRA-AVGNSLFASA-N Arg-Leu-Arg Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O LVMUGODRNHFGRA-AVGNSLFASA-N 0.000 description 2
- UHFUZWSZQKMDSX-DCAQKATOSA-N Arg-Leu-Asn Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N UHFUZWSZQKMDSX-DCAQKATOSA-N 0.000 description 2
- UZGFHWIJWPUPOH-IHRRRGAJSA-N Arg-Leu-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N UZGFHWIJWPUPOH-IHRRRGAJSA-N 0.000 description 2
- SSZGOKWBHLOCHK-DCAQKATOSA-N Arg-Lys-Asn Chemical compound NC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CCCN=C(N)N SSZGOKWBHLOCHK-DCAQKATOSA-N 0.000 description 2
- BTJVOUQWFXABOI-IHRRRGAJSA-N Arg-Lys-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CCCNC(N)=N BTJVOUQWFXABOI-IHRRRGAJSA-N 0.000 description 2
- KZXPVYVSHUJCEO-ULQDDVLXSA-N Arg-Phe-Lys Chemical compound NC(=N)NCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CCCCN)C(O)=O)CC1=CC=CC=C1 KZXPVYVSHUJCEO-ULQDDVLXSA-N 0.000 description 2
- ISJWBVIYRBAXEB-CIUDSAMLSA-N Arg-Ser-Glu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(O)=O ISJWBVIYRBAXEB-CIUDSAMLSA-N 0.000 description 2
- JOTRDIXZHNQYGP-DCAQKATOSA-N Arg-Ser-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CCCN=C(N)N)N JOTRDIXZHNQYGP-DCAQKATOSA-N 0.000 description 2
- FRBAHXABMQXSJQ-FXQIFTODSA-N Arg-Ser-Ser Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O FRBAHXABMQXSJQ-FXQIFTODSA-N 0.000 description 2
- UVTGNSWSRSCPLP-UHFFFAOYSA-N Arg-Tyr Natural products NC(CCNC(=N)N)C(=O)NC(Cc1ccc(O)cc1)C(=O)O UVTGNSWSRSCPLP-UHFFFAOYSA-N 0.000 description 2
- 239000004475 Arginine Substances 0.000 description 2
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- DXZNJWFECGJCQR-FXQIFTODSA-N Asn-Asn-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC(=O)N)N DXZNJWFECGJCQR-FXQIFTODSA-N 0.000 description 2
- MSBDSTRUMZFSEU-PEFMBERDSA-N Asn-Glu-Ile Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O MSBDSTRUMZFSEU-PEFMBERDSA-N 0.000 description 2
- DXVMJJNAOVECBA-WHFBIAKZSA-N Asn-Gly-Asn Chemical compound NC(=O)C[C@H](N)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(O)=O DXVMJJNAOVECBA-WHFBIAKZSA-N 0.000 description 2
- GWNMUVANAWDZTI-YUMQZZPRSA-N Asn-Gly-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CC(=O)N)N GWNMUVANAWDZTI-YUMQZZPRSA-N 0.000 description 2
- ACKNRKFVYUVWAC-ZPFDUUQYSA-N Asn-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(=O)N)N ACKNRKFVYUVWAC-ZPFDUUQYSA-N 0.000 description 2
- PLTGTJAZQRGMPP-FXQIFTODSA-N Asn-Pro-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CC(N)=O PLTGTJAZQRGMPP-FXQIFTODSA-N 0.000 description 2
- OOXUBGLNDRGOKT-FXQIFTODSA-N Asn-Ser-Arg Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O OOXUBGLNDRGOKT-FXQIFTODSA-N 0.000 description 2
- MKJBPDLENBUHQU-CIUDSAMLSA-N Asn-Ser-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O MKJBPDLENBUHQU-CIUDSAMLSA-N 0.000 description 2
- VLDRQOHCMKCXLY-SRVKXCTJSA-N Asn-Ser-Phe Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O VLDRQOHCMKCXLY-SRVKXCTJSA-N 0.000 description 2
- AMGQTNHANMRPOE-LKXGYXEUSA-N Asn-Thr-Ser Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O AMGQTNHANMRPOE-LKXGYXEUSA-N 0.000 description 2
- LTDGPJKGJDIBQD-LAEOZQHASA-N Asn-Val-Gln Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O LTDGPJKGJDIBQD-LAEOZQHASA-N 0.000 description 2
- IXIWEFWRKIUMQX-DCAQKATOSA-N Asp-Arg-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](N)CC(O)=O IXIWEFWRKIUMQX-DCAQKATOSA-N 0.000 description 2
- GHODABZPVZMWCE-FXQIFTODSA-N Asp-Glu-Glu Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O GHODABZPVZMWCE-FXQIFTODSA-N 0.000 description 2
- WYOSXGYAKZQPGF-SRVKXCTJSA-N Asp-His-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)NC(=O)[C@H](CC(=O)O)N WYOSXGYAKZQPGF-SRVKXCTJSA-N 0.000 description 2
- UMHUHHJMEXNSIV-CIUDSAMLSA-N Asp-Leu-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC(O)=O UMHUHHJMEXNSIV-CIUDSAMLSA-N 0.000 description 2
- KRQFMDNIUOVRIF-KKUMJFAQSA-N Asp-Phe-His Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)NC(=O)[C@H](CC(=O)O)N KRQFMDNIUOVRIF-KKUMJFAQSA-N 0.000 description 2
- WMLFFCRUSPNENW-ZLUOBGJFSA-N Asp-Ser-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O WMLFFCRUSPNENW-ZLUOBGJFSA-N 0.000 description 2
- KGHLGJAXYSVNJP-WHFBIAKZSA-N Asp-Ser-Gly Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O KGHLGJAXYSVNJP-WHFBIAKZSA-N 0.000 description 2
- HRVQDZOWMLFAOD-BIIVOSGPSA-N Asp-Ser-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CO)NC(=O)[C@H](CC(=O)O)N)C(=O)O HRVQDZOWMLFAOD-BIIVOSGPSA-N 0.000 description 2
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 2
- 101001011741 Bos taurus Insulin Proteins 0.000 description 2
- 101100314454 Caenorhabditis elegans tra-1 gene Proteins 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 108010009685 Cholinergic Receptors Proteins 0.000 description 2
- 108091033380 Coding strand Proteins 0.000 description 2
- 108060005980 Collagenase Proteins 0.000 description 2
- 102000029816 Collagenase Human genes 0.000 description 2
- 108091035707 Consensus sequence Proteins 0.000 description 2
- CPTUXCUWQIBZIF-ZLUOBGJFSA-N Cys-Asn-Ser Chemical compound SC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O CPTUXCUWQIBZIF-ZLUOBGJFSA-N 0.000 description 2
- ZXCAQANTQWBICD-DCAQKATOSA-N Cys-Lys-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CS)N ZXCAQANTQWBICD-DCAQKATOSA-N 0.000 description 2
- PGBLJHDDKCVSTC-CIUDSAMLSA-N Cys-Met-Gln Chemical compound CSCC[C@H](NC(=O)[C@@H](N)CS)C(=O)N[C@@H](CCC(N)=O)C(O)=O PGBLJHDDKCVSTC-CIUDSAMLSA-N 0.000 description 2
- BSGXXYRIDXUEOM-IHRRRGAJSA-N Cys-Phe-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)[C@H](CS)N BSGXXYRIDXUEOM-IHRRRGAJSA-N 0.000 description 2
- JRZMCSIUYGSJKP-ZKWXMUAHSA-N Cys-Val-Asn Chemical compound SC[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O JRZMCSIUYGSJKP-ZKWXMUAHSA-N 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 229920002271 DEAE-Sepharose Polymers 0.000 description 2
- 230000004544 DNA amplification Effects 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 2
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 2
- 102100024785 Fibroblast growth factor 2 Human genes 0.000 description 2
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 2
- MAGNEQBFSBREJL-DCAQKATOSA-N Gln-Glu-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCC(=O)N)N MAGNEQBFSBREJL-DCAQKATOSA-N 0.000 description 2
- GQZDDFRXSDGUNG-YVNDNENWSA-N Gln-Ile-Gln Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(N)=O)C(O)=O GQZDDFRXSDGUNG-YVNDNENWSA-N 0.000 description 2
- JRHPEMVLTRADLJ-AVGNSLFASA-N Gln-Lys-Lys Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCC(=O)N)N JRHPEMVLTRADLJ-AVGNSLFASA-N 0.000 description 2
- LPIKVBWNNVFHCQ-GUBZILKMSA-N Gln-Ser-Leu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O LPIKVBWNNVFHCQ-GUBZILKMSA-N 0.000 description 2
- SYZZMPFLOLSMHL-XHNCKOQMSA-N Gln-Ser-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CO)NC(=O)[C@H](CCC(=O)N)N)C(=O)O SYZZMPFLOLSMHL-XHNCKOQMSA-N 0.000 description 2
- VLOLPWWCNKWRNB-LOKLDPHHSA-N Gln-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCC(=O)N)N)O VLOLPWWCNKWRNB-LOKLDPHHSA-N 0.000 description 2
- SJMJMEWQMBJYPR-DZKIICNBSA-N Gln-Tyr-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H](CCC(=O)N)N SJMJMEWQMBJYPR-DZKIICNBSA-N 0.000 description 2
- CVPXINNKRTZBMO-CIUDSAMLSA-N Glu-Arg-Asn Chemical compound C(C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CCC(=O)O)N)CN=C(N)N CVPXINNKRTZBMO-CIUDSAMLSA-N 0.000 description 2
- CGYDXNKRIMJMLV-GUBZILKMSA-N Glu-Arg-Glu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O CGYDXNKRIMJMLV-GUBZILKMSA-N 0.000 description 2
- NLKVNZUFDPWPNL-YUMQZZPRSA-N Glu-Arg-Gly Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O NLKVNZUFDPWPNL-YUMQZZPRSA-N 0.000 description 2
- ZXQPJYWZSFGWJB-AVGNSLFASA-N Glu-Cys-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CCC(=O)O)N ZXQPJYWZSFGWJB-AVGNSLFASA-N 0.000 description 2
- ILGFBUGLBSAQQB-GUBZILKMSA-N Glu-Glu-Arg Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O ILGFBUGLBSAQQB-GUBZILKMSA-N 0.000 description 2
- AIGROOHQXCACHL-WDSKDSINSA-N Glu-Gly-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](C)C(O)=O AIGROOHQXCACHL-WDSKDSINSA-N 0.000 description 2
- NJPQBTJSYCKCNS-HVTMNAMFSA-N Glu-His-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](CCC(=O)O)N NJPQBTJSYCKCNS-HVTMNAMFSA-N 0.000 description 2
- ZCOJVESMNGBGLF-GRLWGSQLSA-N Glu-Ile-Ile Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O ZCOJVESMNGBGLF-GRLWGSQLSA-N 0.000 description 2
- VMKCPNBBPGGQBJ-GUBZILKMSA-N Glu-Leu-Asn Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CCC(=O)O)N VMKCPNBBPGGQBJ-GUBZILKMSA-N 0.000 description 2
- IOUQWHIEQYQVFD-JYJNAYRXSA-N Glu-Leu-Tyr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O IOUQWHIEQYQVFD-JYJNAYRXSA-N 0.000 description 2
- BCYGDJXHAGZNPQ-DCAQKATOSA-N Glu-Lys-Glu Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(O)=O BCYGDJXHAGZNPQ-DCAQKATOSA-N 0.000 description 2
- YGLCLCMAYUYZSG-AVGNSLFASA-N Glu-Lys-His Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 YGLCLCMAYUYZSG-AVGNSLFASA-N 0.000 description 2
- SOEPMWQCTJITPZ-SRVKXCTJSA-N Glu-Met-Lys Chemical compound CSCC[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCC(=O)O)N SOEPMWQCTJITPZ-SRVKXCTJSA-N 0.000 description 2
- JZJGEKDPWVJOLD-QEWYBTABSA-N Glu-Phe-Ile Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O JZJGEKDPWVJOLD-QEWYBTABSA-N 0.000 description 2
- ALMBZBOCGSVSAI-ACZMJKKPSA-N Glu-Ser-Asn Chemical compound C(CC(=O)O)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(=O)N)C(=O)O)N ALMBZBOCGSVSAI-ACZMJKKPSA-N 0.000 description 2
- ZAPFAWQHBOHWLL-GUBZILKMSA-N Glu-Ser-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(=O)O)N ZAPFAWQHBOHWLL-GUBZILKMSA-N 0.000 description 2
- HMJULNMJWOZNFI-XHNCKOQMSA-N Glu-Ser-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CO)NC(=O)[C@H](CCC(=O)O)N)C(=O)O HMJULNMJWOZNFI-XHNCKOQMSA-N 0.000 description 2
- QEJKKJNDDDPSMU-KKUMJFAQSA-N Glu-Tyr-Met Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCSC)C(O)=O QEJKKJNDDDPSMU-KKUMJFAQSA-N 0.000 description 2
- VXEFAWJTFAUDJK-AVGNSLFASA-N Glu-Tyr-Ser Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CCC(=O)O)N)O VXEFAWJTFAUDJK-AVGNSLFASA-N 0.000 description 2
- QIZJOTQTCAGKPU-KWQFWETISA-N Gly-Ala-Tyr Chemical compound [NH3+]CC(=O)N[C@@H](C)C(=O)N[C@H](C([O-])=O)CC1=CC=C(O)C=C1 QIZJOTQTCAGKPU-KWQFWETISA-N 0.000 description 2
- AIJAPFVDBFYNKN-WHFBIAKZSA-N Gly-Asn-Asp Chemical compound C([C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)CN)C(=O)N AIJAPFVDBFYNKN-WHFBIAKZSA-N 0.000 description 2
- NZAFOTBEULLEQB-WDSKDSINSA-N Gly-Asn-Glu Chemical compound C(CC(=O)O)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)CN NZAFOTBEULLEQB-WDSKDSINSA-N 0.000 description 2
- CUYLIWAAAYJKJH-RYUDHWBXSA-N Gly-Glu-Tyr Chemical compound NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 CUYLIWAAAYJKJH-RYUDHWBXSA-N 0.000 description 2
- GDOZQTNZPCUARW-YFKPBYRVSA-N Gly-Gly-Glu Chemical compound NCC(=O)NCC(=O)N[C@H](C(O)=O)CCC(O)=O GDOZQTNZPCUARW-YFKPBYRVSA-N 0.000 description 2
- PDUHNKAFQXQNLH-ZETCQYMHSA-N Gly-Lys-Gly Chemical compound NCCCC[C@H](NC(=O)CN)C(=O)NCC(O)=O PDUHNKAFQXQNLH-ZETCQYMHSA-N 0.000 description 2
- VEPBEGNDJYANCF-QWRGUYRKSA-N Gly-Lys-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CCCCN VEPBEGNDJYANCF-QWRGUYRKSA-N 0.000 description 2
- CSMYMGFCEJWALV-WDSKDSINSA-N Gly-Ser-Gln Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(N)=O CSMYMGFCEJWALV-WDSKDSINSA-N 0.000 description 2
- SOEGEPHNZOISMT-BYPYZUCNSA-N Gly-Ser-Gly Chemical compound NCC(=O)N[C@@H](CO)C(=O)NCC(O)=O SOEGEPHNZOISMT-BYPYZUCNSA-N 0.000 description 2
- POJJAZJHBGXEGM-YUMQZZPRSA-N Gly-Ser-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)CN POJJAZJHBGXEGM-YUMQZZPRSA-N 0.000 description 2
- JQFILXICXLDTRR-FBCQKBJTSA-N Gly-Thr-Gly Chemical compound NCC(=O)N[C@@H]([C@H](O)C)C(=O)NCC(O)=O JQFILXICXLDTRR-FBCQKBJTSA-N 0.000 description 2
- FFALDIDGPLUDKV-ZDLURKLDSA-N Gly-Thr-Ser Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O FFALDIDGPLUDKV-ZDLURKLDSA-N 0.000 description 2
- 108010031186 Glycoside Hydrolases Proteins 0.000 description 2
- 102000005744 Glycoside Hydrolases Human genes 0.000 description 2
- JBCLFWXMTIKCCB-UHFFFAOYSA-N H-Gly-Phe-OH Natural products NCC(=O)NC(C(O)=O)CC1=CC=CC=C1 JBCLFWXMTIKCCB-UHFFFAOYSA-N 0.000 description 2
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 2
- ZPVJJPAIUZLSNE-DCAQKATOSA-N His-Arg-Ser Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(O)=O ZPVJJPAIUZLSNE-DCAQKATOSA-N 0.000 description 2
- JWTKVPMQCCRPQY-SRVKXCTJSA-N His-Asn-Leu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O JWTKVPMQCCRPQY-SRVKXCTJSA-N 0.000 description 2
- ZZLWLWSUIBSMNP-CIUDSAMLSA-N His-Asp-Ser Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O ZZLWLWSUIBSMNP-CIUDSAMLSA-N 0.000 description 2
- IWXMHXYOACDSIA-PYJNHQTQSA-N His-Ile-Val Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(O)=O IWXMHXYOACDSIA-PYJNHQTQSA-N 0.000 description 2
- SKYULSWNBYAQMG-IHRRRGAJSA-N His-Leu-Arg Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O SKYULSWNBYAQMG-IHRRRGAJSA-N 0.000 description 2
- AIPUZFXMXAHZKY-QWRGUYRKSA-N His-Leu-Gly Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O AIPUZFXMXAHZKY-QWRGUYRKSA-N 0.000 description 2
- TWROVBNEHJSXDG-IHRRRGAJSA-N His-Leu-Val Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O TWROVBNEHJSXDG-IHRRRGAJSA-N 0.000 description 2
- JMSONHOUHFDOJH-GUBZILKMSA-N His-Ser-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC1=CN=CN1 JMSONHOUHFDOJH-GUBZILKMSA-N 0.000 description 2
- VIJMRAIWYWRXSR-CIUDSAMLSA-N His-Ser-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC1=CN=CN1 VIJMRAIWYWRXSR-CIUDSAMLSA-N 0.000 description 2
- ILUVWFTXAUYOBW-CUJWVEQBSA-N His-Ser-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC1=CN=CN1)N)O ILUVWFTXAUYOBW-CUJWVEQBSA-N 0.000 description 2
- RXKFKJVJVHLRIE-XIRDDKMYSA-N His-Ser-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC3=CN=CN3)N RXKFKJVJVHLRIE-XIRDDKMYSA-N 0.000 description 2
- DQZCEKQPSOBNMJ-NKIYYHGXSA-N His-Thr-Glu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(O)=O DQZCEKQPSOBNMJ-NKIYYHGXSA-N 0.000 description 2
- LQSBBHNVAVNZSX-GHCJXIJMSA-N Ile-Ala-Asn Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CC(=O)N)C(=O)O)N LQSBBHNVAVNZSX-GHCJXIJMSA-N 0.000 description 2
- JRHFQUPIZOYKQP-KBIXCLLPSA-N Ile-Ala-Glu Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCC(O)=O JRHFQUPIZOYKQP-KBIXCLLPSA-N 0.000 description 2
- ATXGFMOBVKSOMK-PEDHHIEDSA-N Ile-Arg-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)O)N ATXGFMOBVKSOMK-PEDHHIEDSA-N 0.000 description 2
- KUHFPGIVBOCRMV-MNXVOIDGSA-N Ile-Gln-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC(C)C)C(=O)O)N KUHFPGIVBOCRMV-MNXVOIDGSA-N 0.000 description 2
- LKACSKJPTFSBHR-MNXVOIDGSA-N Ile-Gln-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCCCN)C(=O)O)N LKACSKJPTFSBHR-MNXVOIDGSA-N 0.000 description 2
- GVKKVHNRTUFCCE-BJDJZHNGSA-N Ile-Leu-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)O)N GVKKVHNRTUFCCE-BJDJZHNGSA-N 0.000 description 2
- FFAUOCITXBMRBT-YTFOTSKYSA-N Ile-Lys-Ile Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O FFAUOCITXBMRBT-YTFOTSKYSA-N 0.000 description 2
- BJECXJHLUJXPJQ-PYJNHQTQSA-N Ile-Pro-His Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N BJECXJHLUJXPJQ-PYJNHQTQSA-N 0.000 description 2
- VGSPNSSCMOHRRR-BJDJZHNGSA-N Ile-Ser-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)O)N VGSPNSSCMOHRRR-BJDJZHNGSA-N 0.000 description 2
- PXKACEXYLPBMAD-JBDRJPRFSA-N Ile-Ser-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)O)N PXKACEXYLPBMAD-JBDRJPRFSA-N 0.000 description 2
- HJDZMPFEXINXLO-QPHKQPEJSA-N Ile-Thr-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)O)N HJDZMPFEXINXLO-QPHKQPEJSA-N 0.000 description 2
- 102000016844 Immunoglobulin-like domains Human genes 0.000 description 2
- 108050006430 Immunoglobulin-like domains Proteins 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 108010065920 Insulin Lispro Proteins 0.000 description 2
- 108010002350 Interleukin-2 Proteins 0.000 description 2
- 101710177504 Kit ligand Proteins 0.000 description 2
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 2
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 2
- SENJXOPIZNYLHU-UHFFFAOYSA-N L-leucyl-L-arginine Natural products CC(C)CC(N)C(=O)NC(C(O)=O)CCCN=C(N)N SENJXOPIZNYLHU-UHFFFAOYSA-N 0.000 description 2
- CNNQBZRGQATKNY-DCAQKATOSA-N Leu-Arg-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CS)C(=O)O)N CNNQBZRGQATKNY-DCAQKATOSA-N 0.000 description 2
- HASRFYOMVPJRPU-SRVKXCTJSA-N Leu-Arg-Glu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCC(O)=O)C(O)=O HASRFYOMVPJRPU-SRVKXCTJSA-N 0.000 description 2
- UCOCBWDBHCUPQP-DCAQKATOSA-N Leu-Arg-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(O)=O UCOCBWDBHCUPQP-DCAQKATOSA-N 0.000 description 2
- IGUOAYLTQJLPPD-DCAQKATOSA-N Leu-Asn-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N IGUOAYLTQJLPPD-DCAQKATOSA-N 0.000 description 2
- OIARJGNVARWKFP-YUMQZZPRSA-N Leu-Asn-Gly Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O OIARJGNVARWKFP-YUMQZZPRSA-N 0.000 description 2
- FMEICTQWUKNAGC-YUMQZZPRSA-N Leu-Gly-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(O)=O FMEICTQWUKNAGC-YUMQZZPRSA-N 0.000 description 2
- CCQLQKZTXZBXTN-NHCYSSNCSA-N Leu-Gly-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H]([C@@H](C)CC)C(O)=O CCQLQKZTXZBXTN-NHCYSSNCSA-N 0.000 description 2
- KEVYYIMVELOXCT-KBPBESRZSA-N Leu-Gly-Phe Chemical compound CC(C)C[C@H]([NH3+])C(=O)NCC(=O)N[C@H](C([O-])=O)CC1=CC=CC=C1 KEVYYIMVELOXCT-KBPBESRZSA-N 0.000 description 2
- YWYQSLOTVIRCFE-SRVKXCTJSA-N Leu-His-Asp Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(O)=O)C(O)=O YWYQSLOTVIRCFE-SRVKXCTJSA-N 0.000 description 2
- DPURXCQCHSQPAN-AVGNSLFASA-N Leu-Pro-Pro Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 DPURXCQCHSQPAN-AVGNSLFASA-N 0.000 description 2
- IRMLZWSRWSGTOP-CIUDSAMLSA-N Leu-Ser-Ala Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O IRMLZWSRWSGTOP-CIUDSAMLSA-N 0.000 description 2
- KZZCOWMDDXDKSS-CIUDSAMLSA-N Leu-Ser-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O KZZCOWMDDXDKSS-CIUDSAMLSA-N 0.000 description 2
- VHTIZYYHIUHMCA-JYJNAYRXSA-N Leu-Tyr-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCC(N)=O)C(O)=O VHTIZYYHIUHMCA-JYJNAYRXSA-N 0.000 description 2
- XZNJZXJZBMBGGS-NHCYSSNCSA-N Leu-Val-Asn Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O XZNJZXJZBMBGGS-NHCYSSNCSA-N 0.000 description 2
- YQFZRHYZLARWDY-IHRRRGAJSA-N Leu-Val-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCCN YQFZRHYZLARWDY-IHRRRGAJSA-N 0.000 description 2
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 2
- BTSXLXFPMZXVPR-DLOVCJGASA-N Lys-Ala-His Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CCCCN)N BTSXLXFPMZXVPR-DLOVCJGASA-N 0.000 description 2
- ZQCVMVCVPFYXHZ-SRVKXCTJSA-N Lys-Asn-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(O)=O)CCCCN ZQCVMVCVPFYXHZ-SRVKXCTJSA-N 0.000 description 2
- LZWNAOIMTLNMDW-NHCYSSNCSA-N Lys-Asn-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCCCN)N LZWNAOIMTLNMDW-NHCYSSNCSA-N 0.000 description 2
- ZAWOJFFMBANLGE-CIUDSAMLSA-N Lys-Cys-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CCCCN)N ZAWOJFFMBANLGE-CIUDSAMLSA-N 0.000 description 2
- NDSNUWJPZKTFAR-DCAQKATOSA-N Lys-Cys-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)[C@H](CS)NC(=O)[C@@H](N)CCCCN NDSNUWJPZKTFAR-DCAQKATOSA-N 0.000 description 2
- DRCILAJNUJKAHC-SRVKXCTJSA-N Lys-Glu-Arg Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O DRCILAJNUJKAHC-SRVKXCTJSA-N 0.000 description 2
- VQXAVLQBQJMENB-SRVKXCTJSA-N Lys-Glu-Met Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCSC)C(O)=O VQXAVLQBQJMENB-SRVKXCTJSA-N 0.000 description 2
- NKKFVJRLCCUJNA-QWRGUYRKSA-N Lys-Gly-Lys Chemical compound NCCCC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCCN NKKFVJRLCCUJNA-QWRGUYRKSA-N 0.000 description 2
- QKXZCUCBFPEXNK-KKUMJFAQSA-N Lys-Leu-His Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 QKXZCUCBFPEXNK-KKUMJFAQSA-N 0.000 description 2
- LJADEBULDNKJNK-IHRRRGAJSA-N Lys-Leu-Val Chemical compound CC(C)C[C@H](NC(=O)[C@@H](N)CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O LJADEBULDNKJNK-IHRRRGAJSA-N 0.000 description 2
- WBSCNDJQPKSPII-KKUMJFAQSA-N Lys-Lys-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(O)=O WBSCNDJQPKSPII-KKUMJFAQSA-N 0.000 description 2
- ATNKHRAIZCMCCN-BZSNNMDCSA-N Lys-Lys-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)N ATNKHRAIZCMCCN-BZSNNMDCSA-N 0.000 description 2
- YXPJCVNIDDKGOE-MELADBBJSA-N Lys-Lys-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)N)C(=O)O YXPJCVNIDDKGOE-MELADBBJSA-N 0.000 description 2
- SVSQSPICRKBMSZ-SRVKXCTJSA-N Lys-Pro-Gln Chemical compound [H]N[C@@H](CCCCN)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(O)=O SVSQSPICRKBMSZ-SRVKXCTJSA-N 0.000 description 2
- CNGOEHJCLVCJHN-SRVKXCTJSA-N Lys-Pro-Glu Chemical compound NCCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O CNGOEHJCLVCJHN-SRVKXCTJSA-N 0.000 description 2
- PDIDTSZKKFEDMB-UWVGGRQHSA-N Lys-Pro-Gly Chemical compound [H]N[C@@H](CCCCN)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O PDIDTSZKKFEDMB-UWVGGRQHSA-N 0.000 description 2
- DNWBUCHHMRQWCZ-GUBZILKMSA-N Lys-Ser-Gln Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(N)=O DNWBUCHHMRQWCZ-GUBZILKMSA-N 0.000 description 2
- JMNRXRPBHFGXQX-GUBZILKMSA-N Lys-Ser-Glu Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(O)=O JMNRXRPBHFGXQX-GUBZILKMSA-N 0.000 description 2
- YCJCEMKOZOYBEF-OEAJRASXSA-N Lys-Thr-Phe Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O YCJCEMKOZOYBEF-OEAJRASXSA-N 0.000 description 2
- 239000004472 Lysine Substances 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 102000000422 Matrix Metalloproteinase 3 Human genes 0.000 description 2
- QXEVZBXTDTVPCP-GMOBBJLQSA-N Met-Asn-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCSC)N QXEVZBXTDTVPCP-GMOBBJLQSA-N 0.000 description 2
- IZLCDZDNZFEDHB-DCAQKATOSA-N Met-Cys-Lys Chemical compound CSCC[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCCN)C(=O)O)N IZLCDZDNZFEDHB-DCAQKATOSA-N 0.000 description 2
- VAGCEUUEMMXFEX-GUBZILKMSA-N Met-Met-Asn Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(O)=O VAGCEUUEMMXFEX-GUBZILKMSA-N 0.000 description 2
- VQILILSLEFDECU-GUBZILKMSA-N Met-Pro-Ala Chemical compound [H]N[C@@H](CCSC)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O VQILILSLEFDECU-GUBZILKMSA-N 0.000 description 2
- MIXPUVSPPOWTCR-FXQIFTODSA-N Met-Ser-Ser Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O MIXPUVSPPOWTCR-FXQIFTODSA-N 0.000 description 2
- ZBLSZPYQQRIHQU-RCWTZXSCSA-N Met-Thr-Val Chemical compound CSCC[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(O)=O ZBLSZPYQQRIHQU-RCWTZXSCSA-N 0.000 description 2
- LPNWWHBFXPNHJG-AVGNSLFASA-N Met-Val-Lys Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCCN LPNWWHBFXPNHJG-AVGNSLFASA-N 0.000 description 2
- NTNWOCRCBQPEKQ-YFKPBYRVSA-N N(omega)-methyl-L-arginine Chemical compound CN=C(N)NCCC[C@H](N)C(O)=O NTNWOCRCBQPEKQ-YFKPBYRVSA-N 0.000 description 2
- XZFYRXDAULDNFX-UHFFFAOYSA-N N-L-cysteinyl-L-phenylalanine Natural products SCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 XZFYRXDAULDNFX-UHFFFAOYSA-N 0.000 description 2
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 2
- 108010079364 N-glycylalanine Proteins 0.000 description 2
- 108010002311 N-glycylglutamic acid Proteins 0.000 description 2
- 108010087066 N2-tryptophyllysine Proteins 0.000 description 2
- 102000001068 Neural Cell Adhesion Molecules Human genes 0.000 description 2
- 239000000020 Nitrocellulose Substances 0.000 description 2
- 102000003992 Peroxidases Human genes 0.000 description 2
- NAOVYENZCWFBDG-BZSNNMDCSA-N Phe-His-His Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC=1NC=NC=1)C(O)=O)C1=CC=CC=C1 NAOVYENZCWFBDG-BZSNNMDCSA-N 0.000 description 2
- DVOCGBNHAUHKHJ-DKIMLUQUSA-N Phe-Ile-Leu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(O)=O DVOCGBNHAUHKHJ-DKIMLUQUSA-N 0.000 description 2
- RMKGXGPQIPLTFC-KKUMJFAQSA-N Phe-Lys-Asn Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O RMKGXGPQIPLTFC-KKUMJFAQSA-N 0.000 description 2
- WZEWCHQHNCMBEN-PMVMPFDFSA-N Phe-Lys-Trp Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC2=CNC3=CC=CC=C32)C(=O)O)N WZEWCHQHNCMBEN-PMVMPFDFSA-N 0.000 description 2
- RTUWVJVJSMOGPL-KKUMJFAQSA-N Phe-Met-Glu Chemical compound CSCC[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N RTUWVJVJSMOGPL-KKUMJFAQSA-N 0.000 description 2
- MCIXMYKSPQUMJG-SRVKXCTJSA-N Phe-Ser-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O MCIXMYKSPQUMJG-SRVKXCTJSA-N 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 108010004729 Phycoerythrin Proteins 0.000 description 2
- 108091036407 Polyadenylation Proteins 0.000 description 2
- IHCXPSYCHXFXKT-DCAQKATOSA-N Pro-Arg-Glu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O IHCXPSYCHXFXKT-DCAQKATOSA-N 0.000 description 2
- BNBBNGZZKQUWCD-IUCAKERBSA-N Pro-Arg-Gly Chemical compound NC(N)=NCCC[C@@H](C(=O)NCC(O)=O)NC(=O)[C@@H]1CCCN1 BNBBNGZZKQUWCD-IUCAKERBSA-N 0.000 description 2
- VCYJKOLZYPYGJV-AVGNSLFASA-N Pro-Arg-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(O)=O VCYJKOLZYPYGJV-AVGNSLFASA-N 0.000 description 2
- TXPUNZXZDVJUJQ-LPEHRKFASA-N Pro-Asn-Pro Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CC(=O)N)C(=O)N2CCC[C@@H]2C(=O)O TXPUNZXZDVJUJQ-LPEHRKFASA-N 0.000 description 2
- ODPIUQVTULPQEP-CIUDSAMLSA-N Pro-Gln-Asn Chemical compound NC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@@H]1CCCN1 ODPIUQVTULPQEP-CIUDSAMLSA-N 0.000 description 2
- FISHYTLIMUYTQY-GUBZILKMSA-N Pro-Gln-Gln Chemical compound NC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H]1CCCN1 FISHYTLIMUYTQY-GUBZILKMSA-N 0.000 description 2
- KIPIKSXPPLABPN-CIUDSAMLSA-N Pro-Glu-Asn Chemical compound NC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H]1CCCN1 KIPIKSXPPLABPN-CIUDSAMLSA-N 0.000 description 2
- UIMCLYYSUCIUJM-UWVGGRQHSA-N Pro-Gly-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H]1CCCN1 UIMCLYYSUCIUJM-UWVGGRQHSA-N 0.000 description 2
- YXHYJEPDKSYPSQ-AVGNSLFASA-N Pro-Leu-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H]1CCCN1 YXHYJEPDKSYPSQ-AVGNSLFASA-N 0.000 description 2
- QCMYJBKTMIWZAP-AVGNSLFASA-N Pro-Met-Lys Chemical compound CSCC[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@@H]1CCCN1 QCMYJBKTMIWZAP-AVGNSLFASA-N 0.000 description 2
- JLMZKEQFMVORMA-SRVKXCTJSA-N Pro-Pro-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 JLMZKEQFMVORMA-SRVKXCTJSA-N 0.000 description 2
- RFWXYTJSVDUBBZ-DCAQKATOSA-N Pro-Pro-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 RFWXYTJSVDUBBZ-DCAQKATOSA-N 0.000 description 2
- GOMUXSCOIWIJFP-GUBZILKMSA-N Pro-Ser-Arg Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O GOMUXSCOIWIJFP-GUBZILKMSA-N 0.000 description 2
- RNEFESSBTOQSAC-DCAQKATOSA-N Pro-Ser-His Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O RNEFESSBTOQSAC-DCAQKATOSA-N 0.000 description 2
- 102000004022 Protein-Tyrosine Kinases Human genes 0.000 description 2
- 108090000412 Protein-Tyrosine Kinases Proteins 0.000 description 2
- 239000012614 Q-Sepharose Substances 0.000 description 2
- 101150040459 RAS gene Proteins 0.000 description 2
- 101500027531 Rattus norvegicus Transforming growth factor alpha Proteins 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- 208000021063 Respiratory fume inhalation disease Diseases 0.000 description 2
- MWMKFWJYRRGXOR-ZLUOBGJFSA-N Ser-Ala-Asn Chemical compound N[C@H](C(=O)N[C@H](C(=O)N[C@H](C(=O)O)CC(N)=O)C)CO MWMKFWJYRRGXOR-ZLUOBGJFSA-N 0.000 description 2
- YQHZVYJAGWMHES-ZLUOBGJFSA-N Ser-Ala-Ser Chemical compound OC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O YQHZVYJAGWMHES-ZLUOBGJFSA-N 0.000 description 2
- JPIDMRXXNMIVKY-VZFHVOOUSA-N Ser-Ala-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O JPIDMRXXNMIVKY-VZFHVOOUSA-N 0.000 description 2
- VQBLHWSPVYYZTB-DCAQKATOSA-N Ser-Arg-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CO)N VQBLHWSPVYYZTB-DCAQKATOSA-N 0.000 description 2
- VAUMZJHYZQXZBQ-WHFBIAKZSA-N Ser-Asn-Gly Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O VAUMZJHYZQXZBQ-WHFBIAKZSA-N 0.000 description 2
- ZOHGLPQGEHSLPD-FXQIFTODSA-N Ser-Gln-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O ZOHGLPQGEHSLPD-FXQIFTODSA-N 0.000 description 2
- LALNXSXEYFUUDD-GUBZILKMSA-N Ser-Glu-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O LALNXSXEYFUUDD-GUBZILKMSA-N 0.000 description 2
- GRSLLFZTTLBOQX-CIUDSAMLSA-N Ser-Glu-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CO)N GRSLLFZTTLBOQX-CIUDSAMLSA-N 0.000 description 2
- VQBCMLMPEWPUTB-ACZMJKKPSA-N Ser-Glu-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O VQBCMLMPEWPUTB-ACZMJKKPSA-N 0.000 description 2
- WBINSDOPZHQPPM-AVGNSLFASA-N Ser-Glu-Tyr Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CO)N)O WBINSDOPZHQPPM-AVGNSLFASA-N 0.000 description 2
- WSTIOCFMWXNOCX-YUMQZZPRSA-N Ser-Gly-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CO)N WSTIOCFMWXNOCX-YUMQZZPRSA-N 0.000 description 2
- WEQAYODCJHZSJZ-KKUMJFAQSA-N Ser-His-Tyr Chemical compound C([C@H](NC(=O)[C@H](CO)N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CN=CN1 WEQAYODCJHZSJZ-KKUMJFAQSA-N 0.000 description 2
- RIAKPZVSNBBNRE-BJDJZHNGSA-N Ser-Ile-Leu Chemical compound OC[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(O)=O RIAKPZVSNBBNRE-BJDJZHNGSA-N 0.000 description 2
- MOINZPRHJGTCHZ-MMWGEVLESA-N Ser-Ile-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CO)N MOINZPRHJGTCHZ-MMWGEVLESA-N 0.000 description 2
- HDBOEVPDIDDEPC-CIUDSAMLSA-N Ser-Lys-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O HDBOEVPDIDDEPC-CIUDSAMLSA-N 0.000 description 2
- XUDRHBPSPAPDJP-SRVKXCTJSA-N Ser-Lys-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CO XUDRHBPSPAPDJP-SRVKXCTJSA-N 0.000 description 2
- UGGWCAFQPKANMW-FXQIFTODSA-N Ser-Met-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](C)C(O)=O UGGWCAFQPKANMW-FXQIFTODSA-N 0.000 description 2
- ZSLFCBHEINFXRS-LPEHRKFASA-N Ser-Met-Pro Chemical compound CSCC[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CO)N ZSLFCBHEINFXRS-LPEHRKFASA-N 0.000 description 2
- MQUZANJDFOQOBX-SRVKXCTJSA-N Ser-Phe-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(O)=O MQUZANJDFOQOBX-SRVKXCTJSA-N 0.000 description 2
- ADJDNJCSPNFFPI-FXQIFTODSA-N Ser-Pro-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CO ADJDNJCSPNFFPI-FXQIFTODSA-N 0.000 description 2
- QPPYAWVLAVXISR-DCAQKATOSA-N Ser-Pro-His Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CO)N)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O QPPYAWVLAVXISR-DCAQKATOSA-N 0.000 description 2
- GZGFSPWOMUKKCV-NAKRPEOUSA-N Ser-Pro-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CO GZGFSPWOMUKKCV-NAKRPEOUSA-N 0.000 description 2
- NMZXJDSKEGFDLJ-DCAQKATOSA-N Ser-Pro-Lys Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CO)N)C(=O)N[C@@H](CCCCN)C(=O)O NMZXJDSKEGFDLJ-DCAQKATOSA-N 0.000 description 2
- FLONGDPORFIVQW-XGEHTFHBSA-N Ser-Pro-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CO FLONGDPORFIVQW-XGEHTFHBSA-N 0.000 description 2
- BMKNXTJLHFIAAH-CIUDSAMLSA-N Ser-Ser-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O BMKNXTJLHFIAAH-CIUDSAMLSA-N 0.000 description 2
- CUXJENOFJXOSOZ-BIIVOSGPSA-N Ser-Ser-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CO)NC(=O)[C@H](CO)N)C(=O)O CUXJENOFJXOSOZ-BIIVOSGPSA-N 0.000 description 2
- PYTKULIABVRXSC-BWBBJGPYSA-N Ser-Ser-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O PYTKULIABVRXSC-BWBBJGPYSA-N 0.000 description 2
- KKKVOZNCLALMPV-XKBZYTNZSA-N Ser-Thr-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(O)=O KKKVOZNCLALMPV-XKBZYTNZSA-N 0.000 description 2
- ZWSZBWAFDZRBNM-UBHSHLNASA-N Ser-Trp-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CO)C(O)=O ZWSZBWAFDZRBNM-UBHSHLNASA-N 0.000 description 2
- FGBLCMLXHRPVOF-IHRRRGAJSA-N Ser-Tyr-Arg Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O FGBLCMLXHRPVOF-IHRRRGAJSA-N 0.000 description 2
- BEBVVQPDSHHWQL-NRPADANISA-N Ser-Val-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O BEBVVQPDSHHWQL-NRPADANISA-N 0.000 description 2
- MFQMZDPAZRZAPV-NAKRPEOUSA-N Ser-Val-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CO)N MFQMZDPAZRZAPV-NAKRPEOUSA-N 0.000 description 2
- JGUWRQWULDWNCM-FXQIFTODSA-N Ser-Val-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O JGUWRQWULDWNCM-FXQIFTODSA-N 0.000 description 2
- STGXWWBXWXZOER-MBLNEYKQSA-N Thr-Ala-His Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 STGXWWBXWXZOER-MBLNEYKQSA-N 0.000 description 2
- VUVCRYXYUUPGSB-GLLZPBPUSA-N Thr-Gln-Glu Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N)O VUVCRYXYUUPGSB-GLLZPBPUSA-N 0.000 description 2
- ONNSECRQFSTMCC-XKBZYTNZSA-N Thr-Glu-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O ONNSECRQFSTMCC-XKBZYTNZSA-N 0.000 description 2
- UBDDORVPVLEECX-FJXKBIBVSA-N Thr-Gly-Met Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CCSC)C(O)=O UBDDORVPVLEECX-FJXKBIBVSA-N 0.000 description 2
- AYCQVUUPIJHJTA-IXOXFDKPSA-N Thr-His-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(C)C)C(O)=O AYCQVUUPIJHJTA-IXOXFDKPSA-N 0.000 description 2
- XYFISNXATOERFZ-OSUNSFLBSA-N Thr-Ile-Val Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)O)NC(=O)[C@H]([C@@H](C)O)N XYFISNXATOERFZ-OSUNSFLBSA-N 0.000 description 2
- WKGAAMOJPMBBMC-IXOXFDKPSA-N Thr-Ser-Phe Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O WKGAAMOJPMBBMC-IXOXFDKPSA-N 0.000 description 2
- WPSKTVVMQCXPRO-BWBBJGPYSA-N Thr-Ser-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O WPSKTVVMQCXPRO-BWBBJGPYSA-N 0.000 description 2
- UQCNIMDPYICBTR-KYNKHSRBSA-N Thr-Thr-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O UQCNIMDPYICBTR-KYNKHSRBSA-N 0.000 description 2
- KZTLZZQTJMCGIP-ZJDVBMNYSA-N Thr-Val-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O KZTLZZQTJMCGIP-ZJDVBMNYSA-N 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 102100023935 Transmembrane glycoprotein NMB Human genes 0.000 description 2
- 229920004890 Triton X-100 Polymers 0.000 description 2
- 239000013504 Triton X-100 Substances 0.000 description 2
- JZSLIZLZGWOJBJ-PMVMPFDFSA-N Trp-Phe-Lys Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC2=CNC3=CC=CC=C32)N JZSLIZLZGWOJBJ-PMVMPFDFSA-N 0.000 description 2
- MICSYKFECRFCTJ-IHRRRGAJSA-N Tyr-Arg-Asp Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC(=O)O)C(=O)O)N)O MICSYKFECRFCTJ-IHRRRGAJSA-N 0.000 description 2
- WZQZUVWEPMGIMM-JYJNAYRXSA-N Tyr-Gln-Lys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCCCN)C(=O)O)N)O WZQZUVWEPMGIMM-JYJNAYRXSA-N 0.000 description 2
- LHTGRUZSZOIAKM-SOUVJXGZSA-N Tyr-Glu-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC2=CC=C(C=C2)O)N)C(=O)O LHTGRUZSZOIAKM-SOUVJXGZSA-N 0.000 description 2
- KOVXHANYYYMBRF-IRIUXVKKSA-N Tyr-Glu-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)N)O KOVXHANYYYMBRF-IRIUXVKKSA-N 0.000 description 2
- WQOHKVRQDLNDIL-YJRXYDGGSA-N Tyr-Thr-Ser Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O WQOHKVRQDLNDIL-YJRXYDGGSA-N 0.000 description 2
- RVGVIWNHABGIFH-IHRRRGAJSA-N Tyr-Val-Ser Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O RVGVIWNHABGIFH-IHRRRGAJSA-N 0.000 description 2
- 101800003344 Vaccinia growth factor Proteins 0.000 description 2
- GXAZTLJYINLMJL-LAEOZQHASA-N Val-Asn-Gln Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N GXAZTLJYINLMJL-LAEOZQHASA-N 0.000 description 2
- OUUBKKIJQIAPRI-LAEOZQHASA-N Val-Gln-Asn Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O OUUBKKIJQIAPRI-LAEOZQHASA-N 0.000 description 2
- VFOHXOLPLACADK-GVXVVHGQSA-N Val-Gln-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](C(C)C)N VFOHXOLPLACADK-GVXVVHGQSA-N 0.000 description 2
- BRPKEERLGYNCNC-NHCYSSNCSA-N Val-Glu-Arg Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N BRPKEERLGYNCNC-NHCYSSNCSA-N 0.000 description 2
- CVIXTAITYJQMPE-LAEOZQHASA-N Val-Glu-Asn Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O CVIXTAITYJQMPE-LAEOZQHASA-N 0.000 description 2
- OQWNEUXPKHIEJO-NRPADANISA-N Val-Glu-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CO)C(=O)O)N OQWNEUXPKHIEJO-NRPADANISA-N 0.000 description 2
- OVBMCNDKCWAXMZ-NAKRPEOUSA-N Val-Ile-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](C(C)C)N OVBMCNDKCWAXMZ-NAKRPEOUSA-N 0.000 description 2
- DJQIUOKSNRBTSV-CYDGBPFRSA-N Val-Ile-Val Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)O)NC(=O)[C@H](C(C)C)N DJQIUOKSNRBTSV-CYDGBPFRSA-N 0.000 description 2
- KTEZUXISLQTDDQ-NHCYSSNCSA-N Val-Lys-Asp Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(=O)O)C(=O)O)N KTEZUXISLQTDDQ-NHCYSSNCSA-N 0.000 description 2
- YDVDTCJGBBJGRT-GUBZILKMSA-N Val-Met-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CO)C(=O)O)N YDVDTCJGBBJGRT-GUBZILKMSA-N 0.000 description 2
- GBIUHAYJGWVNLN-AEJSXWLSSA-N Val-Ser-Pro Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CO)C(=O)N1CCC[C@@H]1C(=O)O)N GBIUHAYJGWVNLN-AEJSXWLSSA-N 0.000 description 2
- GBIUHAYJGWVNLN-UHFFFAOYSA-N Val-Ser-Pro Natural products CC(C)C(N)C(=O)NC(CO)C(=O)N1CCCC1C(O)=O GBIUHAYJGWVNLN-UHFFFAOYSA-N 0.000 description 2
- UJMCYJKPDFQLHX-XGEHTFHBSA-N Val-Ser-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](C(C)C)N)O UJMCYJKPDFQLHX-XGEHTFHBSA-N 0.000 description 2
- UQMPYVLTQCGRSK-IFFSRLJSSA-N Val-Thr-Gln Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](C(C)C)N)O UQMPYVLTQCGRSK-IFFSRLJSSA-N 0.000 description 2
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 2
- 102000034337 acetylcholine receptors Human genes 0.000 description 2
- 238000013019 agitation Methods 0.000 description 2
- 235000012538 ammonium bicarbonate Nutrition 0.000 description 2
- 239000001099 ammonium carbonate Substances 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 2
- 235000009697 arginine Nutrition 0.000 description 2
- 108010060035 arginylproline Proteins 0.000 description 2
- 229960001230 asparagine Drugs 0.000 description 2
- 235000009582 asparagine Nutrition 0.000 description 2
- 108010010430 asparagine-proline-alanine Proteins 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 238000004061 bleaching Methods 0.000 description 2
- IXIBAKNTJSCKJM-BUBXBXGNSA-N bovine insulin Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@H]1CSSC[C@H]2C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3C=CC(O)=CC=3)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3NC=NC=3)NC(=O)[C@H](CO)NC(=O)CNC1=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O)=O)CSSC[C@@H](C(N2)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](NC(=O)CN)[C@@H](C)CC)C(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C=CC=CC=1)C(C)C)C1=CN=CN1 IXIBAKNTJSCKJM-BUBXBXGNSA-N 0.000 description 2
- 201000008274 breast adenocarcinoma Diseases 0.000 description 2
- 210000000845 cartilage Anatomy 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 239000013553 cell monolayer Substances 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000011210 chromatographic step Methods 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 229960002424 collagenase Drugs 0.000 description 2
- 210000004922 colonic epithelial cell Anatomy 0.000 description 2
- 238000004132 cross linking Methods 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 210000004207 dermis Anatomy 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- BFMYDTVEBKDAKJ-UHFFFAOYSA-L disodium;(2',7'-dibromo-3',6'-dioxido-3-oxospiro[2-benzofuran-1,9'-xanthene]-4'-yl)mercury;hydrate Chemical compound O.[Na+].[Na+].O1C(=O)C2=CC=CC=C2C21C1=CC(Br)=C([O-])C([Hg])=C1OC1=C2C=C(Br)C([O-])=C1 BFMYDTVEBKDAKJ-UHFFFAOYSA-L 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 210000002744 extracellular matrix Anatomy 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- 108010063718 gamma-glutamylaspartic acid Proteins 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- 108010055341 glutamyl-glutamic acid Proteins 0.000 description 2
- 108010026195 glycanase Proteins 0.000 description 2
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 2
- 108010089804 glycyl-threonine Proteins 0.000 description 2
- 108010020688 glycylhistidine Proteins 0.000 description 2
- 108010081551 glycylphenylalanine Proteins 0.000 description 2
- 108010077515 glycylproline Proteins 0.000 description 2
- 238000003306 harvesting Methods 0.000 description 2
- 210000002216 heart Anatomy 0.000 description 2
- 108010040030 histidinoalanine Proteins 0.000 description 2
- 108010025306 histidylleucine Proteins 0.000 description 2
- 238000002169 hydrotherapy Methods 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- 239000012133 immunoprecipitate Substances 0.000 description 2
- 238000001114 immunoprecipitation Methods 0.000 description 2
- 230000001976 improved effect Effects 0.000 description 2
- 238000007901 in situ hybridization Methods 0.000 description 2
- 239000000411 inducer Substances 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 230000000977 initiatory effect Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 208000014674 injury Diseases 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- 210000000936 intestine Anatomy 0.000 description 2
- 108010078274 isoleucylvaline Proteins 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- 229960000318 kanamycin Drugs 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 2
- 229930182823 kanamycin A Natural products 0.000 description 2
- 208000017169 kidney disease Diseases 0.000 description 2
- 239000010410 layer Substances 0.000 description 2
- 108010034529 leucyl-lysine Proteins 0.000 description 2
- 108010000761 leucylarginine Proteins 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 239000012139 lysis buffer Substances 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 235000019341 magnesium sulphate Nutrition 0.000 description 2
- 210000004216 mammary stem cell Anatomy 0.000 description 2
- 230000035800 maturation Effects 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 108010085203 methionylmethionine Proteins 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 125000004123 n-propyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])* 0.000 description 2
- 230000009826 neoplastic cell growth Effects 0.000 description 2
- 230000001537 neural effect Effects 0.000 description 2
- 229920001220 nitrocellulos Polymers 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 231100000590 oncogenic Toxicity 0.000 description 2
- 230000002246 oncogenic effect Effects 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 210000000578 peripheral nerve Anatomy 0.000 description 2
- 108040007629 peroxidase activity proteins Proteins 0.000 description 2
- -1 phenylthiohydantoinyl Chemical group 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 229920003023 plastic Polymers 0.000 description 2
- 229920002401 polyacrylamide Polymers 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 2
- 229920000053 polysorbate 80 Polymers 0.000 description 2
- 239000003755 preservative agent Substances 0.000 description 2
- 108010070643 prolylglutamic acid Proteins 0.000 description 2
- 108010053725 prolylvaline Proteins 0.000 description 2
- 230000006337 proteolytic cleavage Effects 0.000 description 2
- 238000011536 re-plating Methods 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 210000004116 schwann cell Anatomy 0.000 description 2
- 238000007790 scraping Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000004017 serum-free culture medium Substances 0.000 description 2
- 108010071207 serylmethionine Proteins 0.000 description 2
- 230000019491 signal transduction Effects 0.000 description 2
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 108091007196 stromelysin Proteins 0.000 description 2
- 125000000341 threoninyl group Chemical group [H]OC([H])(C([H])([H])[H])C([H])(N([H])[H])C(*)=O 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 239000003053 toxin Substances 0.000 description 2
- 231100000765 toxin Toxicity 0.000 description 2
- 108700012359 toxins Proteins 0.000 description 2
- 229910021654 trace metal Inorganic materials 0.000 description 2
- 108091007466 transmembrane glycoproteins Proteins 0.000 description 2
- 229940108519 trasylol Drugs 0.000 description 2
- 238000011269 treatment regimen Methods 0.000 description 2
- 239000012137 tryptone Substances 0.000 description 2
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 2
- 239000004474 valine Substances 0.000 description 2
- 125000002987 valine group Chemical group [H]N([H])C([H])(C(*)=O)C([H])(C([H])([H])[H])C([H])([H])[H] 0.000 description 2
- 239000011782 vitamin Substances 0.000 description 2
- 229940088594 vitamin Drugs 0.000 description 2
- 235000013343 vitamin Nutrition 0.000 description 2
- 229930003231 vitamin Natural products 0.000 description 2
- 150000003722 vitamin derivatives Chemical class 0.000 description 2
- VHJLVAABSRFDPM-UHFFFAOYSA-N 1,4-dithiothreitol Chemical compound SCC(O)C(O)CS VHJLVAABSRFDPM-UHFFFAOYSA-N 0.000 description 1
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 1
- RNAMYOYQYRYFQY-UHFFFAOYSA-N 2-(4,4-difluoropiperidin-1-yl)-6-methoxy-n-(1-propan-2-ylpiperidin-4-yl)-7-(3-pyrrolidin-1-ylpropoxy)quinazolin-4-amine Chemical compound N1=C(N2CCC(F)(F)CC2)N=C2C=C(OCCCN3CCCC3)C(OC)=CC2=C1NC1CCN(C(C)C)CC1 RNAMYOYQYRYFQY-UHFFFAOYSA-N 0.000 description 1
- JEPVUMTVFPQKQE-AAKCMJRZSA-N 2-[(1s,2s,3r,4s)-1,2,3,4,5-pentahydroxypentyl]-1,3-thiazolidine-4-carboxylic acid Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C1NC(C(O)=O)CS1 JEPVUMTVFPQKQE-AAKCMJRZSA-N 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- AZKSAVLVSZKNRD-UHFFFAOYSA-M 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Chemical compound [Br-].S1C(C)=C(C)N=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=CC=C1 AZKSAVLVSZKNRD-UHFFFAOYSA-M 0.000 description 1
- UMCMPZBLKLEWAF-BCTGSCMUSA-N 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulfonate Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCC[N+](C)(C)CCCS([O-])(=O)=O)C)[C@@]2(C)[C@@H](O)C1 UMCMPZBLKLEWAF-BCTGSCMUSA-N 0.000 description 1
- QFVHZQCOUORWEI-UHFFFAOYSA-N 4-[(4-anilino-5-sulfonaphthalen-1-yl)diazenyl]-5-hydroxynaphthalene-2,7-disulfonic acid Chemical compound C=12C(O)=CC(S(O)(=O)=O)=CC2=CC(S(O)(=O)=O)=CC=1N=NC(C1=CC=CC(=C11)S(O)(=O)=O)=CC=C1NC1=CC=CC=C1 QFVHZQCOUORWEI-UHFFFAOYSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- WKGGTEFWVLVCGO-UHFFFAOYSA-N 8-(2,5-dioxo-3-sulfopyrrolidin-1-yl)oxy-8-oxooctanoic acid Chemical compound OC(=O)CCCCCCC(=O)ON1C(=O)CC(S(O)(=O)=O)C1=O WKGGTEFWVLVCGO-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- 208000003918 Acute Kidney Tubular Necrosis Diseases 0.000 description 1
- 208000036832 Adenocarcinoma of ovary Diseases 0.000 description 1
- 208000004804 Adenomatous Polyps Diseases 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 108010033760 Amphiregulin Proteins 0.000 description 1
- 102100038778 Amphiregulin Human genes 0.000 description 1
- 101000904962 Arabidopsis thaliana Histone H2B.6 Proteins 0.000 description 1
- MAISCYVJLBBRNU-DCAQKATOSA-N Arg-Asn-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCCN=C(N)N)N MAISCYVJLBBRNU-DCAQKATOSA-N 0.000 description 1
- JCROZIFVIYMXHM-GUBZILKMSA-N Arg-Met-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CCSC)NC(=O)[C@@H](N)CCCN=C(N)N JCROZIFVIYMXHM-GUBZILKMSA-N 0.000 description 1
- XMZZGVGKGXRIGJ-JYJNAYRXSA-N Arg-Tyr-Val Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C(C)C)C(O)=O XMZZGVGKGXRIGJ-JYJNAYRXSA-N 0.000 description 1
- 206010003571 Astrocytoma Diseases 0.000 description 1
- 208000010061 Autosomal Dominant Polycystic Kidney Diseases 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- 206010004992 Bladder adenocarcinoma stage unspecified Diseases 0.000 description 1
- 206010006802 Burns second degree Diseases 0.000 description 1
- 206010006803 Burns third degree Diseases 0.000 description 1
- 102100023703 C-C motif chemokine 15 Human genes 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 102000016289 Cell Adhesion Molecules Human genes 0.000 description 1
- 108010067225 Cell Adhesion Molecules Proteins 0.000 description 1
- 102000000844 Cell Surface Receptors Human genes 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- 101710172503 Chemokine-binding protein Proteins 0.000 description 1
- 208000017667 Chronic Disease Diseases 0.000 description 1
- 206010009900 Colitis ulcerative Diseases 0.000 description 1
- 208000011231 Crohn disease Diseases 0.000 description 1
- LYSHSHHDBVKJRN-JBDRJPRFSA-N Cys-Ile-Ala Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C)C(=O)O)NC(=O)[C@H](CS)N LYSHSHHDBVKJRN-JBDRJPRFSA-N 0.000 description 1
- CAXGCBSRJLADPD-FXQIFTODSA-N Cys-Pro-Asn Chemical compound [H]N[C@@H](CS)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(O)=O CAXGCBSRJLADPD-FXQIFTODSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 230000004543 DNA replication Effects 0.000 description 1
- 208000037162 Ductal Breast Carcinoma Diseases 0.000 description 1
- 208000006402 Ductal Carcinoma Diseases 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 101150029707 ERBB2 gene Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 206010014989 Epidermolysis bullosa Diseases 0.000 description 1
- 108050007372 Fibroblast Growth Factor Proteins 0.000 description 1
- 102000018233 Fibroblast Growth Factor Human genes 0.000 description 1
- 108090000386 Fibroblast Growth Factor 1 Proteins 0.000 description 1
- 102100031706 Fibroblast growth factor 1 Human genes 0.000 description 1
- 102100028071 Fibroblast growth factor 7 Human genes 0.000 description 1
- 108090000385 Fibroblast growth factor 7 Proteins 0.000 description 1
- 201000008808 Fibrosarcoma Diseases 0.000 description 1
- 241001200922 Gagata Species 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- ITVBKCZZLJUUHI-HTUGSXCWSA-N Glu-Phe-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O ITVBKCZZLJUUHI-HTUGSXCWSA-N 0.000 description 1
- IWAXHBCACVWNHT-BQBZGAKWSA-N Gly-Asp-Arg Chemical compound NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N IWAXHBCACVWNHT-BQBZGAKWSA-N 0.000 description 1
- 102000006771 Gonadotropins Human genes 0.000 description 1
- 108010086677 Gonadotropins Proteins 0.000 description 1
- 108010017080 Granulocyte Colony-Stimulating Factor Proteins 0.000 description 1
- 102000004269 Granulocyte Colony-Stimulating Factor Human genes 0.000 description 1
- 102000009465 Growth Factor Receptors Human genes 0.000 description 1
- 108010009202 Growth Factor Receptors Proteins 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 241000711549 Hepacivirus C Species 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- 102400001369 Heparin-binding EGF-like growth factor Human genes 0.000 description 1
- 101800001649 Heparin-binding EGF-like growth factor Proteins 0.000 description 1
- 241000700721 Hepatitis B virus Species 0.000 description 1
- 241001622557 Hesperia Species 0.000 description 1
- 229920000209 Hexadimethrine bromide Polymers 0.000 description 1
- 229940122957 Histamine H2 receptor antagonist Drugs 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000809450 Homo sapiens Amphiregulin Proteins 0.000 description 1
- 101000947120 Homo sapiens Beta-casein Proteins 0.000 description 1
- 101000978376 Homo sapiens C-C motif chemokine 15 Proteins 0.000 description 1
- 101001109800 Homo sapiens Pro-neuregulin-1, membrane-bound isoform Proteins 0.000 description 1
- 101000756346 Homo sapiens RE1-silencing transcription factor Proteins 0.000 description 1
- 102000003839 Human Proteins Human genes 0.000 description 1
- 108090000144 Human Proteins Proteins 0.000 description 1
- 206010062767 Hypophysitis Diseases 0.000 description 1
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 1
- VAXBXNPRXPHGHG-BJDJZHNGSA-N Ile-Ala-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)O)N VAXBXNPRXPHGHG-BJDJZHNGSA-N 0.000 description 1
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 1
- 102000004218 Insulin-Like Growth Factor I Human genes 0.000 description 1
- 108010002352 Interleukin-1 Proteins 0.000 description 1
- 108010002386 Interleukin-3 Proteins 0.000 description 1
- 108090001005 Interleukin-6 Proteins 0.000 description 1
- 108090001007 Interleukin-8 Proteins 0.000 description 1
- 102100020880 Kit ligand Human genes 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- LJBVRCDPWOJOEK-PPCPHDFISA-N Leu-Thr-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O LJBVRCDPWOJOEK-PPCPHDFISA-N 0.000 description 1
- 108010074338 Lymphokines Proteins 0.000 description 1
- 102000008072 Lymphokines Human genes 0.000 description 1
- NTSPQIONFJUMJV-AVGNSLFASA-N Lys-Arg-Val Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(O)=O NTSPQIONFJUMJV-AVGNSLFASA-N 0.000 description 1
- 231100000002 MTT assay Toxicity 0.000 description 1
- 238000000134 MTT assay Methods 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 108010016160 Matrix Metalloproteinase 3 Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 206010054949 Metaplasia Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 102000014171 Milk Proteins Human genes 0.000 description 1
- 108010011756 Milk Proteins Proteins 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- MQUQNUAYKLCRME-INIZCTEOSA-N N-tosyl-L-phenylalanyl chloromethyl ketone Chemical compound C1=CC(C)=CC=C1S(=O)(=O)N[C@H](C(=O)CCl)CC1=CC=CC=C1 MQUQNUAYKLCRME-INIZCTEOSA-N 0.000 description 1
- 101150108866 Ndf gene Proteins 0.000 description 1
- 206010056677 Nerve degeneration Diseases 0.000 description 1
- 102100023616 Neural cell adhesion molecule L1-like protein Human genes 0.000 description 1
- 108010006232 Neuraminidase Proteins 0.000 description 1
- 102000005348 Neuraminidase Human genes 0.000 description 1
- 206010061328 Ovarian epithelial cancer Diseases 0.000 description 1
- 206010061535 Ovarian neoplasm Diseases 0.000 description 1
- 238000009004 PCR Kit Methods 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 241000609499 Palicourea Species 0.000 description 1
- 206010033701 Papillary thyroid cancer Diseases 0.000 description 1
- 102000000447 Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase Human genes 0.000 description 1
- 108010055817 Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase Proteins 0.000 description 1
- 208000010886 Peripheral nerve injury Diseases 0.000 description 1
- 208000007913 Pituitary Neoplasms Diseases 0.000 description 1
- 229920000954 Polyglycolide Polymers 0.000 description 1
- 108010021757 Polynucleotide 5'-Hydroxyl-Kinase Proteins 0.000 description 1
- 102000008422 Polynucleotide 5'-hydroxyl-kinase Human genes 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 201000004681 Psoriasis Diseases 0.000 description 1
- 101710138270 PspA protein Proteins 0.000 description 1
- 241000219492 Quercus Species 0.000 description 1
- 102100022940 RE1-silencing transcription factor Human genes 0.000 description 1
- 239000013614 RNA sample Substances 0.000 description 1
- 239000012979 RPMI medium Substances 0.000 description 1
- 238000010240 RT-PCR analysis Methods 0.000 description 1
- 108090000244 Rat Proteins Proteins 0.000 description 1
- 241000700157 Rattus norvegicus Species 0.000 description 1
- 101000809448 Rattus norvegicus Amphiregulin Proteins 0.000 description 1
- 101100501691 Rattus norvegicus Erbb2 gene Proteins 0.000 description 1
- 206010038540 Renal tubular necrosis Diseases 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- IYCBDVBJWDXQRR-FXQIFTODSA-N Ser-Ala-Met Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CCSC)C(O)=O IYCBDVBJWDXQRR-FXQIFTODSA-N 0.000 description 1
- 108010079105 Shope fibroma virus growth factor Proteins 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 241000327799 Thallomys paedulcus Species 0.000 description 1
- IMULJHHGAUZZFE-MBLNEYKQSA-N Thr-Gly-Ile Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H]([C@@H](C)CC)C(O)=O IMULJHHGAUZZFE-MBLNEYKQSA-N 0.000 description 1
- ZESGVALRVJIVLZ-VFCFLDTKSA-N Thr-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N1CCC[C@@H]1C(=O)O)N)O ZESGVALRVJIVLZ-VFCFLDTKSA-N 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 108010022394 Threonine synthase Proteins 0.000 description 1
- 108091005906 Type I transmembrane proteins Proteins 0.000 description 1
- 208000025865 Ulcer Diseases 0.000 description 1
- 201000006704 Ulcerative Colitis Diseases 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 206010073696 Wallerian degeneration Diseases 0.000 description 1
- SXEHKFHPFVVDIR-UHFFFAOYSA-N [4-(4-hydrazinylphenyl)phenyl]hydrazine Chemical compound C1=CC(NN)=CC=C1C1=CC=C(NN)C=C1 SXEHKFHPFVVDIR-UHFFFAOYSA-N 0.000 description 1
- 230000001594 aberrant effect Effects 0.000 description 1
- 201000010272 acanthosis nigricans Diseases 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 description 1
- 125000003172 aldehyde group Chemical group 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000008485 antagonism Effects 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- 230000002494 anti-cea effect Effects 0.000 description 1
- 238000011319 anticancer therapy Methods 0.000 description 1
- 239000002518 antifoaming agent Substances 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 239000012736 aqueous medium Substances 0.000 description 1
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 description 1
- 229910052786 argon Inorganic materials 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 229940009098 aspartate Drugs 0.000 description 1
- 239000012131 assay buffer Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 208000022185 autosomal dominant polycystic kidney disease Diseases 0.000 description 1
- 210000000270 basal cell Anatomy 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 235000019445 benzyl alcohol Nutrition 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- 239000003124 biologic agent Substances 0.000 description 1
- 201000006587 bladder adenocarcinoma Diseases 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000005013 brain tissue Anatomy 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 230000005907 cancer growth Effects 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 208000002458 carcinoid tumor Diseases 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000011712 cell development Effects 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 230000004709 cell invasion Effects 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 230000008619 cell matrix interaction Effects 0.000 description 1
- 230000011748 cell maturation Effects 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 230000009134 cell regulation Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000030570 cellular localization Effects 0.000 description 1
- 230000036755 cellular response Effects 0.000 description 1
- 229920002301 cellulose acetate Polymers 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 210000003679 cervix uteri Anatomy 0.000 description 1
- 238000010382 chemical cross-linking Methods 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000000973 chemotherapeutic effect Effects 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 230000005757 colony formation Effects 0.000 description 1
- 238000010293 colony formation assay Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 230000008602 contraction Effects 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 238000011461 current therapy Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 210000005220 cytoplasmic tail Anatomy 0.000 description 1
- 230000001120 cytoprotective effect Effects 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 230000003831 deregulation Effects 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000011026 diafiltration Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 229940111685 dibasic potassium phosphate Drugs 0.000 description 1
- 102000004419 dihydrofolate reductase Human genes 0.000 description 1
- 239000013024 dilution buffer Substances 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 230000003292 diminished effect Effects 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- VHJLVAABSRFDPM-ZXZARUISSA-N dithioerythritol Chemical compound SC[C@H](O)[C@H](O)CS VHJLVAABSRFDPM-ZXZARUISSA-N 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 208000000718 duodenal ulcer Diseases 0.000 description 1
- 210000001198 duodenum Anatomy 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 210000004696 endometrium Anatomy 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
- 210000005081 epithelial layer Anatomy 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 210000003238 esophagus Anatomy 0.000 description 1
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 1
- 229960005542 ethidium bromide Drugs 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 239000012526 feed medium Substances 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 206010016629 fibroma Diseases 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000000834 fixative Substances 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 238000010230 functional analysis Methods 0.000 description 1
- 201000006585 gastric adenocarcinoma Diseases 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 239000003365 glass fiber Substances 0.000 description 1
- 230000002518 glial effect Effects 0.000 description 1
- 208000005017 glioblastoma Diseases 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 108010085617 glycopeptide alpha-N-acetylgalactosaminidase Proteins 0.000 description 1
- 239000002622 gonadotropin Substances 0.000 description 1
- 239000011544 gradient gel Substances 0.000 description 1
- 230000009422 growth inhibiting effect Effects 0.000 description 1
- 210000003780 hair follicle Anatomy 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 1
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 1
- 102000043494 human AREG Human genes 0.000 description 1
- 102000051957 human ERBB2 Human genes 0.000 description 1
- 102000055650 human NRG1 Human genes 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 125000001165 hydrophobic group Chemical group 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-M hydroxide Chemical compound [OH-] XLYOFNOQVPJJNP-UHFFFAOYSA-M 0.000 description 1
- 230000003463 hyperproliferative effect Effects 0.000 description 1
- 238000010191 image analysis Methods 0.000 description 1
- 229940127121 immunoconjugate Drugs 0.000 description 1
- 238000010185 immunofluorescence analysis Methods 0.000 description 1
- 238000011532 immunohistochemical staining Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000008611 intercellular interaction Effects 0.000 description 1
- 210000002490 intestinal epithelial cell Anatomy 0.000 description 1
- 210000004347 intestinal mucosa Anatomy 0.000 description 1
- 230000004068 intracellular signaling Effects 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 230000000302 ischemic effect Effects 0.000 description 1
- 125000000741 isoleucyl group Chemical group [H]N([H])C(C(C([H])([H])[H])C([H])([H])C([H])([H])[H])C(=O)O* 0.000 description 1
- YWXYYJSYQOXTPL-SLPGGIOYSA-N isosorbide mononitrate Chemical compound [O-][N+](=O)O[C@@H]1CO[C@@H]2[C@@H](O)CO[C@@H]21 YWXYYJSYQOXTPL-SLPGGIOYSA-N 0.000 description 1
- 210000000738 kidney tubule Anatomy 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 210000003140 lateral ventricle Anatomy 0.000 description 1
- 231100000636 lethal dose Toxicity 0.000 description 1
- 125000001909 leucine group Chemical group [H]N(*)C(C(*)=O)C([H])([H])C(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000012577 media supplement Substances 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 230000015689 metaplastic ossification Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 235000021239 milk protein Nutrition 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 239000003226 mitogen Substances 0.000 description 1
- 230000002297 mitogenic effect Effects 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- 229940111688 monobasic potassium phosphate Drugs 0.000 description 1
- 238000004264 monolayer culture Methods 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 210000002161 motor neuron Anatomy 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 210000000663 muscle cell Anatomy 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 108010079250 myxoma virus growth factor Proteins 0.000 description 1
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 1
- 210000004897 n-terminal region Anatomy 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 230000001613 neoplastic effect Effects 0.000 description 1
- 230000000926 neurological effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000002777 nucleoside Substances 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 230000008266 oncogenic mechanism Effects 0.000 description 1
- 230000006548 oncogenic transformation Effects 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 208000013371 ovarian adenocarcinoma Diseases 0.000 description 1
- 201000006588 ovary adenocarcinoma Diseases 0.000 description 1
- 238000012261 overproduction Methods 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 210000001428 peripheral nervous system Anatomy 0.000 description 1
- 108010083127 phage repressor proteins Proteins 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 150000004633 phorbol derivatives Chemical class 0.000 description 1
- 239000002644 phorbol ester Substances 0.000 description 1
- XLCISDOVNFLSGO-VONOSFMSSA-N phorbol-12-myristate Chemical compound C([C@]1(O)C(=O)C(C)=C[C@H]1[C@@]1(O)[C@H](C)[C@H]2OC(=O)CCCCCCCCCCCCC)C(CO)=C[C@H]1[C@H]1[C@]2(O)C1(C)C XLCISDOVNFLSGO-VONOSFMSSA-N 0.000 description 1
- DHRLEVQXOMLTIM-UHFFFAOYSA-N phosphoric acid;trioxomolybdenum Chemical compound O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.OP(O)(O)=O DHRLEVQXOMLTIM-UHFFFAOYSA-N 0.000 description 1
- 210000003635 pituitary gland Anatomy 0.000 description 1
- 208000010916 pituitary tumor Diseases 0.000 description 1
- 230000004983 pleiotropic effect Effects 0.000 description 1
- 229920000747 poly(lactic acid) Polymers 0.000 description 1
- 208000030761 polycystic kidney disease Diseases 0.000 description 1
- 239000004633 polyglycolic acid Substances 0.000 description 1
- 239000004626 polylactic acid Substances 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 239000000244 polyoxyethylene sorbitan monooleate Substances 0.000 description 1
- 210000004896 polypeptide structure Anatomy 0.000 description 1
- 229940068968 polysorbate 80 Drugs 0.000 description 1
- 238000010837 poor prognosis Methods 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000037452 priming Effects 0.000 description 1
- 238000000159 protein binding assay Methods 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 229940024999 proteolytic enzymes for treatment of wounds and ulcers Drugs 0.000 description 1
- 108010015255 protransforming growth factor alpha Proteins 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 102000037983 regulatory factors Human genes 0.000 description 1
- 108091008025 regulatory factors Proteins 0.000 description 1
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 238000002271 resection Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 210000004918 root sheath Anatomy 0.000 description 1
- 210000003079 salivary gland Anatomy 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 231100000241 scar Toxicity 0.000 description 1
- 230000036573 scar formation Effects 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 201000008407 sebaceous adenocarcinoma Diseases 0.000 description 1
- 201000003385 seborrheic keratosis Diseases 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 239000006152 selective media Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 description 1
- 230000033962 signal peptide processing Effects 0.000 description 1
- 238000012868 site-directed mutagenesis technique Methods 0.000 description 1
- 231100000444 skin lesion Toxicity 0.000 description 1
- 206010040882 skin lesion Diseases 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- HRZFUMHJMZEROT-UHFFFAOYSA-L sodium disulfite Chemical compound [Na+].[Na+].[O-]S(=O)S([O-])(=O)=O HRZFUMHJMZEROT-UHFFFAOYSA-L 0.000 description 1
- 229940001584 sodium metabisulfite Drugs 0.000 description 1
- 235000010262 sodium metabisulphite Nutrition 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 210000003594 spinal ganglia Anatomy 0.000 description 1
- 238000013223 sprague-dawley female rat Methods 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 239000003270 steroid hormone Substances 0.000 description 1
- 210000000434 stratum corneum Anatomy 0.000 description 1
- 210000000437 stratum spinosum Anatomy 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000004114 suspension culture Methods 0.000 description 1
- 201000010965 sweat gland carcinoma Diseases 0.000 description 1
- 238000012353 t test Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 230000036962 time dependent Effects 0.000 description 1
- 230000017423 tissue regeneration Effects 0.000 description 1
- 230000007838 tissue remodeling Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 230000004565 tumor cell growth Effects 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 231100000397 ulcer Toxicity 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 238000012762 unpaired Student’s t-test Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 230000002485 urinary effect Effects 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 230000008734 wallerian degeneration Effects 0.000 description 1
- 239000003357 wound healing promoting agent Substances 0.000 description 1
- 239000010930 yellow gold Substances 0.000 description 1
- 229910001097 yellow gold Inorganic materials 0.000 description 1
- 235000021247 β-casein Nutrition 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/4756—Neuregulins, i.e. p185erbB2 ligands, glial growth factor, heregulin, ARIA, neu differentiation factor
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- This invention relates to novel polypeptides (herein referred to as neu differentiation factors), produced by recombinant DNA methods, which interact with and stimulate the neu receptor and modulate cellular function.
- the invention includes analogs and
- oncogenic transformation can be induced by constitutive production of growth regulatory factors or by altered forms of their cognate receptors (Yarden, Y., and Ullrich, A., Ann . Rev. Biochem . 57:443-448, 1988) .
- the oncogenic receptors include a family of
- transmembrane glycoproteins that share a common
- the neu proto-oncogene also known as HER-2 or c-erbB-2
- This pl85 neu glycoprotein is known to be present in many epithelial and neural tissues
- pl85 neu Although adult tissues generally express less p185 neu than corresponding fetal tissues, pl85 neu
- pl85 neu or neu gene amplification occurs in approximately twenty percent of breast, stomach, bladder and ovarian adenocarcinomas (Lofts, F.J., and Gullick, W.J., Cancer Treat . Res .
- Increased p185 neu expression also occurs in certain non-malignant neoplasias, such as adenomatous polyps (D'Emilia, J.K. et al., Oncogene 4:1233-1239, 1989; Cohen, J.A. et al., Oncogene 4:1233-1239 1989), Barrett's esophagous (Jankowski, J.G. et al., Gut 33:1033-1038, 1992), and polycystic kidneys (Herrera, G., Kidney Int . 40:509-513, 1991).
- adenomatous polyps D'Emilia, J.K. et al., Oncogene 4:1233-1239, 1989; Cohen, J.A. et al., Oncogene 4:1233-1239 1989
- Barrett's esophagous Jankowski, J.G. et al., Gut 33:1033-1038, 1992
- polycystic kidneys Herrera,
- Phosphorylation of the pl85 neu receptor on tyrosine residues can be stimulated by proteins purified from several sources (Peles, E. et al., Cell 69: 205-216, 1992; Lupu, R. et al.. Science 249:1552-1555, 1990; Holmes, W.E. et al.. Science
- neu The 44 kDa rat factor, named neu
- NDF NDF differentiation factor
- NDF transmembrane glycoprotein precursor
- the recombinant version of this rat NDF was found to interact with pl85 neu and stimulate tyrosine phosphorylation of the receptor in human tumor cells of breast, colon and neuronal origin (Peles, E. et al., EMBO J. 12:461-971, 1993).
- In situ hybridization with a NDF probe
- polypeptides add to the expanding knowledge of NDF-related molecules, which now also includes glial growth factors (Marchionni, M.A. et al.. Nature 362:312-318, 1993), as well as chicken acetylcholine receptor
- non-naturally occurring polypeptides encoded by human nucleotide sequences which function as stimulators and inducers of neu (or Her-2, or c-erbB-2) receptor activities.
- These polypeptides include
- recombinant DNA-derived neu receptor stimulating factors expressed from cDNA clones isolated from human tissues and cell lines.
- Such polypeptides possess an ability to stimulate human p185 neu tyrosine phosphorylation.
- These recombinant polypeptides can be termed neu receptor stimulating factors, but are preferably referred to herein as neu differentiation factors (or "NDFs"), based on their ability to induce a differentiated phenotype in certain cell lines.
- NDFs neu differentiation factors
- polypeptides are encoded by nucleotide sequences which are also described in detail herein.
- the isolated DNA sequences provided by the present invention are useful in securing expression of the polypeptides in procaryotic and/or eucaryotic host cells.
- the present invention specifically provides DNA sequences encoding all or part of the unprocessed amino acid sequences (proNDFs) as well as DNA sequences encoding all or part of the processed (mature) forms of the NDFs and novel recombinant analogs thereof.
- Such DNA sequences include:
- cDNA complementary DNA
- genomic DNA sequences encoding variant forms of human NDF, manufactured DNA sequences (e.g., as by solid phase chemical synthesis) encoding human NDF, fragments of human NDF, and analogs of human NDF.
- the DNA sequences may incorporate codons facilitating transcription and translation of messenger RNA in host cells.
- vectors containing such DNA sequences and host cells transformed or transfected with such vectors. Additionally, the invention includes methods of producing biologically active human NDF polypeptides by recombinant DNA techniques, and methods of treating disorders using recombinant NDFs.
- compositions containing recombinant human NDF polypeptides and antibodies generated with such polypeptides are also encompassed by this invention.
- Figure 1 depicts a high pressure liquid chromatogram of the trypsin digest of "naturally- occurring" neu receptor stimulating factor purified from media conditioned by ras-transformed rat fibroblasts according to the methods of Peles, E. et al., Cell, 1992, above. The elution profile is shown, and the amino acid sequences obtained from the fractions corresponding to each peak are indicated in conventional single letter designation.
- the amino acid in parentheses represents an asparagine-linked glycosylation site, while the dotted line represents a longer sequence in which the amino acids were not identified.
- the inset shows
- FIG. 2 shows the structure of the mammalian
- COS-7 cell expression vector pJT-2/NDF COS-7 cell expression vector pJT-2/NDF.
- the rat NDF cDNA insert is the clone 44 cDNA sequence of Figure 5 [SEQ ID NO: 21].
- Figure 3 demonstrates stimulation of human neu receptor tyrosine phosphorylation by recombinant rat NDF.
- Human MDA-MB-453 breast carcinoma cells were incubated with concentrated conditioned media from COS-7 cells transfected with the indicated partially purified cDNA clones (left panel) or with fully purified cDNA clones (right panel). Positive controls comprised
- COS-7 cells untransfected COS-7 cells (lane designated "COS"), or by cells transfected with pJT-2 plasmids containing
- Figure 4 shows the nucleotide sequence of rat NDF cDNA [SEQ ID NO: 19] and a deduced amino acid sequence [SEQ ID NO: 20]. Depicted is a combined nucleotide sequence from four rat cDNA clones. The beginning of the clone 44 DNA sequence in particular is indicated by an arrow. Nucleotide numbers and amino acid numbers are given in the left and right columns, respectively. Potential sites of N-linked glycoprotein are marked by asterisks, and cysteine residues found in the presumed extracellular domain are encircled. The overlining indicates peptide sequences that were
- transmembrane region whereas the dashed underlining indicates the polyadenylation sites.
- the portions of the protein sequence that contain an immunoglobulin (Ig) homology unit and an epidermal growth factor (EGF)-like motif are indicated on the right hand side.
- Figure 5 shows the nucleotide sequence of rat NDF cDNA clone 44 [SEQ ID NO: 21], separately, and a deduced amino acid sequence [SEQ ID NO: 22]. Nucleotide numbers are given in the right hand column.
- Figure 6 depicts the hydropathy profile of the precursor of the rat neu receptor stimulating factor encoded by rat cDNA clone 44.
- Figure 7 shows the alignment of the amino acid sequence [SEQ ID NO: 23] of the EGF-like domain and the flanking carboxyl terminal sequence of rat NDF from clone 44 with representative members of the EGF family. Alignment and numbering begin at the most amino terminal cysteine residue of the EGF motifs. Amino acid residues are indicated by the single letter code. Dashes
- NDF rat transforming growth factor ⁇ [SEQ ID NO: 24] (TGF ⁇ ; Marquardt, H. et al., Science
- MEF myxoma virus growth factor
- human heparin-binding EGF [SEQ ID NO: 27] (HB-EGF; Higashiyama, S. et al.,
- Figure 8 shows the alignment of the immunoglobulin (Ig)-like domain of rat NDF [SEQ ID NO: 1]
- SEQ ID NO: 33 (NCAM; Barthels, D. et al., EMBO J. 6; 907-914, 1987), a representative protein of the C2-set of the immunoglobulin superfamily. Residues that are highly conserved in the C2-set are indicated on the lower line, and positions conserved across the
- Figure 9 is a schematic presentation of the presumed secondary structure and membrane orientation of the precursor of the neu receptor simulating factor from rat cDNA clone 44. Positions corresponding to the immunoglobulin (Ig)-domain and epidermal growth factor (EGF) motif are shown by thick lines and their cysteine residues are indicated by circles. The disulfide linkage of the Ig domain was directly demonstrated by amino acid sequence analysis (Example ID, below), and the secondary structure of the EGF-domain is based on the homology with the EGF family. Also shown are the three cysteine residues found in the transmembrane domain. Arrows mark the processing sites of the immunoglobulin (Ig)-domain and epidermal growth factor (EGF) motif are shown by thick lines and their cysteine residues are indicated by circles. The disulfide linkage of the Ig domain was directly demonstrated by amino acid sequence analysis (Example ID, below), and the secondary structure of the EGF-domain is based on the homology with the EGF family. Also shown are
- N-glycosylation site and the short vertical lines represent presumed sites of O-glycosylation.
- FIG 10 depicts a receptor competition assay using naturally-occurring neu receptor stimulating factor purified from ras-transformed rat fibroblast conditioned media (Peles, E. et al., Cell, 1992, above) which has been radiolabelled (" 125 I-NDF”).
- the radio-labelled NDF was incubated with human MDA-MB-453 breast carcinoma cells in the absence ("NONE") or presence of conditioned media from COS-7 cells expressing either recombinant neu receptor stimulating factor from rat cDNA clone 44 ("C-NDF") or recombinant TGF ⁇ ("C-TGF ⁇ ”) .
- C-NDF recombinant neu receptor stimulating factor from rat cDNA clone 44
- C-TGF ⁇ recombinant TGF ⁇
- Figure 11 depicts another receptor competition assay. 125 I-labeled naturally-occurring NDF was
- C-TGF ⁇ ⁇ -TGF ⁇
- Figure 12 shows autoradiograms obtained from Northern blots of mRNA isolated from cultured cells (panels A and C) or freshly isolated tissue of an adult rat (panel B), using rat NDF cDNA clone 44 as a probe. The autoradiograms were obtained after a three-hour
- Figure 13 shows the nucleotide sequence of rat
- NDF cDNA clone 4 [SEQ ID NO: 34] and a deduced partial rat (proNDF-ß3) amino acid sequence [SEQ ID NO: 35]. Nucleotide numbers are given in the left hand column and amino acids numbers on the right hand column. Linker sequences added to the 5' and 3' ends of the cDNA to facilitate cloning are included. The amino acid
- Figure 14 shows the nucleotide sequence of rat NDF cDNA clone 19 [SEQ ID NO: 36] and a deduced rat (proNDF- ⁇ 2b) amino acid sequence [SEQ ID NO: 37].
- Nucleotide numbers are given in the left hand column and amino acids numbers on the right hand column. Linker sequences added to the 3' ends of the cDNA to facilitate cloning are included.
- Figure 15 shows the nucleotide sequence of rat NDF cDNA clone 20 [SEQ ID NO: 38] and a deduced rat (proNDF- ⁇ 2b) amino acid sequence [SEQ ID NO: 39].
- Nucleotide numbers are given in the left hand column and amino acids numbers on the right hand column. Linker sequences added to the 3' ends of the cDNA to facilitate cloning are included.
- Figure 16 shows the nucleotide sequence of rat NDF cDNA clone 22 [SEQ ID NO: 40] and a deduced rat (proNDF-ß2a) amino acid sequence [SEQ ID NO: 41].
- Nucleotide numbers are given in the left hand column and amino acids numbers on the right hand column. Linker sequences added to the 3' ends of the cDNA to facilitate cloning are included.
- Figure 17 shows the nucleotide sequence of rat
- NDF cDNA clone 38 [SEQ ID NO: 42] and a deduced rat (proNDF- ⁇ 2a) amino acid sequence [SEQ ID NO: 43].
- Nucleotide numbers are given in the left hand column and amino acids numbers on the right hand column. Linker sequences added to the 3' ends of the cDNA to facilitate cloning are included.
- Figure 18 shows the nucleotide sequence of rat NDF cDNA clone 40 [SEQ ID NO: 44] and a deduced partial rat (proNDF-ß2) amino acid sequence [SEQ ID NO: 45] . Nucleotide numbers are given in the left hand column and amino acids numbers on the right hand column. Linker sequences added to the 3' ends of the cDNA to facilitate cloning are included.
- Figure 19 shows the nucleotide sequence of rat NDF cDNA clone 41 [SEQ ID NO: 46] and a deduced rat (proNDF-ß2a) amino acid sequence [SEQ ID NO: 47].
- Nucleotide numbers are given in the left hand column and amino acids numbers on the right hand column. Linker sequences added to the 3' ends of the cDNA to facilitate cloning are included.
- Figure 20 shows the nucleotide sequence of rat NDF cDNA clone 42A [SEQ ID NO: 48] and a deduced rat (proNDF-ß4a) amino acid sequence [SEQ ID NO: 49].
- Nucleotide numbers are given in the left hand column and amino acids numbers on the right hand column. Linker sequences added to the 3' ends of the cDNA to facilitate cloning are included.
- Figure 21 shows the nucleotide sequence of rat NDF cDNA clone 42B [SEQ ID NO: 50] and a deduced rat (proNDF- ⁇ 2a) amino acid sequence [SEQ ID NO: 51].
- Nucleotide numbers are given in the left hand column and amino acids numbers on the right hand column. Linker sequences added to the 3' ends of the cDNA to facilitate cloning are included.
- Figure 22 shows rat proNDF (precursor) structures predicted from the rat cDNA sequences of Figures 4, 5, and 13-21. Boxed areas indicate protein coding regions. "Ig”, “EGF” and “TM” indicate the immunoglobulin-like, EGF-like and transmembrane domains, respectively. The number of amino acid residues in each predicted precursor sequence is given in the right hand column. Dashed lines represent divergent 3'
- Figure 23 shows the amino acid sequences encoded by different rat proNDF cDNAs.
- the complete proNDF- ⁇ 2a amino acid sequence from Figure 17 is shown [SEQ ID NO: 43].
- Divergent sequences in proNDF variants are aligned with the proNDF- ⁇ 2a sequence.
- ProNDF-ß2a [SEQ ID NO: 126], ß3 [SEQ ID NO: 127] and ß4a [SEQ ID NO: 52] structures were deduced from Rat-1-EJ cDNAs (Table 2, below, and Figures 16, 13, and 20,
- the NDF-ß1 sequence [SEQ ID NO: 52] was obtained from cDNA amplified from rat brain and spinal cord (See Example 9 and Figure 28, below). Asterisks, carboxyl-terminal amino acids; dashes, gaps introduced to facilitate sequence alignment; overline, putative transmembrane domain; dots, cysteine residues in predicted extracellular domain (the two N-terminal cysteine residues are part of the immunoglobulin-like domain; the six other marked cysteine residues reside in the EGF-like growth factor domain).
- Figure 24 shows autoradiograms of anti-phosphotyrosine Western blots, using the neu receptor tyrosine phosphorylation assay procedure of Example 3, below. The results with recombinant rat NDF proteins expressed in COS-7 cells from the indicated cDNA clones are shown.
- COS-7(-) is medium from untransfected COS-7 cells.
- Figure 25 shows the transient expression of recombinant proNDFs from rat cDNA clones.
- COS-7 cells were transfected with expression plasmids containing cDNAs encoding the indicated NDF isoforms and were labeled with 35 S-methionine/cysteine.
- Conditioned media and cell lysates were immunoprecipitated with an affinity-purified rabbit anti-NDF antibody and analyzed by electrophoresis. The positions of molecular weight standards are shown on the right.
- Figure 26 shows endoglycosidase treatment of recombinant rat proNDFs expressed in COS-7 cells.
- COS-7 cells were transfected with proNDF-ß3 and proNDF- ⁇ 2c cDNA expression plasmids and labeled for seventeen hours with 35 S-methionine/cysteine.
- Radiolabeled COS-7 cell media and lysates were immunoprecipitated with affinity-purified rabbit antibody raised against recombinant rat NDF- ⁇ 2 14-241 .
- the immunoprecipitates were treated with endoglycosidases as indicated and analyzed by
- Figure 27 shows the reverse transcription-polymerase chain reaction (RT-PCR) analysis of NDF mRNAs expressed in various rat tissues. Reverse transcription was used to prepare first-strand cDNA from mRNA samples. NDF cDNAs were amplified in PCR reactions with primers specific for the NDF EGF-like domain. Lanes are as follows: 1, Rat-1-EJ; 2, heart; 3, skin; 4, ovary; 5, lung; 6, stomach; 7, spleen; 8, liver; 9, muscle; 10, kidney; 11, brain; 12, spinal cord; 13, cDNA clone 20 (proNDF- ⁇ 2b) 14, cDNA clone 40 (proNDF-ß2); 15, cDNA clone 42A (proNDF-ß4). Positions of DNA size markers (in base pairs) are indicated.
- Figure 28 shows the nucleotide sequence [SEQ ID NO: 55] of the PCR products obtained from rat spinal cord and brain, and a deduced partial rat NDF-ß1 amino acid sequence [SEQ ID NO: 56]. Nucleotide numbers are given in the left hand column, and amino acid numbers are given in the right hand column. The amino acid numbering corresponds to the amino acid numbering in Figure 23.
- Figure 29 shows a Northern blot analysis of several human cell lines screened for the presence of NDF-related mRNA with rat NDF clone 44 cDNA probe.
- Figure 30 shows the nucleotide sequence of human NDF cDNA clone PI [SEQ ID NO: 3] and a deduced partial amino acid sequence for human proNDF- ⁇ 1a [SEQ ID NO: 4]. Nucleotide numbers are given in the left hand column and amino acid numbers on the right hand column. Amino acid numbering corresponds to the amino acid numbering of Figure 38 (composite figure for human
- Figure 31 shows the composite nucleotide sequence of human proNDF- ⁇ 2b cDNA [SEQ ID NO: 5] and a deduced amino acid sequence [SEQ ID NO: 6]. This sequence was derived from the sequences of clone 43 [SEQ ID NO: 7] and clone 17, shown in Figures 32 and 33, respectively. Nucleotide numbers are given in the left hand column and amino acid numbers on the right hand column. Linker sequences added to the 3' end of the cDNA to facilitate cloning are included.
- Figure 32 shows the nucleotide sequence of human NDF cDNA clone 43 and a deduced human proNDF- ⁇ 2b amino acid sequence [SEQ ID NO: 8]. Nucleotide numbers are given in the left hand column and amino acid numbers on the right hand column. Linker sequences added to the 3' end of the cDNA to facilitate cloning are included.
- Figure 33 shows the nucleotide sequence of human NDF cDNA clone 17 [SEQ ID NO: 9] and a deduced partial amino acid sequence of human proNDF- ⁇ 2b [SEQ ID NO: 10]. Nucleotide numbers are given in the left hand column and amino acid numbers on the right hand column. Amino acid numbering corresponds to the amino acid numbering of Figure 38. Linker sequences added to the 5' and 3' ends of the cDNA to facilitate cloning are included.
- Figure 34 shows the nucleotide sequence of human NDF cDNA clone 19 [SEQ ID NO: 11] and a deduced partial amino acid sequence of human proNDF- ⁇ 3 [SEQ ID NO: 12]. Nucleotide numbers are given in the left hand column and amino acid numbers on the right hand column. Amino acid numbering corresponds to the amino acid numbering of Figure 38 (composite figure for human NDFs). Linker sequences added to the 5' and 3 ' ends of the cDNA to facilitate cloning are included .
- Figure 35 shows the nucleotide sequence of human NDF cDNA clone P13 [SEQ ID NO: 13] and a deduced partial amino acid sequence for human NDF-ß1a [SEQ ID NO: 14]. Nucleotide numbers are given in the left hand column and amino acid numbers on the right hand column. Amino acid numbering corresponds to the amino acid numbering of Figure 38 (composite figure for human
- Figure 36 shows the nucleotide sequence of human NDF cDNA clone 294-8 [SEQ ID NO: 15] and a deduced partial amino acid sequence for human proNDF-ß2 [SEQ ID NO: 16]. Nucleotide numbers are given in the left hand column and amino acid numbers on the right hand column. Amino acid numbering corresponds to the amino acid numbering of Figure 38 (composite figure for human
- Figure 37 shows the nucleotide sequence of human NDF cDNA clone 33 [SEQ ID NO: 17] and a deduced partial amino acid sequence for human proNDF-ß3 [SEQ ID NO: 18]. Nucleotide numbers are given in the left hand column and amino acid numbers on the right hand column. Amino acid numbering corresponds to the amino acid numbering of Figure 38 (composite figure for human NDFs). Linker sequences added to the 5' and 3' ends of the cDNA to facilitate cloning are included.
- Figure 38 is a composite of the amino acid sequences encoded by different human proNDF cDNAs
- Figure 39 is a schematic diagram of a mammalian cell vector for expression of a chimeric NDF gene comprised of rat and human sequences.
- Figure 40 shows the induction of tyrosine phosphorylation in MDA-MB-453 cells (using the assay procedure of Example 3) with conditioned medium from COS-7 cells which had been transfected with expression plasmids described in Example 11, below, containing various (human, rat, and human-rat chimera) NDF cDNA clones: Lane 1, control, 100 ⁇ l 10x concentrated conditioned medium from untransfected COS-7 cells; lanes 2 and 3, hNDF- ⁇ 2b; lanes 4 and 5, h-rNDF- ⁇ 2c; lanes 6 and 7, h-rNDF-ß1c; lanes 8 and 9, h-rNDF-ß2c; lanes 10 and 11, h-rNDF- ⁇ 1c; lanes 12 and 13, rNDF- ⁇ 2c; lane 14, positive control of human met-NDF- ⁇ 2 14-241 purified from E. coli . Even-numbered lanes represent 100 ⁇ l 10x concentrated conditioned medium from untransfected COS-7
- conditioned media, and odd-numbered lanes represent 100 ⁇ l 10x concentrated media.
- Figure 41 shows SDS-PAGE analysis of a recombinant rat NDF- ⁇ 2 purified from media conditioned by CHO cells with the pDSR ⁇ 2/rNDF- ⁇ 2c expression plasmid described in Example 11. The sizes of molecular weight markers in the left hand lane are indicated in
- Figure 42 shows the stimulation of MDA-MB-453 cell neu receptor tyrosine phosphorylation by various amounts of recombinant rat NDF- ⁇ 2 purified from media conditioned by transfected CHO cells.
- the assay shows the stimulation of MDA-MB-453 cell neu receptor tyrosine phosphorylation by various amounts of recombinant rat NDF- ⁇ 2 purified from media conditioned by transfected CHO cells. The assay
- Example 3 Procedure of Example 3 was used.
- PBSA phosphate buffered saline, or PBS, with 0.1% bovine serum albumin, or BSA
- BSA bovine serum albumin
- Figure 43 shows the stimulation of MDA-MB-453 neu receptor tyrosine phosphorylation by recombinant human and rat NDF isoforms purified from E. coli .
- PBSA was used as a negative control.
- Figure 44 shows histograms depicting gold-to-red fluorescence ratios for BT-474 cells treated for seven days in culture with recombinant human met-NDF- ⁇ 2 14-241 purified from E. coli and stained with Nile Red.
- Treatment regimens included the following concentrations of the recombinant NDF: A, 0 ng/ml (media control); B, 100 ng/ml; C, 20 ng/ml; and D, 4 ng/ml. The results show that recombinant human NDF induces accumulation of neutral lipids.
- Figure 45 shows histograms depicting gold-to-red fluorescence ratios for BT-474 cells treated for seven days in culture with recombinant rat NDF- ⁇ 2 produced by CHO cells and stained with Nile Red.
- Treatment regimens included the following concentrations of the recombinant NDF: A, 0 ng/ml (media control); B, 100 ng/ml; C, 20 ng/ml; and D, 4 ng/ml.
- A 0 ng/ml (media control); B, 100 ng/ml; C, 20 ng/ml; and D, 4 ng/ml.
- B 100 ng/ml
- C 20 ng/ml
- D 4 ng/ml
- Figure 46 shows the growth stimulatory effects of recombinant human met-NDF- ⁇ 2 14-241 on BT-474 cell proliferation in vitro . Cells were treated with
- Figure 47 shows the growth inhibitory effects of recombinant human met-NDF- ⁇ 2 14-241 on MDA-MB-468 cell proliferation in vitro . Cells were treated with
- Figure 48 illustrates the effect in vitro of rat met-NDF- ⁇ 2 14-241 on the adhesion on colon epithelial cells. Intestinal crypts were isolated from mouse colons and plated on collagen type IV coated plates.
- Figure 49 shows indirect immunofluorescence analysis of LIM 1215 colon carcinoma cells cultured in the absence (panel A), or presence (panel B) of 50 ng/ml recombinant rat NDF- ⁇ 2 purified from media conditioned by transfected CHO cells. Treated cells were sectioned, then stained with monoclonal anti-CEA antibodies
- Figure 50 shows indirect immunofluorescent analysis of LIM 1863 colon carcinoma cells cultured in the absence (panel A), or presence (panel B) of 50 ng/ml recombinant rat NDF- ⁇ 2 purified from media conditioned by transfected CHO cells. Treated cells were sectioned, then stained with rabbit anti-TIMP-2 antibodies followed with phycoerythrin-labeled goat anti-rabbit IgG
- Figure 51 shows measurements of new epithelium in rabbit ear wounds treated with different doses of recombinant human met-NDF- ⁇ 2 14-241 ,
- Figure 52 shows measurements of the area of new epithelium covering rabbit ear wounds treated with different doses of recombinant human met-NDF- ⁇ 214-241,
- Figure 53 shows measurements of the number and percentage of proliferating (BrdU-positive) basal and suprabasal keratinocytes in rabbit ear wounds treated with different doses of recombinant human met-NDF- ⁇ 2 14-241 .
- DNA sequences of this invention are valuable as products useful in effecting the large scale synthesis of human NDFs by a variety of recombinant techniques.
- DNA sequences provided by the invention are useful in generating new and useful viral and circular plasmid DNA vectors, new and useful transformed and transfected procaryotic and eucaryotic host cells (including bacterial and yeast cells and mammalian cells grown in culture), and new and useful methods for cultured growth of such host cells capable of the expression of recombinant NDFs and their related products.
- DNA sequences of the invention are also suitable materials for use as probes in isolating additional human cDNAs and genomic DNA encoding NDFs and other genes encoding related proteins.
- the DNA sequences of the invention are also suitable materials for use as probes in isolating additional human cDNAs and genomic DNA encoding NDFs and other genes encoding related proteins.
- sequences may also be useful in various alternative methods of protein synthesis (e.g., in insect host cells) or in genetic therapy in humans and other
- DNA sequences of the invention are expected to be useful in developing transgenic mammalian species which may serve as eucaryotic "hosts" for production of NDFs and NDF products in quantity. See, generally, Palmiter et al., Science 222, 809-814 (1983).
- Diagnostic applications of NDF DNA sequences of this invention are also possible, such as for the detection of alterations of genes and of mRNA structure and expression levels.
- the recombinant human NDF polypeptides of this invention are characterized by being the products of procaryotic or eucaryotic host expression (e.g., by bacterial, yeast, higher plant, insect or mammalian cells in culture) of exogenous DNA sequences obtained by genomic or cDNA cloning or by gene synthesis,
- Saccharomyces cerevisiae Saccharomyces cerevisiae
- procaryote e.g., E. coli
- non-human mammalian such as COS or CHO, and avian
- COS non-human mammalian
- avian e.g., non-human mammalian, such as COS or CHO, and avian
- recombinant NDFs of the invention may be glycosylated with mammalian or other eucaryotic carbohydrates or may be non-glycosylated.
- the recombinant NDFs of the invention may also include an additional methionine amino acid residue at the amino terminus.
- the present invention also embraces products such as polypeptide analogs of human NDFs encoded by naturally-occurring mRNAs.
- Such analogs include
- fragments of NDF or of NDF precursors fragments of NDF or of NDF precursors (proNDFs).
- NDF NDF polypeptide products
- modifications of cDNA and genomic nucleotide sequences can be readily accomplished by well-known site-directed mutagenesis techniques and employed to generate analogs and derivatives of the described NDFs.
- Such products share one or more of the biological properties of NDF polypeptide products encoded by naturally-occurring mRNAs.
- inventions include those which are foreshortened, for example, by deletions; or those which are more stable to hydrolysis (and, therefore, may have more pronounced or longer-lasting effects); or which have been altered to delete one or more potential sites for O-glycosylation and/or N-glycosylation or which have one or more
- cysteine residues deleted or replaced by other residues e.g., alanine or serine residues, and are potentially more easily isolated in active form from microbial systems; or which have one or more tyrosine residues replaced by phenylalanine and bind more or less readily to target proteins or to receptors on target cells.
- polypeptide fragments duplicating only a part of the continuous amino acid sequence are also included.
- any one or more of the products of the invention may have therapeutic utility or utility in other contexts, such as in antagonism of naturally-occurring NDFs.
- Competitive antagonists may be quite useful in, for example, cases of overproduction of NDF.
- the present invention also includes that class of polypeptides encoded by portions of the DNA
- compositions useful in treating are also encompassed by the invention.
- solubilizers useful in recombinant NDF therapy.
- a "therapeutically effective amount” as used herein refers to that amount which provides therapeutic effect for a given condition and administration regimen.
- Such compositions include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents (e.g.,
- Tween 80, Polysorbate 80 Tween 80, Polysorbate 80
- anti-oxidants e.g., ascorbic acid, sodium metabisulfite
- preservatives e.g., sodium metabisulfite
- Thimerosol, benzyl alcohol) and bulking substances e.g., lactose, mannitol
- covalent attachment of polymers to the polypeptide to prolong in vivo half-life and to enhance potency for instance, water-soluble polymers as polyethylene glycol, polypropylene glycol and copolymers of polyethylene glycol and polypropylene glycol, see, e.g., Davis et al., U.S. Patent No.
- particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc., or into liposomes.
- Such compositions will influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of recombinant NDF.
- the recombinant NDF polypeptides of this invention are expected to be useful, alone or in combination with other therapy, in treating diseases and conditions involving cells which express the neu receptor on their surfaces.
- the neu receptor on their surfaces.
- recombinant NDFs of this invention are useful as biological agents for modulating cellular proliferation and differentiation and, accordingly, are applicable for the therapeutic treatments described below.
- the neu receptor is expressed by human epithelial cells of the gastrointestinal, respiratory, urinary and reproductive tracts (Press, M. et al., Oncogene, 1990, above; Gullick, W. et al., Int. J.
- recombinant NDFs can be used to promote reepithelialization and restoration of mature, functionally differentiated phenotypes in the new epithelia.
- recombinant NDFs can be used alone or in combination with cytotoxic agents to inhibit abnormal cellular proliferation.
- Recombinant NDF polypeptides are expected to be particularly useful the treatment of dermal wounds, cancer, gastrointestinal disorders, kidney diseases, Barrett's esophagus, and diseased or damaged lung.
- Recombinant neu differentiation factors are useful in the repair of diseased and damaged skin through their ability to stimulate tyrosine
- Recombinant NDFs can be used to accelerate healing of dermal wounds, such as acute and chronic dermal wounds, excisional wounds, second and third degree burns (partial and full thickness) and
- Epidermolysis bullosa is a defect in adherence of the epidermis to the underlying dermis, resulting in frequent open, painful blisters, which can cause severe morbidity. Accelerated reepithelialization of these lesions would result in less risk of infection, diminished pain, and less. wound care.
- Recombinant neu differentiation factors are useful in cancer therapy for inhibiting proliferation of certain cancer cells.
- recombinant NDFs can restore a more mature and functionally differentiated phenotype in certain cancer cells, thereby restoring normal cellular
- recombinant NDFs can inhibit tumor cell invasion and metastasis by induction of intercellular and extracellular matrix adhesion
- Cancer cells that are susceptible to NDF treatment are those that express the neu receptor.
- Tumors in which neu receptor expression or neu gene amplification have been detected include cancers of the breast, ovary, endometrium, cervix, salivary gland, esophagus, lung, stomach, pancreas, colon, kidney, prostate, and bladder, as well as squamous cell
- carcinomas of the head and neck carcinoid tumors of the gut, glioblastomas, astrocytomas, papillary thyroid carcinomas, sebaceous and sweat gland carcinomas, and hepatocellular carcinomas.
- NDF proteins such as lung and colon cancers
- lung and colon cancers will be particularly susceptible to recombinant NDF therapy, since normal NDF levels can be restored with recombinant NDF polypeptides.
- tumors with mutated NDF genes will be responsive to NDF therapy.
- Additional inhibition of tumor growth can be achieved through enhancement of chemotherapy by recombinant NDFs.
- Antibodies and ligands that interact with the neu receptor and with the related EGF receptor enhance tumor cell sensitivity to the chemotherapeutic agent cisplatin (Aboud-Pirak, E. et al. J. Natl . Cancer Inst. 80:1605-1611 1988; Christen, R.D. et al., J. Clin . Invest 86:1632-1640, 1990; Hancock, M. C. et al. Cancer Res . 51:4575-4580 1991; Nishikawa, K. et al.. Cancer Res . 52:4758-4765, 1992). Phorbol esters, which
- NDF mRNA and the neu receptor messenger in normal gastrointestinal tissues predicts that recombinant NDFs will have activities on normal, diseased and injured stomach and intestine.
- recombinant NDF is a potent inducer of adherence for normal colonic epithelial cells. It is expected that recombinant NDFs can be useful in
- recombinant NDFs can offer a significant therapeutic improvement in the treatment of gastric ulcers.
- Duodenal ulcers like gastric ulcers, are treatable, but recombinant NDF therapy to more fully and more rapidly regenerate the mucosal lining of the duodenum would be an important advance.
- Inflammatory bowel diseases such as Crohn's disease (affecting primarily the small intestine) and ulcerative colitis (affecting primarily the large bowel) are chronic diseases of unknown etiology which result in the destruction of the mucosal surface, inflammation, scar and adhesion formation during repair, and
- Recombinant NDF therapy to stimulate resurfacing of the mucosal surface can be of benefit in controlling progression of disease.
- Gut toxicity is a major limiting factor in radiation and chemotherapy treatment regimes.
- Pretreatment with recombinant NDFs may have a
- cytoprotective affect on the small intestinal mucosa allowing increased dosages of such therapies while reducing potential fatal side effects of gut toxicity.
- Barrett's esophagus (a precursor of adenocarcinoma) exhibits esophageal columnar metaplasia with frequent overexpression of the neu receptor
- Smoke inhalation is a significant cause of morbidity and mortality in the week following a burn injury due to necrosis of the bronchiolar epithelium and the alveoli.
- Recombinant NDFs are expected to stimulate proliferation and differentiation of the bronchiolar epithelial cells, which express the neu receptor, thereby enhancing repair and regeneration of the lung epithelium damaged by smoke inhalation.
- kidney diseases with abnormal cell growth and differentiation e.g. autosomal-dominant polycystic kidney disease, acquired dialysis-associated cystic disease, and non-cystic end stage kidneys
- Recombinant NDF therapy may restore the normal differentiated phenotype to the diseased tissue and also inhibit abnormal proliferation.
- Recombinant NDFs through stimulation of the neu receptor present in epithelial cells lining kidney tubules, will also be useful in restoring normal kidney epithelium following ischemic acute tubular necrosis.
- Neu receptor expression in the liver can be detected in certain hepatitis B virus and hepatitis C virus infectious lesions of the liver (Brunt, E.M., and Swanson, P.E., Am. J. Clin . Pathol . 97:53-61, 1992).
- Recombinant NDFs can be used to treat such lesions, either alone or in combination with cytotoxic agents.
- Peripheral nerves can be used to treat such lesions, either alone or in combination with cytotoxic agents.
- Neu receptor expression is found in Schwann cells associated with transected peripheral nerves undergoing Wallerian degeneration (Cohen, J.A. et al., J. Neurosci . Res . , 1992, above). Recombinant NDFs can be used to regulate Schwann cell proliferation and maturation in cases of peripheral nerve injury or degeneration.
- NDF DNAs Altered expression of naturally-occurring NDF in diseased tissues (e.g., malignant or premalignant tissues) can be found with the NDF DNAs, NDF mRNA detection methods, anti-NDF antibodies, and NDF
- Perturbations in the expression of naturally-occurring NDFs are expected to alter normal cell growth and differentiation with pathological consequences such as neoplasia.
- Other methods can also be used to detect aberrant NDF expression in human tissues. These methods, known to those skilled in the art of analyzing protein and mRNA expression, include immunohistochemical assays, enzyme-linked immunoabsorbent assays, and polymerase chain reaction assays. The identification of premalignant and malignant cells with abnormal NDF expression is particularly useful for the diagnosis of cancer.
- the NDFs of this invention may also be combined with substances such as radiolabeled molecules, toxins, cytokines, and other compounds useful in tumor treatment, in order to increase localization of these substances on human tumors expressing high levels of the neu receptor.
- substances such as radiolabeled molecules, toxins, cytokines, and other compounds useful in tumor treatment, in order to increase localization of these substances on human tumors expressing high levels of the neu receptor.
- an effective amount of recombinant NDF or analog or derivative is an amount that is effective to alter cellular proliferation and differentiation, or to prevent, lessen the worsening of, alleviate or cure the condition for which the polypeptide is administered.
- the activity of the present polypeptide may be enhanced or supplemented with use of one or more additional biologically active or cytotoxic agents which are known to be useful in treating the same condition for which the polypeptide of the invention is being administered, e.g., IL-2 or chemotherapeutic agents for cancer
- platelet-derived growth factors for wound healing, and so forth.
- epidermal growth factor for wound healing
- Biological materials employed in these Examples were obtained as follows.
- the monoclonal antibody (Ab-3) to the carboxyl terminus of the neu receptor was obtained from Oncogene Science (Uniondale, NY).
- a monoclonal antibody to phosphotyrosine, PY20, was obtained from Amersham (Arlington Heights, IL).
- a mouse monoclonal antibody to human ß-casein was obtained through Dr. R.C. Coombes from the Ludwig Institute in London (Earl, H.M., and McIlhinney, R.A.J., Mol . Immunol . 22: 981-991,
- Rat1-EJ cell line (ATCC CRL 10984) was generated by transfection of the human EJ ras oncogene into Ratl fibroblasts as described by Peles, E. et al. in Cell, 1992, above, and by Land, H. et al., in Nature 304:596-602, 1983.
- the following cell lines were obtained from the American Type Culture Collection
- MDA-MB-231 (ATCC HTB 26), MDA-MB-453 (ATCC HTB 131), Hs 294T (ATCC HTB 140), SK-BR-3 (ATCC HTB 30), HT-1080 (ATCC CCL 121), BALB/c 3T3 (ATCC CRL 6587) and COS-7 (ATCC CRL 1651).
- COS-7 cells were cultured in Dulbecco's modified Eagle medium (GIBCO, Grand Island, NY) supplemented with 10% fetal bovine serum (Hyclone, Logan, Utah).
- MDA-MB-453 cells were grown in RPMI Medium 1640 with 15% fetal bovine serum.
- ras-transformed rat fibroblasts has been described by Peles, E. et al., in Cell, 1992, above). In order to obtain more amino acid sequence information for the design of independent
- oligonucleotide probes 300 picomoles of the purified rat protein were subjected to partial proteolysis with trypsin, as follows: ten micrograms of the protein were reconstituted in 200 ⁇ l of 0.1 M ammonium bicarbonate buffer (pH 7.8); digestion was conducted with L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (Serva) at 37°C for eighteen hours, using an enzyme-to-substrate ratio of 1:10.
- the resulting peptide mixture was separated by reversed phase HPLC and monitored at 215 nm using a Vydac C4 micro column (2.1 mm i.d. ⁇ 15 cm, 300 A) and a Hewlett-Packard 1090 liquid chromatographic system equipped with a diode-array detector and a workstation ( Figure 1).
- the column was equilibrated with 0.1% trifluoroacetic acid (mobile phase A) and elution was effected with a linear gradient from 0-55% mobile phase B (90% acetonitrile in 0.1% trifluoroacetic acid) over seventy minutes.
- the flow rate was 0.2 ml/min and the column temperature was controlled at 25°C.
- amino acid sequences of the peptides corresponding to the three major eluted fractions were determined by automated Edman degradation. Amino acid sequence analyses of the peptides were performed with a Model 477 protein sequencer (Applied Biosystems, Inc., Foster City, CA) equipped with an on-line
- PTH phenylthiohydantoinyl amino acid analyzer
- Model 900 data analysis system Haunkapiller, M.W.
- cDNA was synthesized with the "Superscript" kit (GIBCO BRL Life Technologies, Inc., Gaithersburg, MD). Column-fractionated double-strand cDNA was ligated into a SalI- and NotI-digested pJT-2 plasmid vector, to yield plasmids containing cDNA inserts such as depicted in
- the pJT-2 plasmid vector was derived from the V19.8 vector (ATCC 68124). In particular, the HindIII and SacII cloning sites of V19.8 were changed into SalI and NotI sites by using synthetic oligonucleotide linkers to yield the PJT-2 vector. Plasmids containing cDNA inserts were transformed into DH10B E. coli cells by electroporation (Dower, W.J. et al., Nucleic Acids Res . 16:6127-6145, 1988). B. Preparation of cDNA Probes and Isolation of Clones
- N-terminal region of NDF as described in Example 1C specifically, residues 5-24 (RGSRGKPGPAEGDPSPALPP) [SEQ ID NO: 71], and residues 7-12 of the T40.4 tryptic peptide (GEYMCK) [SEQ ID NO: 72].
- RGSRGKPGPAEGDPSPALPP residues 5-24
- GEYMCK tryptic peptide
- the synthetic oligonucleotides were end-labeled with ⁇ - 32 p-ATP with T4 polynucleotide kinase and used to screen replicate sets of nitrocellulose filters.
- the hybridization solution contained 6 ⁇ SSC, 50 mM sodium-phosphate (pH 6.8), 0.1% sodium-pyrophosphate, 2 ⁇ Denhardt's solution, 50 ⁇ g/ml salmon sperm DNA and 20% formamide (for probe 1) or no formamide (for probe 2). Hybridization was carried out for fourteen hours at 42°C (for probe 1) or 37°C (for probe 2).
- the filters were washed at either 50°C with 0.5 ⁇ SSC/ 0.2% SDS/ 2 mM EDTA (for probe 1) or at 37°C with 2 ⁇ SSC/ 0.2% SDS/ 2 mM EDTA (for probe 2). Autoradiography of the filters gave ten clones that hybridized with both probes. These clones were purified by re-plating and probe
- the clones were transiently expressed in COS-7 cells.
- the cDNA clones were inserted into the pJT-2 eukaryotic expression vector under the control of the SV40 promoter and 3'-flanked with SV40 termination and poly-adenylation signals.
- the resulting plasmids including the pJT-2/NDF plasmid containing clone 44 cDNA (Fig. 2), were used to transfect COS-7 cells by
- electroporation as follows. 6 ⁇ 10 6 cells in 0.8 ml Dulbecco's modified Eagle medium (DMEM) and 10% fetal bovine serum were transferred to a 0.4 cm cuvette and mixed with 20 ⁇ g of plasmid DNA in 10 ⁇ L of TE solution (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). Electroporation was performed at room temperature, 1600 volts and 25 ⁇ F, using a BioRad Gene Pulser apparatus with the pulse controller unit set at 200 ohms. The cells were then diluted into 20 ml of DMEM/10% fetal bovine serum and transferred into a T75 flask (Falcon). After fourteen hours of incubation at 37°C, the medium was replaced with DMEM/1% fetal bovine serum, and the incubation was continued for an additional forty eight hours.
- DMEM Dulbecco's modified Eagle medium
- Falcon T75 flask
- the resulting conditioned media were then evaluated for their ability to stimulate tyrosine phosphorylation of the neu receptor in MDA-MB-453 human breast tumor cells as follows.
- the conditioned media were filtered through a 0.2-micron sterile filter unit
- clone 44 cDNA was completely purified by re-plating and by filter
- clone 44 was selected for further analysis by DNA sequencing.
- Rat clone 44 cDNA was primarily sequenced using a 373A automated DNA sequencer and "Taq DyeDeoxyTM Terminator" cycle sequencing kits from Applied
- a 594 base-long untranslated stretch Downstream of the 3' end of this reading frame is a 594 base-long untranslated stretch.
- the latter includes a poly (A) tail preceded by a polyadenylation signal (AAATAAA) . Because no stop codon and recognizable signal peptide sequence were found at the amino terminus of the longest open reading frame, the nucleotide sequences of three other independent positive cDNA clones were analyzed in a similar manner. All three clones included an
- the combined nucleotide sequence of the rat cDNA clones [SEQ ID NO: 19] is presented in Figure 4, and the clone 44 cDNA sequence is given in Figure 5.
- the combined sequence spans 2,186 base pairs, including a poly (A) tail, and contains an open reading frame of four hundred and twenty-two residues [SEQ ID NO: 20] if the amino terminal methionine is considered to be the initiator codon.
- Hydropathy analysis ( Figure 6) revealed no prominent hydrophobic sequence at the amino terminus of the protein that could function as a signal peptide for protein secretion (von Hijne, G.,
- extracellular domain contains all of the peptide sequences that were determined directly from the purified protein (overlined in Fig. 4). They all perfectly matched the sequence deduced from the cDNA except for threonine residue 137, which was assigned as isoleucine in peptide T27.3 (Fig. 1). Five different cDNA clones encoded a threonine residue in the
- the NDF precursor is expected to accumulate on the surface of cells expressing high levels of NDF mRNAs.
- Membrane-bound precursor proteins have been found for other members of the EGF growth factor family.
- Brachmann, R., et al. demonstrated the presence of transforming growth factor ⁇ on the surface of cells expressing transforming growth factor ⁇ mRNA.
- Molecules that specifically bind proNDF could selectively direct therapeutic molecules to cells overexpressing NDF.
- monoclonal antibodies raised against recombinant NDF and conjugated to therapeutic molecules could selectively localize the therapeutic molecule on the surface of cells expressing high levels of membrane-bound proNDF.
- Such cells would include tumor cells with an activated ras gene.
- Antibody conjugates could be constructed using
- chemotherapeutic compounds include cytokines, toxins, and others.
- lymphokines or radionuclides are lymphokines or radionuclides.
- ⁇ means less than
- Excellulose GF-5 desalting column (Pierce).
- the specific activity of the radiolabeled naturally-occurring NDF was 3 ⁇ 10 6 cpm/ng.
- the radiolabeled NDF (10 picomolar) was incubated for sixty minutes at 4°C with monolayers of MDA-MB-453 cells that were grown in a 24-well dish (Costar). This incubation was also performed in the presence of conditioned media from transfected COS-7 cells. Unbound 125 I-NDF was removed by three washes with phosphate-buffered saline (PBS), and the cells were solubilized in 0.1 N NaOH solution containing 0.1% SDS. Radioactivity was determined with a ⁇ -counter.
- PBS phosphate-buffered saline
- conditioned medium containing recombinant NDF expressed from the pJT-2/NDF expression vector (Fig. 2) in COS-7 cells reduced the total 125I-NDF binding by approximately 50%.
- COS-7 conditioned medium from cells transfected with a pJT-2 expression vector containing a rat TGF ⁇ cDNA (Blasband, A. et al., Mol . Cell Biol . 10 : 2111-2121, 1990) was employed.
- the TGF ⁇ conditioned medium did not inhibit 125I-NDF binding.
- the partial inhibitory effect of the recombinant rat NDF conditioned medium may be attributed either to its relatively low concentration in the binding assay, or to high non-specific ligand binding.
- the 1.9 kb-long cDNA insert of rat clone 44 was labeled with ⁇ - 32 P-dCTP by the random priming method (Feinberg, A.P., and Vogelstein, B., Anal. Biochem 232:6-13, 1983).
- the conditions of hybridization were as follows: 6 ⁇ SSC, 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 2 ⁇
- Denhardt's solution 50 ⁇ g/ml salmon sperm DNA and 50% formamide. Hybridization was carried out for fourteen hours at 42°C, followed by washing for thirty minutes at 60°C with 0.2 ⁇ SSC, 0.1% SDS and 2 mM EDTA. The filters were exposed to Kodak XAR x-ray film with an intensifier screen at -70°C for the indicated periods of time.
- Figure 12 shows that three bands were visualized in the Northern blots with poly (A) selected RNA of Rat-1-EJ fibroblasts. Their molecular sizes corresponded to 6.8, 2.6, and 1.7 kilobases.
- panel B demonstrates tissue-specific regulation of NDF mRNA expression levels in adult rat tissues.
- the highest NDF mRNA expression was observed in the spinal cord.
- NDF mRNAs were also detected in brain tissue. In addition to the mRNAs described above, these tissues express a variant mRNA that is 3.4 kb in size.
- Other positive tissues include lung, ovary and stomach. Relatively low amounts of the middle-size transcript were displayed by the skin, kidney and heart. The liver, spleen and muscle did not contain detectable NDF mRNA.
- the Northern blot also shows tissue-specific differences in the relative proportion of the different NDF mRNA species. Each variant mRNA could encode variant NDF proteins with different biological properties.
- Naturally-occurring NDF may regulate cell proliferation and differentiation through tissue-specific variations in NDF mRNA structure and expression.
- FIG. 12 panel C, shows that human tumor cells can overexpress NDF mRNA.
- An initial survey of human tumor cell lines found that HT-1080 fibrosarcoma cells, Hs 294T melanoma cells and MDA-MB-231 mammary adenocarcinoma cells express elevated NDF mRNA levels.
- the nucleotide sequences for both strands of the NDF coding region were determined for all ten cDNAs using synthetic oligonucleotide primers with fluorescence-based dideoxy-DNA sequencing as described in Example 4.
- the cDNAs ranged in size from 1.1 kb to 3.2 kb and, presumably, are derived from the 6.8, 2.6 and 1.7 kb NDF mRNAs found in Rat-1-EJ cells (See
- Example 6 DNA sequence analyses revealed that these ten cDNAs encode six distinct NDF precursor proteins (proNDFs) (Table 2, and Figures 5 and 13-21). The open reading frames in the ten cDNAs ranged from 0.7 kb
- clone 4 to 2.0 kb (clone 42A).
- Two of the cDNAs were partial cDNA clones.
- the clone 4 sequence begins at proNDF codon 11.
- Clone 40 is truncated at codon 36 of the intracellular domain.
- Clone 44 has a complete open reading frame, but has a truncated 5'-untranslated sequence.
- the ten cDNAs encode six homologous NDF precursor proteins comprised of 241 to 662 amino acid residues, which are designated proNDF- ⁇ 2a [SEQ ID NO: 43], proNDF- ⁇ 2b, proNDF- ⁇ 2c, proNDF- ⁇ 2a, proNDF- ⁇ 3 and proNDF ⁇ -4a ( Figures 22, 23 and Table 2).
- the six predicted proNDFs are identical in the first 213 amino acid residues. This identical region includes: a basic N-terminus which is proteolytically processed at amino acid 14 in naturally occurring rat NDF; an
- ProNDF cDNAs do not encode N-terminal hydrophobic signal peptide sequences. However, with the exception of proNDF- ⁇ 3, a 23-amino acid hydrophobic region is present in the proNDFs . Carboxy-terminal to this putative transmembrane domain is a cytoplasmic domain that is invariant in the first 157 amino acid residues.
- variable region of the proNDF ectodomain begins in the EGF-like domain at amino acid position 213.
- the ⁇ / ⁇ variation alters the
- the ⁇ / ⁇ sequence variation may alter receptor activation in different cellular contexts.
- the NDF isoforms may differentially bind receptor heterodimers formed between the NDF receptor and related receptor molecules, such as the 170 kDa EGF receptor (Wada, T. et al., Cell 61:1339-1347, 1990; Spivak-Kroizman, T. et al., J.
- pl60erbB3 Plowman, G.D. et al., Mol . Cell Biol . 10 : 1969-1981 , 1990
- p180 erbB4 Plowman, G.D. et al., Proc. Natl . Acad. Sci . USA 90 : 1746-1750 , 1993.
- proNDF- ⁇ proteins share a common sequence at positions 213 to 230 that is distinct from the corresponding NDF- ⁇ sequence. Variation between members of the proNDF- ⁇ subfamily occurs carboxy-terminal to the EGF-like domain, where the ⁇ 1 and ⁇ 4a proteins contain additional amino acid residues. ProNDF- ⁇ 3 cDNA encodes a stop codon in this region, resulting in a smaller protein that lacks the transmembrane and cytoplasmic domains.
- the different proNDF sequences may alter proteolytic release of 44 kDa isoforms from the larger NDF precursor molecules.
- the proNDF proteins differ significantly in the number of charged residues located between the EGF-like domain and the hydrophobic
- the 27-amino acid sequence that is unique to the ⁇ 4a isoform includes nine basic residues and five acidic residues. This sequence introduces several dibasic residues that are potential proteolytic cleavage sites.
- Cytoplasmic domains with 374, 196 and 157 amino acid residues distinguish proNDFs ⁇ 2a, ⁇ 2b and ⁇ 2c, respectively ( Figures 5, 14, 15, 17, 21, 22, and
- the proNDF- ⁇ 2 a, b and c isoforms are identical in the first four hundred and twenty-two amino acid
- the proNDF- ⁇ 2a cytoplasmic domain contains two hundred and seventeen additional amino acid
- ProNDF- ⁇ 2b cDNA encodes a different 39-amino acid sequence
- ProNDF- ⁇ 2c has the shortest cytoplasmic domain, with the cDNA having a stop codon at codon position 423 followed by a different 3'-untranslated sequence.
- NDF proteins in transfected COS-7 was also analyzed by immunoprecipitation of 35 S-labeled proteins.
- antibodies were raised by immunizing rabbits with recombinant rat met-NDF- ⁇ 2 14- 241 purified from E. coli as described in Example 14 (Section I), below. Rabbits were immunized with an initial injection of 200 ⁇ g of recombinant rat met-NDF- ⁇ 2 14-241 . The antigen was emulsified with complete
- the resulting purified anti-NDF antibody was used to immunoprecipitate recombinant 35 S-labeled NDF proteins from COS-7 cell cultures expressing the various recombinant precursor proteins.
- COS-7 cells transfected with proNDF cDNA expression plasmids were grown in 60-mm dishes in DMEM plus 10% FBS at 37°C for forty eight hours. Cells were washed and placed in 1.5 ml DMEM minus methionine and cysteine with 1% dialyzed FBS.
- the cell lysates were clarified by centrifugation and diluted 1:4 in immunoprecipitation dilution buffer (1.25% Triton X-100; 190 mM NaCl; 60 mM Tris-HCl, pH 7.4; 6 mM EDTA; 10 units/liter trasylol).
- the radiolabeled COS-7 cell conditioned media and diluted cell lysates were pretreated at 4°C for two hours with 20 ⁇ l of rabbit normal serum and 20 ⁇ l of protein A-Sepharose CL-4B (Sigma) diluted 1:1 in PBS. The protein A-Sepharose was then removed by centrifugation.
- the pretreated samples were gently agitated at 4°C overnight with affinity-purified anti-NDF antibody (20 ⁇ g/ml) and 20 ⁇ l protein A-Sepharose.
- the protein A-Sepharose beads were pelleted and washed four times in a washing solution (0.1% Triton X-100; 0.02% SDS; 150 mM NaCl; 50 mM Tris-HCl, pH 7.5; 5 mM EDTA; 10 units/liter trasylol).
- the washed beads were heated with 60 ⁇ l of SDS gel electrophoresis loading buffer at 100°C for three minutes.
- NDF proteins were undetectable in cell lysates and in conditioned media from COS-7 cells transfected with irrelevant cDNAs, and also when samples were immunoprecipitated in the presence of excess unlabelled recombinant NDF (not shown in Figures).
- the immunoprecipitated proteins correspond in size to the processed form of NDF purified from media conditioned by Rat-1-EJ cells.
- the 40 to 45 kDa recombinant NDF proteins were found in both cell lysates and conditioned media. Larger NDF precursor proteins were detected as high molecular weight proteins immunoprecipitable from the cell lysates, but not from conditioned media.
- the NDF ⁇ 2a, ⁇ 2a, and ⁇ 4a precursor proteins are significantly larger than the NDF ⁇ 2b and ⁇ 2c precursor proteins.
- immunoprecipitated proteins are presumably the full-length, glycosylated NDF precursor proteins.
- NDF- ⁇ 2c precursor proteins were found at higher levels than the other precursor isoforms, suggesting somewhat less efficient processing for this variant.
- proNDF- ⁇ 3 cDNA directed the synthesis of 30- to 35-kDa proteins. These proteins were found in the COS-7 cell lysate, but not in the conditioned medium ( Figure 25).
- Prestained molecular weight markers were from Novex. Enzymatic deglycoslyation of the 40-44 kDa and 60-75 kDa NDF- ⁇ 2c recombinant proteins reduced their size
- glycoslyations are more abundant than N-linked
- NDF- ⁇ 3 is apparently modified prior to proteolytic processing of the proNDF.
- NDF- ⁇ 3 was resistant to glycanase treatment. This result, and the exclusively intracellular localization of NDF- ⁇ 3, indicates that NDF- ⁇ 3 protein does not enter the proNDF.
- Amino terminal signal peptide sequences are notably absent from all of the predicted NDF precursor proteins.
- the different isoforms of proNDF (with the exception of proNDF- ⁇ 3) are all glycoslyated and processed to 40 to 45 kDa biologically active NDF glycoproteins. These proteins are found in the
- ProNDF- ⁇ 3 which accumulates as an unglycosylated intracellular protein, has essentially the entire proNDF extracellular domain.
- rat clone 40 cDNA encodes a truncated proNDF- ⁇ 2
- the intracellular, nonsecreted NDF- ⁇ 3 protein may be
- NDF- ⁇ 3 may function as a sequestered growth factor that is released when tissues are injured.
- IL-1 ⁇ A similar role has been proposed for IL-1 ⁇ , which also lacks a signal peptide sequence (Mitzutani, H. et al., J. Clin . Invest . 87:1066-1071, 1991).
- NDF- ⁇ 3 could have intracellular functions.
- NDF cDNA sequences encoding the variable portion of the EGF-like domain (e.g. proNDF- ⁇ 2a codon positions 200-241) were amplified using the synthetic oligonueleotide primers 5' -GCGTCTAGATGAAGGACCTGTCAAAOCC-3' (sense) [SEQ ID NO: 75] and 5'- GCGGGATCCCTTCTGGTAGAGTTCCTCC-3' (antisense) [SEQ ID NO: 75] and 5'- GCGGGATCCCTTCTGGTAGAGTTCCTCC-3' (antisense) [SEQ ID NO: 75] and 5'- GCGGGATCCCTTCTGGTAGAGTTCCTCC-3' (antisense) [SEQ ID NO: 75] and 5'- GCGGGATCCCTTCTGGTAGAGTTCCTCC-3' (antisense) [SEQ ID NO: 75] and 5'- GCGGGATCCCTTCTGGTAGAGTTCCTCC-3' (antisense) [SEQ ID NO
- RNA samples (1 ⁇ g) from normal adult female Sprague-Dawley rat (Charles River Laboratory, Wilmington, MA) tissues and from the Rat-1 and Rat-1-EJ cell lines were reverse transcribed to generate first strand cDNA with cDNA
- PCR reactions employed Perkin Elmer Cetus GeneAmpTM PCR kits. The reactions were in a final volume of 50 ⁇ l and contained 10% of the reverse transcription reaction products or approximately 100 ng of cloned cDNAs as positive controls. Twenty-five cycles in a Perkin Elmer-Cetus GeneAmpTM 9600
- thermocycler amplified the PCR products. Each cycle included twenty seconds at 94°C, twenty seconds at 55°C, and twenty seconds at 72°C. Ten ⁇ l aliquots of each reaction mixture were analyzed by electrophoresis in 2% agarose gels, followed by ethidium bromide staining.
- NDF- ⁇ 1 is homologous to human heregulin ⁇ 1 (Holmes et al. Science, 1992, above).
- Tissue-specific NDF variants were not detected by PCR and DNA sequencing with the exception of
- a NDF- ⁇ 1 related cDNA (designated ARIA-1) was cloned from an embryonic chick brain cDNA library (Falls et al.. Cell, 1993, above). Recombinant ARIA-1
- NDF and ARIA in situ hybridizations with NDF and ARIA probes detect expression in spinal cord motor neurons, intestinal and dorsal root ganglia, and neuroepithelium lining lateral ventricles of the brain (See, also, Orr-Urtreger, et al., Proc. Natl . Acad. Sci . USA, 1993, above).
- the conserved 374 amino acid cytoplasmic domain found in proNDFs ⁇ 2a, ⁇ 2a and ⁇ 4a is more than 85% identical to the proHRG (Holmes et al.. Science, 1992, above) and proARIA-1 (Falls, D.L. et al.. Cell, 1993, above) cytoplasmic domains. All of these cytoplasmic domains have a carboxy-terminal valine residue that may be important for proteolytic processing.
- ProTGF- ⁇ has a carboxy-terminal valine that is critical for regulated release of TGF- ⁇ (Bosenberg, M.W. et al.. Cell 71:1157- 1165, 1992). Leucine or isoleucine can substitute for the terminal TGF- ⁇ valine in directing cleavage.
- ProNDF- ⁇ 2b has a 196 amino acid residue cytoplasmic domain with a carboxy-terminal leucine and is processed efficiently to mature NDF- ⁇ 2.
- the 157 amino acid proNDF- ⁇ 2c cytoplasmic domain ends with two hydrophilic residues. Accumulation of proNDF- ⁇ 2c within transfected COS-7 cells suggests that this precursor isoform is less efficiently processed. However, processing to mature NDF- ⁇ 2 does occur, demonstrating that NDF release from COS-7 cells does not require a terminal hydrophobic residue. In nontransfected cells, alternative carboxy-termini may be more critical for mature NDF release.
- RNAs were transferred onto nitrocellulose filters and probed with 32 P-labeled rat NDF clone 44 cDNA (See
- Example 6 a human kidney adenocarcinoma cell line, A-704 (American Type Culture Collection, ATCC HTB 45) exhibited the highest level of NDF mRNA
- Double-strand cDNA was synthesized from poly (A) + RNA extracted from A-704 cells. Column-fractionated cDNA was ligated to SalI and NotI digested plasmid vector pJT-2 and transformed into E. coli strain DH10B (GIBCO BRL) or E. coli strain MC1061 (BioRad) by electroporation, using a cDNA kit and procedure
- Hybridization was at medium stringency (30% formamide , 6x SSC, 2x Denhardt's solution, 100 ⁇ g/ml salmon sperm DNA, 1 mM EDTA, 0.2% SDS, 0.1% sodium pyrophosphate, 50 mM NaH 2 PO 4 , pH 6.8) at 42°C overnight. Filters were washed in a solution of 0.5x SSC, 2 mM EDTA, and 0.2% SDS at 23°C for thirty minutes; then at 42°C for ninety minutes. The filters were exposed overnight to X-ray film at -75°C.
- NDF- ⁇ 2b human NDF- ⁇ 2b [SEQ ID NO: 7] ( Figure 32, clone 43), and two cDNAs encoding partial human NDF sequences, designated as NDF- ⁇ 2b [SEQ ID NO: 7] ( Figure 32, clone 43), and two cDNAs encoding partial human NDF sequences, designated as NDF- ⁇ 2b [SEQ ID NO: 7] ( Figure 32, clone 43), and two cDNAs encoding partial human NDF sequences, designated as NDF- ⁇ 2b [SEQ ID NO: 7] ( Figure 32, clone 43), and two cDNAs encoding partial human NDF sequences, designated as NDF- ⁇ 2b [SEQ ID NO: 7] ( Figure 32, clone 43), and two cDNAs encoding partial human NDF sequences, designated as NDF- ⁇ 2b [SEQ ID NO: 7] ( Figure 32, clone 43), and two cDNAs encoding partial human NDF
- a cDNA library was synthesized with poly (A) + RNA extracted from A-704 cells. Double-strand cDNA was ligated to BstXI linkers and ligated into the BstXI site on plasmid vector pCDNA II (Invitrogen, San Diego, CA). The ligation mixture was used to transform E. coli strain DH10B.
- Denhardt's solution 100 ⁇ g/ml salmon sperm DNA, 1 mM EDTA, 0.2% SDS, 0.1% sodium pyrophosphate, 50 mM NaH 2 PO 4 , pH 6.8) at 42°C overnight.
- the filters were washed in a solution made of 0.1x SSC, 2 mM EDTA, 0.2% SDS, at 23°C for 30 min; then at 70°C for 60 min.
- the filters were then exposed to X-ray films at -75°C overnight.
- NDF cDNA sequences ( ⁇ 1 kb)
- This region contains: the spacer domain; the variable EGF-like domain ( ⁇ and ⁇ forms); the variable sequences between the EGF-like domain and the transmembrane domain; the conserved transmembrane domain; and a portion of the cytoplasmic tail.
- the sense primer was 5' -AGG AAA TGA CAG TGC CTC T-3' [SEQ ID NO: 78].
- the antisense primer was 5'-TCT CTG GCA TGC CTG AGG-3' [SEQ ID NO: 79]. PCR was carried out for 40 cycles. The reaction conditions were: 94°C, 1 minute; 50°C,
- the human proNDF cDNA clones are summarized in Table 4, and the composite amino acid sequence for the different human proNDF isoforms is shown in Figure 38.
- Rat cDNA clone 44 (proNDF- ⁇ 2c, Figure 5) was subjected to PCR using two primers. The 37-base
- oligonucleotide sense primer [SEQ ID NO: 80] had the sequence: 5'-CGG TCT AGA AGC TTC CAC CAT GTC TGA GCG CAA AGA A-3'. It included 5' XbaI and HindIII restriction enzyme cloning sites followed by the Kozak consensus sequence CCACC, as well as the initial 18 bases of the rat NDF coding sequences.
- the 30-base oligonucleotide antisense primer [SEQ ID NO: 81] had the sequence: 5'-GCC GTC GAC CTA TTA CCT TTC GCT ATG AGG-3 '.
- Rat clone 42A cDNA ( Figure 20) encodes proNDF-ß4a, with a unique XbaI site at amino acid positions 244-245.
- Example 11 included the same 37-base sense primer described in Example 11 [SEQ ID NO: 80] for expression of rat NDF- ⁇ 2c.
- This primer contained XbaI and HindIII sites, the Kozak consensus sequence CCACC, and the 18 bases
- the 27-base antisense primer had the following sequence: 5'-GCT CTA GAG GCT TCT CTG TTT CTT GCC-3' [SEQ ID NO: 82] and contained an XbaI site and 19 bases of the upstream sequences adjacent to the XbaI site mentioned above.
- the rat proNDF-ß4a (clone 42A) cDNA was subjected to PCR using this pair of primers.
- the PCR product was
- the second pair of PCR primers included a 33-base sense primer with the following sequences: 5'-GCT CTA GAA AGA AAA TTG GAT CAT AGC CTT GTG-3' [SEQ ID NO: 83], containing an XbaI site and 25 bases of the adjacent sequences downstream from the XbaI site in NDF.
- the 33-base antisense primer had the sequence 5'-GCC GTC GAC CTA TTA TAC AGC AAT AGG GTC TTG-3' [SEQ ID NO: 84], and contained a SalI site, two tandem translation stop codons and 18 bases complementary to sequences encoding the proNDF-ß4a carboxyl terminus.
- the rat proNDF-ß4a cDNA was subjected to PCR using this second pair of primers.
- the PCR product was digested with XbaI and SalI to generate an approximately 1.3 kb DNA fragment which encoded amino acids 245-662.
- the approximately 730 bp HindIII-XbaI fragment and this approximately 1.3 kb Xba-SalI fragment were ligated with the expression vector pDSR ⁇ 2 which was digested with HindIII and SalI.
- the resulting plasmid was designated pDSR ⁇ 2/rNDF-ß4a.
- the initial plasmid made to express recombinant human NDF- ⁇ 2b was constructed following the strategy described above for expresson of rat NDF- ⁇ 2c.
- Human NDF- ⁇ 2b clone 43 cDNA ( Figure 32) was used as the template DNA for PCR.
- the two primers used had the following sequences: sense primer (37-mer): 5'-CGG TCT AGA AGC TTC CAC CAT GTC CGA GCG CAA AGA A-3' [SEQ ID NO: 85]; antisense primer (30-mer): 5'-GCC GTC GAC CTA TTA GAG AAT GAA GCC CAA-3 ' [SEQ ID NO: 86].
- transfected cell clones expressing recombinant rat proNDF- ⁇ 2c consistently secreted higher levels of NDF protein than the transfected cell clones expressing recombinant human proNDF- ⁇ 2b.
- some of the human proNDF- ⁇ 2b expressing cell clones had NDF mRNA levels comparable to that of rat NDF- ⁇ 2c clones, but produced much lower levels of NDF protein. Since the cytoplasmic portion of the rat proNDF- ⁇ 2c amino acid sequence somehow augments recombinant NDF production (i.e., affects proNDF stability or enhances the
- NDF- ⁇ 2b DNA was engineered in this way, many clones were isolated which produced increased amounts of
- the chimeric human-rat proNDF- ⁇ 2c expression vector was constructed by the following method.
- Human NDF- ⁇ 2b (clone 43) cDNA ( Figure 32) was subjected to PCR using two PCR primers.
- the sense primer (39-mer) had the sequence: 5' -GCC GAA GAC GGT CAT GAA GCT TCT GCC GCT GTT TCT TGG-'3 [SEQ ID NO: 87], and included an unique internal XhoI site;
- the antisense primer (33-mer) had the sequence: 5'-CCT TTC AAA CCC CTC GAG ATA CTT GTG CAA GTG-3' [SEQ ID NO: 88] and included several base changes to create a HindIII site.
- the resulting PCR product of approximately two hundred and ten base pairs, encoding proNDF- ⁇ 2b amino acids 206-274 and including the
- transmembrane domain (amino acids 243-265), was digested with XhoI and HindIII and subcloned into plasmid
- the resulting plasmid was a chimeric molecule encoding the human proNDF- ⁇ 2b extracellular domain, the conserved transmembrane domain (TM) , and the rat proNDF- ⁇ 2c cytoplasmic domain.
- This plasmid was designated as pDSR ⁇ 2/h-rNDF- ⁇ 2c. e. Chimeric human-rat NDF- ⁇ 1c, ß1c and ß2c
- NDF- ⁇ 2 from a chimeric rat-human DNA construct was also used for expressing recombinant human NDF- ⁇ 1, -ß1 and -ß2 isoforms. Briefly, cDNAs individually encoding human proNDF- ⁇ l, -ß1 and -ß2 were subjected to PCR using the same primers ⁇ described above for generating the chimeric human-rat NDF- ⁇ 2c DNA.
- the -210 bp XhoI- HindIII PCR fragments generated from human NDF cDNA clone PI as the template represents sequences from human NDF- ⁇ 1; the human NDF cDNA clone P-13 template gave the human NDF-ß1 sequence; and the human NDF cDNA clone 294- 8 template yielded the human NDF-B2 sequence.
- the XhoI-HindIII fragments were first subcloned into pGEM7Zf(-).
- COS-7 cells ATCC CRL 1651
- pDSR ⁇ 2/hNDF- ⁇ 2b ATCC CRL 1651
- pDSR ⁇ 2/h-rNDF- ⁇ 2c pDSR ⁇ 2/h-rNDF-ßlc
- pDSR ⁇ 2/h-rNDF-ß2c pDSR ⁇ 2/h-rNDF-ß2c
- Transfected COS-7 cells were plated at 2 ⁇ 10 6 cells per 60 mm plate in DMEM with 10% FBS.
- Conditioned medium (CM) from twenty-four to seventy-two hours post-transfection were collected from the plates and tested in the neu receptor tyrosine phosphorylation assay using MDA-MB-453 carcinoma cells as described in Example 3. As shown in Figure 40, all of the CM samples tested at two concentrations (100 ⁇ l unconcentrated CM, or 100 ⁇ l of 10-fold concentrated CM) were active in the assay.
- Conditioned medium from untransfected COS-7 cells was negative (Fig. 40, lane 1). EXAMPLE 12
- the CHO D- cell line requires proline for growth and is routinely grown in high glucose DMEM, supplemented with non-essential amino acids (NEAA) , 16 ⁇ M thymidine,
- Plasmid DNAs were introduced into the CHO D- cells by calcium phosphate precipitation. Approximately 0.8 ⁇ 10 6 cells in 60 mm plates were exposed to 2 to 3 ⁇ g of plasmid DNA together with sufficient mouse spleen DNA to total 10 ⁇ g. The medium was changed the next day. After incubation for another twenty-four hours, the cells were split into eight 100-mm plates containing selective medium (high glucose DMEM with NEAA and 5% dialyzed FBS but without nucleosides). After ten to fourteen days, clones were picked and serum-free
- Example 3 or analyzed by SDS/polyacrylamide gel
- recombinant clones were grown in a spinner bottle in a 1:1 mixture of high glucose DMEM and F12 with NEAA, 5% FBS and 2 mM glutamine. Cells were then transferred to 850 cm 2 roller bottles containing the same medium with 2 ⁇ 10 7 cells per bottle. After three to four days at 37°C, the cell monolayers were washed with PBS and re-fed with 150 to 200 ml fresh medium per bottle (as above but lacking FBS). Conditioned medium was
- Recombinant rat NDF- ⁇ 2 expressed by CHO cells transfected with pDSR ⁇ 2/rNDF- ⁇ 2c was purified by the following procedure. Pooled serum-free conditioned media from harvests of roller bottles containing the transfected CHO cells expressing
- recombinant rat NDF- ⁇ 2c were cleared by filtration through 0.2 ⁇ filters and concentrated with a Pellicon diafiltration system with 10-kDa molecular size cut-off membrane. Concentrated material was directly loaded onto a column of heparin-Sepharose, pre-equilibrated with 20 mM sodium phosphate buffer pH 7.210.1)
- the dialyzed, clarified sample was then loaded onto a DEAE Sepharose 6B fast flow column which had been pre-equilibrated with dialysis buffer described above.
- the column was extensively washed until no absorbance at 280 nm could be detected.
- the column was then developed with a 200 ml gradient of 0.02 M to 0.5M NaCl in 20 mM sodium phosphate buffer, pH 7.210.1. Fractions were collected and assayed for NDF content. Ammonium sulfate was added to the pooled NDF fraction obtained from DEAE Sepharose chromatography to achieve a concentration of 2 M.
- the material was loaded on a Phenyl-Sepharose 4B column (Pharmacia) pre-equilibrated with 20 mM sodium phosphate, pH 7.210.1 containing 2 M ammonium sulfate. After loading, the column was washed with starting buffer and developed with a gradient of ammonium sulfate (from 2 M to no salt) in 20 mM sodium phosphate, pH 7.110.1. The main peak of activity was pooled and extensively dialyzed against PBS buffer and concentrated by centrifugation using a Centriprep 10 cartridge. IV. Characterization of the purified recombinant rat NDF- ⁇ 2 from CHO cells
- the purified recombinant rat NDF- ⁇ 2 isoform migrates at a molecular weight of about forty to forty-four kilodaltons, identical to that of the
- Expression plasmids, pCFM1656 and pCFM3106, used in the following examples can be derived from
- plasmid pCFM836 (a detailed description of pCFM836 is contained in U.S. Patent No. 4,710,473, incorporated
- Plasmid pCFM1656 can be derived from pCFM836 plasmid by destroying the two endogenous Ndel restriction sites by end filling with T4 polymerase enzyme followed by blunt end ligation, by replacing the DNA sequence between the unique Aatll and Clal
- Plasmid pCFM3106 can be derived from pCFM1656 by making a series of site-directed base changes by PCR
- the base pair changes are as follows: plasmid bp # bp in PCFM1656 bp changed to in
- Table 5 shows the PCR primer sequences used to amplify NDF coding sequences for insertion into pCFM1656 and pCFM3106.
- NDF- ⁇ 2 14-241 a DNA fragment encoding NDF- ⁇ 2 14-241 was first constructed. This fragment contained the native cDNA sequence corresponding to amino acid positions 14-241 of human NDF- ⁇ 2.
- the oligonucleotide primers 409-27 [SEQ ID NO: 97] and 409-28 [SEQ ID NO: 98] were used to amplify the NDF coding sequence from human NDF- ⁇ 2 cDNA plasmid clone 43 ( Figure 32).
- the 5' forward (sense) primer 409-27 provided an XbaI site, a ribosome-binding sequence, and a methionine start codon followed by the NDF- ⁇ 2 gene sequence starting from codon 14.
- the 3 1 backward (antisense) primer 409-28 contained a BamHI site and the complementary sequence of a TAA stop codon and the NDF- ⁇ 2 3' coding sequence ending at codon 241.
- the PCR fragment amplified from cDNA clone 43 was ligated into the XbaI and BamHI sites of plasmid
- E. coli strain FM5 ATCC #53911 which is a derivative of strain K-12 with a temperature sensitive ⁇ repressor gene, CI857 (Sussman et al., C. R. Acad. Sci. 254:
- the transformant was designated Strain 1664.
- the met-huNDF- ⁇ 2 expression level of Strain 1664 was subsequently improved by optimizing the first 12 codons of the protein coding sequence.
- a fragment with NdeI and SacII cohesive ends was generated by annealing two chemically synthesized oligonucleotides (436-23 and 436-24) [SEQ ID NOS: 100 and 101,
- NDF cDNAs isolated from cDNA libraries that encode different human NDF isoforms differ in the sequences 3' to an unique XhoI site in the EGF-like coding region, which makes the XhoI site a convenient cloning site for the construction of different
- Oligonucleotide 502-10 [SEQ ID NO: 106] containing sequences 5' to the XhoI site was used as the 5' primer, and oligonucleotide 502-11 [SEQ ID NO: 107], which provided the BamHI site and TAA stop codon sequence, was the 3' primer.
- the resulting PCR fragment amplified from human NDF- ⁇ 1 cDNA clone P1 ( Figure 30) was used to replace the XhoI-BamHI fragment in plasmid pCFM1656-methuNDF- ⁇ 2 14-241 , and thus generated the NDF- ⁇ l expression plasmid pCFM1656-met-huNDF- ⁇ 1 14-249 .
- Strain 1854 was the designation assigned to the FM5 strain containing this plasmid.
- d Recombinant human met-NDF- ⁇ 1 14-246
- the primers 502-10 and 502-11 [SEQ ID NOS: 106 and 107, respectively] were also used for PCR synthesis of a NDF- ⁇ 1 gene.
- the XhoI-BamHI fragment in pCFM1656-methuNDF- ⁇ 2 14-241 was replaced by the PCR fragment amplified from pCFM1656-met-huNDF- ⁇ 1 177-246 (described below in Section e).
- the FM5 strain harboring the resulting construct pCFM1656-met-huNDF- ⁇ 1 14-246 was designated
- Clone 294-1 is a partial cDNA clone for NDF- ⁇ 1 which encodes amino acids 119 to 410 ( Figure 38) . It is isolated by PCR as described in Example 10 (Section IV). Strain 1769 was assigned to this FM5 strain, bearing pCFM1656-met-huNDF- ⁇ 1 177-246 . f. Recombinant human NDF- ⁇ 2 177-241
- the ⁇ 2 isoform counterpart of pCFM1656-methuNDF- ⁇ 2 177-241 was constructed similarly with PCR primers 437-29 [SEQ ID NO: 102] and 443-1 [SEQ ID NO: 103] from human cDNA clone 43.
- the expression level of this resulting strain 1697 with pCFM1656-met-huNDF- ⁇ 2 177- 241 was unsatisfactory. Therefore, secretion was considered as an alternative to produce NDF- ⁇ 2 177-241 .
- An ompA secretion signal peptide (A) was fused to the cDNA sequence encoding human NDF- ⁇ 2 amino acids 177-241.
- Oligonucleotide 502-24 [SEQ ID NO: 108] containing an XbaI cloning site, a ribosome-binding sequence, a methionine start codon, degenerate ompA codons, and part of the NDF 5' coding sequences beginning at codon 177 was used as the sense primer together with antisense primer 502-25 [SEQ ID NO: 109] containing sequence around the XhoI site. Using these two primers,
- fragments containing the ompA signal peptide codons and the NDF- ⁇ 2 coding sequence were PCR synthesized from pCFM1656-met-huNDF- ⁇ 2 177-241 .
- the fragments were used to substitute the XbaI-XhoI fragment of pCFM1656-met-huNDF- ⁇ 2 177-241 .
- the FM5 secretion strain harboring pCFM1656-A1-huNDF- ⁇ 2 177-241 was designated Strain 1776.
- the expression level of secretion strain 1776 was greatly improved .
- the ratio of the processed huNDF- ⁇ 2 177-241 vs . non-processed preprotein was designated
- the nucleotide sequence of human NDF- ⁇ 3 differs from human NDF- ⁇ 2 only in the region downstream from the unique XhoI site in the EGF-like domain.
- the ⁇ 3 segment was PCR amplified with primers 580-31 and 583-16 [SEQ ID NOS: 113 and 114, respectively] from cDNA clone 19.
- the resulting fragment was then used to replace the XhoI-BamHI fragment of pCFM1656-met-huNDF- ⁇ 2 14-241 in which the N-terminal codons of NDF have been optimized.
- the new construct, pCFM1656-met-huNDF- ⁇ 3 14- 247 was transformed into FM5, and thus generated Strain 1910.
- cDNA fragment encoding rat NDF- ⁇ 2 14-241 was PCR amplified from rat cDNA plasmid clone 44 with primers 260-19 [SEQ ID NO: 96] and 425-26 [SEQ ID NO: 99] and cloned into the XbaI and XhoI sites in pCFM1656.
- the primer design and cloning strategy are similar to the ones used for the cloning of the met-huNDF- ⁇ 2 14-241 sequence described.
- the FM5 strain containing the resulting expression plasmid pCFM1656-met-ratNDF- ⁇ 2 14-241 was designated as Strain 1685. II. Protein expression a. Fermentor
- Biolafitte fermentor which contained eight liters of batch medium (80 g of yeast extract, 42 g of ammonium sulfate, 28 g of dibasic potassium phosphate, 32 g of monobasic potassium phosphate, 5 g of sodium chloride, 40 g of glucose, 32 ml of 1 M magnesium sulfate, 5 ml of antifoam Dow P2000, 16 ml of trace metal solution and 16 ml of vitamin solution.
- the culture was allowed to grow to an optical density of 4-5 at 600 nm before starting phase I feeding.
- the feed one medium contains 100 g of Bacto tryptone, 100 g of yeast extract, 900 g of glucose, 70 ml of 1 M magnesium sulfate, 20 ml of trace metal solution and 20 ml of vitamin solution in a total volume of two liters.
- the phase one feed rate was initiated at 13 ml/hr and then adjusted every two hours according to the cell mass determined by optical density.
- the medium fed into the fermentor allowed the E. coli cells to grow at an exponential rate under a glucose limited condition so that the amount of toxic by-products accumulating in the cell culture was minimal.
- the temperature during the entire growth phase was set at 30°C to ensure the complete suppression of the transcription from the lambda P L promoter and the tight control of plasmid amplification.
- the culture pH was kept at 7 with phosphoric acid and ammonium
- the desired dissolved oxygen level was maintained by adjusting the agitation, air- and oxygen-input rates, and back pressure in the fermentor. As the optical density of the culture reached 30, the
- the first feed medium was replaced with a second medium (800 g of Bacto tryptone, 400 g of yeast extract, and 440 g of glucose in a total volume of four liters) at a constant feed rate of 250-300 ml/hr to provide enough glucose as energy and amino acids as precursors for the synthesis of recombinant NDF.
- a second medium 800 g of Bacto tryptone, 400 g of yeast extract, and 440 g of glucose in a total volume of four liters
- E. coli Strain 1776 was inoculated into a 250 ml flask which contained 100 ml Luria broth plus
- Recombinant rat met-NDF- ⁇ 2 14-241 was purified from E. coli Strain 1685 by subjecting a clarified cell lysate to anion exchange, cation exchange, hydrophobic interaction, and hydroxyapatite column chromatography.
- E. coli cell paste was disrupted in 5 mM EDTA, pH 8.5, by two passages through a Niro-Soavi Homogenizer at 14,000 psi.
- the cell lysate was adjusted to pH 8.5 and centrifuged for one hour at 4,100 rpm in a J6-B centrifuge (Beckman), using a JS-4.2 rotor.
- S-Sepharose fractions containing a prominent recombinant NDF protein of about 34 kDa were identified by SDS-PAGE analysis using a 10% gel (Novex, San Diego) followed by Coomassie blue staining. These fractions were pooled, diluted with an equal volume of water, and adjusted to 1.2 M ammonium sulfate, pH 6.5. The sample was then loaded onto a column containing TSKGEL Butyltoyopearl 650M (TosoHaas, Montgomeryville, PA). This column was previously equilibrated with 1.2 M ammonium sulfate in PBS, pH 6.5. After washing with
- the column was eluted with a gradient of 1.2 to 0 M ammonium sulfate in 1x PBS, pH 6.5. Individual fractions were collected. Column fractions containing the recombinant NDF protein were identified by SDS-PAGE. The first NDF peak eluted contained less aggregates and more monomer. Fractions containing this first peak were pooled and dialyzed overnight at 4oC against two changes of 10 mM sodium phosphate, pH 8.5. The dialyzed sample was loaded onto a S-Sepharose Fast Flow column previously equilibrated with 10 mM sodium phosphate, pH 8.5. The column was washed with 10 mM sodium phosphate, pH 8.5, then eluted with a gradient of 0 to 1 M NaCl in 10 mM sodium
- a hydroxyapatite (IBF Biotechnics, Columbia, MD) column was used as a fourth chromatography step.
- the column was equilibrated with 10 mM sodium phosphate, pH 6.8. After loading the dialyzed S-Sepharose
- Protein concentrations for recombinant rat met-NDF- ⁇ 2 14-241 were determined by ultraviolet absorption at 280 nm with a Beckman DU-650 spectrophotometer and an extinction coefficient of 0.54 mg -1 cm -1 ml.
- Reversed-phase HPLC analysis using a HP1090 HPLC with a Vydac C 4 column (Hewlett-Packard Co., Palo Alto, CA) showed that the purified and refolded NDF preparation had a single peak with a retention time of 47.5 minutes in the absence and presence of 6 M guanidineHCl.
- the addition of both guanidineHCl and dithiothreitol caused the NDF to become reduced and unfolded, observed by the shift in retention time to 50.2 minutes.
- Recombinant human NDF- ⁇ 2 1-241 was purified from E. coli Strain 1784 by subjecting a clarified cell lysate to anion exchange, cation exchange, and
- E. coli cell paste was disrupted in 5 mM EDTA, pH 7.5 by two passages through a Niro-Soavi Homogenizer at 14,000 psi.
- the cell lysate was centrifuged one hour at 4,100 rpm in a J6-B centrifuge (Beckman), using a JS-4.2 rotor.
- the clarified supernatant was adjusted to pH 8.5 and passed through Q-Sepharose Fast Flow
- Tris-Cl, pH 8.5 Tris-Cl, pH 8.5.
- the flow-through fraction from the Q-Sepharose column was loaded onto a S-Sepharose Fast Flow column. Column loading, equilibration and elution conditions were as described for recombinant human met-NDF- ⁇ 2 14-241 .
- S-Sepharose fractions containing a prominent monomeric recombinant NDF protein were identified by SDS-PAGE analysis, pooled, and loaded onto a column containing TSKGEL Butyl-toyopearl 650M equilibrated with 1.2 M ammonium sulfate, pH 6.5. The column loading and elution conditions were the same as described for rat met-NDF- ⁇ 2 14-241 . Column fractions containing the monomeric recombinant NDF protein were identified by SDS-PAGE, pooled, and dialyzed against three changes of 1x PBS, pH 8.5 at 4oC overnight.
- the dialyzed sample was loaded onto a S-Sepharose Fast Flow column previously equilibrated with 1x PBS, pH 8.5.
- the column was washed with 1x PBS, pH 8.5, and then eluted with a gradient of 0 to 1 M NaCl in 1x PBS, pH 8.5.
- Column fractions primarily
- the protein concentration of recombinant human NDF- ⁇ 2 1-241 was determined by ultraviolet absorption at 280 nm using a Beckman DU-650 spectrophotometer with an extinction coefficient of 0.48 mg 1- cm -1 ml. Reverse phase HPLC analysis showed that the purified and refolded NDF preparation had a single peak with a retention time of 45.8 minutes. Addition of
- Recombinant human met-NDF- ⁇ 2 14-241 was purified from E. coli Strain 1664 by subjecting a clarified cell lysate to anion exchange, cation exchange, hydrophobic interaction, and hydroxyapatite column chromatography.
- E. coli cell paste was disrupted in of 5 mM EDTA, pH 8.5 by two passages through a Niro-Soavi
- the cell lysate was adjusted to pH 8.5 and centrifuged one hour at 4,100 rpm in a J6-B centrifuge (Beckman), using a JS-4.2 rotor. The clarified supernatant was then passed through
- S-Sepharose fractions containing a prominent recombinant NDF protein of about 35 kDa were identified by SDS-PAGE analysis using a 10% gel (Novex, San Diego) followed by coomassie blue staining. These fractions were pooled, diluted with an equal volume of H 2 O, and adjusted to 1.4 M ammonium sulfate, pH 6.5. The sample was then loaded onto a column containing TSKGEL Butyltoyopearl 650M, equilibrated with 1.4 M ammonium
- a hydroxyapatite (IBF Biotechnics, Columbia, MD) column was used as a fourth chromatography step.
- the column was equilibrated with 10 mM sodium phosphate, pH 6.8. After loading the dialyzed butyl-toyo fraction, the column was washed with 10 mM sodium phosphate
- the sample was loaded onto a S-Sepharose Fast Flow column previously equilibrated with 1x PBS, pH 8.5.
- the column was washed with 1x PBS, pH 8.5, and then eluted with a gradient of 0 to 1 M NaCl in 1x PBS, pH 8.5.
- Protein concentrations for recombinant human met-NDF- ⁇ 14-241 were determined by ultraviolet absorption at 280 nm using a Beckman DU-650 spectrophotometer with an extinction coeffiecient of 0.54 mg -1 cm -1 ml.
- Vydac C4 or Synchropak RP-4 columns were used with a HP1090 HPLC (Hewlett-Packard, Palo Alto, CA). The columns were equilibrated with 97% buffer A (0.1% trifluoroacetic acid in HPLC water) and 3% buffer B (90% acetonitrile, 0.1% trifluoroacetic acid in HPLC water).
- Recombinant NDF protein (10-50 ⁇ g) was injected in a total volume of 250 ⁇ l.
- a 40 ⁇ l sample was first treated with 200 ⁇ l of 0 . 1 M Tris-6 M guanidineHCl, pH 8 and 10 ⁇ l of 0.2 M dithiothreitol for thirty minutes at 23°C.
- the final concentrations of guanidineHCl and dithiothreitol were 4.8 M and 8 mM, respectively.
- the columns were eluted with a linear gradient of 3-50% buffer B for the first sixty minutes. The gradient was increased to 95% buffer B for the next ten minutes. Elution was continued for another ten to fifteen minutes with 95% buffer B.
- the analysis showed that the purified and refolded NDF preparation had a single peak with a retention time of 46.4 minutes in the presence and absence of 6 M
- guanidineHCl The addition of both guanidineHCl and dithiothreitol caused the NDF to become reduced and unfolded, observed by the shift in retention time to 49.6 minutes.
- the N-terminal amino acid sequence analysis for the first ten residues (MKKKERGSGK) [SEQ ID NO: 122] matched the sequence predicted from DNA and included the N-terminal methionine used to initiate translation in E. coli . VII. Purification of recombinant human NDF- ⁇ 2 177-241
- Recombinant human NDF- ⁇ 2 177-241 was purified from E. coli Strain 1776 by renaturing recombinant NDF protein from a cell lysate pellet fraction, followed by cation exchange, hydrophobic and anion exchange column chromatography. E. coli cell paste was disrupted in 5 mM EDTA, pH 6.5, by two passages through a Microfluidizer
- the cell lysate was centrifuged for one hour at 4,100 rpm in a J6-B centrifuge (Beckman). The pellet was resuspended in 5 mM EDTA, pH 6.5, and centrifuged again for one hour. The pellet was solubilized by mixing with eight volumes of 8 M urea, 20 mM Tris-HCl, pH 8.6, for thirty minutes at room temperature. Reduced glutathione was added to 10 mM and the sample was stirred at room temperature, pH 8.8, for an additional thirty minutes.
- the denatured, reduced inclusion body protein was diluted 1:20 into a buffer containing 20 mM Tris-HCl, 1 mM EDTA, 0.2 M L-arginine, 1 mM reduced glutathione, 1 mM oxidized glutathione (pH 8.8). The solution was left for sixty minutes at room temperature, then at 4oC for sixteen hours without further stirring.
- the renatured sample was diluted with two volumes of water and adjusted to pH 4.6 by adding citric acid to 5 mM followed by 5 M HCl. After one hour of centifugation at 4,100, rpm in a J6-B centrifuge, the supernatant was loaded onto a S-Sepharose Fast Flow column. The column was previously equilibrated with 20 mM citric acid, pH 4.6. After loading, the column was washed first with equilibration buffer, then with 20 mM sodium phosphate, pH 6.8. Bound protein was stepwise eluted with 0.5 M NaCl in 1x PBS, pH 7.0.
- S-Sepharose fractions containing a prominent recombinant NDF protein of about 8 kDa were identified by SDS-PAGE analysis using a 4-20% gradient gel (Novex, San Diego) followed by coomassie blue staining. These fractions were pooled, diluted to 1 mg/ml with 10 mM citric acid, pH 5.0, and adjusted to 1.5 M ammonium sulfate. The sample was then loaded onto a column containing TSKGEL Butyl-toyopearl 650M equilibrated with 1.5 M ammonium sulfate, 10 mM citric acid, pH 5. The column was washed with the equilibration buffer and eluted with a gradient of 1.5 to 0 M ammonium sulfate in 10 mM citric acid, pH 5. Column fractions containing the monomeric
- recombinant NDF protein were identified by SDS-PAGE and pooled. After concentration in a stirred cell with a YM3 ultrafiltration membrane (Amicon, Danvers, MA), the sample was dialyzed against three changes of 1x PBS overnight at 4oC.
- the dialyzed sample was diluted with two volumes of water, adjusted to pH 6.9, and passed through a Q-Sepharose Fast Flow column previously equilibrated with 20 mM sodium phosphate, pH 6.9.
- the flow-through fraction was adjusted to pH 4.5 with 3 M citric acid, then loaded onto a S-Sepharose Fast Flow column
- Protein concentrations for recombinant human NDF- ⁇ 2 177-241 were determined by ultraviolet absorption at 280 nm using a Beckman DU-650 spectrophotometer with an extinction coefficient of 0.389 mg -1 cm -1 ml. Reverse phase HPLC analysis showed that the purified and refolded NDF preparation had a single peak with a retention time of 34.5 minutes. The addition of both guanidineHCl and dithiothreitol caused the NDF to become reduced and unfolded, which was observed by a shift in retention time to 43.5 minutes.
- the N-terminal amino acid sequence analysis for the first fifteen residues [SEQ ID NO: 123] matches the sequence predicted from DNA. IX. Purification of recombinant human met-NDF- ⁇ 1 14-249
- Recombinant human met-NDF- ⁇ 1 14-249 was purified from E. coli Strain 1854 by renaturing recombinant NDF protein from a cell lysate pellet fraction, followed by cation exchange and hydrophobic column chromatography.
- E. coli cell paste was disrupted in 1 mM EDTA, 10 mM Tris-Cl, pH 7.0 by two passages through a Niro-Soavi Homogenizer at 14,000 psi. The cell lysate was centrifuged for one hour at 4,100 rpm in a J6-B
- the denatured, reduced inclusion body protein was dropped into 50 volumes of 20 mM Tris-HCl, 1 mM EDTA, 0.2 M L-arginine, 1 mM reduced glutathione, 1 mM oxidized glutathione (pH 8.8). The stirring was continued for ten minutes at room temperature. The solution was then left at 4oC overnight.
- the renatured sample was diluted with one volume of water and adjusted to pH 7.0. After one hour of centifugation at 4,100 rpm in a J6-B centrifuge, the supernatant was loaded onto a S-Sepharose Fast Flow column, previously equilibrated with 1x PBS, pH 7.0.
- the column was first washed with 1x PBS, pH 7.0, then with 1x PBS, pH 7.0 containing an
- the dialyzed sample was loaded onto a S- Sepharose Fast Flow column previously equilibrated with 1x PBS, pH 7.0. Bound protein was eluted by a gradient of 0.15-0.80 M NaCl in 1x PBS, pH 7.0.
- Protein concentrations for recombinant human met-NDF- ⁇ 1 14-249 were determined by ultraviolet absorption at 280 nm with an extinction efficient of 0.486 mg -1 cm -1 ml. Reverse phase HPLC analysis showed that the
- residues matched the sequence predicted from DNA and included the N-terminal methionine residue added to initiate translation in E. coli .
- sequence analysis also showed that amino-terminal processing occurred at amino acid residue 15 with the ratio of 4.8%.
- E. coli cell paste was disrupted in ten volumes of 10mM tris-HCl 5mM EDTA, pH 8.0. The cell lysate was centrifuged, and the pellet was solubilized in five volumes of 8M Urea, pH 8.6, for thirty minutes at room temprature. Dithiothreitol was added to 5mM to complete the reduction. The sample was stirred at room temperature, pH 8.6, for an additional thirty minutes. The solublized, reduced inclusion body protein was added dropwise to fifty volumes of 20 mM Tris-Cl, 1 mM EDTA, 0.2 M L-arginine, 2 M urea, 1 mM reduced glutathione, 1 mM oxidized glutathione (pH 8.6). The stirring was continued for 10 minutes at room temperature. The solution was then left at 4°C overnight.
- the protein concentration of recombinant human met-NDF- ⁇ 1 14-246 was determined by absorption at
- Recombinant human met-NDF- ⁇ 1 177-246 was purified from E. coli Strain 1769 by renaturing recombinant NDF protein from a cell lysate pellet fraction, followed by cation exchange and reverse phase column chromatography.
- E. coli cell paste was disrupted in ten volumes of 5mM EDTA, 10mM tris-HCl, pH 8.0. The cell lysate was centrifuged, the pellet was resuspended in 5 mM EDTA, 10 mM Tris-Cl, pH 8.0 and centrifuged again, then solubilized by mixing with five volumes of 8 M urea, pH 8.5, at room temperature for thirty minutes. Reduced glutathione was added to lOmM, and the sample was stirred at room temperature, pH 8.7, for an
- the solublized, reduced inclusion body protein was added dropwise to fifty volumes of thirty mM Tris-HCl, 1 mM EDTA, 0.2 M
- CM-Sepharose fractions containing a prominent ⁇ 7 kD recombinant NDF protein were identified by SDS-PAGE analysis. Pooled fractions containing the product were diluted with two volumes of water. The pH was adjusted to 4.0, and the material was loaded onto a
- Vydac C 4 column from The Separations Group, Hesperia, CA.
- the C 4 column was previously packed in 80% ethanol, pH 4.0, and equilibrated with 20mM citric acid, pH 4.0. After loading, the column was washed with 20 mM citric acid, pH 4.0, then with 20% ethanol in 20mM citric acid, pH 4.0. Bound proteins were eluted by a gradient of 20-80% ethanol in 20 mM citric acid, pH 4.0.
- the dialyzed sample was loaded onto a S-Sepharose Fast Flow (Pharmacia) column previously
- This final product was designated recombinant human met- NDF- ⁇ 1 177-246 .
- the protein concentration of recombinant human met-NDF- ⁇ 1 177-246 was determined by absorption at 280 nm with an extinction coefficient of 0.66 mg -1 cm -1 ml.
- Recombinant human met-NDF- ⁇ 3 14-247 was purified from E. coli Strain 1910 by renaturing recombinant NDF protein from a cell lysate pellet traction, followed by cation exchange, hydrophobic extraction and anion exchange column chromatography.
- E. coli cell paste was disrupted in ten volumes of 5mM tris-HCl, 1mM EDTA, pH 8.0.
- the cell lysate was centrifuged, and the pellet was solubilized in ten volumes of 8M urea, 2mM EDTA, 5mM Dithioerythritol (2, 3-Dihydroxybutane-1.4-dithiol), pH 9 for ninety minutes at room temperature.
- the solubilized, reduced inclusion body protein was added slowly into twenty five volumes of 20 mM tris-HCl, 0.4 M L-arginine, ImM EDTA, 0.5mM reduced glutathione, pH 8.7. The stirring was continued for five minutes at room temperature. The sample was then left at room
- the renatured sample was adjusted to pH 4.7 by adding citric acid to 10 mM followed by 5M HCl. After centrifugation, the supernatant was loaded onto a s-sepharose fast flow column. The column was previously equilibrated with 20mM citric acid pH 4.7. After
- recombinant NDF protein were identified by SDS-PAGE and pooled.
- the pooled sample was dialyzed against three changes of 1 ⁇ PBS pH 7.5.
- the dialyzed sample was passed through a
- the protein concentration of recombinant human met-NDF- ⁇ 3 14-247 was determined by absorption at 280 nm with an extinction coefficient of 0.446 mg -1 cm -1 ml. SDS-PAGE analysis using a 10% NOVEX gel followed by
- NDF preparation had a single peak with a retention time of 47.9 minutes.
- the addition of both guanidineHCl and dithiothreitol caused the NDF protein to become reduced and unfolded, observed by a shift in retention time to 51.0 minutes.
- the N-terminal amino sequence analysis of the first nineteen residues matched the sequence predicted from DNA and included the
- NDF- ⁇ 2 and Recombinant Rat NDF- ⁇ 2 Phenotypic changes usually associated with "functional" differentiation of breast epithelium include the synthesis of milk components and lipids (Topper, Y.J, and Freeman, C.S., Physiol . Rev. 60:1049-1106, 1980; Stampfer, M.R., and Yaswen, P.,
- BT-474 cells (ATCC HTB 20) were obtained from the
- BT-474 cells were seeded in 6-well flat-bottom dishes in 2 mL of "complete" media at a density of 1 ⁇ 10 5 cells per well. After incubation for twenty-four hours at 37°C in a humidified air/5% CO2 chamber, media were removed and replaced with samples containing either rHuNDF- ⁇ 2 or rRtNDF- ⁇ 2. Plates were re-incubated using the
- rHuNDF- ⁇ 2 14-241 370 ⁇ g/mL; rRtNDF- ⁇ 2, 492 ⁇ g/mL
- Diluent consisted of a 1:1 mixture of DMEM (GIBCO-BRL) :F-12
- GEBCO BRL with 100 ⁇ M non-essential amino acids (GIBCO BRL) and 2mM L-glutamine (GIBCO BRL).
- Cells were refed with fresh media (with or without recombinant NDF- ⁇ 2) after three and five days of incubation. On day 7, cells were detached from the substratum using 0.25 mL 0.1% trypsin/1.1 mM EDTA (GIBCO BRL) in Dulbecco's phosphate-buffered saline (D-PBS) (GIBCO BRL) at 37°C.
- D-PBS Dulbecco's phosphate-buffered saline
- the FACStar PLUS (Becton Dickinson Immunocytometry Systems, San Jose, CA) was equipped with a 5 W argon laser operated at 488 nm. Gold fluorescence was collected using a narrow band pass filter at 575 ⁇ 13 nm (Becton Dickinson) while red fluorescence was collected beyond 610 nm with a long pass filter (Omega Optical, Brattleboro, VT). Data were acquired and analyzed using the software package Lysis II Version 1.0 (Beckton Dickinson).
- Nile Red fluorescence can vary:
- BT-474 cells were treated with recombinant NDF- ⁇ 2 samples for seven days prior to staining with Nile Red and analysis by flow cytometry. Histograms depicting the gold-to-red fluorescence ratio measurements are presented in Figures 44 and 45.
- Recombinant NDF- ⁇ 2 induced changes in the distribution of cells from the low ratio peak (i.e., "nondifferentiated" cells with few neutral lipid vesicles) to the high ratio peak (i.e.,
- E. coli/human refers to recombinant human met-NDF- ⁇ 2 14-241 purified from E. coli ;
- CHO/rat refers to recombinant rat NDF- ⁇ 2 purified from CHO cell supernatents.
- the in vitro growth of cancer cells can be modified by numerous growth stimulatory and inhibitory factors (Aaronson, S.A., Science 254:1146-1153, 1991). Some of these, such as EGF (Imai, Y. et al., Cancer Res . 42:4394-4398, 1982), bFGF (Karey, K.P., and Sirbasku, D.A., Cancer Res . 48:4083-4092, 1988), IGF-I (Karey, K.P., and Sirbasku, D.A., Cancer Res . , above) and TGF-ß ⁇ (Knabbe, C. et al.. Cell 48 :411-428, 1987), can regulate the proliferation of rodent and mammalian breast
- rHuNDF- ⁇ 2 14-241 recombinant human met-NDF- ⁇ 2 14-241 (rHuNDF- ⁇ 2) stimulates, retards or has no affect on the in vitro growth of two human mammary cell lines: BT-474 cells, a ductal carcinoma line that overexpresses the neu/Her-2 receptor, and MDA-MB-468 cells, an
- rHuNDF- ⁇ 2 was purified from E. coli and re-folded in an active configuration (Example 14).
- BT-474 ATCC HTB 20
- MDA-MB-468 ATCC HTB 132 cells were obtained from the American Type Culture Collection (Rockville, MD). Cells were
- MDA-MB-468 were cultured in the same media without L-glutamine and insulin as supplements.
- Cells were seeded in 96-well flat-bottom microtiter dishes in 100 ⁇ L of cell-specific "complete" media at low density [i.e., 3,000 (BT-474) or 2,000 (MDA-MB-468) cells per well]. After incubation for twenty-four hours at 37°C in a humidified air/5% CO 2 chamber, 100 ⁇ L aliquots with samples of rHuNDF- ⁇ 2 were added which was followed by incubation of the plates using the
- rHuNDF- ⁇ 2 dilutions were prepared from a stock solution (rHuNDF- ⁇ 2 14-241 , 370 ⁇ g/mL) in diluent (1:1 mix of DMEM (GIBCO):F-12 (GIBCO) with 100 ⁇ M non-essential amino acids (GIBCO) and 2 mM L-glutamine (GIBCO)). All dilutions were tested in quadruplicate. Just prior to analysis, sample media were replaced with 50 ⁇ L per well phenol red-free RPMI 1640 (GIBCO) containing 1 mg/mL MTT (Sigma). Plates were then re-incubated for an additional three hours. The formazan precipitate was solubilized by adding
- Total absorbance i.e., the result of subtracting background absorbance at 690 nm from
- Mouse colon crypts were prepared as described by Whitehead et al. ( In Vitro Cellular & Developmental Biology, Vol. 23, Number 6, Pages 436-442, 1987). Mice were sacrificed with a lethal dose of CO2, and large intestines were isolated. The large intestine was cut longitudinally, rinsed with buffer A(1x PBS containing 0.3 mg/ml of L-Glutamine, 100 units/ml of penicillin, 100 units/ml of streptomycin), and sliced into 0.5 cm pieces. These pieces were washed again with buffer A in a 50 ml conical tube several times. Clean tissue was washed with extraction buffer (0.5mM DTT, 2mM EDTA in buffer A) and incubated with 10 ml of extraction buffer for one hour. The extraction buffer was then
- Crypts were harvested by shaking the tissue in 5 ml of Solution A. Crypts were transferred to a 15-ml tube.
- the crypts were distributed in collagen type IV coated 6-well plates (Collaborative Biomedical, Cambridge, MA) at a density of five hundred crypts per well.
- the medium (RPMI 1640, 0.3 mg/ml L-Glutamine, 100 units/ml penicillin, 100 units/ml streptomycin) contained either 1% or 10% fetal bovine serum (FBS). After twenty-four hours of incubation at 37°C, colonies of attached cells were stained with Gram Crystal Violet and counted. Formation of colonies was FBS dependent with 3-5 fold more colonies in medium containing 10% FBS than in media containing 1% FBS. The increase of colony formation was not due to the general increase of protein in the medium, since the same effect was not observed when BSA was used to substitute FBS.
- IL-1, IL-2, IL-3, IL-6, IL-8, PDGF, SCF, EGF, TGF- ⁇ , bFGF, aFGF, KGF, G-CSF, and NDF were comparison tested at 10 ng/ml in medium containing 1% FBS. Among these factors, only NDF gave results similar to the stimulation obtained with 10% FBS in this assay. The assay was then repeated with various
- Colorectal carcinoma is among the most common malignancies in Western societies.
- the carcinogenic target is the colonic epithelium, which is a highly proliferative organ that undergoes constant growth renewal and differentiation. Tumors are thought to arise from undifferentiated progenitor cells which harbor mutations that disrupt normal growth control mechanisms. Tumors begin as a small benign growth, or adenoma, which later acquire more aggressive growth characteristics and eventually metastasize.
- Current therapies include surgical resection and stringent chemotherapy in a combined modality prior to metastasis. Unfortunately, tumors that are not cured by surgery respond poorly to follow-up chemotherapy.
- the HER-2/neu proto-oncogene encodes a type I transmembrane protein with intrinsic tyrosine kinase activity, which is generally believed to be a growth factor receptor that transmits mitogenic signals.
- HER-2/neu is expressed in a variety of tissues including those enriched in epithelial surfaces such as breast, lung and the intestine.
- the HER-2 gene has previously been shown to be amplified in breast cancer, suggesting its role in tumorigenesis of epithelial cell types
- NDF Neu-Differentiation Factor
- CEA carcinoembryonic antigen
- the LIM 1215 colorectal carcinoma cell line was provided by Robert Whitehead (Ludwig Institute for Cancer Research, Melbourne) and maintained as adherent monolayer cultures in RPMI 1640 growth media containing 5% FBS, 1 ⁇ g/ml insulin, 10 ⁇ g/ml hydrocortisone and
- the embedded frozen pellets were sectioned using a cryostat fitted with a glass knife, and the frozen sections placed on glass slides.
- the sections were fixed in absolute acetone at -20°C for six minutes, then air dried.
- the fixed sections were rehydrated in PBS containing 0.3 ⁇ g/ml BSA (antibody diluent) then incubated with 1 ⁇ g/ml mouse monoclonal antibody to human CEA (MAB 425, Chemicon International, Inc.) in antibody diluent for one hour at 23°C.
- the slides were washed three times with antibody diluent, then incubated with 1 ⁇ g/ml FITC conjugated sheep anti-mouse IgG antibody (Amersham, Inc.) for approximately thirty minutes at room temperature.
- the slides were washed, and the area containing the stained section was covered with PBS solution containing 4% n-propyl galate (Sigma Chemical Co., # P-3130) and 90% glycerol to minimize fluorochrome bleaching.
- the slides were analyzed under a fluorescent microscope at UV 320 nm and photographed using Kodak Gold ASA 100 color film (Figure 49).
- CEA is detected at significant levels only at the apical portion of the colonic crypt (Benchimol, S. et al, Cell, 1989, above). Intestinal epithelial cells are known to differentiate as they migrate from the basal to apical crypt compartments (Cheng, H., and
- NDF is capable of regulating cell growth and differentiation control in HER-2 expressing colon carcinoma in vitro .
- NDF may therefore modulate CEA expression in normal and transformed colonic epithelial cells in vivo . This may prove to be useful in treatment of colon carcinoma by regulating carcinoma cell-cell and cell-matrix interactions by decreasing the mortality and morbidity associated with metastatic colon carcinoma.
- Tumor metastasis is a complex, multi-step process that begins when individual tumor cells leave the primary site of transformation by degrading their resident extracellular matrix.
- Matrix degradation is mediated by several classes of proteolytic enzymes, including members of the Zn + -dependent metalloproteinases, such as type IV collagenase and stromelysin (transin) (Liotta, L.A., et al., Cancer Research
- the type IV collagenase and stromelysin genes are known to be induced following initiation of the growth response, and to be
- TIMP-2 tissue inhibitors of metalloproteinase
- LIM 1863 the metastatic ileoceacal carcinoma cell line LIM 1863.
- LIM 1863 cells were treated with recombinant rat NDF to determine its effects on TIMP-2 protein expression and found that NDF treatment dramatically increased TIMP-2 expression and secretion. Since NDF is believed to play a role in differentiation of epithelial cell types, NDF-induced expression of TIMP-2 may have a role in inducing
- NDF treatment may alter the metastatic properties of LIM 1863 cell in vitro, and of other human colon cancers in vivo.
- the LIM 1863 ileoceacal carcinoma cell line (Whitehead, R.H. et al, Cancer Research 47: 2704-2713 , 1987) was provided by Robert Whitehead (Ludwig Institute for Cancer Research, Melbourne) and maintained in suspension cultures in RPMI 1640 growth media containing 5% FBS, 1 ⁇ g/ml insulin, 10 ⁇ g/ml hydrocortisone and 10 ⁇ M ⁇ -thioglycerol. Confluent cultures are
- the medium was removed at this time and replaced with 500 ml of fresh serum-free media, then allowed to incubate overnight.
- the cells were then harvested, counted, diluted to a concentration of about six hundred organoids per ml, then cultured in the presence or absence of 50 ng/ml of recombinant rat NDF (CHO-derived NDF- ⁇ 2), for one hundred and twenty hours. After treatment, the cells were harvested and collected by centrifugation. The cell pellet was surrounded by optimum cooling temperature (OCT) embedding compound (#4583, Miles, Inc.) and flash-frozen in liquid
- OCT optimum cooling temperature
- the embedded frozen pellets were sectioned using a cryostat fitted with a glass knife, and the frozen sections were placed on glass slides. The sections were fixed in absolute acetone at -20°C for six minutes, then air dried. The fixed sections were rehydrated in PBS containing 0.3 mg/ml BSA (antibody diluent), then incubated with a 1:300 dilution of rabbit polyclonal anti-TIMP-2 in antibody diluent for one hour at room temperature.
- the TIMP-2 antiserum provided by Helen Hockmen (Amgen, Inc.), was raised against
- the slides were washed, and the area containing the stained section was covered with PBS solution containing 4% n-propyl galate and 90% glycerol to minimize fluorochrome bleaching.
- the slides were analyzed under a fluorescent microscope at UV 320 nm and photographed using Kodak Gold ASA 100 color film.
- the LIM 1863 cell line spontaneously forms organoids in suspension consisting of a polarized layer of epithelial cells surrounding a central lumen
- TIMP-2 expression in cells expressing HER-2 receptors implies that NDF treatment would result in the downstream modulation of metalloproteinase activity in various compartments of the body. This may be useful in regulating tissue remodeling and repair in injured organs. More
- TIMP-2 HER-2 receptor positive colon cancer cells by NDF treatment may inhibit their ability to metastasize and subsequently invade neighboring tissues. This effect may be difficult to mimic by the addition of even large amounts of recombinant TIMP-2 protein and may decrease the mortality and morbidity associated with metastatic colon carcinoma.
- the area of epithelium produced in treated and untreated wounds was calculated from cross-sectional bisection for each dose group.
- a one-way ANOVA and Dunnett's t-test was run for each dose against the control group.
- Paraffin-embedded 3 ⁇ m sections of tissue were stained using anti-BrdU (Dako Corp., Carpinteria, CA), Avidin-Biotin Complex (Vector Laboratories, Inc., Burlingame, CA) , and diaminobenzidine substrate (DAB; Sigma Chemical Co., St. Louis, MO). Sections were digested with 0.1% protease solution, followed by treatment with 2N HCl. Endogenous peroxidase was quenched by exposure to 3% hydrogen peroxide solution. Slides were blocked with a 10% solution of normal horse serum in phosphate buffered saline (PBS), then incubated with anti-BrdU diluted 1:400 in 1% bovine serum albumin.
- PBS phosphate buffered saline
- Sections were counterstained with hematoxylin.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Cell Biology (AREA)
- Immunology (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Non-naturally occurring polypeptides are described which stimulate neu receptor phosphorylation and can be prepared by recombinant DNA methods from nucleotide sequences obtained from human cells and tissues. These polypeptides are useful to treat various human conditions involving cells that express the neu receptor and respond to modulation by the polypeptides. Also described are DNA molecules encoding the polypeptides, or analogs of the polypeptides, methods of recombinant DNA production of the polypeptides or analogs from the DNA molecules, biological materials useful in these methods such as expression vectors and transformed or transfected host cells, and pharmaceutical compositions containing the polypeptides.
Description
RECOMBINANT NEU DIFFERENTIATION FACTORS
Cross-Reference to Related Application
This application is a continuation-in-part of copending application number 07/877,431, filed April 29, 1992, which is incorporated by reference herein in its entirety.
Field Of The Invention
This invention relates to novel polypeptides (herein referred to as neu differentiation factors), produced by recombinant DNA methods, which interact with and stimulate the neu receptor and modulate cellular function. The invention includes analogs and
derivatives of such polypeptides, as well as nucleotide sequences encoding the polypeptides, analogs and
derivatives, and to methods of use for the polypeptides and nucleotide sequences.
Background Of The Invention Cell growth and differentiation are regulated in part by extracellular signals that are mediated by polypeptide molecules (Aaronson, S.A., Science
254:1146-1152, 1991). The interaction between these factors and specific cell surface receptors initiates a biochemical cascade culminating in nuclear events that regulate gene expression and DNA replication
(Ullrich, A. and Schlessinger, J., Cell 61:203-212, 1990). This mechanism of cell regulation has
implications for the determination of cell fate during development, and breakdown of this mechanism may lead to
oncogenic transformation. The latter can be induced by constitutive production of growth regulatory factors or by altered forms of their cognate receptors (Yarden, Y., and Ullrich, A., Ann . Rev. Biochem . 57:443-448, 1988) . The oncogenic receptors include a family of
transmembrane glycoproteins that share a common
catalytic function in their cytoplasmic domains, namely, a tyrosine-specific protein kinase activity
(Hanks, S.K., Cur. Op . Struct . Biol . 1:364-383, 1991).
The neu proto-oncogene (also known as HER-2 or c-erbB-2) encodes a 185 kilodalton (kDa) receptor tyrosine kinase. This pl85neu glycoprotein is known to be present in many epithelial and neural tissues
(Maguire, H.C. et al., J. Invest . Dermatol . 92 : 186-190 , 1989; Press, M.F. et al., Oncogene 5:953-962, 1990;
Gullick, W.J. et al.. Int . J. Cancer 40:246-254, 1987; Quirke, P.A. et al., Br. J. Cancer 60:64-69, 1989;
Kokai, Y. et al., Proc . Natl . Acad. Sci . USA 84 : 8498-8501, 1987; Natali, P.G. et al., Int . J. Cancer 45:457-461, 1990; Cohen, J.A. et al., J. Neurosci . Res . 31 : 622-634, 1992; Mori, S. et al., Lab . Invest . 61:93-97,
1989). Although adult tissues generally express less p185neu than corresponding fetal tissues, pl85neu
expression levels are frequently elevated in neoplastic adult tissues. Overexpression of pl85neu or neu gene amplification occurs in approximately twenty percent of breast, stomach, bladder and ovarian adenocarcinomas (Lofts, F.J., and Gullick, W.J., Cancer Treat . Res .
61 : 161-119, 1992), and is associated with poor prognosis for breast and ovarian cancers (Slamon, D.J. et al.,
Science 244 : 707-712 , 1989). Increased p185neu expression also occurs in certain non-malignant neoplasias, such as adenomatous polyps (D'Emilia, J.K. et al., Oncogene 4:1233-1239, 1989; Cohen, J.A. et al., Oncogene 4:1233-1239 1989), Barrett's esophagous (Jankowski, J.G.
et al., Gut 33:1033-1038, 1992), and polycystic kidneys (Herrera, G., Kidney Int . 40:509-513, 1991).
Polypeptide molecules that activate receptor tyrosine kinases induce receptor tyrosine
phosphorylation, thereby initiating intracellular signaling leading to cellular responses (Cantley, L.C. et al., Cell 64:281-302, 1991). Phosphorylation of the pl85neu receptor on tyrosine residues can be stimulated by proteins purified from several sources (Peles, E. et al., Cell 69: 205-216, 1992; Lupu, R. et al.. Science 249:1552-1555, 1990; Holmes, W.E. et al.. Science
256:1205-1210, 1992; Huang, S.S., and Huang, J.S., J. Biol . Chem . 267:11508-11512, 1992; Dobashi, K. et al., Proc . Natl . Acad . Sci . USA 88:8582-8586, 1991) .
Purification of rat and human pl85neu stimulatory proteins led to the isolation of cDNAs encoding novel epidermal growth factor (EGF) -related proteins (Peles, E. et al., Cell . 1992, above; Wen, D. et al., Cell
69:559-572, 1992; Holmes, W.E. et al., Science, 1992, above). The 44 kDa rat factor, named neu
differentiation factor (NDF), stimulates p185neu tyrosine phosphorylation and induces the production of milk components (casein and lipids) in certain breast
carcinoma cell lines (Peles, E. et al., Cell . 1992, above). The NDF cDNA sequences were predictive of a transmembrane glycoprotein precursor (proNDF) with an EGF-like domain, an immunoglobulin homology unit, and a 157 amino acid cytoplasmic domain. The recombinant version of this rat NDF was found to interact with pl85neu and stimulate tyrosine phosphorylation of the receptor in human tumor cells of breast, colon and neuronal origin (Peles, E. et al., EMBO J. 12:461-971, 1993). In situ hybridization with a NDF probe
identified the central and peripheral nervous systems as prominent sites of NDF expression in mouse embryos (Orr-
Urtreger, A. et al., Proc. Natl . Acad. Sci . USA 90:1867-1871, 1993).
As part of the invention now described herein, a number of human NDF cDNA clones and human proNDF polypeptides encoded by them have been discovered.
Various recombinant human NDF polypeptides have been made using bacterial and mammalian expression systems and characterized as to biological activities, as described in the following text. These human
polypeptides add to the expanding knowledge of NDF-related molecules, which now also includes glial growth factors (Marchionni, M.A. et al.. Nature 362:312-318, 1993), as well as chicken acetylcholine receptor
inducing activity, or "ARIA" (Falls, D.L. et al., Cell 72:801-815, 1993). See, also, published PCT application WO 92/18627, which describes Schwann cell-mitogenic factors of 30-36 kDa and 55-63 kDa.
Summary of the Invention
In accordance with this invention, non-naturally occurring polypeptides encoded by human nucleotide sequences are provided which function as stimulators and inducers of neu (or Her-2, or c-erbB-2) receptor activities. These polypeptides include
recombinant DNA-derived neu receptor stimulating factors expressed from cDNA clones isolated from human tissues and cell lines. Such polypeptides possess an ability to stimulate human p185neu tyrosine phosphorylation. These recombinant polypeptides can be termed neu receptor stimulating factors, but are preferably referred to herein as neu differentiation factors (or "NDFs"), based on their ability to induce a differentiated phenotype in certain cell lines. The polypeptides are also
characterized by their ability to stimulate or inhibit
proliferation of certain cells. These polypeptides are encoded by nucleotide sequences which are also described in detail herein.
The neu differentiation factors of the present invention are also characterized by the inclusion in all instances of either of the following two amino acid sequences in their polypeptide structure:
"Alpha" Form [SEQ ID NO: 1]
CysAlaGluLysGluLysThrPheCysValAsnGlyGlyGluCysPheMetVal LysAspLeuSerAsnProSerArgTyrLeuCysLysCysGlnProGlyPheThr GlyAlaArgCys
"Beta" Form [SEQ ID NO: 2]
CysAlaGluLysGluLysThrPheCysValAsnGlyGlyGluCysPheMetVal LysAspLeuSerAsnProSerArgTyrLeuCysLysCysProAsnGluPheThr GlyAspArgCys
The isolated DNA sequences provided by the present invention are useful in securing expression of the polypeptides in procaryotic and/or eucaryotic host cells. The present invention specifically provides DNA sequences encoding all or part of the unprocessed amino acid sequences (proNDFs) as well as DNA sequences encoding all or part of the processed (mature) forms of the NDFs and novel recombinant analogs thereof. Such DNA sequences include:
(a) the DNA sequences set out in
Figures 30, 31, 32, 33, 34, 35, 36, 37 [SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15 and 17, respectively] and their complementary strands;
(b) DNA sequences which hybridize to the DNA sequences defined in (a) or fragments thereof; and
(c) DNA sequences which, but for the degeneracy of the genetic code, would hybridize to the DNA sequences defined in (a) and (b).
Specifically included in parts (b) and (c) are complementary DNA (cDNA) and genomic DNA sequences encoding variant forms of human NDF, manufactured DNA sequences (e.g., as by solid phase chemical synthesis) encoding human NDF, fragments of human NDF, and analogs of human NDF. The DNA sequences may incorporate codons facilitating transcription and translation of messenger RNA in host cells.
Also provided are vectors containing such DNA sequences, and host cells transformed or transfected with such vectors. Additionally, the invention includes methods of producing biologically active human NDF polypeptides by recombinant DNA techniques, and methods of treating disorders using recombinant NDFs.
Pharmaceutical compositions containing recombinant human NDF polypeptides and antibodies generated with such polypeptides are also encompassed by this invention.
Brief Description Of The Figures
Figure 1 depicts a high pressure liquid chromatogram of the trypsin digest of "naturally- occurring" neu receptor stimulating factor purified from media conditioned by ras-transformed rat fibroblasts according to the methods of Peles, E. et al., Cell, 1992, above. The elution profile is shown, and the amino acid sequences obtained from the fractions corresponding to each peak are indicated in conventional single letter designation. The amino acid in parentheses represents an asparagine-linked glycosylation site, while the dotted
line represents a longer sequence in which the amino acids were not identified. The inset shows
re-chromatographic separation of the 27.3-minute peak after dithiothreitol reduction.
Figure 2 shows the structure of the mammalian
COS-7 cell expression vector pJT-2/NDF. The rat NDF cDNA insert is the clone 44 cDNA sequence of Figure 5 [SEQ ID NO: 21].
Figure 3 demonstrates stimulation of human neu receptor tyrosine phosphorylation by recombinant rat NDF. Human MDA-MB-453 breast carcinoma cells were incubated with concentrated conditioned media from COS-7 cells transfected with the indicated partially purified cDNA clones (left panel) or with fully purified cDNA clones (right panel). Positive controls comprised
10 ng/ml (left lane) and 100 ng/ml of purified
naturally-occurring rat NDF. Negative controls
comprised concentrated media conditioned by
untransfected COS-7 cells (lane designated "COS"), or by cells transfected with pJT-2 plasmids containing
unrelated cDNA (lanes marked as "clone 27" and
"clone 29"), and no addition ("NONE"). Lysates were prepared from the stimulated MDA-MB-453 cells and subjected to SDS-PAGE in a 10% gel (Novex). Shown are autoradiograms of anti-phosphotyrosine Western blots, with the locations of molecular weight marker proteins given in kilodaltons. The following volumes of the COS-7 supernatants were used in a total volume of
0.125 ml PBS containing 0.1% BSA: 0.01 ml (left lane) and 0.1 ml (right lane) for clones 4, 19 and 44. Assays of the purified clones were performed in 0.25 ml total volume with 0.2 ml of cell supernatants or as indicated.
Figure 4 shows the nucleotide sequence of rat NDF cDNA [SEQ ID NO: 19] and a deduced amino acid sequence [SEQ ID NO: 20]. Depicted is a combined
nucleotide sequence from four rat cDNA clones. The beginning of the clone 44 DNA sequence in particular is indicated by an arrow. Nucleotide numbers and amino acid numbers are given in the left and right columns, respectively. Potential sites of N-linked glycoprotein are marked by asterisks, and cysteine residues found in the presumed extracellular domain are encircled. The overlining indicates peptide sequences that were
determined directly from purified naturally-occurring NDF. The underlining indicates the potential
transmembrane region, whereas the dashed underlining indicates the polyadenylation sites. The portions of the protein sequence that contain an immunoglobulin (Ig) homology unit and an epidermal growth factor (EGF)-like motif are indicated on the right hand side.
Figure 5 shows the nucleotide sequence of rat NDF cDNA clone 44 [SEQ ID NO: 21], separately, and a deduced amino acid sequence [SEQ ID NO: 22]. Nucleotide numbers are given in the right hand column.
Figure 6 depicts the hydropathy profile of the precursor of the rat neu receptor stimulating factor encoded by rat cDNA clone 44. The method of Kyte and Doolittle, J. Mol . Biol . 157:105-132 (1982), was used with a window size of nine amino acid residues.
Positive values indicate increasing hydrophobicity.
Amino acid numbers are given below the profile.
Figure 7 shows the alignment of the amino acid sequence [SEQ ID NO: 23] of the EGF-like domain and the flanking carboxyl terminal sequence of rat NDF from clone 44 with representative members of the EGF family. Alignment and numbering begin at the most amino terminal cysteine residue of the EGF motifs. Amino acid residues are indicated by the single letter code. Dashes
indicate gaps that were introduced for maximal
alignment. Residues are boxed to indicate their
identity with the corresponding amino acids of NDF. The underlines indicate the amino terminal portions of the putative transmembrane domains that flank some of the EGF motifs at their carboxy terminal side. Asterisks show the carboxyl termini. The following proteins are compared to NDF: rat transforming growth factor α [SEQ ID NO: 24] (TGFα; Marquardt, H. et al., Science
223:1079-1082, 1984), human amphiregulin [SEQ ID NO: 25] (AR; Shoyab, M. et al.. Science 243:1074-1076, 1989), sheep fibroma virus growth factor [SEQ ID NO: 30] (SFGF; Chang, W. et al., Λfol. Cell . Biol . 7:535-540 1987);
myxoma virus growth factor [SEQ ID NO: 31] (MGF;
Upton, C. et al., J. Virol . 51:1271-1275, 1987); mouse EGF [SEQ ID NO: 26] (Gray, A. et al., Nature
303:722-725, 1983; Scott, J. et al., Science
221 : 236-240, 1983); human heparin-binding EGF [SEQ ID NO: 27] (HB-EGF; Higashiyama, S. et al.,
Science 252:936-939, 1991); rat schwannoma-derived growth factor [SEQ ID NO: 28] (SDGF; Kimura, H. et al., Nature 348:257-260, 1990); and vaccinia virus growth factor [SEQ ID NO: 29] (VGF; Blomquist, M.D. et al., Proc . Natl . Acad . Sci . USA 81:7363-7367, 1984).
Figure 8 shows the alignment of the immunoglobulin (Ig)-like domain of rat NDF [SEQ ID
NO: 32] from cDNA clone 44 with the fourth Ig-related sequence of the murine neural cell adhesion molecule
[SEQ ID NO: 33] (NCAM; Barthels, D. et al., EMBO J. 6; 907-914, 1987), a representative protein of the C2-set of the immunoglobulin superfamily. Residues that are highly conserved in the C2-set are indicated on the lower line, and positions conserved across the
superfamily (according to Williams, A., and Barclay, A., Rev. Immunol . 6:381-405, 1988) are overlined. Stretches of amino acids that may be involved in β-strand
formation are heavily underlined and labeled B through
F, in analogy with the immunoglobulin domains. Boxes indicate identical residues in NDF and NCAM. Dashes (gaps) were introduced for maximal alignment.
Figure 9 is a schematic presentation of the presumed secondary structure and membrane orientation of the precursor of the neu receptor simulating factor from rat cDNA clone 44. Positions corresponding to the immunoglobulin (Ig)-domain and epidermal growth factor (EGF) motif are shown by thick lines and their cysteine residues are indicated by circles. The disulfide linkage of the Ig domain was directly demonstrated by amino acid sequence analysis (Example ID, below), and the secondary structure of the EGF-domain is based on the homology with the EGF family. Also shown are the three cysteine residues found in the transmembrane domain. Arrows mark the processing sites of the
precursor protein at the amino terminus (based on
N-terminal sequencing of rat NDF protein) and a putative proteolytic site close to the plasma membrane. The branched line indicates the location of a proven
N-glycosylation site and the short vertical lines represent presumed sites of O-glycosylation.
Figure 10 depicts a receptor competition assay using naturally-occurring neu receptor stimulating factor purified from ras-transformed rat fibroblast conditioned media (Peles, E. et al., Cell, 1992, above) which has been radiolabelled ("125I-NDF"). The radio-labelled NDF was incubated with human MDA-MB-453 breast carcinoma cells in the absence ("NONE") or presence of conditioned media from COS-7 cells expressing either recombinant neu receptor stimulating factor from rat cDNA clone 44 ("C-NDF") or recombinant TGFα ("C-TGFα") . The cell-bound radioactivity is shown a≤ average ± S.D. (n=3).
Figure 11 depicts another receptor competition assay. 125I-labeled naturally-occurring NDF was
incubated with human MDA-MB-453 cells in the absence ("NONE") or presence of unlabeled "cold" naturally-occurring NDF ("NDF"; 200 ng/ml), or media conditioned by COS-7 cells that were transfected either with the NDF expression plasmid of Fig. 2 ("C-NDF"), containing rat cDNA clone 44, or with a TGFα-encoding vector
("C-TGFα"). Following crosslinking with BS3, the cells were lysed and the neu receptor protein was
immunoprecipitated by using a monoclonal antibody.
Shown is an autoradiogram (3-day exposure) of the polyacrylamide gel-separated immunocomplexes. Molecular weights of marker proteins are indicated in kilodaltons. Mostly the presumed receptor dimer was radiolabeled.
Figure 12 shows autoradiograms obtained from Northern blots of mRNA isolated from cultured cells (panels A and C) or freshly isolated tissue of an adult rat (panel B), using rat NDF cDNA clone 44 as a probe. The autoradiograms were obtained after a three-hour
(panel A) or twenty-four hour (panels B and C) exposure. Molecular weight estimation (in kilobases) was performed by using a mixture of marker molecules from GIBCO BRL Life Technologies, Inc. (Gaithersburg, MD).
Figure 13 shows the nucleotide sequence of rat
NDF cDNA clone 4 [SEQ ID NO: 34] and a deduced partial rat (proNDF-ß3) amino acid sequence [SEQ ID NO: 35]. Nucleotide numbers are given in the left hand column and amino acids numbers on the right hand column. Linker sequences added to the 5' and 3' ends of the cDNA to facilitate cloning are included. The amino acid
numbering corresponds to the amino acid numbering of Figure 23 (composite figure for rat NDFs).
Figure 14 shows the nucleotide sequence of rat NDF cDNA clone 19 [SEQ ID NO: 36] and a deduced rat
(proNDF-α2b) amino acid sequence [SEQ ID NO: 37].
Nucleotide numbers are given in the left hand column and amino acids numbers on the right hand column. Linker sequences added to the 3' ends of the cDNA to facilitate cloning are included.
Figure 15 shows the nucleotide sequence of rat NDF cDNA clone 20 [SEQ ID NO: 38] and a deduced rat (proNDF-α2b) amino acid sequence [SEQ ID NO: 39].
Nucleotide numbers are given in the left hand column and amino acids numbers on the right hand column. Linker sequences added to the 3' ends of the cDNA to facilitate cloning are included.
Figure 16 shows the nucleotide sequence of rat NDF cDNA clone 22 [SEQ ID NO: 40] and a deduced rat (proNDF-ß2a) amino acid sequence [SEQ ID NO: 41].
Nucleotide numbers are given in the left hand column and amino acids numbers on the right hand column. Linker sequences added to the 3' ends of the cDNA to facilitate cloning are included.
Figure 17 shows the nucleotide sequence of rat
NDF cDNA clone 38 [SEQ ID NO: 42] and a deduced rat (proNDF-α2a) amino acid sequence [SEQ ID NO: 43].
Nucleotide numbers are given in the left hand column and amino acids numbers on the right hand column. Linker sequences added to the 3' ends of the cDNA to facilitate cloning are included.
Figure 18 shows the nucleotide sequence of rat NDF cDNA clone 40 [SEQ ID NO: 44] and a deduced partial rat (proNDF-ß2) amino acid sequence [SEQ ID NO: 45] . Nucleotide numbers are given in the left hand column and amino acids numbers on the right hand column. Linker sequences added to the 3' ends of the cDNA to facilitate cloning are included.
Figure 19 shows the nucleotide sequence of rat NDF cDNA clone 41 [SEQ ID NO: 46] and a deduced rat
(proNDF-ß2a) amino acid sequence [SEQ ID NO: 47].
Nucleotide numbers are given in the left hand column and amino acids numbers on the right hand column. Linker sequences added to the 3' ends of the cDNA to facilitate cloning are included.
Figure 20 shows the nucleotide sequence of rat NDF cDNA clone 42A [SEQ ID NO: 48] and a deduced rat (proNDF-ß4a) amino acid sequence [SEQ ID NO: 49].
Nucleotide numbers are given in the left hand column and amino acids numbers on the right hand column. Linker sequences added to the 3' ends of the cDNA to facilitate cloning are included.
Figure 21 shows the nucleotide sequence of rat NDF cDNA clone 42B [SEQ ID NO: 50] and a deduced rat (proNDF-α2a) amino acid sequence [SEQ ID NO: 51].
Nucleotide numbers are given in the left hand column and amino acids numbers on the right hand column. Linker sequences added to the 3' ends of the cDNA to facilitate cloning are included.
Figure 22 shows rat proNDF (precursor) structures predicted from the rat cDNA sequences of Figures 4, 5, and 13-21. Boxed areas indicate protein coding regions. "Ig", "EGF" and "TM" indicate the immunoglobulin-like, EGF-like and transmembrane domains, respectively. The number of amino acid residues in each predicted precursor sequence is given in the right hand column. Dashed lines represent divergent 3'
untranslated DNA sequences.
Figure 23 shows the amino acid sequences encoded by different rat proNDF cDNAs. The complete proNDF-α2a amino acid sequence from Figure 17 is shown [SEQ ID NO: 43]. Divergent sequences in proNDF variants are aligned with the proNDF-α2a sequence. ProNDF-ß2a [SEQ ID NO: 126], ß3 [SEQ ID NO: 127] and ß4a [SEQ ID NO: 52] structures were deduced from Rat-1-EJ cDNAs
(Table 2, below, and Figures 16, 13, and 20,
respectively). The NDF-ß1 sequence [SEQ ID NO: 52] was obtained from cDNA amplified from rat brain and spinal cord (See Example 9 and Figure 28, below). Asterisks, carboxyl-terminal amino acids; dashes, gaps introduced to facilitate sequence alignment; overline, putative transmembrane domain; dots, cysteine residues in predicted extracellular domain (the two N-terminal cysteine residues are part of the immunoglobulin-like domain; the six other marked cysteine residues reside in the EGF-like growth factor domain).
Figure 24 shows autoradiograms of anti-phosphotyrosine Western blots, using the neu receptor tyrosine phosphorylation assay procedure of Example 3, below. The results with recombinant rat NDF proteins expressed in COS-7 cells from the indicated cDNA clones are shown. COS-7(-) is medium from untransfected COS-7 cells.
Figure 25 shows the transient expression of recombinant proNDFs from rat cDNA clones. COS-7 cells were transfected with expression plasmids containing cDNAs encoding the indicated NDF isoforms and were labeled with 35S-methionine/cysteine. Conditioned media and cell lysates were immunoprecipitated with an affinity-purified rabbit anti-NDF antibody and analyzed by electrophoresis. The positions of molecular weight standards are shown on the right.
Figure 26 shows endoglycosidase treatment of recombinant rat proNDFs expressed in COS-7 cells. COS-7 cells were transfected with proNDF-ß3 and proNDF-α2c cDNA expression plasmids and labeled for seventeen hours with 35S-methionine/cysteine. Radiolabeled COS-7 cell media and lysates were immunoprecipitated with affinity-purified rabbit antibody raised against recombinant rat NDF-α214-241. The immunoprecipitates were treated with
endoglycosidases as indicated and analyzed by
electrophoresis. The positions of the molecular weight markers are shown on the right.
Figure 27 shows the reverse transcription-polymerase chain reaction (RT-PCR) analysis of NDF mRNAs expressed in various rat tissues. Reverse transcription was used to prepare first-strand cDNA from mRNA samples. NDF cDNAs were amplified in PCR reactions with primers specific for the NDF EGF-like domain. Lanes are as follows: 1, Rat-1-EJ; 2, heart; 3, skin; 4, ovary; 5, lung; 6, stomach; 7, spleen; 8, liver; 9, muscle; 10, kidney; 11, brain; 12, spinal cord; 13, cDNA clone 20 (proNDF-α2b) 14, cDNA clone 40 (proNDF-ß2); 15, cDNA clone 42A (proNDF-ß4). Positions of DNA size markers (in base pairs) are indicated.
Figure 28 shows the nucleotide sequence [SEQ ID NO: 55] of the PCR products obtained from rat spinal cord and brain, and a deduced partial rat NDF-ß1 amino acid sequence [SEQ ID NO: 56]. Nucleotide numbers are given in the left hand column, and amino acid numbers are given in the right hand column. The amino acid numbering corresponds to the amino acid numbering in Figure 23.
Figure 29 shows a Northern blot analysis of several human cell lines screened for the presence of NDF-related mRNA with rat NDF clone 44 cDNA probe.
Figure 30 shows the nucleotide sequence of human NDF cDNA clone PI [SEQ ID NO: 3] and a deduced partial amino acid sequence for human proNDF-α1a [SEQ ID NO: 4]. Nucleotide numbers are given in the left hand column and amino acid numbers on the right hand column. Amino acid numbering corresponds to the amino acid numbering of Figure 38 (composite figure for human
NDFs). Linker sequences added to the 5' and 3' ends of the cDNA to facilitate cloning are included.
Figure 31 shows the composite nucleotide sequence of human proNDF-α2b cDNA [SEQ ID NO: 5] and a deduced amino acid sequence [SEQ ID NO: 6]. This sequence was derived from the sequences of clone 43 [SEQ ID NO: 7] and clone 17, shown in Figures 32 and 33, respectively. Nucleotide numbers are given in the left hand column and amino acid numbers on the right hand column. Linker sequences added to the 3' end of the cDNA to facilitate cloning are included.
Figure 32 shows the nucleotide sequence of human NDF cDNA clone 43 and a deduced human proNDF-α2b amino acid sequence [SEQ ID NO: 8]. Nucleotide numbers are given in the left hand column and amino acid numbers on the right hand column. Linker sequences added to the 3' end of the cDNA to facilitate cloning are included.
Figure 33 shows the nucleotide sequence of human NDF cDNA clone 17 [SEQ ID NO: 9] and a deduced partial amino acid sequence of human proNDF-α2b [SEQ ID NO: 10]. Nucleotide numbers are given in the left hand column and amino acid numbers on the right hand column. Amino acid numbering corresponds to the amino acid numbering of Figure 38. Linker sequences added to the 5' and 3' ends of the cDNA to facilitate cloning are included.
Figure 34 shows the nucleotide sequence of human NDF cDNA clone 19 [SEQ ID NO: 11] and a deduced partial amino acid sequence of human proNDF-α3 [SEQ ID NO: 12]. Nucleotide numbers are given in the left hand column and amino acid numbers on the right hand column. Amino acid numbering corresponds to the amino acid numbering of Figure 38 (composite figure for human NDFs). Linker sequences added to the 5' and 3 ' ends of the cDNA to facilitate cloning are included .
Figure 35 shows the nucleotide sequence of human NDF cDNA clone P13 [SEQ ID NO: 13] and a deduced
partial amino acid sequence for human NDF-ß1a [SEQ ID NO: 14]. Nucleotide numbers are given in the left hand column and amino acid numbers on the right hand column. Amino acid numbering corresponds to the amino acid numbering of Figure 38 (composite figure for human
NDFs). Linker sequences added to the 5' and 3' ends of the cDNA to facilitate cloning are included.
Figure 36 shows the nucleotide sequence of human NDF cDNA clone 294-8 [SEQ ID NO: 15] and a deduced partial amino acid sequence for human proNDF-ß2 [SEQ ID NO: 16]. Nucleotide numbers are given in the left hand column and amino acid numbers on the right hand column. Amino acid numbering corresponds to the amino acid numbering of Figure 38 (composite figure for human
NDFs). Flanking PCR primer sequences are included.
Figure 37 shows the nucleotide sequence of human NDF cDNA clone 33 [SEQ ID NO: 17] and a deduced partial amino acid sequence for human proNDF-ß3 [SEQ ID NO: 18]. Nucleotide numbers are given in the left hand column and amino acid numbers on the right hand column. Amino acid numbering corresponds to the amino acid numbering of Figure 38 (composite figure for human NDFs). Linker sequences added to the 5' and 3' ends of the cDNA to facilitate cloning are included.
Figure 38 is a composite of the amino acid sequences encoded by different human proNDF cDNAs
(similar to Figure 23 for the rat clones). The complete human proNDF-α2b amino acid sequence [SEQ ID NO: 8] from Figure 32 is shown. Divergent sequences in human proNDF isoforms are aligned with the proNDF-α2b sequence.
Figure 39 is a schematic diagram of a mammalian cell vector for expression of a chimeric NDF gene comprised of rat and human sequences.
Figure 40 shows the induction of tyrosine phosphorylation in MDA-MB-453 cells (using the assay
procedure of Example 3) with conditioned medium from COS-7 cells which had been transfected with expression plasmids described in Example 11, below, containing various (human, rat, and human-rat chimera) NDF cDNA clones: Lane 1, control, 100 μl 10x concentrated conditioned medium from untransfected COS-7 cells; lanes 2 and 3, hNDF-α2b; lanes 4 and 5, h-rNDF-α2c; lanes 6 and 7, h-rNDF-ß1c; lanes 8 and 9, h-rNDF-ß2c; lanes 10 and 11, h-rNDF-α1c; lanes 12 and 13, rNDF-α2c; lane 14, positive control of human met-NDF-α214-241 purified from E. coli . Even-numbered lanes represent 100 μl
conditioned media, and odd-numbered lanes represent 100 μl 10x concentrated media.
Figure 41 shows SDS-PAGE analysis of a recombinant rat NDF-α2 purified from media conditioned by CHO cells with the pDSRα2/rNDF-α2c expression plasmid described in Example 11. The sizes of molecular weight markers in the left hand lane are indicated in
kilodaltons. The indicated amounts of purified rat NDF-α2 were electrophoresed in the gel which was stained with Coomassie blue.
Figure 42 shows the stimulation of MDA-MB-453 cell neu receptor tyrosine phosphorylation by various amounts of recombinant rat NDF-α2 purified from media conditioned by transfected CHO cells. The assay
procedure of Example 3 was used. PBSA (phosphate buffered saline, or PBS, with 0.1% bovine serum albumin, or BSA) was used as a negative control.
Figure 43 shows the stimulation of MDA-MB-453 neu receptor tyrosine phosphorylation by recombinant human and rat NDF isoforms purified from E. coli . PBSA was used as a negative control.
Figure 44 shows histograms depicting gold-to-red fluorescence ratios for BT-474 cells treated for seven days in culture with recombinant human met-NDF-
α214-241 purified from E. coli and stained with Nile Red. Treatment regimens included the following concentrations of the recombinant NDF: A, 0 ng/ml (media control); B, 100 ng/ml; C, 20 ng/ml; and D, 4 ng/ml. The results show that recombinant human NDF induces accumulation of neutral lipids.
Figure 45 shows histograms depicting gold-to-red fluorescence ratios for BT-474 cells treated for seven days in culture with recombinant rat NDF-α2 produced by CHO cells and stained with Nile Red.
Treatment regimens included the following concentrations of the recombinant NDF: A, 0 ng/ml (media control); B, 100 ng/ml; C, 20 ng/ml; and D, 4 ng/ml. The results show that recombinant rat NDF induces accumulation of neutral lipids.
Figure 46 shows the growth stimulatory effects of recombinant human met-NDF-α214-241 on BT-474 cell proliferation in vitro . Cells were treated with
different concentrations of the recombinant NDF purified from E. coli and processed on days 5, 6, and 7 using a modified MTT bioassay protocol. Absorbance measurements at 560 nm (minus the reference absorbance at 690 nm) for each treatment are presented as the arithmetic mean ± standard deviation (n=4).
Figure 47 shows the growth inhibitory effects of recombinant human met-NDF-α214-241 on MDA-MB-468 cell proliferation in vitro . Cells were treated with
different concentrations of the recombinant NDF purified from E. coli and processed on days 5, 6, and 7 using a modified MTT bioassay protocol. Absorbance measurements at 560 nm (minus the reference absorbance at 690 nm) for each treatment are presented as the arithmetic mean ± standard deviation (n=4).
Figure 48 illustrates the effect in vitro of rat met-NDF-α214-241 on the adhesion on colon epithelial
cells. Intestinal crypts were isolated from mouse colons and plated on collagen type IV coated plates.
After twenty-four hours at 37°C with varying
concentrations of recombinant NDF, colonies of attached cells were stained and counted.
Figure 49 shows indirect immunofluorescence analysis of LIM 1215 colon carcinoma cells cultured in the absence (panel A), or presence (panel B) of 50 ng/ml recombinant rat NDF-α2 purified from media conditioned by transfected CHO cells. Treated cells were sectioned, then stained with monoclonal anti-CEA antibodies
followed with FITC-labeled sheep anti-mouse IgG
antibodies.
Figure 50 shows indirect immunofluorescent analysis of LIM 1863 colon carcinoma cells cultured in the absence (panel A), or presence (panel B) of 50 ng/ml recombinant rat NDF-α2 purified from media conditioned by transfected CHO cells. Treated cells were sectioned, then stained with rabbit anti-TIMP-2 antibodies followed with phycoerythrin-labeled goat anti-rabbit IgG
antibodies.
Figure 51 shows measurements of new epithelium in rabbit ear wounds treated with different doses of recombinant human met-NDF-α214-241,
Figure 52 shows measurements of the area of new epithelium covering rabbit ear wounds treated with different doses of recombinant human met-NDF-α214-241,
Figure 53 shows measurements of the number and percentage of proliferating (BrdU-positive) basal and suprabasal keratinocytes in rabbit ear wounds treated with different doses of recombinant human met-NDF-α214-241.
Detailed Description Of The Invention
The DNA sequences of this invention are valuable as products useful in effecting the large scale synthesis of human NDFs by a variety of recombinant techniques. Put another way, DNA sequences provided by the invention are useful in generating new and useful viral and circular plasmid DNA vectors, new and useful transformed and transfected procaryotic and eucaryotic host cells (including bacterial and yeast cells and mammalian cells grown in culture), and new and useful methods for cultured growth of such host cells capable of the expression of recombinant NDFs and their related products.
DNA sequences of the invention are also suitable materials for use as probes in isolating additional human cDNAs and genomic DNA encoding NDFs and other genes encoding related proteins. The DNA
sequences may also be useful in various alternative methods of protein synthesis (e.g., in insect host cells) or in genetic therapy in humans and other
mammals. DNA sequences of the invention are expected to be useful in developing transgenic mammalian species which may serve as eucaryotic "hosts" for production of NDFs and NDF products in quantity. See, generally, Palmiter et al., Science 222, 809-814 (1983).
Diagnostic applications of NDF DNA sequences of this invention are also possible, such as for the detection of alterations of genes and of mRNA structure and expression levels.
The recombinant human NDF polypeptides of this invention are characterized by being the products of procaryotic or eucaryotic host expression (e.g., by bacterial, yeast, higher plant, insect or mammalian cells in culture) of exogenous DNA sequences obtained by
genomic or cDNA cloning or by gene synthesis,
particularly with use of the exogenous DNA sequence carried on an autonomously replicating DNA plasmid or viral vector in accordance with conventional techniques. The product of expression in typical yeast (e.g.,
Saccharomyces cerevisiae) or procaryote (e.g., E. coli) host cells is free of association with any mammalian proteins. The product of expression in vertebrate
(e.g., non-human mammalian, such as COS or CHO, and avian) cells is free of association with any human proteins. Depending upon the host employed, the
recombinant NDFs of the invention may be glycosylated with mammalian or other eucaryotic carbohydrates or may be non-glycosylated. The recombinant NDFs of the invention may also include an additional methionine amino acid residue at the amino terminus.
The present invention also embraces products such as polypeptide analogs of human NDFs encoded by naturally-occurring mRNAs. Such analogs include
fragments of NDF or of NDF precursors (proNDFs).
Following known procedures, one can readily design and manufacture genes encoding related polypeptides having primary structures which differ from that herein
specified for in terms of the identity or location of one or more residues (e.g., substitutions, terminal and intermediate additions and deletions). Alternately, modifications of cDNA and genomic nucleotide sequences can be readily accomplished by well-known site-directed mutagenesis techniques and employed to generate analogs and derivatives of the described NDFs. Such products share one or more of the biological properties of NDF polypeptide products encoded by naturally-occurring mRNAs. As examples, polypeptide products of the
invention include those which are foreshortened, for example, by deletions; or those which are more stable to
hydrolysis (and, therefore, may have more pronounced or longer-lasting effects); or which have been altered to delete one or more potential sites for O-glycosylation and/or N-glycosylation or which have one or more
cysteine residues deleted or replaced by other residues, e.g., alanine or serine residues, and are potentially more easily isolated in active form from microbial systems; or which have one or more tyrosine residues replaced by phenylalanine and bind more or less readily to target proteins or to receptors on target cells.
Also included are polypeptide fragments duplicating only a part of the continuous amino acid sequence or
secondary conformations within the recombinant NDFs and NDF precursors described herein, which fragments may possess one property of such NDFs and not others. It is noteworthy that the described activities are not
necessary for any one or more of the products of the invention to have therapeutic utility or utility in other contexts, such as in antagonism of naturally-occurring NDFs. Competitive antagonists may be quite useful in, for example, cases of overproduction of NDF.
The present invention also includes that class of polypeptides encoded by portions of the DNA
complementary to the protein-coding strand of the human cDNA or genomic DNA sequences of the described NDFs.
Also encompassed by the invention are pharmaceutical compositions useful in treating
biological disorders associated with neu receptor expression, comprising therapeutically effective amounts of a polypeptide product or products of the invention together with suitable diluents, preservatives,
solubilizers, emulsifiers, adjuvants and/or carriers useful in recombinant NDF therapy. A "therapeutically effective amount" as used herein refers to that amount which provides therapeutic effect for a given condition
and administration regimen. Such compositions include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents (e.g.,
Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g.,
Thimerosol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol); covalent attachment of polymers to the polypeptide to prolong in vivo half-life and to enhance potency (for instance, water-soluble polymers as polyethylene glycol, polypropylene glycol and copolymers of polyethylene glycol and polypropylene glycol, see, e.g., Davis et al., U.S. Patent No.
4,179,337); incorporation of the material into
particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc., or into liposomes. Such compositions will influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of recombinant NDF.
The recombinant NDF polypeptides of this invention are expected to be useful, alone or in combination with other therapy, in treating diseases and conditions involving cells which express the neu receptor on their surfaces. In particular, the
recombinant NDFs of this invention are useful as biological agents for modulating cellular proliferation and differentiation and, accordingly, are applicable for the therapeutic treatments described below.
The neu receptor is expressed by human epithelial cells of the gastrointestinal, respiratory, urinary and reproductive tracts (Press, M. et al., Oncogene, 1990, above; Gullick, W. et al., Int. J.
Cancer Res . , 1987, above; Quirke, P. et al., Br. J. Cancer 1989, above). For injured tissues that express the neu receptor, recombinant NDFs can be used to
promote reepithelialization and restoration of mature, functionally differentiated phenotypes in the new epithelia. For diseased tissues expressing the neu receptor, recombinant NDFs can be used alone or in combination with cytotoxic agents to inhibit abnormal cellular proliferation. Recombinant NDF polypeptides are expected to be particularly useful the treatment of dermal wounds, cancer, gastrointestinal disorders, kidney diseases, Barrett's esophagus, and diseased or damaged lung.
Dermal wound healing:
Recombinant neu differentiation factors are useful in the repair of diseased and damaged skin through their ability to stimulate tyrosine
phosphorylation of neu receptors present in the basal and squamous layers of human skin. Increased expression of the neu receptor is associated with the more
differentiated epithelium of the surface and the external root sheath of hair follicles (Maguire, H.C. et al., J. Invest . Dermatol . , 1990, above), indicating that recombinant NDFs can be used to induce a more mature and functionally differentiated phenotype in hyperproliferative skin lesions such as psoriasis vulgaris, seborrheic keratoses, acanthosis nigricans, icthyosis.
Recombinant NDFs can be used to accelerate healing of dermal wounds, such as acute and chronic dermal wounds, excisional wounds, second and third degree burns (partial and full thickness) and
epidemolysis bullosa. Epidermolysis bullosa is a defect in adherence of the epidermis to the underlying dermis, resulting in frequent open, painful blisters, which can cause severe morbidity. Accelerated reepithelialization
of these lesions would result in less risk of infection, diminished pain, and less. wound care.
Cancer:
Recombinant neu differentiation factors are useful in cancer therapy for inhibiting proliferation of certain cancer cells. In addition to inhibiting tumor cell growth, recombinant NDFs can restore a more mature and functionally differentiated phenotype in certain cancer cells, thereby restoring normal cellular
functions. For example, recombinant NDFs can inhibit tumor cell invasion and metastasis by induction of intercellular and extracellular matrix adhesion
molecules and tissue inhibitors of metalloproteinases.
Cancer cells that are susceptible to NDF treatment are those that express the neu receptor.
Tumors in which neu receptor expression or neu gene amplification have been detected include cancers of the breast, ovary, endometrium, cervix, salivary gland, esophagus, lung, stomach, pancreas, colon, kidney, prostate, and bladder, as well as squamous cell
carcinomas of the head and neck, carcinoid tumors of the gut, glioblastomas, astrocytomas, papillary thyroid carcinomas, sebaceous and sweat gland carcinomas, and hepatocellular carcinomas.
Tumors showing reduced levels of naturally occurring NDF proteins, such as lung and colon cancers (Park, J.W., et al. Proc. Am . Assoc. Cancer Res . 34:521 1993), will be particularly susceptible to recombinant NDF therapy, since normal NDF levels can be restored with recombinant NDF polypeptides. Likewise, tumors with mutated NDF genes will be responsive to NDF therapy.
Additional inhibition of tumor growth can be achieved through enhancement of chemotherapy by
recombinant NDFs. Antibodies and ligands that interact with the neu receptor and with the related EGF receptor enhance tumor cell sensitivity to the chemotherapeutic agent cisplatin (Aboud-Pirak, E. et al. J. Natl . Cancer Inst. 80:1605-1611 1988; Christen, R.D. et al., J. Clin . Invest . 86:1632-1640, 1990; Hancock, M. C. et al. Cancer Res . 51:4575-4580 1991; Nishikawa, K. et al.. Cancer Res . 52:4758-4765, 1992). Phorbol esters, which
activate the signal transduction pathway downstream from receptor tyrosine kinases, also enhance cisplatin cytotoxicity (Isohishi, S., Andrews, P. A., and Howell, S. B., J. Biol . Chem. 5:3623-3627, 1990). Through analogous mechanisms, recombinant NDF can enhance the cytotoxicity of cisplatin and other chemotherapeutic agents toward tumor cells expressing the neu receptor.
Gastrointestinal diseases
The distribution of NDF mRNA and the neu receptor messenger in normal gastrointestinal tissues predicts that recombinant NDFs will have activities on normal, diseased and injured stomach and intestine.
Immunohistochemical studies show that the neu receptor localizes to epithelial cells in these tissues.
Furthermore, recombinant NDF is a potent inducer of adherence for normal colonic epithelial cells. It is expected that recombinant NDFs can be useful in
restoring normal gastrointestinal epithelia following disease or injury.
Gastric ulcers, although treatable by H2 antagonists, show significant morbidity and recurrence, and heal by scar formation of the mucosal lining. The ability to regenerate mucosa more rapidly with
recombinant NDFs can offer a significant therapeutic improvement in the treatment of gastric ulcers.
Duodenal ulcers, like gastric ulcers, are treatable, but
recombinant NDF therapy to more fully and more rapidly regenerate the mucosal lining of the duodenum would be an important advance.
Inflammatory bowel diseases, such as Crohn's disease (affecting primarily the small intestine) and ulcerative colitis (affecting primarily the large bowel) are chronic diseases of unknown etiology which result in the destruction of the mucosal surface, inflammation, scar and adhesion formation during repair, and
significant morbidity to the affected individuals.
Therapy at present is designed to quiet the
inflammation. Recombinant NDF therapy to stimulate resurfacing of the mucosal surface, resulting in faster healing, can be of benefit in controlling progression of disease.
Gut toxicity is a major limiting factor in radiation and chemotherapy treatment regimes.
Pretreatment with recombinant NDFs may have a
cytoprotective affect on the small intestinal mucosa allowing increased dosages of such therapies while reducing potential fatal side effects of gut toxicity.
Barrett's esophagus
Barrett's esophagus (a precursor of adenocarcinoma) exhibits esophageal columnar metaplasia with frequent overexpression of the neu receptor
(Jankowski, J., et al.. Gut, 1992, above). Recombinant NDF therapy is expected to restore the normal
differentiated phenotype to the diseased tissue and inhibit abnormal cellular proliferation. Lung
Smoke inhalation is a significant cause of morbidity and mortality in the week following a burn injury due to necrosis of the bronchiolar epithelium and
the alveoli. Recombinant NDFs are expected to stimulate proliferation and differentiation of the bronchiolar epithelial cells, which express the neu receptor, thereby enhancing repair and regeneration of the lung epithelium damaged by smoke inhalation.
Kidney
A variety of kidney diseases with abnormal cell growth and differentiation (e.g. autosomal-dominant polycystic kidney disease, acquired dialysis-associated cystic disease, and non-cystic end stage kidneys) have frequent overexpression of the neu receptor (Herrera, G.A., Kidney. Int . , 1991, above). Recombinant NDF therapy may restore the normal differentiated phenotype to the diseased tissue and also inhibit abnormal proliferation.
Recombinant NDFs, through stimulation of the neu receptor present in epithelial cells lining kidney tubules, will also be useful in restoring normal kidney epithelium following ischemic acute tubular necrosis.
Liver
Neu receptor expression in the liver can be detected in certain hepatitis B virus and hepatitis C virus infectious lesions of the liver (Brunt, E.M., and Swanson, P.E., Am. J. Clin . Pathol . 97:53-61, 1992). Recombinant NDFs can be used to treat such lesions, either alone or in combination with cytotoxic agents. Peripheral nerves
Neu receptor expression is found in Schwann cells associated with transected peripheral nerves undergoing Wallerian degeneration (Cohen, J.A. et al., J. Neurosci . Res . , 1992, above). Recombinant NDFs can be used to regulate Schwann cell proliferation and
maturation in cases of peripheral nerve injury or degeneration.
Altered expression of naturally-occurring NDF in diseased tissues (e.g., malignant or premalignant tissues) can be found with the NDF DNAs, NDF mRNA detection methods, anti-NDF antibodies, and NDF
polypeptide detection methods of this invention.
Perturbations in the expression of naturally-occurring NDFs are expected to alter normal cell growth and differentiation with pathological consequences such as neoplasia. Other methods can also be used to detect aberrant NDF expression in human tissues. These methods, known to those skilled in the art of analyzing protein and mRNA expression, include immunohistochemical assays, enzyme-linked immunoabsorbent assays, and polymerase chain reaction assays. The identification of premalignant and malignant cells with abnormal NDF expression is particularly useful for the diagnosis of cancer.
The NDFs of this invention may also be combined with substances such as radiolabeled molecules, toxins, cytokines, and other compounds useful in tumor treatment, in order to increase localization of these substances on human tumors expressing high levels of the neu receptor.
The polypeptides of the invention will be formulated and dosed according to the specific disorder to be treated, the condition of the individual patient, the site of delivery of the factor, the method of administration, and other circumstances known to the skilled practitioner. Thus, for the purposes herein, an effective amount of recombinant NDF or analog or derivative is an amount that is effective to alter cellular proliferation and differentiation, or to prevent, lessen the worsening of, alleviate or cure the
condition for which the polypeptide is administered. The activity of the present polypeptide may be enhanced or supplemented with use of one or more additional biologically active or cytotoxic agents which are known to be useful in treating the same condition for which the polypeptide of the invention is being administered, e.g., IL-2 or chemotherapeutic agents for cancer
therapy, platelet-derived growth factors, epidermal growth factor, fibroblast growth factors, and the like for wound healing, and so forth.
Description of the Specific Embodiments
The invention is illustrated in the following Examples, which are not intended to be limiting.
Biological materials employed in these Examples were obtained as follows. The monoclonal antibody (Ab-3) to the carboxyl terminus of the neu receptor was obtained from Oncogene Science (Uniondale, NY). A monoclonal antibody to phosphotyrosine, PY20, was obtained from Amersham (Arlington Heights, IL). A mouse monoclonal antibody to human ß-casein was obtained through Dr. R.C. Coombes from the Ludwig Institute in London (Earl, H.M., and McIlhinney, R.A.J., Mol . Immunol . 22: 981-991,
1985). AU-565 human breast carcinoma cells were
obtained from the Cell Culture Laboratory, Naval Supply Center (Oakland, CA). The Rat1-EJ cell line (ATCC CRL 10984) was generated by transfection of the human EJ ras oncogene into Ratl fibroblasts as described by Peles, E. et al. in Cell, 1992, above, and by Land, H. et al., in Nature 304:596-602, 1983. The following cell lines were obtained from the American Type Culture Collection
(Rockville, MD) : MDA-MB-231 (ATCC HTB 26), MDA-MB-453 (ATCC HTB 131), Hs 294T (ATCC HTB 140), SK-BR-3 (ATCC HTB 30), HT-1080 (ATCC CCL 121), BALB/c 3T3 (ATCC CRL
6587) and COS-7 (ATCC CRL 1651). COS-7 cells were cultured in Dulbecco's modified Eagle medium (GIBCO, Grand Island, NY) supplemented with 10% fetal bovine serum (Hyclone, Logan, Utah). MDA-MB-453 cells were grown in RPMI Medium 1640 with 15% fetal bovine serum.
EXAMPLE 1
Amino Acid Sequence Analysis of Naturally-Occurring Rat NDF
A. Tryptic Digestion
The purification and amino-terminal sequencing of the approximately 44-kilodalton naturally-occurring NDF glycoprotein from media conditioned by
ras-transformed rat fibroblasts (Rat-1-EJ cells) has been described by Peles, E. et al., in Cell, 1992, above). In order to obtain more amino acid sequence information for the design of independent
oligonucleotide probes, 300 picomoles of the purified rat protein were subjected to partial proteolysis with trypsin, as follows: ten micrograms of the protein were reconstituted in 200 μl of 0.1 M ammonium bicarbonate buffer (pH 7.8); digestion was conducted with L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (Serva) at 37°C for eighteen hours, using an enzyme-to-substrate ratio of 1:10.
B. Separation of Trvptic Digests by HPLC
The resulting peptide mixture was separated by reversed phase HPLC and monitored at 215 nm using a Vydac C4 micro column (2.1 mm i.d. × 15 cm, 300 A) and a Hewlett-Packard 1090 liquid chromatographic system equipped with a diode-array detector and a workstation (Figure 1). The column was equilibrated with 0.1%
trifluoroacetic acid (mobile phase A) and elution was effected with a linear gradient from 0-55% mobile phase B (90% acetonitrile in 0.1% trifluoroacetic acid) over seventy minutes. The flow rate was 0.2 ml/min and the column temperature was controlled at 25°C. Three absorbance peaks eluted from the column very early (retention time less than five minutes), suggesting that these fractions correspond to short-length peptides. Three other, major fractions were recovered for amino acid sequence determination: a first fraction eluted after thirteen minutes (T13.3), a second after twenty- one minutes (T21.8), and a third after twenty-seven minutes (T27.3). C. Sequencing of Eluted Peptide Fractions
The amino acid sequences of the peptides corresponding to the three major eluted fractions were determined by automated Edman degradation. Amino acid sequence analyses of the peptides were performed with a Model 477 protein sequencer (Applied Biosystems, Inc., Foster City, CA) equipped with an on-line
phenylthiohydantoinyl (PTH) amino acid analyzer and a Model 900 data analysis system (Hunkapiller, M.W.
et al.. Methods of Protein Microcharacterization, Humana Press, Clifton, NJ, pages 223-247, 1986). The protein was loaded onto a trifluoroacetic acid-treated glass fiber disc precycled with polybrene and NaCl. The PTH-amino acid analysis was performed with a micro liquid chromatography system (Model 120) using dual syringe pumps and reversed phase (C-18) narrow bore columns (Applied Biosystems, 2.1 mm × 250 mm). The first fraction (T13.3) yielded two distinguishable major sequence signals (signal ratio 5:1) with the length of three [SEQ ID NO: 65] and four amino acids [SEQ ID
NO: 66] (the sequences are indicated in Fig. 1). The
second fraction (T21.8) gave a single sequence [SEQ ID NO: 67], starting at residue number 9 of the previously determined primary N-terminal amino acid sequence (Peles et al., Cell, above; the eighth residue is arginine). The sequence of peptide T21.8 thus confirmed the information obtained from the N-terminal sequence analysis of the whole protein and also assigned an aspartate to position 17, that was previously reported as an unassigned residue. The third fraction (T27.3) gave three distinct sequencing signals up to cycle 12 of Edman sequencing, and one clear sequence from cycles 13 through 24, suggesting that this third peptide is even longer. D. Re-Chromatographing and Sequencing of Co-Eluted Peptides.
To precisely determine the amino acid sequences of the co-eluting peptides in fraction T27.3, an aliquot of that fraction was treated as follows. A seventy percent aliquot of the peptide fraction was dried in vacuo and reconstituted in 100 μl of 0.2 M ammonium bicarbonate buffer (pH 7.8). Dithiothreitol (final concentration 2 mM) was added to the solution which was then incubated at 37°C for thirty minutes. The reduced peptide mixture was then separated by reversed phase HPLC using a Vydac column (2.1 mm i.d. × 15 cm). Elution conditions and flow rate were identical to those described previously.
Two major peptide peaks were recovered and sequenced by automated Edman degradation as described above, namely T34.4, an 11-residue arginine peptide [SEQ ID NO: 68] and T40.4, a 12-amino acid long lysine peptide [SEQ ID NO: 69], (Fig. 1, inset). Residue 1 of peptide T34.4 and cycle 11 from T40.4 remained
unassigned, suggesting that they may be cysteine
residues related to the disulfide bond of peptides in fraction T27.3. This possibility was confirmed by electro-spray mass spectrometric analysis using an API-III mass spectrometer (SCIEX, Toronto, Canada). Both peptides T34.4 and T40.4 were analyzed, resulting in average mass figures of 1261.5 and 1274.0, respectively. It was therefore concluded that these two peptides are held together in the naturally-occurring protein by a disulfide linkage. On the basis of these data, the mixed sequence signals of peak T27.3 could be re-examined. When the sequences of peptides T34.4 and T40.4 were subtracted from the mixed signals of fraction T27.3, the sequence of the longer third peptide became apparent also for the first twelve cycles. The deduced 24-amino acid long sequence [SEQ ID NO: 70] of this peptide is indicated in Figure 1. The 9th amino acid in this peptide sequence was assigned as an asparagine but at a much lower yield (about 10% of the sequence
signal), suggesting a site for N-linked glycosylation.
In view of the molecular weight of naturally-occurring NDF and the estimation that approximately one-quarter of its molecular mass is contributed by sugar moieties (Peles, E. et al., Cell, 1992, above), it was unexpected that only a few peptides were recovered after partial proteolysis. One possibility is that the naturally-occurring protein contains a protease-resistance core region that remained intact during proteolysis, but underwent denaturation and therefore was not resolved by HPLC chromatography.
EXAMPLE 2
Cloning of cDNA Encoding Rat NDF
A. Construction of a cDNA Library
RNA was isolated from Rat-1-EJ cells by standard procedures (Maniatis, T. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory, Cold Spring Harbor, NY, 1982) and poly (A)+ mRNA was selected using an "mRNA Separator" kit
(Clontech Laboratories, Inc., Palo Alto, CA). cDNA was synthesized with the "Superscript" kit (GIBCO BRL Life Technologies, Inc., Gaithersburg, MD). Column-fractionated double-strand cDNA was ligated into a SalI- and NotI-digested pJT-2 plasmid vector, to yield plasmids containing cDNA inserts such as depicted in
Figure 2. The pJT-2 plasmid vector was derived from the V19.8 vector (ATCC 68124). In particular, the HindIII and SacII cloning sites of V19.8 were changed into SalI and NotI sites by using synthetic oligonucleotide linkers to yield the PJT-2 vector. Plasmids containing cDNA inserts were transformed into DH10B E. coli cells by electroporation (Dower, W.J. et al., Nucleic Acids Res . 16:6127-6145, 1988). B. Preparation of cDNA Probes and Isolation of Clones
Approximately 5 × 105 primary transformants were screened with two oligonucleotide probes that were based on the combined amino acid sequences of the
N-terminal region of NDF as described in Example 1C, specifically, residues 5-24 (RGSRGKPGPAEGDPSPALPP) [SEQ ID NO: 71], and residues 7-12 of the T40.4 tryptic peptide (GEYMCK) [SEQ ID NO: 72]. Their respective sequences (shown in antisense) were as follows ("N" indicates all four nucleotides):
(1) 5'-ATA GGG AAG GGC GGG GGA AGG GTC NCC CTC NGC AGG
A T GCC GGG CTT GCC TCT GGA GCC TCT-3' [SEQ ID NO: 73] (2) 5'-TTT ACA CAT ATA TTC NCC-3' [SEQ ID NO: 74]
C G G C
The synthetic oligonucleotides were end-labeled with α-32p-ATP with T4 polynucleotide kinase and used to screen replicate sets of nitrocellulose filters. The hybridization solution contained 6 × SSC, 50 mM sodium-phosphate (pH 6.8), 0.1% sodium-pyrophosphate, 2 × Denhardt's solution, 50 μg/ml salmon sperm DNA and 20% formamide (for probe 1) or no formamide (for probe 2). Hybridization was carried out for fourteen hours at 42°C (for probe 1) or 37°C (for probe 2). The filters were washed at either 50°C with 0.5 × SSC/ 0.2% SDS/ 2 mM EDTA (for probe 1) or at 37°C with 2 × SSC/ 0.2% SDS/ 2 mM EDTA (for probe 2). Autoradiography of the filters gave ten clones that hybridized with both probes. These clones were purified by re-plating and probe
hybridization as previously described.
EXAMPLE 3
Expression of Recombinant Rat NDF by Transfected
COS-7 Cells
In order to verify that the cDNA clones detected in the primary screening procedure described in Example 2B corresponded to NDF-specific transcripts, the clones were transiently expressed in COS-7 cells. For that purpose the cDNA clones were inserted into the pJT-2 eukaryotic expression vector under the control of the SV40 promoter and 3'-flanked with SV40 termination and poly-adenylation signals. The resulting plasmids,
including the pJT-2/NDF plasmid containing clone 44 cDNA (Fig. 2), were used to transfect COS-7 cells by
electroporation as follows. 6 × 106 cells in 0.8 ml Dulbecco's modified Eagle medium (DMEM) and 10% fetal bovine serum were transferred to a 0.4 cm cuvette and mixed with 20 μg of plasmid DNA in 10 μL of TE solution (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). Electroporation was performed at room temperature, 1600 volts and 25 μF, using a BioRad Gene Pulser apparatus with the pulse controller unit set at 200 ohms. The cells were then diluted into 20 ml of DMEM/10% fetal bovine serum and transferred into a T75 flask (Falcon). After fourteen hours of incubation at 37°C, the medium was replaced with DMEM/1% fetal bovine serum, and the incubation was continued for an additional forty eight hours.
The resulting conditioned media were then evaluated for their ability to stimulate tyrosine phosphorylation of the neu receptor in MDA-MB-453 human breast tumor cells as follows. The conditioned media were filtered through a 0.2-micron sterile filter unit
(Costar, Cambridge, MA) and concentrated sixteen-fold by using a Centriprep 10 unit (Amicon, Beverly, MA) . The concentrated media were diluted in phosphate-buffered saline (PBS) that contained 0.1% bovine serum albumin and were then added to individual wells of a forty eight-well dish that contained 3 × 105 MDA-MB-453 cells per well. Following five minutes of incubation at 37°C, the media were aspirated and the cells were processed for Western blotting using a monoclonal antibody to phosphotyrosine (PY20). The protocol used for cell lysis and Western blotting is described by Peles, E. et al., Cell, 1992, above.
The results of the analysis are depicted in Figure 3. Although the medium of untransfected COS-7 cells induced a slight increase in tyrosine
phosphorylation, the medium harvested from clone 44 (i.e., pJT-2/NDF plasmid) transfected cells was
significantly more active. Therefore, clone 44 cDNA was completely purified by re-plating and by filter
hybridization screening. Figure 3 (right panel)
compares the activity of the completely purified
clone 44 with the activity of control clones 27 and 29 that were randomly selected. Evidently, after
transfection into COS-7 cells the completely purified clone 44 cDNA elicited higher activity than the control plasmids or the partially purified clones, including partially purified clone 44. On the basis of its hybridization to two independent oligonucleotide probes (Example 2B) and its ability to direct the synthesis of a biologically active NDF, clone 44 was selected for further analysis by DNA sequencing.
EXAMPLE 4
Sequencing of Rat NDF cDNA
Rat clone 44 cDNA was primarily sequenced using a 373A automated DNA sequencer and "Taq DyeDeoxy™ Terminator" cycle sequencing kits from Applied
Biosystems, Inc. (Foster City, CA) , in accordance with the manufacturer's instructions. Some of the sequencing was performed using 35s-dATP (Amersham) and "Sequenase™" kits from United States Biochemicals (Cleveland, Ohio), following manufacturer's instructions. Both strands of the cDNA of clone 44 cDNA were sequenced using synthetic oligonucleotides as primers. Nucleotide sequence analysis of the cDNA insert of clone 44 yielded a 1894 bp sequence [SEQ ID NO: 21] that contained a 436 amino acid long open reading frame [SEQ ID NO: 22] that extends to the 5' end of the cDNA. Downstream of the 3' end of this reading frame is a 594 base-long
untranslated stretch. The latter includes a poly (A) tail preceded by a polyadenylation signal (AAATAAA) . Because no stop codon and recognizable signal peptide sequence were found at the amino terminus of the longest open reading frame, the nucleotide sequences of three other independent positive cDNA clones were analyzed in a similar manner. All three clones included an
additional 5' 292 bp sequence with an in-frame stop codon, suggesting that clone 44 is a 5' truncated cDNA.
The combined nucleotide sequence of the rat cDNA clones [SEQ ID NO: 19] is presented in Figure 4, and the clone 44 cDNA sequence is given in Figure 5. The combined sequence spans 2,186 base pairs, including a poly (A) tail, and contains an open reading frame of four hundred and twenty-two residues [SEQ ID NO: 20] if the amino terminal methionine is considered to be the initiator codon. Hydropathy analysis (Figure 6) revealed no prominent hydrophobic sequence at the amino terminus of the protein that could function as a signal peptide for protein secretion (von Hijne, G.,
J. Mol . Biol . 184:99-105, 1985). However, this analysis showed the presence of a highly hydrophobic stretch of twenty three amino acids (underlined in Fig. 4) that qualifies as a potential transmembrane domain. This region bisects the presumed precursor of NDF (proNDF) and defines a putative cytoplasmic domain of one hundred and fifty seven amino acids. The predicted
extracellular domain contains all of the peptide sequences that were determined directly from the purified protein (overlined in Fig. 4). They all perfectly matched the sequence deduced from the cDNA except for threonine residue 137, which was assigned as isoleucine in peptide T27.3 (Fig. 1). Five different cDNA clones encoded a threonine residue in the
corresponding position, indicating that this change may
not be due to an isoform of the protein. Instead, reexamination of the protein sequence data suggested that the isoleucine signal was carried over from the previous Edman cycle, and that the threonine escaped detection due to O-glycosylation. Significantly, all of these peptides sequenced are located in the amino-terminal half of the presumed ectodomain. The other half contains six cysteine residues that comprise an epidermal growth factor (EGF)-like domain (Figure 7), which is known to be resistant to proteolysis due to its very compact structure (reviewed in Massague, J.,
J. Biol . Chem. 265:21393-21396, 1990). The other two cysteine residues of the ectodomain, that appear to be disulfide-linked in NDF (described in Example 1D), define an immunoglobulin (Ig) homology unit (Figure 8). In addition, the predicted protein sequence includes four potential sites for N-linked glycosylation, all of which reside amino-terminally to the transmembrane region (indicated by asterisks in Fig. 4). A predicted overall structure for the rat NDF precursor is presented in Figure 9.
By virtue of the transmembrane domain depicted in Figure 9, the NDF precursor is expected to accumulate on the surface of cells expressing high levels of NDF mRNAs. Membrane-bound precursor proteins have been found for other members of the EGF growth factor family. For example, Brachmann, R., et al. (Cell 56: 691-700, 1989) demonstrated the presence of transforming growth factor α on the surface of cells expressing transforming growth factor α mRNA. Molecules that specifically bind proNDF could selectively direct therapeutic molecules to cells overexpressing NDF. For example, monoclonal antibodies raised against recombinant NDF and conjugated to therapeutic molecules could selectively localize the therapeutic molecule on the surface of cells expressing
high levels of membrane-bound proNDF. Such cells would include tumor cells with an activated ras gene.
Antibody conjugates could be constructed using
chemotherapeutic compounds, cytokines, toxins,
lymphokines or radionuclides.
EXAMPLE 5
Functional Analyses of Recombinant Rat NDF Expressed bv Transfected COS-7 Cells
It has been previously reported that homogeneously purified, naturally-occurring NDF secreted from ras-transformec rat fibroblasts inhibits the growth of cultured human breast carcinoma cells and induces them to produce milk components indicative of cell
differentiation (Peles, E. et al., Cell, 1992, above). In order to directly correlate these activities with the cloned DNA, the effects of a recombinant NDF derived from rat clone 44 on cultured breast carcinoma cells were examined. More specifically, cultures of AU-565 or MDA-MB-453 cells were treated with sixteen-fold
concentrated conditioned medium from COS-7 cells that had been transfected with either the clone 44 pJT-2/NDF expression vector (Fig. 2) or a control pJT-2 plasmid that contained an unrelated cDNA insert (clone 27). Both media were used at 1:50 final dilution and incubated with the cells at 37°C for three days. Cell numbers were then determined with a hemocytometer and the nuclear area was measured by computerized image analysis. Staining for lipids and casein was performed as described in
Bacus, S., et al., Mol. Carcinog. 3:350-362, 1990. The experiment was repeated three times and yielded
qualitatively similar results, which are set forth in Table 1.
TABLE 1
Cells with Cell Number Nuclear Area Cells with Lipid Droplets (104/cm2) (μ2) Casein (%) AU-565 Cells
Control 10% 4 × 104 106 <2 Recombinant NRST 74% 1.3 × 104 215.8 >90
MDA-MB-453 Cells
Control 9% 3.6 × 104 79.8 >1 Recombinant NRSF 56% 1.8 × 104 115.7 78
> means greater than
< means less than
The results show that AU-565 and MDA-MB-453 cultures that were treated with the control-conditioned medium displayed mostly an immature morphology, whereas most of the cells treated with conditioned medium containing the recombinant NDF from rat clone 44 displayed a characteristic mature morphology that included large nuclei and the appearance of lipid droplets in the cytoplasm. Immunohistochemical staining specific for human β-casein indicated that most of these cells also synthesized casein, unlike the control-treated cultures. In conclusion, despite the fact that rat clone 44 lacks part of the 5' end of the NDF cDNA, this cDNA clone directs synthesis of a functionally active mammalian NDF which is produced by the
transfected COS-7 cells.
This conclusion was further supported by the ability of recombinant NDF to compete with naturally-occurring NDF in ligand displacement analyses. Purified naturally-occurring rat NDF was radiolabeled with 1 mCi of Nai25ι using lodogen (Pierce, Rockford, IL) according to the manufacturer's instructions. Unreacted iodine was separated from the protein by gel filtration on
Excellulose GF-5 desalting column (Pierce). The specific activity of the radiolabeled naturally-occurring NDF (125I-NDF) was 3 × 106 cpm/ng. The radiolabeled NDF (10 picomolar) was incubated for sixty minutes at 4°C with monolayers of MDA-MB-453 cells that were grown in a 24-well dish (Costar). This incubation was also performed in the presence of conditioned media from transfected COS-7 cells. Unbound 125I-NDF was removed by three washes with phosphate-buffered saline (PBS), and the cells were solubilized in 0.1 N NaOH solution containing 0.1% SDS. Radioactivity was determined with a γ-counter.
As depicted in Figure 10, conditioned medium containing recombinant NDF expressed from the pJT-2/NDF expression vector (Fig. 2) in COS-7 cells reduced the total 125I-NDF binding by approximately 50%. For a negative control, COS-7 conditioned medium from cells transfected with a pJT-2 expression vector containing a rat TGFα cDNA (Blasband, A. et al., Mol . Cell Biol . 10 : 2111-2121, 1990) was employed. In contrast to the recombinant NDF conditioned medium, the TGFα conditioned medium did not inhibit 125I-NDF binding. The partial inhibitory effect of the recombinant rat NDF conditioned medium, as compared with the purified, natural, unlabeled rat NDF (Peles, E. et al., Cell, 1992, above), may be attributed either to its relatively low concentration in the binding assay, or to high non-specific ligand binding.
To overcome this problem and also demonstrate direct interaction with the neu receptor, a covalent crosslinking assay was employed. This was performed by allowing MDA-MB-453 cells to bind 125ι-NDF in the
presence of transfected COS-7 cell conditioned media, as above. The chemical crosslinking reagent bis
(sulfosuccinimidyl) suberate (BS3, Pierce) was added at 1 mM final concentration after one hour of binding at 4°C, followed by a brief wash with PBS. Following forty-five minutes of incubation at 22°C, the monolayers were incubated for ten minutes with quenching buffer (100 mM glycine in PBS, pH 7.4). The cells were then washed twice with ice-cold PBS, lysed in lysis buffer, and the neu protein was immunoprecipitated with
monoclonal antibody Ab-3 according to the protocol described in Peles, E. et al., EMBO J. 10: 2077-2086 , 1991). The extensively washed immunocomplexes were resolved by gel electrophoresis (6% acrylamide) and autoradiography. The results of this analysis
(Figure 11) indicated that the recombinant NDF, unlike recombinant TGFα, was able to displace most of the receptor-bound I25ι-NDF. This result confirmed that rat clone 44 cDNA encodes a functional NDF molecule.
EXAMPLE 6
Northern Blot Analyses of NDF Expression
To determine the size and tissue distribution of the NDF mRNA, Northern blot hybridization experiments were carried out using the cDNA insert of clone 44 as a hybridization probe. Tissues were obtained through surgery of adult female rats, their RNA was extracted and poly (A) + RNA was selected using standard methods (Manniatis, T. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring
Harbor, New York, 1982). The 1.9 kb-long cDNA insert of rat clone 44 was labeled with α-32P-dCTP by the random priming method (Feinberg, A.P., and Vogelstein, B., Anal. Biochem 232:6-13, 1983). The conditions of hybridization were as follows: 6 × SSC, 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 2 ×
Denhardt's solution, 50 μg/ml salmon sperm DNA and 50% formamide. Hybridization was carried out for fourteen hours at 42°C, followed by washing for thirty minutes at 60°C with 0.2 × SSC, 0.1% SDS and 2 mM EDTA. The filters were exposed to Kodak XAR x-ray film with an intensifier screen at -70°C for the indicated periods of time.
Figure 12 (panel A) shows that three bands were visualized in the Northern blots with poly (A) selected RNA of Rat-1-EJ fibroblasts. Their molecular sizes corresponded to 6.8, 2.6, and 1.7 kilobases.
After further autoradiography, two additional species of mRNA became visible (not shown). The relative level of
expression of NDF in normal Rat1 or 3T3 fibroblasts was significantly lower than in the ras-transformed cells, in agreement with the earlier observation that
correlated the neu receptor stimulatory activity with transformation by an oncogenic ras gene (Yarden, Y., and Weinberg, R., Proc . Natl . Acad. Sci . USA 86: 3179-3188, 1989).
Figure 12, panel B demonstrates tissue-specific regulation of NDF mRNA expression levels in adult rat tissues. The highest NDF mRNA expression was observed in the spinal cord. NDF mRNAs were also detected in brain tissue. In addition to the mRNAs described above, these tissues express a variant mRNA that is 3.4 kb in size. Other positive tissues include lung, ovary and stomach. Relatively low amounts of the middle-size transcript were displayed by the skin, kidney and heart. The liver, spleen and muscle did not contain detectable NDF mRNA. The Northern blot also shows tissue-specific differences in the relative proportion of the different NDF mRNA species. Each variant mRNA could encode variant NDF proteins with different biological properties. Naturally-occurring NDF may regulate cell proliferation and differentiation through tissue-specific variations in NDF mRNA structure and expression.
Figure 12, panel C, shows that human tumor cells can overexpress NDF mRNA, An initial survey of human tumor cell lines found that HT-1080 fibrosarcoma cells, Hs 294T melanoma cells and MDA-MB-231 mammary adenocarcinoma cells express elevated NDF mRNA levels.
EXAMPLE 7
Characterization of cDNA Clones Isolated from a Rat-1-EJ cDNA Library that Encode Additional NDF Isoforms The isolation of rat NDF cDNA clone 44 and nine additional independent rat NDF cDNA clones from a cDNA library prepared from Rat-1-EJ cell poly (A)+ RNA is described in Example 2. These ten independent cDNA clones have been designated as clones 4, 19, 20, 22, 38, 40, 41, 42A, 42B, and 44 (Table 2). Clones 44 and 42B were deposited with American Type Culture collection, Rockville, Maryland, under accession number 69303 and 69301, respectively. The nucleotide sequences for both strands of the NDF coding region were determined for all ten cDNAs using synthetic oligonucleotide primers with fluorescence-based dideoxy-DNA sequencing as described in Example 4. The cDNAs ranged in size from 1.1 kb to 3.2 kb and, presumably, are derived from the 6.8, 2.6 and 1.7 kb NDF mRNAs found in Rat-1-EJ cells (See
Example 6). DNA sequence analyses revealed that these ten cDNAs encode six distinct NDF precursor proteins (proNDFs) (Table 2, and Figures 5 and 13-21). The open reading frames in the ten cDNAs ranged from 0.7 kb
(clone 4) to 2.0 kb (clone 42A). Two of the cDNAs were partial cDNA clones. The clone 4 sequence begins at proNDF codon 11. Clone 40 is truncated at codon 36 of the intracellular domain. Clone 44 has a complete open reading frame, but has a truncated 5'-untranslated sequence.
The ten cDNAs encode six homologous NDF precursor proteins comprised of 241 to 662 amino acid residues, which are designated proNDF-α2a [SEQ ID NO: 43], proNDF-α2b, proNDF-α2c, proNDF-β2a, proNDF-β3 and proNDFβ-4a (Figures 22, 23 and Table 2). The six predicted proNDFs are identical in the first 213 amino
acid residues. This identical region includes: a basic N-terminus which is proteolytically processed at amino acid 14 in naturally occurring rat NDF; an
immunoglobulin-like domain; a spacer domain with sites for N- and O-linked glycosylations; and the first two disulfide loops of the EGF-like domain (See Example 4 and Figure 9). ProNDF cDNAs do not encode N-terminal hydrophobic signal peptide sequences. However, with the exception of proNDF-β3, a 23-amino acid hydrophobic region is present in the proNDFs . Carboxy-terminal to this putative transmembrane domain is a cytoplasmic domain that is invariant in the first 157 amino acid residues.
Differences in NDF precursor proteins localized to two regions. The variable region of the proNDF ectodomain begins in the EGF-like domain at amino acid position 213. The α/β variation alters the
sequence between the fifth and sixth cysteines of this domain. The homologous regions of EGF and TGF-α are essential for activity and are thought to interact directly with the EGF receptor. Since the neu receptor stimulatory activities reside in the EGF-like domains (e.g., NDF-α2177_241, Example 15 and Figure 43), the α/β sequence variation may alter receptor activation in different cellular contexts. Alternatively, the NDF isoforms may differentially bind receptor heterodimers formed between the NDF receptor and related receptor molecules, such as the 170 kDa EGF receptor (Wada, T. et al., Cell 61:1339-1347, 1990; Spivak-Kroizman, T. et al., J. Biol . Chem. 267:8056-8063, 1992), pl60erbB3 (Plowman, G.D. et al., Mol . Cell Biol . 10 : 1969-1981 , 1990) or p180erbB4 (Plowman, G.D. et al., Proc. Natl . Acad. Sci . USA 90 : 1746-1750 , 1993).
As can be seen from Figure 23, the proNDF-β proteins share a common sequence at positions 213 to 230
that is distinct from the corresponding NDF-α sequence. Variation between members of the proNDF-β subfamily occurs carboxy-terminal to the EGF-like domain, where the β1 and β4a proteins contain additional amino acid residues. ProNDF-β3 cDNA encodes a stop codon in this region, resulting in a smaller protein that lacks the transmembrane and cytoplasmic domains.
The different proNDF sequences may alter proteolytic release of 44 kDa isoforms from the larger NDF precursor molecules. The proNDF proteins differ significantly in the number of charged residues located between the EGF-like domain and the hydrophobic
transmembrane domain. For example, the 27-amino acid sequence that is unique to the β4a isoform includes nine basic residues and five acidic residues. This sequence introduces several dibasic residues that are potential proteolytic cleavage sites.
Cytoplasmic domains with 374, 196 and 157 amino acid residues distinguish proNDFs α2a, α2b and α2c, respectively (Figures 5, 14, 15, 17, 21, 22, and
23). The proNDF-α2 a, b and c isoforms are identical in the first four hundred and twenty-two amino acid
residues. The proNDF-α2a cytoplasmic domain contains two hundred and seventeen additional amino acid
residues. This extended carboxyl-terminus is found in proNDFs β2a and β4a (Figures 16, 19, and 20). ProNDF-α2b cDNA encodes a different 39-amino acid sequence
following position 422 that results from a DNA insert of one hundred and forty-six base pairs in the proNDF-α2b cDNA sequence. ProNDF-α2c has the shortest cytoplasmic domain, with the cDNA having a stop codon at codon position 423 followed by a different 3'-untranslated sequence.
TABLE 2. Rat-1-EJ cDNA Clones cDNA Size proNDF Bioassay Figure
Clone (kb) Isoform Resultsa Number
4b 1.1 proNDF-β3 - 13
19 2.9 proNDF-α2b + 14
20 2.5 proNDF-α2b + 15
22c 3.4 proNDF-β2a + 16
38 2.4 proNDF-α2a + 17
40d 1.3 truncated + 18 proNDF-β2
41 2.7 proNDF-β2a + 19
42A 2.4 proNDF-β4a + 20
42Bc 3.2 proNDF-α2a + 21
44 1.9 proNDF-α2c + 5
a Media conditioned by transfected COS-7 cells were assayed for stimulation of neu receptor tyrosine phosphorylation using the procedure described in Example 3.
b Truncated at 5' end, starts at codon 11. The 5' end was
replaced with the 5' end of clone 44 for expression studies. c The original cDNA clones had two inserts. Data on the NDFrelated sequences are presented.
d Truncated at codon 36 of intracellular domain.
EXAMPLE 8
Expression of Recombinant Rat NDF Proteins in COS-7 Cells
Transient COS-7 cell expression of proNDF cDNAs was employed to determine if all ten cDNA clones encode biologically active NDF proteins. Utilizing the methods of Example 3, the proNDF cDNA clones were
inserted into the pJT-2 eukaryotic plasmid vector under the control of the SV40 early promoter with 3 '-flanking SV40 termination and polyadenylation signals, and
transfected into COS-7 cells. Media conditioned by the transfected COS-7 cells were incubated with human MDA-MB-453 cells expressing the neu receptor. The MDA-MB-453 cells were lysed, and the lysates were analyzed in Western blots for tyrosine phosphorylation (Figure 24). Nine of the ten cDNA clones directed the
synthesis and secretion of biologically active
recombinant NDF into the COS-7 cell conditioned media (Table 2) . Clone 4, which encodes proNDF-β3, did not produce a biologically active conditioned media.
Synthesis of NDF proteins in transfected COS-7 was also analyzed by immunoprecipitation of 35S-labeled proteins. For these experiments, antibodies were raised by immunizing rabbits with recombinant rat met-NDF-α214- 241 purified from E. coli as described in Example 14 (Section I), below. Rabbits were immunized with an initial injection of 200 μg of recombinant rat met-NDF-α214-241. The antigen was emulsified with complete
Freund's adjuvant and injected subcutaneously at
multiple sites. At four week intervals the rabbits were boosted with 200 μg of antigen in incomplete Freund's adjuvant injected at multiple subcutaneous sites. Sera were collected by ear vein bleed ten days after each injection and evaluated for reactivity with recombinant rat met-NDF-α214_241 by an ELISA. Anti-NDF antibodies
were purified by affinity chromatography using an
Actigel ALD kit as recommended by the manufacturer (Sterogene Bioseparations, Inc, Arcadia, CA). The affinity gel was made by overnight coupling of 2 mg of recombinant rat met-NDF-α214-241 to 3 ml monoaldehydeagarose. Unreacted aldehyde groups were deactivated with 0.1 M ethanolamine. Five milliliters of antiserum were passed through a 1 × 10 cm column containing the affinity gel. The column was washed with 50 ml PBS, and eluted with 3 mM MgCl2, 80 mM HEPES, pH 6.0, 25% ethylene glycol. The eluted antibodies were dialyzed overnight against PBS at 4°C and concentrated with a Centriprep unit (Amicon) to ~1 mg/ml concentration.
The resulting purified anti-NDF antibody was used to immunoprecipitate recombinant 35S-labeled NDF proteins from COS-7 cell cultures expressing the various recombinant precursor proteins. COS-7 cells transfected with proNDF cDNA expression plasmids were grown in 60-mm dishes in DMEM plus 10% FBS at 37°C for forty eight hours. Cells were washed and placed in 1.5 ml DMEM minus methionine and cysteine with 1% dialyzed FBS.
After sixty minutes, 200 μCi 35S methionine/cysteine (Tran35S-label, ICN) was added and cells were incubated overnight for approximately seventeen hours. Media were collected, made 1 mM in phenylmethyl sulfonylfluoride (PMSF), and clarified of cell debris in a microfuge. The cells were harvested from culture dishes by scraping them into 1 ml PBS. The cells were recovered by centrifugation and lysed by addition of 200 μl of 1% SDS, 1 mM PMSF and heating at 100°C for three minutes. The cell lysates were clarified by centrifugation and diluted 1:4 in immunoprecipitation dilution buffer (1.25% Triton X-100; 190 mM NaCl; 60 mM Tris-HCl, pH 7.4; 6 mM EDTA; 10 units/liter trasylol). The radiolabeled COS-7 cell conditioned media and diluted
cell lysates were pretreated at 4°C for two hours with 20 μl of rabbit normal serum and 20 μl of protein A-Sepharose CL-4B (Sigma) diluted 1:1 in PBS. The protein A-Sepharose was then removed by centrifugation. The pretreated samples were gently agitated at 4°C overnight with affinity-purified anti-NDF antibody (20 μg/ml) and 20 μl protein A-Sepharose. The protein A-Sepharose beads were pelleted and washed four times in a washing solution (0.1% Triton X-100; 0.02% SDS; 150 mM NaCl; 50 mM Tris-HCl, pH 7.5; 5 mM EDTA; 10 units/liter trasylol). The washed beads were heated with 60 μl of SDS gel electrophoresis loading buffer at 100°C for three minutes. The beads were removed by centrifugation and the supernatants analyzed by electrophoresis in 10% SDS-PAGE gels (Novex). Prestained protein molecular weight markers were from Novex. NDF proteins were undetectable in cell lysates and in conditioned media from COS-7 cells transfected with irrelevant cDNAs, and also when samples were immunoprecipitated in the presence of excess unlabelled recombinant NDF (not shown in Figures). The affinity-purified antibody
immunoprecipitated 40 to 45 kDa recombinant NDF proteins from cells transfected with cDNAs encoding proNDF proteins α2a, α2b, α2c, β2a and β4a (Figure 25). The immunoprecipitated proteins correspond in size to the processed form of NDF purified from media conditioned by Rat-1-EJ cells. The 40 to 45 kDa recombinant NDF proteins were found in both cell lysates and conditioned media. Larger NDF precursor proteins were detected as high molecular weight proteins immunoprecipitable from the cell lysates, but not from conditioned media. As predicted from proNDF cDNA structures (Fig. 23), the NDF α2a, β2a, and β4a precursor proteins are significantly larger than the NDF α2b and α2c precursor proteins.
These immunoprecipitated proteins are presumably the
full-length, glycosylated NDF precursor proteins.
NDF-α2c precursor proteins were found at higher levels than the other precursor isoforms, suggesting somewhat less efficient processing for this variant. In contrast to other clones, proNDF-β3 cDNA directed the synthesis of 30- to 35-kDa proteins. These proteins were found in the COS-7 cell lysate, but not in the conditioned medium (Figure 25).
The diffuse nature and the size of the immunoprecipitated recombinant NDF proteins suggests that they are glycosylated. A large percentage of the mass of naturally occurring rat NDF is due to N- and O- linked glycosylations (Peles, E. et al., Cell, 1992, above). Five microliters of immunoprecipitated recombinant NDF-ß3 and -α2c proteins in SDS gel loading buffer were diluted into 20 μl H2O with CHAPS added to a 2% final concentration. The samples were incubated at 37°C for two hours after adding 0.5 unit of N-glycanase, O-glycanase or neuraminidase (Genzyme). The digested proteins were mixed with 5 μl of 4X SDS gel loading buffer, heated at 100°C for three minutes and analyzed by electrophoresis in a 10% SDS-PAGE gel (Novex)
Prestained molecular weight markers were from Novex. Enzymatic deglycoslyation of the 40-44 kDa and 60-75 kDa NDF-α2c recombinant proteins reduced their size
approximately 10 kDa (Figure 26). The mobility shifts with the glycanases indicate that O-linked
glycoslyations are more abundant than N-linked
glycosylations on both the recombinant NDF-α2c precursor proteins and processed 40-44 kDa NDF-α2c recombinant proteins. Glycosylation sites found in the proNDF
"spacer" region are apparently modified prior to proteolytic processing of the proNDF. NDF-β3, however, was resistant to glycanase treatment. This result, and the exclusively intracellular localization of NDF-β3,
indicates that NDF-β3 protein does not enter the
secretory pathway. This is consistent with the absence of a significant hydrophobic domain in NDF-β3.
Amino terminal signal peptide sequences are notably absent from all of the predicted NDF precursor proteins. However, the different isoforms of proNDF (with the exception of proNDF-β3) are all glycoslyated and processed to 40 to 45 kDa biologically active NDF glycoproteins. These proteins are found in the
transfected COS-7 cell culture media. The capacity for secretion localizes to the internal 23-amino acid hydrophobic domain. ProNDF-β3, which accumulates as an unglycosylated intracellular protein, has essentially the entire proNDF extracellular domain. In contrast, rat clone 40 cDNA encodes a truncated proNDF-β2
comprised of the the extracellular domain, the 23-amino acid hydrophobic domain, and the first thirty-six amino acid residues of the cytoplasmic domain. This truncated cDNA clone directs the expression of biologically active NDF that accumulates in conditioned medium. The intracellular, nonsecreted NDF-β3 protein may be
released from cells by a different mechanism. The NDF-β3 isoform may function as a sequestered growth factor that is released when tissues are injured. A similar role has been proposed for IL-1β, which also lacks a signal peptide sequence (Mitzutani, H. et al., J. Clin . Invest . 87:1066-1071, 1991). Alternatively, NDF-β3 could have intracellular functions. EXAMPLE 9
PCR Analyses of Rat ProNDF mRNA and Cloning of an
Additional Rat NDF cDNA
PCR was used to analyze expression of proNDF mRNAs that encode the alternative EGF-like domains.
Eleven rat tissues were analyzed for tissue-specific expression of mRNAs encoding variant proNDF proteins (Figure 27). NDF cDNA sequences encoding the variable portion of the EGF-like domain (e.g. proNDF-α2a codon positions 200-241) were amplified using the synthetic oligonueleotide primers 5' -GCGTCTAGATGAAGGACCTGTCAAAOCC-3' (sense) [SEQ ID NO: 75] and 5'- GCGGGATCCCTTCTGGTAGAGTTCCTCC-3' (antisense) [SEQ ID
NO: 76]. The underlined sequences add XbaI or BamHI restriction sites to the 5' ends of the primers. RNA samples (1 μg) from normal adult female Sprague-Dawley rat (Charles River Laboratory, Wilmington, MA) tissues and from the Rat-1 and Rat-1-EJ cell lines were reverse transcribed to generate first strand cDNA with cDNA
Cycle™ kits (Invitrogen). PCR reactions employed Perkin Elmer Cetus GeneAmp™ PCR kits. The reactions were in a final volume of 50 μl and contained 10% of the reverse transcription reaction products or approximately 100 ng of cloned cDNAs as positive controls. Twenty-five cycles in a Perkin Elmer-Cetus GeneAmp™ 9600
thermocycler amplified the PCR products. Each cycle included twenty seconds at 94°C, twenty seconds at 55°C, and twenty seconds at 72°C. Ten μl aliquots of each reaction mixture were analyzed by electrophoresis in 2% agarose gels, followed by ethidium bromide staining.
DNA molecular weight markers were from Boehringer
Mannheim Corp. (Indianapolis, IN). Most of the eleven tissues yielded cDNA that comigrated with products obtained from cloned proNDF-α2 and proNDF-β2 cDNAs. The slightly lrger PCR products from brain and spinal cord (lanes 11 and 12) were cloned into the pSPORT vector (GIBCO BRL) and the resulting plasmids transformed into E. coli . Plasmids from colonies that hybridized to the oligonucleotide probe 5'-TGAAGGACCTGTCAAACCC-3' [SEQ ID NO: 77], which corresponds to the sense oligonucleotide
PCR primer without a XbaI sequence, were analyzed by DNA sequencing. These cDNAs encoded a new NDF amino acid sequence corresponding to a NDF-β2 sequence with eight additional amino acids following codon position 231 (Figures 23 and 28). This sequence, herein termed
NDF-β1, is homologous to human heregulin β1 (Holmes et al. Science, 1992, above).
Tissue-specific NDF variants were not detected by PCR and DNA sequencing with the exception of
preferential β1 isoform expression in brain and spinal cord, which suggests a neurological role for the NDF-βl isoform. A NDF-β1 related cDNA (designated ARIA-1) was cloned from an embryonic chick brain cDNA library (Falls et al.. Cell, 1993, above). Recombinant ARIA-1
increases the number of acetylcholine receptors and Na+ channels in muscle cells. In addition, NDF and ARIA in situ hybridizations with NDF and ARIA probes detect expression in spinal cord motor neurons, intestinal and dorsal root ganglia, and neuroepithelium lining lateral ventricles of the brain (See, also, Orr-Urtreger, et al., Proc. Natl . Acad. Sci . USA, 1993, above).
The conserved 374 amino acid cytoplasmic domain found in proNDFs α2a, β2a and β4a is more than 85% identical to the proHRG (Holmes et al.. Science, 1992, above) and proARIA-1 (Falls, D.L. et al.. Cell, 1993, above) cytoplasmic domains. All of these cytoplasmic domains have a carboxy-terminal valine residue that may be important for proteolytic processing. ProTGF-α has a carboxy-terminal valine that is critical for regulated release of TGF-α (Bosenberg, M.W. et al.. Cell 71:1157- 1165, 1992). Leucine or isoleucine can substitute for the terminal TGF-α valine in directing cleavage.
ProNDF-α2b has a 196 amino acid residue cytoplasmic domain with a carboxy-terminal leucine and is processed efficiently to mature NDF-α2. In contrast, the 157
amino acid proNDF-α2c cytoplasmic domain ends with two hydrophilic residues. Accumulation of proNDF-α2c within transfected COS-7 cells suggests that this precursor isoform is less efficiently processed. However, processing to mature NDF-α2 does occur, demonstrating that NDF release from COS-7 cells does not require a terminal hydrophobic residue. In nontransfected cells, alternative carboxy-termini may be more critical for mature NDF release.
EXAMPLE 10
Isolation of Human NDF cDNA Clones
I. Initial cDNA Library Screening
Several human cell lines were screened for the presence of NDF-related mRNAs by Northern analysis with rat clone 44 NDF cDNA probe. Poly (A)+ RNA was isolated from the cell lines indicated in Figure 29 and separated by agarose gel electrophoresis (5μg mRNA per lane).
RNAs were transferred onto nitrocellulose filters and probed with 32P-labeled rat NDF clone 44 cDNA (See
Example 6). Among them, a human kidney adenocarcinoma cell line, A-704 (American Type Culture Collection, ATCC HTB 45) exhibited the highest level of NDF mRNA
expression .
Double-strand cDNA was synthesized from poly (A)+ RNA extracted from A-704 cells. Column-fractionated cDNA was ligated to SalI and NotI digested plasmid vector pJT-2 and transformed into E. coli strain DH10B (GIBCO BRL) or E. coli strain MC1061 (BioRad) by electroporation, using a cDNA kit and procedure
recommended by the manufacturer (GIBCO BRL) .
Approximately 700,000 primary transformants were
screened with the 32P-labelled rat clone 44 NDF cDNA
probe. Hybridization was at medium stringency (30% formamide , 6x SSC, 2x Denhardt's solution, 100 μg/ml salmon sperm DNA, 1 mM EDTA, 0.2% SDS, 0.1% sodium pyrophosphate, 50 mM NaH2PO4, pH 6.8) at 42°C overnight. Filters were washed in a solution of 0.5x SSC, 2 mM EDTA, and 0.2% SDS at 23°C for thirty minutes; then at 42°C for ninety minutes. The filters were exposed overnight to X-ray film at -75°C.
Seven positive clones were identified after several rounds of colony hybridization. Plasmid DNA was purified from each clone and cDNA sequences were
determined by the methods of Example 4.
Among the seven positive human cDNA clones were one cDNA encoding a full length NDF-related
molecule, designated as human NDF-α2b [SEQ ID NO: 7] (Figure 32, clone 43), and two cDNAs encoding partial human NDF sequences, designated as NDF-α2b [SEQ ID
NO: 9] (Figure 33, clone 17) and NDF-α3 [SEQ ID NO: 11] (Figure 34, clone 19).
II. Isolation of Additional Human NDF cDNA Clones
A cDNA library was synthesized with poly (A)+ RNA extracted from A-704 cells. Double-strand cDNA was ligated to BstXI linkers and ligated into the BstXI site on plasmid vector pCDNA II (Invitrogen, San Diego, CA). The ligation mixture was used to transform E. coli strain DH10B.
Approximately 260,000 primary transformants were screened with a 32P-labeled human NDFα2b cDNA probe (clone 43, Fig. 32) using the procedure described by Martin et al. ( Cell, 63:203-211, 1990). Hybridization was at high stringency (50% formamide, 6x SSC, 2x
Denhardt's solution, 100 μg/ml salmon sperm DNA, 1 mM EDTA, 0.2% SDS, 0.1% sodium pyrophosphate, 50 mM NaH2PO4,
pH 6.8) at 42°C overnight. The filters were washed in a solution made of 0.1x SSC, 2 mM EDTA, 0.2% SDS, at 23°C for 30 min; then at 70°C for 60 min. The filters were then exposed to X-ray films at -75°C overnight.
Six positive clones were identified after several rounds of colony hybridization. Plasmid DNA was purified from the clones and the cDNA sequences were determined by the method previously described. Among the positive cDNA clones was a 920 bp cDNA encoding a partial human NDF-β3 [SEQ ID NO: 17] (Figure 37,
clone 33).
III. Screening of human pituitary cDNA libraries Two human pituitary cDNA libraries (Clontech,
Palo Alto, CA) were screened with the human NDF-α 2b clone 43 cDNA probe. One library was made from normal pituitary gland (Cat.# HL1139a) while the second was made from a gonadotropin producing adenoma (Cat.#
HL1096v). The library screening procedures were the same as mentioned above.
Among ten positive cDNA clones identified from the normal pituitary library, DNA sequencing data showed that one partial cDNA clone encoded human NDF-α1a sequences [SEQ ID NO: 3] (Figure 30, clone PI) and another partial cDNA clone encoded human NDF-β3
sequences.
Among four positive cDNA clones identified from the pituitary tumor library, one partial cDNA encoded human NDF-β1a sequences [SEQ ID NO: 13]
(Figure 35, clone P13).
IV. PCR Cloning of Additional Human NDF cDNA Sequences
To identify additional independent human NDF cDNA clones, a PCR cloning strategy was used to amplify NDF-specific DNA sequences from tumor cell lines that express NDF mRNAs (See Fig. 29). Five human tumor cell lines were used: HT-1080, Hs 294T, 5637 (ATCC HTB9) , MDA-MB-231 and A-704. Poly (A)+ RNAs were extracted from the five cell lines and reverse-transcribed to generate first-strand cDNA templates using a cDNA kit from GIBCO BRL. NDF cDNA sequences (~1 kb)
corresponding to the region containing amino acids 119 to 410 in human NDF-α2b (Figure 31) were amplified.
This region contains: the spacer domain; the variable EGF-like domain (α and β forms); the variable sequences between the EGF-like domain and the transmembrane domain; the conserved transmembrane domain; and a portion of the cytoplasmic tail. The sense primer was 5' -AGG AAA TGA CAG TGC CTC T-3' [SEQ ID NO: 78]. The antisense primer was 5'-TCT CTG GCA TGC CTG AGG-3' [SEQ ID NO: 79]. PCR was carried out for 40 cycles. The reaction conditions were: 94°C, 1 minute; 50°C,
2 minutes; 72°C, 3 minutes. The PCR amplified DNA fragments were subcloned into the pSPORT plasmid vector (GIBCO BRL) and the DNA sequences were determined. The results are listed in Table 3. In addition to the previously identified human NDF-α2, -ß1 and -ß3
sequences, DNA encoding an additional NDF isoform (ß2) was identified. The sequence of proNDF-ß2 (clone 294-8) [SEQ ID NO: 15] is shown in Figure 36.
Table 3.
Frequency of Alternative NDF Sequences in NDF cDNA
Amplified from Five Different Human Tumor Cell Lines
Human NDF Isoforms
α2 β1 β2 β3
A-704 19 1 3
MDA-MB-231 5
Hs294T 16 2 2
HT1080 2 1
5637 1
The human proNDF cDNA clones are summarized in Table 4, and the composite amino acid sequence for the different human proNDF isoforms is shown in Figure 38.
TABLE 4 . Human proNDF cDNA Clones
CDNA Size proNDF Figure ATCC Clone (kb) Isoform Number Number
P1* 1.1 NDF-α1a 30 69305
43* 1.8 NDF-α2b 32 69307
17 1.6 NDF-α2b 33 ╌
19* 0.9 NDF-α3 34 69308
P13* 1.8 NDF-ß1a 35 69302
294-8* 0.8 NDF-ß2 36 69304
33* 0.9 NDF-ß3 37 69306
*Deposited with American Type Culture Collection, Rockville, M.D .
EXAMPLE 11
Expression of Recombinant NDFs in Mammalian Cells I. Construction of pDSR-α2 expression plasmids a. Rat NDF-α2c expression vector
Rat cDNA clone 44 (proNDF-α2c, Figure 5) was subjected to PCR using two primers. The 37-base
oligonucleotide sense primer [SEQ ID NO: 80] had the sequence: 5'-CGG TCT AGA AGC TTC CAC CAT GTC TGA GCG CAA AGA A-3'. It included 5' XbaI and HindIII restriction enzyme cloning sites followed by the Kozak consensus sequence CCACC, as well as the initial 18 bases of the rat NDF coding sequences. The 30-base oligonucleotide antisense primer [SEQ ID NO: 81] had the sequence: 5'-GCC GTC GAC CTA TTA CCT TTC GCT ATG AGG-3 '. It included a SalI site, two tandem translation stop codons, and 15 bases complementary to the sequences encoding the carboxyl end of rat proNDF-α2c. The PCR product was digested with XbaI and SalI to generate a ~1.4 kb DNA fragment containing sequences encoding the entire rat proNDF-α2c. This fragment was then subcloned into the expression vector pDSRα2 (published European patent application A20398753) that had been cut with XbaI and SalI. The final plasmid was designated as pDSRα2/rNDF-α2c. b. Rat NDF-ß4 expression vector
Rat clone 42A cDNA (Figure 20) encodes proNDF-ß4a, with a unique XbaI site at amino acid positions 244-245. To express recombinant rat NDF-ß4, two pairs of PCR primers were used to amplify different portions of the proNDF-ß4a coding region. The first pair
included the same 37-base sense primer described in
Example 11 [SEQ ID NO: 80] for expression of rat NDF-α2c. This primer contained XbaI and HindIII sites, the Kozak consensus sequence CCACC, and the 18 bases
encoding amino-terminal rat proNDF-ßa sequences. The 27-base antisense primer had the following sequence: 5'-GCT CTA GAG GCT TCT CTG TTT CTT GCC-3' [SEQ ID NO: 82] and contained an XbaI site and 19 bases of the upstream sequences adjacent to the XbaI site mentioned above. The rat proNDF-ß4a (clone 42A) cDNA was subjected to PCR using this pair of primers. The PCR product was
digested with HindIII and XbaI to generate an
approximately 730 bp DNA fragment that encoded amino acids 1-244. The second pair of PCR primers included a 33-base sense primer with the following sequences: 5'-GCT CTA GAA AGA AAA TTG GAT CAT AGC CTT GTG-3' [SEQ ID NO: 83], containing an XbaI site and 25 bases of the adjacent sequences downstream from the XbaI site in NDF. The 33-base antisense primer had the sequence 5'-GCC GTC GAC CTA TTA TAC AGC AAT AGG GTC TTG-3' [SEQ ID NO: 84], and contained a SalI site, two tandem translation stop codons and 18 bases complementary to sequences encoding the proNDF-ß4a carboxyl terminus. The rat proNDF-ß4a cDNA was subjected to PCR using this second pair of primers. The PCR product was digested with XbaI and SalI to generate an approximately 1.3 kb DNA fragment which encoded amino acids 245-662. The approximately 730 bp HindIII-XbaI fragment and this approximately 1.3 kb Xba-SalI fragment were ligated with the expression vector pDSRα2 which was digested with HindIII and SalI. The resulting plasmid was designated pDSRα2/rNDF-ß4a. c. Human NDF-α2b expression vector
The initial plasmid made to express recombinant human NDF-α2b was constructed following the strategy described above for expresson of rat NDF-α2c.
Human NDF-α2b clone 43 cDNA (Figure 32) was used as the template DNA for PCR. The two primers used had the following sequences: sense primer (37-mer): 5'-CGG TCT AGA AGC TTC CAC CAT GTC CGA GCG CAA AGA A-3' [SEQ ID NO: 85]; antisense primer (30-mer): 5'-GCC GTC GAC CTA TTA GAG AAT GAA GCC CAA-3 ' [SEQ ID NO: 86]. The -1.4 kb PCR product, after cutting with XbaI and SalI, was subcloned into expression vector pDSRα2. This plasmid was designated as pDSRα2/hNDF-α2b. d. Chimeric human-rat NDFa2c expression vector
Using methods described in Example 12 below, it was observed that transfected cell clones expressing recombinant rat proNDF-α2c consistently secreted higher levels of NDF protein than the transfected cell clones expressing recombinant human proNDF-α2b. Moreover, some of the human proNDF-α2b expressing cell clones had NDF mRNA levels comparable to that of rat NDF-α2c clones, but produced much lower levels of NDF protein. Since the cytoplasmic portion of the rat proNDF-α2c amino acid sequence somehow augments recombinant NDF production (i.e., affects proNDF stability or enhances the
proteolytic cleavage rate) to generate more of the mature active recombinant NDF than the "b" isoform of human proNDF, a chimeric gene was constructed. The 197-amino acid cytoplasmic domain of human proNDF-α2b was substituted with the 157-amino acid cytoplasmic domain of rat proNDF-α2c. Figure 39 illustrates the structure of such a chimeric proNDF plasmid. When the human
NDF-α2b DNA was engineered in this way, many clones were isolated which produced increased amounts of
biologically active recombinant human NDF-α2.
The chimeric human-rat proNDF-α2c expression vector was constructed by the following method. Human NDF-α2b (clone 43) cDNA (Figure 32) was subjected to PCR
using two PCR primers. The sense primer (39-mer) had the sequence: 5' -GCC GAA GAC GGT CAT GAA GCT TCT GCC GCT GTT TCT TGG-'3 [SEQ ID NO: 87], and included an unique internal XhoI site; the antisense primer (33-mer) had the sequence: 5'-CCT TTC AAA CCC CTC GAG ATA CTT GTG CAA GTG-3' [SEQ ID NO: 88] and included several base changes to create a HindIII site. The resulting PCR product of approximately two hundred and ten base pairs, encoding proNDF-α2b amino acids 206-274 and including the
transmembrane domain (amino acids 243-265), was digested with XhoI and HindIII and subcloned into plasmid
pGEM7Zf(-) (Promega) between the XhoI and HindIII cloning sites. This DNA fragment of approximately two hundred and ten base pairs was then ligated to two other DNA fragments, one of them being a ~5.1 kb NotI-XhoI fragment retrieved from the plasmid pDSRα2/hNDF-α2b, the other a -3.6 kb HindIII-NotI fragment was derived from the plasmid pDSRα2/rNDF-α2c. The resulting plasmid was a chimeric molecule encoding the human proNDF-α2b extracellular domain, the conserved transmembrane domain (TM) , and the rat proNDF-α2c cytoplasmic domain. This plasmid was designated as pDSRα2/h-rNDF-α2c. e. Chimeric human-rat NDF-α1c, ß1c and ß2c
The strategy used to express recombinant human
NDF-α2 from a chimeric rat-human DNA construct was also used for expressing recombinant human NDF-α1, -ß1 and -ß2 isoforms. Briefly, cDNAs individually encoding human proNDF-αl, -ß1 and -ß2 were subjected to PCR using the same primers` described above for generating the chimeric human-rat NDF-α2c DNA. The -210 bp XhoI- HindIII PCR fragments generated from human NDF cDNA clone PI as the template represents sequences from human NDF-α1; the human NDF cDNA clone P-13 template gave the human NDF-ß1 sequence; and the human NDF cDNA clone 294-
8 template yielded the human NDF-B2 sequence. The XhoI-HindIII fragments were first subcloned into pGEM7Zf(-). They were then ligated to the 5.1 kb NotI-XhoI fragment (from pDSRα2/hNDF-α2b) and the 3.6 kb HindIII-NotI fragment (from pDSRα2/rNDF-α2c) as described above. The resulting expression plasmids were designated as pDSRα2/h-rNDF-αlc, pDSRα2/h-rNDF-ßlc and pDSRα2/h-rNDF-ß2c, respectively. These DNAs were prepared for transfection to express the recombinant human NDF-α1, -ß1 and -ß2 isoforms in COS-7 and CHO cells. I I . Transient expression of human and rat NDFs in CQS-7 cells
The expression plasmids constructed and described above were tested in a transient expression system to assess their ability to produce biologically active recombinant NDFs. COS-7 cells (ATCC CRL 1651) at 2.5 × 106 cells/ml were electroporated with 12.5 μg/ml DNA from (1) pDSRα2/hNDF-α2b, (2) pDSRα2/h-rNDF-α2c, (3) pDSRα2/h-rNDF-ßlc, (4) pDSRα2/h-rNDF-ß2c,
(5) pDSRα2/h-rNDF-αlc, and (6) pDSRα2/rNDF-α2c,
individually at 1600 volts for 0.7 milliseconds.
Transfected COS-7 cells were plated at 2 × 106 cells per 60 mm plate in DMEM with 10% FBS. Conditioned medium (CM) from twenty-four to seventy-two hours post-transfection were collected from the plates and tested in the neu receptor tyrosine phosphorylation assay using MDA-MB-453 carcinoma cells as described in Example 3. As shown in Figure 40, all of the CM samples tested at two concentrations (100 μl unconcentrated CM, or 100 μl of 10-fold concentrated CM) were active in the assay. Conditioned medium from untransfected COS-7 cells was negative (Fig. 40, lane 1).
EXAMPLE 12
Expression of Human and Rat NDFs in CHO Cells
I. Transfection
A Chinese hamster ovary cell line (CHO D-) deficient in dihydrofolate reductase initially described by Urlaub and Chasin (Urlaub, G. and Chasin, L.A., Proc . Natl . Acad. Sci . USA 77:4216-4220, 1980) was used. The CHO D- cell line requires proline for growth and is routinely grown in high glucose DMEM, supplemented with non-essential amino acids (NEAA) , 16 μM thymidine,
100 μM sodium hypoxanthine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 5% fetal bovine serum (FBS). Plasmid DNAs were introduced into the CHO D- cells by calcium phosphate precipitation. Approximately 0.8 × 106 cells in 60 mm plates were exposed to 2 to 3 μg of plasmid DNA together with sufficient mouse spleen DNA to total 10 μg. The medium was changed the next day. After incubation for another twenty-four hours, the cells were split into eight 100-mm plates containing selective medium (high glucose DMEM with NEAA and 5% dialyzed FBS but without nucleosides). After ten to fourteen days, clones were picked and serum-free
conditioned media from individual clones were assayed by the neu receptor tyrosine phosphorylation assay of
Example 3, or analyzed by SDS/polyacrylamide gel
electrophoresis followed by Western blot deletion using rabbit antisera specific for human or rat NDF (rabbits were immunized with purified recombinant NDF-α214-241 expressed in E. coli ; see Examples 8 and 14). Both procedures were used to select the highest NDF-producing clones for generating recombinant NDF-containing
conditioned media for subsequent purifications as described below.
II. NDF/CHO cell growth and production of conditioned medium for purification
To generate conditioned medium from recombinant CHO cells producing NDFs, selected
recombinant clones were grown in a spinner bottle in a 1:1 mixture of high glucose DMEM and F12 with NEAA, 5% FBS and 2 mM glutamine. Cells were then transferred to 850 cm2 roller bottles containing the same medium with 2 × 107 cells per bottle. After three to four days at 37°C, the cell monolayers were washed with PBS and re-fed with 150 to 200 ml fresh medium per bottle (as above but lacking FBS). Conditioned medium was
harvested six days later. Second and third harvests of conditioned media were also produced from the cell monolayer. Conditioned media were harvested and centrifuged at 7,000 × g for twenty minutes at 4°C or filtered using 0.45 μm cellulose acetate filters and stored frozen at -80°C until purification. III. Purification of recombinant mammalian NDF produced in CHO cells
Recombinant rat NDF-α2 expressed by CHO cells transfected with pDSRα2/rNDF-α2c (Example 11) was purified by the following procedure. Pooled serum-free conditioned media from harvests of roller bottles containing the transfected CHO cells expressing
recombinant rat NDF-α2c were cleared by filtration through 0.2 μ filters and concentrated with a Pellicon diafiltration system with 10-kDa molecular size cut-off membrane. Concentrated material was directly loaded onto a column of heparin-Sepharose, pre-equilibrated with 20 mM sodium phosphate buffer pH 7.210.1)
containing 25 mM NaCl. The column was then washed with the same buffer containing 0.25 M NaCl until absorbance
at 280 nm fell below 0.05. Bound proteins were eluted with a continuous gradient of NaCl from 0.25 M to 1 M. Recombinant NDF in the collected fractions was detected with a NDF-specific antibody (Example 8) and by the neu receptor phosphorylation assay. Active fractions were pooled and dialyzed against 25 mM sodium phosphate buffer containing 20 mM NaCl. Insoluble particulates present in the sample were cleared by centrifugation. The purification yield was about 70% at this point.
The dialyzed, clarified sample was then loaded onto a DEAE Sepharose 6B fast flow column which had been pre-equilibrated with dialysis buffer described above. The column was extensively washed until no absorbance at 280 nm could be detected. The column was then developed with a 200 ml gradient of 0.02 M to 0.5M NaCl in 20 mM sodium phosphate buffer, pH 7.210.1. Fractions were collected and assayed for NDF content. Ammonium sulfate was added to the pooled NDF fraction obtained from DEAE Sepharose chromatography to achieve a concentration of 2 M. The material was loaded on a Phenyl-Sepharose 4B column (Pharmacia) pre-equilibrated with 20 mM sodium phosphate, pH 7.210.1 containing 2 M ammonium sulfate. After loading, the column was washed with starting buffer and developed with a gradient of ammonium sulfate (from 2 M to no salt) in 20 mM sodium phosphate, pH 7.110.1. The main peak of activity was pooled and extensively dialyzed against PBS buffer and concentrated by centrifugation using a Centriprep 10 cartridge. IV. Characterization of the purified recombinant rat NDF-α2 from CHO cells
As shown in Figure 41, in SDS-PAGE under reducing conditions, the purified recombinant rat NDF-α2 isoform migrates at a molecular weight of about forty to
forty-four kilodaltons, identical to that of the
purified naturally occurring rat NDF isolated from rat-1-EJ cells described by Peles et al. Cell , 1992, above).
A 5 μg sample was subjected to automated N-terminal
sequence analysis. A single, major amino acid sequence was the only signal detected during analysis. The N-terminal sequence was determined to be:
NH2-Lys-Glu-Gly-Arg-Gly-Lys-Gly-Lys-Gly-Lys- Lys-Lys-Asp-Arg-Gly-Ser-Arg-Gly-... [SEQ ID NO: 89]
This sequence had nine extra amino acids preceding the amino terminal sequence observed for rat
NDF from Rat-1-EJ cells (Peles et al., Cell, 1992,
above). This result indicates that the CHO cell-derived rat NDF-α2 and the Rat-1-EJ cell-derived NDF are
proteolytically processed at different N-terminal sites.
The purified recombinant rat NDF polypeptide of this Example was found to stimulate tyrosine
phosphorylation of the neu receptor protein in the
activity assay shown in Figure 42; it was active at
relatively low concentrations and the stimulatory effect was concentration dependent.
EXAMPLE 13
EXPRESSION OF RECOMBINANT NDF ISOFORMS IN E. COLI
I. Construction of expression plasmids
Expression plasmids, pCFM1656 and pCFM3106, used in the following examples can be derived from
plasmid pCFM836 (a detailed description of pCFM836 is contained in U.S. Patent No. 4,710,473, incorporated
herein by reference). Plasmid pCFM1656 can be derived from pCFM836 plasmid by destroying the two endogenous
Ndel restriction sites by end filling with T4 polymerase enzyme followed by blunt end ligation, by replacing the DNA sequence between the unique Aatll and Clal
restriction sites containing the synthetic PL promoter with a similar fragment obtained from pCFM636 containing the PL promoter, which has the sequence :
AatII PL promoter DNA sequence
5 ' CTAATTCCGCTCTCACCTACCAAACAATGCCCCCCTGCAAAAAATAAATTCATAT¬
3 ' TGCAGATTAAGGCGAGAGTGGATGGTTTGTTACGGGGGGACGTTTTTTATTTAAGTATA¬
-AAAAAACATACAGATAACCATCTGCGGTGATAAATTATCTCTGGCGGTGTTGACATAAA-TTTTTTGTATGTCTATTGGTAGACGCCACTATTTAATAGAGACCGCCACAACTGTATTT¬
-TACCACTGGCGGTGATACTGAGCACAT 3' [SEQ ID NO: 90]
-ATGGTGACCGCCACTATGACTCGTGTAGC 5' [SEQ ID NO: 91]
ClaI and by substituting the small DNA sequence between the unique Clal and Kpnl restriction sites with the
following oligonucleotide:
Clal Kpnl
5' CGATTTGATTCTAGAAGGAGGAATAACATATGGTTAACGCGTTGGAATTCGGTAC 3'
[SEQ ID NO: 92]
3' TAAACTAAGATCTTCCTCCTTATTGTATACCAATTGCGCAACCTTAAGC 5' [SEQ ID NO: 93]
Plasmid pCFM3106 can be derived from pCFM1656 by making a series of site-directed base changes by PCR
overlapping oligonucleotide mutagenesis. These changes can be described by an arbitrary plasmid base pair position reference to a BglII restriction site (plasmid bp # 180) immediately 5' to the plasmid replication promoter PcopB and proceeding toward the plasmid
replication genes. The base pair changes are as follows:
plasmid bp # bp in PCFM1656 bp changed to in
pCFM3106
# 428 A/T G/C
# 509 G/C A/T
# 617 - - insert two G/C bp
# 978 T/A C/G
# 992 G/C A/T
# 1002 A/T C/G
# 1005 C/G T/A
# 1026 A/T T/A
# 1045 C/G T/A
# 1176 G/C T/A
# 1464 G/C T/A
# 2026 G/C bp deletion
# 2186 C/G T/A
# 2479 A/T T/A
# 2498-2501 AGTG GTCA
TCAC CAGT
# 2641-2647 TCCGAGC bps deleted
AGGCTCG
# 3441 G/C A/T
# 3649 A/T T/A
# 4556 ┄ insert bps
5' - GAGCTCACTAGTGTCGACCTGCAG-3'
[SEQ ID NO: 94]
3'- CTCGAGTGATCACAGCTGGACGTC-5' [SEQ ID NO: 95] All recombinant human and rat NDF E. coli expression plasmids were constructed using DNA obtained from PCR amplification of NDF cDNA plasmid clones
according to standard procedures. The source of these plasmid templates, the oligonucleotide primers, the
synthetic DNA fragments, and the resulting constructs
are listed in Table 5. Table 6 shows the PCR primer
sequences used to amplify NDF coding sequences for insertion into pCFM1656 and pCFM3106.
+
a. Recombinant human met-NDF-α214-241
For expression of human NDF isoforms in
E. coli, a DNA fragment encoding NDF-α214-241 was first constructed. This fragment contained the native cDNA sequence corresponding to amino acid positions 14-241 of human NDF-α2. The oligonucleotide primers 409-27 [SEQ ID NO: 97] and 409-28 [SEQ ID NO: 98] were used to amplify the NDF coding sequence from human NDF-α2 cDNA plasmid clone 43 (Figure 32). The 5' forward (sense) primer 409-27 provided an XbaI site, a ribosome-binding sequence, and a methionine start codon followed by the NDF-α2 gene sequence starting from codon 14. The 31 backward (antisense) primer 409-28 contained a BamHI site and the complementary sequence of a TAA stop codon and the NDF-α2 3' coding sequence ending at codon 241. The PCR fragment amplified from cDNA clone 43 was ligated into the XbaI and BamHI sites of plasmid
pCFM3106 (described in Section I above), so that
transcription of the human met-NDF-α214_241 gene was under the control of a PL promoter and ended at the
transcriptional terminator provided by the plasmid vector. The resulting construct was transformed into E. coli strain FM5 (ATCC #53911) which is a derivative of strain K-12 with a temperature sensitive λ repressor gene, CI857 (Sussman et al., C. R. Acad. Sci. 254:
1517-1519, 1962) integrated into the chromosome. The transformant was designated Strain 1664.
The met-huNDF-α2 expression level of Strain 1664 was subsequently improved by optimizing the first 12 codons of the protein coding sequence. A fragment with NdeI and SacII cohesive ends was generated by annealing two chemically synthesized oligonucleotides (436-23 and 436-24) [SEQ ID NOS: 100 and 101,
respectively] with complementary sequences. This synthesized fragment was then used to replace the
NdeI-SacII fragment of plasmid pCFM3106-met-huNDF-α214_241. The coding strand sequence was therefore changed from AAG AAG AAG GAG CGA GGC TCC GGC AAG AAG CCG GAG
[SEQ ID NO: 115] to AAA AAA AAA GAA CGT GGT TCT GGT AAA AAA CCG GAA [SEQ ID NO: 116]. The entire NDF-α2 gene was subcloned into another plasmid expression vector pCFM1656. When transformed into E. coli strain FM5, this new plasmid with the N-terminal codon-optimized met-huNDF-α214-241 gene gave higher expression of NDF-α214- 241, and was used to construct expression plasmids for additional human NDF isoforms. The FM5 strain
containing this plasmid was designated Strain 1679. b. Recombinant human NDF-α21-241
Because the native human NDF cDNA coding sequence starts at codon 1 (methionine), and not at codon 14 (lysine), fragment NDF-α21-241 was PCR
synthesized from human cDNA clone 43. The overlapping forward primers 507-8 [SEQ ID NO: 110] and 507-9 [SEQ ID NO: 111], which introduce a few codon changes, were used with reverse primer 507-10 [SEQ ID NO: 112]. The resulting PCR fragment was cloned into pCFM1656 using the same strategy described above. The coding strand sequence was therefore changed from TCC GAG CGC AAA GAA GGC AGA GGC [SEQ ID NO: 117] to TCT GAA CGT AAA GAA GGT CGT GGT [SEQ ID NO: 118] in the region of codon 2 to codon 9. The FM5 strain with plasmid pCFM1656-huNDF-α21- 241 containing the extended NDF sequence was designated Strain 1784. c. Recombinant human met-NDF-α114-249
The NDF cDNAs isolated from cDNA libraries that encode different human NDF isoforms differ in the sequences 3' to an unique XhoI site in the EGF-like coding region, which makes the XhoI site a convenient
cloning site for the construction of different
expression plasmids for the human NDF isoforms.
Oligonucleotide 502-10 [SEQ ID NO: 106] containing sequences 5' to the XhoI site was used as the 5' primer, and oligonucleotide 502-11 [SEQ ID NO: 107], which provided the BamHI site and TAA stop codon sequence, was the 3' primer. The resulting PCR fragment amplified from human NDF-α1 cDNA clone P1 (Figure 30) was used to replace the XhoI-BamHI fragment in plasmid pCFM1656-methuNDF-α214-241, and thus generated the NDF-αl expression plasmid pCFM1656-met-huNDF-α114-249. Strain 1854 was the designation assigned to the FM5 strain containing this plasmid. d. Recombinant human met-NDF-β114-246
Because human isoforms NDF-α1 and NDF-β1 share a common carboxy-terminal amino acid sequence, the primers 502-10 and 502-11 [SEQ ID NOS: 106 and 107, respectively] were also used for PCR synthesis of a NDF-β1 gene. Using the same cloning strategy used for the NDF-α1 gene, the XhoI-BamHI fragment in pCFM1656-methuNDF-α214-241 was replaced by the PCR fragment amplified from pCFM1656-met-huNDF-β1177-246 (described below in Section e). The FM5 strain harboring the resulting construct pCFM1656-met-huNDF-β114-246 was designated
Strain 1789. e. Recombinant human met-NDF-β1177-246
Expression of smaller recombinant NDF proteins containing the EGF-like domains of isoforms α2 and β1 was also performed in E. coli FM5. A DNA fragment containing the coding sequence for NDF-β1177-246 and necessary cloning and expression elements (cloning sites, ribosome-binding sequence, methionine start codon, and TAA stop codon) was PCR synthesized from the
EGF-like region of huNDF-β1 cDNA clone 294-1 with primers 494-26 [SEQ ID NO: 105] and 487-15 [SEQ ID
NO: 104], and cloned into the XbaI and BamHI sites of pCFM1656. Clone 294-1 is a partial cDNA clone for NDF-β1 which encodes amino acids 119 to 410 (Figure 38) . It is isolated by PCR as described in Example 10 (Section IV). Strain 1769 was assigned to this FM5 strain, bearing pCFM1656-met-huNDF-β1177-246. f. Recombinant human NDF-α2177-241
The α2 isoform counterpart of pCFM1656-methuNDF-α2177-241 was constructed similarly with PCR primers 437-29 [SEQ ID NO: 102] and 443-1 [SEQ ID NO: 103] from human cDNA clone 43. However, the expression level of this resulting strain 1697 with pCFM1656-met-huNDF-α2177- 241 was unsatisfactory. Therefore, secretion was considered as an alternative to produce NDF-α2177-241. An ompA secretion signal peptide (A) was fused to the cDNA sequence encoding human NDF-α2 amino acids 177-241.
Oligonucleotide 502-24 [SEQ ID NO: 108] containing an XbaI cloning site, a ribosome-binding sequence, a methionine start codon, degenerate ompA codons, and part of the NDF 5' coding sequences beginning at codon 177 was used as the sense primer together with antisense primer 502-25 [SEQ ID NO: 109] containing sequence around the XhoI site. Using these two primers,
fragments containing the ompA signal peptide codons and the NDF-α2 coding sequence were PCR synthesized from pCFM1656-met-huNDF-α2177-241. The fragments were used to substitute the XbaI-XhoI fragment of pCFM1656-met-huNDF-α2177-241. One of such clones that showed good
expression and signal peptide processing has the ompA signal sequence A1 (5'-ATG AAG AAG ACC GCG ATT GCA ATT GCG GTA GCG CTG GCG GGT TTT GCG ACC GTT GCG CAG GCG-3') [SEQ ID NO: 119]. The FM5 secretion strain harboring
pCFM1656-A1-huNDF-α2177-241 was designated Strain 1776. The expression level of secretion strain 1776 was greatly improved . However, the ratio of the processed huNDF-α2177-241 vs . non-processed preprotein was
acceptable for cells grown in shake flasks, but not in fermentos rs . g . Recombinant human met-NDF-α314-247
The nucleotide sequence of human NDF-α3 differs from human NDF-α2 only in the region downstream from the unique XhoI site in the EGF-like domain. The α3 segment was PCR amplified with primers 580-31 and 583-16 [SEQ ID NOS: 113 and 114, respectively] from cDNA clone 19. The resulting fragment was then used to replace the XhoI-BamHI fragment of pCFM1656-met-huNDF-α214-241 in which the N-terminal codons of NDF have been optimized. The new construct, pCFM1656-met-huNDF-α314- 247, was transformed into FM5, and thus generated Strain 1910. h. Recombinant rat met-NDF-α214-241
cDNA fragment encoding rat NDF-α214-241 was PCR amplified from rat cDNA plasmid clone 44 with primers 260-19 [SEQ ID NO: 96] and 425-26 [SEQ ID NO: 99] and cloned into the XbaI and XhoI sites in pCFM1656. The primer design and cloning strategy are similar to the ones used for the cloning of the met-huNDF-α214-241 sequence described. The FM5 strain containing the resulting expression plasmid pCFM1656-met-ratNDF-α214-241 was designated as Strain 1685.
II. Protein expression a. Fermentor
E. coli cultures (for all direct expression strains but not the secretion Strain 1776) were
inoculated into a 2 liter Fernbach flask which contained 500 ml Luria broth plus kanamycin at 50 μg/ml. The inoculated flask was then aerated by shaking at 30°C for ten to sixteen hours at 250 rpm. The cells in the flask were aseptically transferred into a 10 liter LSL
Biolafitte fermentor which contained eight liters of batch medium (80 g of yeast extract, 42 g of ammonium sulfate, 28 g of dibasic potassium phosphate, 32 g of monobasic potassium phosphate, 5 g of sodium chloride, 40 g of glucose, 32 ml of 1 M magnesium sulfate, 5 ml of antifoam Dow P2000, 16 ml of trace metal solution and 16 ml of vitamin solution. The culture was allowed to grow to an optical density of 4-5 at 600 nm before starting phase I feeding. The feed one medium contains 100 g of Bacto tryptone, 100 g of yeast extract, 900 g of glucose, 70 ml of 1 M magnesium sulfate, 20 ml of trace metal solution and 20 ml of vitamin solution in a total volume of two liters. The phase one feed rate was initiated at 13 ml/hr and then adjusted every two hours according to the cell mass determined by optical density. The medium fed into the fermentor allowed the E. coli cells to grow at an exponential rate under a glucose limited condition so that the amount of toxic by-products accumulating in the cell culture was minimal. The temperature during the entire growth phase was set at 30°C to ensure the complete suppression of the transcription from the lambda PL promoter and the tight control of plasmid amplification. The culture pH was kept at 7 with phosphoric acid and ammonium
hydroxide. The desired dissolved oxygen level was
maintained by adjusting the agitation, air- and oxygen-input rates, and back pressure in the fermentor. As the optical density of the culture reached 30, the
temperature was raised to 42°C to start the production phase. The maximal protein expression level was
achieved at about six hours afterwards. During the production phase, the first feed medium was replaced with a second medium (800 g of Bacto tryptone, 400 g of yeast extract, and 440 g of glucose in a total volume of four liters) at a constant feed rate of 250-300 ml/hr to provide enough glucose as energy and amino acids as precursors for the synthesis of recombinant NDF. At the end of fermentation, the culture was chilled to 10-15°C. The broth was harvested and centrifuged, and the
resulting cell paste was sealed in plastic bags and stored at -70°C. b. Shake flask
E. coli Strain 1776 was inoculated into a 250 ml flask which contained 100 ml Luria broth plus
kanamycin at 50 μg/ml. The inoculated flask was aerated by shaking at 30°C for 10-16 hours at 250 rpm. Twenty ml of the growth culture was then dispensed into each of ten two-liter Fernbach flasks containing 500 ml Luria broth. The culture continued to grow in the fresh medium at the same temperature and agitation rate. When the cell density reading at OD600 reached 0.6-1.0, the expression of the NDF product was induced by raising the temperature to 42°C. Four hours after induction, the cells were harvested by centrifugation at 4°C. The resulting cell paste was sealed in plastic bags, and stored at -70°C.
EXAMPLE 14
Purification of E. Coli Expressed Isoforms I. Purification of recombinant rat met-NDF-a214-741
Recombinant rat met-NDF-α214-241 was purified from E. coli Strain 1685 by subjecting a clarified cell lysate to anion exchange, cation exchange, hydrophobic interaction, and hydroxyapatite column chromatography.
E. coli cell paste was disrupted in 5 mM EDTA, pH 8.5, by two passages through a Niro-Soavi Homogenizer at 14,000 psi. The cell lysate was adjusted to pH 8.5 and centrifuged for one hour at 4,100 rpm in a J6-B centrifuge (Beckman), using a JS-4.2 rotor. The
clarified supernatant was passed through Q-Sepharose Fast Flow (Pharmacia, Piscataway, NJ) equilibrated with 5 mM Tris-Cl, pH 8.5. The flow-through fraction was loaded onto a S-Sepharose Fast Flow (Pharmacia) column previously equilibrated with 1x Dulbecco's PBS (Gibco BRL, Gaithersburg, MD), adjusted to pH 8.5. The column was washed with PBS, pH 8.5. Bound protein was eluted with a gradient of 0 to 1 M NaCl in 1x PBS, pH 8.5.
Individual fractions were collected.
S-Sepharose fractions containing a prominent recombinant NDF protein of about 34 kDa were identified by SDS-PAGE analysis using a 10% gel (Novex, San Diego) followed by Coomassie blue staining. These fractions were pooled, diluted with an equal volume of water, and adjusted to 1.2 M ammonium sulfate, pH 6.5. The sample was then loaded onto a column containing TSKGEL Butyltoyopearl 650M (TosoHaas, Montgomeryville, PA). This column was previously equilibrated with 1.2 M ammonium sulfate in PBS, pH 6.5. After washing with
equilibration buffer, the column was eluted with a
gradient of 1.2 to 0 M ammonium sulfate in 1x PBS, pH 6.5. Individual fractions were collected. Column fractions containing the recombinant NDF protein were identified by SDS-PAGE. The first NDF peak eluted contained less aggregates and more monomer. Fractions containing this first peak were pooled and dialyzed overnight at 4ºC against two changes of 10 mM sodium phosphate, pH 8.5. The dialyzed sample was loaded onto a S-Sepharose Fast Flow column previously equilibrated with 10 mM sodium phosphate, pH 8.5. The column was washed with 10 mM sodium phosphate, pH 8.5, then eluted with a gradient of 0 to 1 M NaCl in 10 mM sodium
phosphate, pH 8.5. Based on the results of SDS-PAGE, fractions containing primarily the monomer NDF were pooled. This second S-Sepharose pool was dialyzed overnight at 4ºC against two changes of 10 mM sodium phosphate, pH 6.8.
A hydroxyapatite (IBF Biotechnics, Columbia, MD) column was used as a fourth chromatography step. The column was equilibrated with 10 mM sodium phosphate, pH 6.8. After loading the dialyzed S-Sepharose
fraction, the column was washed with 10 mM sodium phosphate, pH 6.8, and eluted with a gradient of 10-500 mM sodium phosphate, pH 6.8. Fractions that contained only a single band on SDS-PAGE corresponding to the monomeric recombinant NDF were pooled and diluted with H2O to reduce conductivity to the level of 1x PBS.
After adjusting the pH to 8.5, the sample was loaded onto a S-Sepharose column previously equilibrated with 1x PBS, pH 8.5. The column was washed with 1x PBS, pH 8.5, and then eluted stepwise with 600mM NaCl in 1x PBS, pH 8.5. Column fractions containing the monomeric recombinant NDF protein were identified by SDS-PAGE, pooled, dialyzed overnight at 4ºC against two changes of 1x PBS, and passed through a 0.2 micron filter
(Nalgene). This final product was designated
recombinant rat met-NDF-α214-241. Characterization of recombinant rat met-NDF-α214-741
Protein concentrations for recombinant rat met-NDF-α214-241 were determined by ultraviolet absorption at 280 nm with a Beckman DU-650 spectrophotometer and an extinction coefficient of 0.54 mg-1cm-1ml. Reversed-phase HPLC analysis using a HP1090 HPLC with a Vydac C4 column (Hewlett-Packard Co., Palo Alto, CA) showed that the purified and refolded NDF preparation had a single peak with a retention time of 47.5 minutes in the absence and presence of 6 M guanidineHCl. The addition of both guanidineHCl and dithiothreitol caused the NDF to become reduced and unfolded, observed by the shift in retention time to 50.2 minutes. N-terminal amino acid sequence analysis of the first twenty residues
(MKKKDRGSRGKPGPAEGDPS) [SEQ ID NO : 120] matched the sequence predicted from the DNA sequence and included the additional N-terminal methionine residue used to initiate translation in E. coli . The sequence analysis also showed that N-terminal processing occurred at amino acid residues 3 and 18, with the ratios of 4% and 8%, respectively.
III. Purification of recombinant human ND F-α21-241
Recombinant human NDF-α21-241 was purified from E. coli Strain 1784 by subjecting a clarified cell lysate to anion exchange, cation exchange, and
hydrophobic interaction column chromatography.
E. coli cell paste was disrupted in 5 mM EDTA, pH 7.5 by two passages through a Niro-Soavi Homogenizer at 14,000 psi. The cell lysate was centrifuged one hour at 4,100 rpm in a J6-B centrifuge (Beckman), using a
JS-4.2 rotor. The clarified supernatant was adjusted to pH 8.5 and passed through Q-Sepharose Fast Flow
(Pharmacia, Piscataway, NJ) equilibrated with 5 mM
Tris-Cl, pH 8.5. The flow-through fraction from the Q-Sepharose column was loaded onto a S-Sepharose Fast Flow column. Column loading, equilibration and elution conditions were as described for recombinant human met-NDF-α214-241.
S-Sepharose fractions containing a prominent monomeric recombinant NDF protein were identified by SDS-PAGE analysis, pooled, and loaded onto a column containing TSKGEL Butyl-toyopearl 650M equilibrated with 1.2 M ammonium sulfate, pH 6.5. The column loading and elution conditions were the same as described for rat met-NDF-α214-241. Column fractions containing the monomeric recombinant NDF protein were identified by SDS-PAGE, pooled, and dialyzed against three changes of 1x PBS, pH 8.5 at 4ºC overnight.
The dialyzed sample was loaded onto a S-Sepharose Fast Flow column previously equilibrated with 1x PBS, pH 8.5. The column was washed with 1x PBS, pH 8.5, and then eluted with a gradient of 0 to 1 M NaCl in 1x PBS, pH 8.5. Column fractions primarily
containing the monomer NDF were identified by SDS-PAGE and pooled. The sample was dialyzed against two changes of 1x PBS at 4°C overnight and passed through a 0.2 micron filter (Nalgene). This final product was
designated recombinant human NDF-α21-241. IV. Characterization of recombinant human NDF-α21-241
The protein concentration of recombinant human NDF-α21-241 was determined by ultraviolet absorption at 280 nm using a Beckman DU-650 spectrophotometer with an extinction coefficient of 0.48 mg1-cm-1ml. Reverse
phase HPLC analysis showed that the purified and refolded NDF preparation had a single peak with a retention time of 45.8 minutes. Addition of
guanidineHCl and dithiothreitol caused the NDF to become reduced and unfolded, which was observed by a shift in retention time to 48.8 minutes. The N-terminal amino acid sequence analysis for the first twenty-five residues (MSERKEGRGKGKGKKKERGSGKKPE) [SEQ ID NO: 12] matched the putative sequence predicted from the DNA sequence. V. Purification of recombinant human met-NDF-α214-241
Recombinant human met-NDF-α214-241 was purified from E. coli Strain 1664 by subjecting a clarified cell lysate to anion exchange, cation exchange, hydrophobic interaction, and hydroxyapatite column chromatography.
E. coli cell paste was disrupted in of 5 mM EDTA, pH 8.5 by two passages through a Niro-Soavi
Homogenizer at 14,000 psi. The cell lysate was adjusted to pH 8.5 and centrifuged one hour at 4,100 rpm in a J6-B centrifuge (Beckman), using a JS-4.2 rotor. The clarified supernatant was then passed through
Q-Sepharose Fast Flow (Pharmacia, Piscataway, NJ) equilibrated with 5 mM Tris-Cl, pH 8.5. The flow-through fraction from the Q-Sepharose column was loaded onto a S-Sepharose Fast Flow (Pharmacia) column
previously equilibrated with 1x Dulbecco's PBS (Gibco BRL, Gaithersburg, MD) adjusted to pH 8.5. The column was washed with PBS, pH 8.5, and eluted with a gradient of 0 to 1 M NaCl in 1x PBS pH 8.5. Individual fractions were collected.
S-Sepharose fractions containing a prominent recombinant NDF protein of about 35 kDa were identified by SDS-PAGE analysis using a 10% gel (Novex, San Diego) followed by coomassie blue staining. These fractions
were pooled, diluted with an equal volume of H2O, and adjusted to 1.4 M ammonium sulfate, pH 6.5. The sample was then loaded onto a column containing TSKGEL Butyltoyopearl 650M, equilibrated with 1.4 M ammonium
sulfate, 20 mM citric acid, pH 6 . 5 . The column was washed with equilibration buffer and eluted with a gradient of 1.4 to 0 M ammonium sulfate in 1x PBS, pH 6.5. Column fractions containing the monomeric NDF protein were identified by SDS-PAGE, pooled, and
dialyzed against 10 mM sodium phosphate, pH 6.8.
A hydroxyapatite (IBF Biotechnics, Columbia, MD) column was used as a fourth chromatography step.
The column was equilibrated with 10 mM sodium phosphate, pH 6.8. After loading the dialyzed butyl-toyo fraction, the column was washed with 10 mM sodium phosphate
buffer, pH 6.8, and eluted with a gradient of 10-600 mM sodium phosphate, pH 6.8. Column fractions containing the monomer NDF protein were identified by SDS-PAGE, pooled, and diluted with H2O to reduce conductivity to the level of 0.15 M NaCl. After adjusting the pH to
8.5, the sample was loaded onto a S-Sepharose Fast Flow column previously equilibrated with 1x PBS, pH 8.5. The column was washed with 1x PBS, pH 8.5, and then eluted with a gradient of 0 to 1 M NaCl in 1x PBS, pH 8.5.
Column fractions containing the monomer NDF protein were identified by SDS-PAGE, pooled, dialyzed against 1x PBS, and passed through a 0.2 micron filter (Nalgene). This final product was designated recombinant human met-NDF-α214-241.
VI. Characterization of recombinant human met-NDF-α21 4-2 4 1
Protein concentrations for recombinant human met-NDF-α14-241 were determined by ultraviolet absorption at 280 nm using a Beckman DU-650 spectrophotometer with
an extinction coeffiecient of 0.54 mg-1cm-1ml. For reverse phase HPLC analysis, Vydac C4 or Synchropak RP-4 columns were used with a HP1090 HPLC (Hewlett-Packard, Palo Alto, CA). The columns were equilibrated with 97% buffer A (0.1% trifluoroacetic acid in HPLC water) and 3% buffer B (90% acetonitrile, 0.1% trifluoroacetic acid in HPLC water). Recombinant NDF protein (10-50 μg) was injected in a total volume of 250 μl. For unfolding and reduction, a 40 μl sample was first treated with 200 μl of 0 . 1 M Tris-6 M guanidineHCl, pH 8 and 10 μl of 0.2 M dithiothreitol for thirty minutes at 23°C. The final concentrations of guanidineHCl and dithiothreitol were 4.8 M and 8 mM, respectively. The columns were eluted with a linear gradient of 3-50% buffer B for the first sixty minutes. The gradient was increased to 95% buffer B for the next ten minutes. Elution was continued for another ten to fifteen minutes with 95% buffer B. The analysis showed that the purified and refolded NDF preparation had a single peak with a retention time of 46.4 minutes in the presence and absence of 6 M
guanidineHCl. The addition of both guanidineHCl and dithiothreitol caused the NDF to become reduced and unfolded, observed by the shift in retention time to 49.6 minutes. The N-terminal amino acid sequence analysis for the first ten residues (MKKKERGSGK) [SEQ ID NO: 122] matched the sequence predicted from DNA and included the N-terminal methionine used to initiate translation in E. coli . VII. Purification of recombinant human NDF-α2177-241
Recombinant human NDF-α2177-241 was purified from E. coli Strain 1776 by renaturing recombinant NDF protein from a cell lysate pellet fraction, followed by cation exchange, hydrophobic and anion exchange column chromatography.
E. coli cell paste was disrupted in 5 mM EDTA, pH 6.5, by two passages through a Microfluidizer
(Microfluidics Corp., Newton, MA) at 14,000 psi. The cell lysate was centrifuged for one hour at 4,100 rpm in a J6-B centrifuge (Beckman). The pellet was resuspended in 5 mM EDTA, pH 6.5, and centrifuged again for one hour. The pellet was solubilized by mixing with eight volumes of 8 M urea, 20 mM Tris-HCl, pH 8.6, for thirty minutes at room temperature. Reduced glutathione was added to 10 mM and the sample was stirred at room temperature, pH 8.8, for an additional thirty minutes. The denatured, reduced inclusion body protein was diluted 1:20 into a buffer containing 20 mM Tris-HCl, 1 mM EDTA, 0.2 M L-arginine, 1 mM reduced glutathione, 1 mM oxidized glutathione (pH 8.8). The solution was left for sixty minutes at room temperature, then at 4ºC for sixteen hours without further stirring.
The renatured sample was diluted with two volumes of water and adjusted to pH 4.6 by adding citric acid to 5 mM followed by 5 M HCl. After one hour of centifugation at 4,100, rpm in a J6-B centrifuge, the supernatant was loaded onto a S-Sepharose Fast Flow column. The column was previously equilibrated with 20 mM citric acid, pH 4.6. After loading, the column was washed first with equilibration buffer, then with 20 mM sodium phosphate, pH 6.8. Bound protein was stepwise eluted with 0.5 M NaCl in 1x PBS, pH 7.0.
S-Sepharose fractions containing a prominent recombinant NDF protein of about 8 kDa were identified by SDS-PAGE analysis using a 4-20% gradient gel (Novex, San Diego) followed by coomassie blue staining. These fractions were pooled, diluted to 1 mg/ml with 10 mM citric acid, pH 5.0, and adjusted to 1.5 M ammonium sulfate. The sample was then loaded onto a column containing TSKGEL Butyl-toyopearl 650M equilibrated with 1.5 M ammonium
sulfate, 10 mM citric acid, pH 5. The column was washed with the equilibration buffer and eluted with a gradient of 1.5 to 0 M ammonium sulfate in 10 mM citric acid, pH 5. Column fractions containing the monomeric
recombinant NDF protein were identified by SDS-PAGE and pooled. After concentration in a stirred cell with a YM3 ultrafiltration membrane (Amicon, Danvers, MA), the sample was dialyzed against three changes of 1x PBS overnight at 4ºC.
The dialyzed sample was diluted with two volumes of water, adjusted to pH 6.9, and passed through a Q-Sepharose Fast Flow column previously equilibrated with 20 mM sodium phosphate, pH 6.9. The flow-through fraction was adjusted to pH 4.5 with 3 M citric acid, then loaded onto a S-Sepharose Fast Flow column
previously equilibrated with 10 mM citric acid, pH 4.5. The column was washed with equilibration buffer, then stepwise eluted with 50 mM sodium phosphate pH 6.9.
Column fractions containing the monomer NDF protein were identified by SDS-PAGE, pooled, dialyzed overnight at 4ºC against three changes of 1x PBS, pH 6.5, and passed through a 0.2 micron filter (Nalgene). This final product was designated recombinant human NDF-α2177-241. VIII. Characterization of recombinant human NDF-α2177-241
Protein concentrations for recombinant human NDF-α2177-241 were determined by ultraviolet absorption at 280 nm using a Beckman DU-650 spectrophotometer with an extinction coefficient of 0.389 mg-1cm-1ml. Reverse phase HPLC analysis showed that the purified and refolded NDF preparation had a single peak with a retention time of 34.5 minutes. The addition of both guanidineHCl and dithiothreitol caused the NDF to become reduced and unfolded, which was observed by a shift in retention time
to 43.5 minutes. The N-terminal amino acid sequence analysis for the first fifteen residues (SHLVKCAEKEKTFCV) [SEQ ID NO: 123] matches the sequence predicted from DNA. IX. Purification of recombinant human met-NDF-α114-249
Recombinant human met-NDF-α114-249 was purified from E. coli Strain 1854 by renaturing recombinant NDF protein from a cell lysate pellet fraction, followed by cation exchange and hydrophobic column chromatography.
E. coli cell paste was disrupted in 1 mM EDTA, 10 mM Tris-Cl, pH 7.0 by two passages through a Niro-Soavi Homogenizer at 14,000 psi. The cell lysate was centrifuged for one hour at 4,100 rpm in a J6-B
centrifuge (Beckman), using a JS-4.2 rotor. The pellet was resuspended in 1 mM EDTA, 10 mM Tris-Cl, pH 7.0 and centrifuged again for one hour. The pellet fraction were solubilized by mixing with five volumes of 8 M urea, 2 mM EDTA, pH 8.6, for thirty minutes at room temperature. Reduced glutathione was added to 10 mM and the sample was stirred at room temperature, pH 8.6, for an additional thirty minutes. The denatured, reduced inclusion body protein was dropped into 50 volumes of 20 mM Tris-HCl, 1 mM EDTA, 0.2 M L-arginine, 1 mM reduced glutathione, 1 mM oxidized glutathione (pH 8.8). The stirring was continued for ten minutes at room temperature. The solution was then left at 4ºC overnight.
The renatured sample was diluted with one volume of water and adjusted to pH 7.0. After one hour of centifugation at 4,100 rpm in a J6-B centrifuge, the supernatant was loaded onto a S-Sepharose Fast Flow column, previously equilibrated with 1x PBS, pH 7.0.
After loading, the column was first washed with 1x PBS, pH 7.0, then with 1x PBS, pH 7.0 containing an
additional 0.15 M NaCl. Bound protein was eluted by a
gradient of 0.15-0.75 M NaCl in 1x PBS, pH 8.0. S-Sepharose fractions containing a prominent recombinant NDF protein of about 34 kDa were identified by SDS-PAGE analysis. These fractions were pooled and mixed with an equal volume of 2.4 M ammonium sulfate. After
adjusting the pH to 6.5, the sample was loaded onto a column containing TSKGEL Butyl-toyopearl 650M
equilibrated with 1.2 M ammonium sulfate, 10 mM sodium phosphate, pH 6.5. The column was washed with the equilibration buffer and eluted with a gradient of 1.2 to 0 M ammonium sulfate in 1x PBS, pH 6.5. Column fractions containing monomeric recombinant NDF protein were identified by SDS-PAGE, pooled, and dialyzed against three changes of 1x PBS overnight at 4ºC.
The dialyzed sample was loaded onto a S- Sepharose Fast Flow column previously equilibrated with 1x PBS, pH 7.0. Bound protein was eluted by a gradient of 0.15-0.80 M NaCl in 1x PBS, pH 7.0. Column
fractions containing the monomer NDF protein were identified by SDS-PAGE, pooled, dialyzed against two changes of 1x PBS, and passed through a 0.2 micron filter (Nalgene). This final product was designated recombinant human met-NDF-α114-249. X. Characterization of recombinant human met-NDF-α114-249
Protein concentrations for recombinant human met-NDF-α114-249 were determined by ultraviolet absorption at 280 nm with an extinction efficient of 0.486 mg-1 cm-1 ml. Reverse phase HPLC analysis showed that the
purified and refolded NDF preparation had a single peak with a retention time of 47.2 minutes. The addition of both guanidineHCl and dithiothreitol caused the NDF to become reduced and unfolded, which was observed by the shift in retention time to 50.4 minutes. The N-terminal
amino acid sequence analysis at the first twenty
residues matched the sequence predicted from DNA and included the N-terminal methionine residue added to initiate translation in E. coli . The sequence analysis also showed that amino-terminal processing occurred at amino acid residue 15 with the ratio of 4.8%.
XI. Purification of recombinant human met-NDF-β114-246 Recombinant human met-NDF-β114-246 was purified from E. coli Strain 1789 by renaturing recombinant NDF protein from a cell lysate pellet fraction, followed by cation exchange and hydrophobic interaction column chromatography.
E. coli cell paste was disrupted in ten volumes of 10mM tris-HCl 5mM EDTA, pH 8.0. The cell lysate was centrifuged, and the pellet was solubilized in five volumes of 8M Urea, pH 8.6, for thirty minutes at room temprature. Dithiothreitol was added to 5mM to complete the reduction. The sample was stirred at room temperature, pH 8.6, for an additional thirty minutes. The solublized, reduced inclusion body protein was added dropwise to fifty volumes of 20 mM Tris-Cl, 1 mM EDTA, 0.2 M L-arginine, 2 M urea, 1 mM reduced glutathione, 1 mM oxidized glutathione (pH 8.6). The stirring was continued for 10 minutes at room temperature. The solution was then left at 4°C overnight.
After diluting the renatured sample with two volumes of water and adjusting the pH to 8.0, the material was loaded onto a CM-Sepharose Fast Flow
(Pharmacia) column, previously equilibrated with 20 mM sodium phosphate, pH 8.0. After loading, the column was washed with 20 mM sodium phosphate pH 8.0, then with 10 mM sodium phosphate, 15mM glycine, pH 8.5. Bound protein was eluted with 0.5 M NaCl in 10 mM sodium phosphate, 15 mM glycine, at pH 8.5.
CM-Seρharose fractions containing a prominent recombinant NDF protein of about 34 kDa were identified by SDS-PAGE analysis. The pooled fractions were
adjusted to 10% (weight/volume) ammonium sulfate and pH 6.5 before loading onto a TSKGEL phenyl-toyopearl
650M column, previously equilibrated with 10% (w/v) ammonium sulfate, pH 6.5. The column was washed with the equilibration buffer and eluted with a gradient of 10% to 0% ammonium sulfate in 20mM sodium phosphate at pH 6.5. Column fractions containing the monomer NDF protein were identified by SDS-PAGE, pooled, and
dialyzed against 50mM sodium phosphate, pH 7.0. The dialyzed sample was loaded onto a S-Sepharose Fast Flow column previously equilibrated with 50mM sodium
phosphate, pH 7.0. The column was washed, with 50mM sodium phosphate, pH 8.0, then stepwise eluted with 0.5M NaCl in 20mM sodium phosphate, 20mM glycine, pH 8.8.
Column fractions containing monomeric recombinant NDF protein were identified by SDS-PAGE, pooled, dialyzed against 1x PBS, and passed through a 0.2-micron filter (Nalgene). This final product was designated
recombinant human met-NDF-β114-246.
XII. Characterization of recombinant human met-NDF-β114-246
The protein concentration of recombinant human met-NDF-β114-246 was determined by absorption at
280nm with an extinction coefficient of 0.59 mg-1cm-1ml. Reverse phase HPLC analysis showed that the purified and refolded recombinant NDF preparation had a single major peak with a retention time of 49.2 minutes. The
addition of both guanidineHCl and dithiothreitol caused the recombinant NDF to become reduced and unfolded, observed by a shift in retention time to 53.2 minutes. The N-terminal amino acid sequence analyses for the
first twenty-five residues (MKKKERGSGKKPESAAGSQSPALPP) [SEQ ID NO: 124] matched the sequence predicted from DNA and included the additional N-terminal methionine used to initiate translation in E. coli . The sequence analysis also revealed that the first two residues were absent from 25% of the material, which has the amino-terminal sequence KKERGSG.
XIII. Purification of recombinant human met-NDF-β1177-246
Recombinant human met-NDF-β1177-246 was purified from E. coli Strain 1769 by renaturing recombinant NDF protein from a cell lysate pellet fraction, followed by cation exchange and reverse phase column chromatography.
E. coli cell paste was disrupted in ten volumes of 5mM EDTA, 10mM tris-HCl, pH 8.0. The cell lysate was centrifuged, the pellet was resuspended in 5 mM EDTA, 10 mM Tris-Cl, pH 8.0 and centrifuged again, then solubilized by mixing with five volumes of 8 M urea, pH 8.5, at room temperature for thirty minutes. Reduced glutathione was added to lOmM, and the sample was stirred at room temperature, pH 8.7, for an
additional thirty minutes. The solublized, reduced inclusion body protein was added dropwise to fifty volumes of thirty mM Tris-HCl, 1 mM EDTA, 0.2 M
L-arginine, 2 M urea, 1 mM reduced glutathione, 1 mM oxidized glutathione (pH 8.6). The stirring was
continued for ten minutes at room temperature. The sample was then left at 4ºC overnight.
After diluting the renatured sample with two volumes of water and adjusting the pH to 4.5 with 3 M citric acid, the material was centrifuged for one hour. The supernatant was loaded onto a CM-Sepharose Fast Flow (Pharmacia) column previously equilibrated with 10 mM citric acid, pH 4.5. After loading, the column was
washed with equilibration buffer. Bound protein was stepwise eluted with 1x PBS, pH 8.5.
CM-Sepharose fractions containing a prominent ~7 kD recombinant NDF protein were identified by SDS-PAGE analysis. Pooled fractions containing the product were diluted with two volumes of water. The pH was adjusted to 4.0, and the material was loaded onto a
Vydac C4 column, from The Separations Group, Hesperia, CA. The C4 column was previously packed in 80% ethanol, pH 4.0, and equilibrated with 20mM citric acid, pH 4.0. After loading, the column was washed with 20 mM citric acid, pH 4.0, then with 20% ethanol in 20mM citric acid, pH 4.0. Bound proteins were eluted by a gradient of 20-80% ethanol in 20 mM citric acid, pH 4.0. Column
fractions containing monomeric recombinant NDF protein were identified by SDS-PAGE, pooled, and dialyzed
against two changes of 20 mM citric acid, pH 4.5, at 4ºC overnight.
The dialyzed sample was loaded onto a S-Sepharose Fast Flow (Pharmacia) column previously
equilibrated with 20 mM citric acid, pH 4.5. The column was washed with the equilibration buffer and stepwise eluted with 1x PBS, pH 8.0. Column fractions containing the monomer NDF protein were identified by SDS-PAGE and pooled. To clarify the pooled material, 0.2 volumes of water and 1/20 volumes of 1 M tris-HCl, pH 8.5, were added. The sample was then dialyzed against 1x PBS, pH 8.0, and passed through a 0.2 micron filter (Nalgene).
This final product was designated recombinant human met- NDF-β1177-246.
XIV. Characterization of recombinant human met-NDF-β1177-246
The protein concentration of recombinant human met-NDF-β1177-246 was determined by absorption at 280 nm
with an extinction coefficient of 0.66 mg-1cm-1ml.
Reverse phase HPLC analysis showed that the purified and refolded recombinant NDF preparation has a single major peak with a retention time of 50.5 minutes. The
addition of both guanidineHCl and dithiothreitol caused the recombinant NDF to become reduced and unfolded, which is observed by a shift in retention time to 53.7 minutes. The N-terminal amino acid sequence analysis of the first fifteen residues revealed a major sequence (MSHLVKCAEKEKTFC) [SEQ ID NO: 125] that matched the sequence predicted from DNA and included the additional N-terminal methionine used to initiate translation in E. coli . XV. Purification of recombinant human met-NDF-α314-247
Recombinant human met-NDF-α314-247 was purified from E. coli Strain 1910 by renaturing recombinant NDF protein from a cell lysate pellet traction, followed by cation exchange, hydrophobic extraction and anion exchange column chromatography. E. coli cell paste was disrupted in ten volumes of 5mM tris-HCl, 1mM EDTA, pH 8.0. The cell lysate was centrifuged, and the pellet was solubilized in ten volumes of 8M urea, 2mM EDTA, 5mM Dithioerythritol (2, 3-Dihydroxybutane-1.4-dithiol), pH 9 for ninety minutes at room temperature. The solubilized, reduced inclusion body protein was added slowly into twenty five volumes of 20 mM tris-HCl, 0.4 M L-arginine, ImM EDTA, 0.5mM reduced glutathione, pH 8.7. The stirring was continued for five minutes at room temperature. The sample was then left at room
temperature for one hour; then at 4°C for eighteen hours without further stirring.
The renatured sample was adjusted to pH 4.7 by adding citric acid to 10 mM followed by 5M HCl. After
centrifugation, the supernatant was loaded onto a s-sepharose fast flow column. The column was previously equilibrated with 20mM citric acid pH 4.7. After
loading, the column was washed first with equilibration buffer, then with 20mM sodium phosphate, 20mM Glycine, pH 8.5; finally with 0.2M NaCl in 20mM sodium phosphate, 20mM glycine, pH 8.5. Bound protein was stepwise eluted with 0.7 M NaCl in 20mM sodium phosphate and 20mM
glycine, pH 8.5. The eluted fractions from S-Sepharose column containing a prominent recombinant NDF protein of about 34 kDa were identified by SDS-PAGE analysis using a 10% NOVEX gel followed by coomassie blue staining.
These fractions were pooled, diluted by water to
approximately lmg/ml, and then adjusted to 1.2M ammonium sulfate and 50mM sodium phosphate pH 6.0. The sample was loaded onto a Butyl-toyopearl 650 M column
equilibrated with 1.2 M ammonium sulfate, 50mM sodium phosphate pH 6.0. After loading the column was washed with the equilibration buffer and eluted with a gradient of 1 M to 0 M ammonium sulfate in 50 mM sodium phosphate pH 6.0. Column fractions containing monomeric
recombinant NDF protein were identified by SDS-PAGE and pooled. The pooled sample was dialyzed against three changes of 1 × PBS pH 7.5.
The dialyzed sample was passed through a
Q-sepharose fast flow column previously equilibrated with 1 × PBS pH 7.5. The flow-through fraction was collected and adjusted to pH 4.7 by 3M citric acid. After adding 0.2M NaCl, the sample was loaded onto a S-Sepharose fast flow column at pH 4.7. The column was then washed with 20mM citric acid, pH 4.7, and with 20mM sodium phosphate, 20mM glycine, pH 8.5. The bound protein was eluted with a gradient of 0 to 1M NaCl in 20mM sodium phosphate, 20mM glycine, pH 8.5. The first peak containing the monomeric recombinant NDF protein was pooled, dialyzed at 4ºC
against three changes of 1x PBS, and passed through a
0.2 micron filter. This final product were designated recombinant human met-NDF-α314-247 XVI. Characterization of recombinant human-met-NDF-α314-247
The protein concentration of recombinant human met-NDF-α314-247 was determined by absorption at 280 nm with an extinction coefficient of 0.446 mg-1cm-1ml. SDS-PAGE analysis using a 10% NOVEX gel followed by
coomassie blue staining showed that the purified NDF protein appeared as a single band approximately 34K
(15μg/lane). In the presence of reducing reagent (30mM dithiothreitol) the reduced form tended to run slower than the non-reduced form in a SDS-PAGE. Reverse phase HPLC analysis showed that the purified and refolded
recombinant NDF preparation had a single peak with a retention time of 47.9 minutes. The addition of both guanidineHCl and dithiothreitol caused the NDF protein to become reduced and unfolded, observed by a shift in retention time to 51.0 minutes. The N-terminal amino sequence analysis of the first nineteen residues matched the sequence predicted from DNA and included the
additional N-terminal methionine used to initiate
translation in E . coli .
EXAMPLE 15
Induction of Her2/nee u Tyrosine Phosphorylation by
Recombinant NDFs from E. coli
Subconfluent monolayers of MDA-MB-453 cells were grown in 48-well plates. The cells were then
treated with the indicated concentrations of recombinant rat or human NDF at 37°C for five minutes . Cell lysates
were prepared and subjected to Western blot analysis with an anti-phosphotyrosine antibody. The results are shown in Figure 43. All of the recombinant rat and human NDF isoforms were active in stimulating tyrosine phosphorylation of the neu receptor in MDA-MB-453 cells at concentrations from 5-20 ng/ml. PBSA is PBS with 0.1% albumin as a negative control.
EXAMPLE 16
Accumulation of Neutral Lipids in Mammary Carcinoma
Cells Cultured in the Presence of Recombinant Human
NDF-α2 and Recombinant Rat NDF-α2 Phenotypic changes usually associated with "functional" differentiation of breast epithelium include the synthesis of milk components and lipids (Topper, Y.J, and Freeman, C.S., Physiol . Rev. 60:1049-1106, 1980; Stampfer, M.R., and Yaswen, P.,
Transformation of Human Epithelial Cells: in Molecular and Oncogenic Mechanisms, G.E. Milo, B.C. Castro and CF. Shuler, eds. ( CRC Press, Ann Arbor, MI) , pp. 117-140, 1992). Specific cultured human breast cell lines can acquire these differentiated characteristics upon treatment with factors such as phorbol 12-myristate
13-acetate (Bacus, S.S. et al., Mol . Carcinog. 3:350-362, 1990). This experiment was designed to determine whether recombinant human met-NDF-α214-241 (rHuNDF-α2) or recombinant rat NDF-α2 (rRtNDF-α2) stimulates, retards or has no affect on the in vitro accumulation of neutral lipids in BT-474 cells, a human mammary ductal carcinoma line which overexpresses the neu receptor. rHuNDF-α2 used herein was purified from E. coli as described in Example 14, whereas rRtNDF-α2 was prepared from CHO cell supernatants as described in Example 12.
I. Materials and Methods
BT-474 cells (ATCC HTB 20) were obtained from the
American Type Culture Collection (Rockville, MD). Cells were routinely cultured in "complete" media containing RPMI 1640 (GIBCO BRL, Gaithersburg, MD) supplemented with 10% heat-inactivated fetal bovine serum (Hyclone Laboratories Inc., Logan UT), 2 mM L-glutamine (GIBCO BRL), and 10 μg/mL bovine insulin (Clonetics Corp., San Diego, CA).
Accumulation of neutral lipids (e.g.,
cytoplasmic triglycerides) was measured using a
modification of the Nile Red bioassay (Smyth, M.J., and Wharton, W., Exp . Cell Res . 199 : 29-38, 1992). BT-474 cells were seeded in 6-well flat-bottom dishes in 2 mL of "complete" media at a density of 1 × 105 cells per well. After incubation for twenty-four hours at 37°C in a humidified air/5% CO2 chamber, media were removed and replaced with samples containing either rHuNDF-α2 or rRtNDF-α2. Plates were re-incubated using the
conditions described above. Recombinant NDF-α2
dilutions were prepared from stock solutions
(rHuNDF-α214-241, 370 μg/mL; rRtNDF-α2, 492 μg/mL) in a 1:1 mixture of "complete" media and diluent. Diluent consisted of a 1:1 mixture of DMEM (GIBCO-BRL) :F-12
(GIBCO BRL) with 100 μM non-essential amino acids (GIBCO BRL) and 2mM L-glutamine (GIBCO BRL). Cells were refed with fresh media (with or without recombinant NDF-α2) after three and five days of incubation. On day 7, cells were detached from the substratum using 0.25 mL 0.1% trypsin/1.1 mM EDTA (GIBCO BRL) in Dulbecco's phosphate-buffered saline (D-PBS) (GIBCO BRL) at 37°C. After each sample was resuspended in a final volume of 2 mL D-PBS, 0.8 mL phosphate-buffered 10% formalin (Baxter, Deerfield, IL) was added (i.e., final
formaldehyde concentration was approximately 1%) as a fixative. Each cell suspension was filtered through Nitex bolting cloth (Tetko Inc., Briarcliff Manor, NY) and stained with 50 ng/mL of Nile Red (Molecular Probes Inc., Eugene, OR). Samples were analyzed (104 events per sample) with a Fluorescence Activated Cell Sorter (FACS) for gold fluorescence, red fluorescence, side-scatter, forward scatter, and a gold-to-red fluorescence ratio. The FACStarPLUS (Becton Dickinson Immunocytometry Systems, San Jose, CA) was equipped with a 5 W argon laser operated at 488 nm. Gold fluorescence was collected using a narrow band pass filter at 575 ± 13 nm (Becton Dickinson) while red fluorescence was collected beyond 610 nm with a long pass filter (Omega Optical, Brattleboro, VT). Data were acquired and analyzed using the software package Lysis II Version 1.0 (Beckton Dickinson).
II. Results and Discussion:
Formation of cytoplasmic lipid vesicles, a phenotypic change normally associated with
differentiating mammary epithelium, can be detected by staining cells with a fluorescent hydrophobic probe known as Nile Red. Nile Red fluorescence can vary:
although not fluorescent in aqueous media, it will fluoresce yellow-gold in a neutral lipid environment (i.e., lipid vesicles) or red if in a polar (i.e., plasma membrane) lipid environment (Fowler, S.D., and Greenspan, P., J. Histochem. Cytochem. 33:833-836, 1985). Therefore, as a cell differentiates and
intracellular neutral lipid deposits accumulate, total gold fluorescence will increase relative to total red fluorescence. Such changes are most readily quantified when gold-to-red fluorescence ratios are monitored
rather than either total gold or total red fluorescence (Smyth, M.J., and Wharton, W., Exp . Cell Res . , above).
BT-474 cells were treated with recombinant NDF-α2 samples for seven days prior to staining with Nile Red and analysis by flow cytometry. Histograms depicting the gold-to-red fluorescence ratio measurements are presented in Figures 44 and 45. Recombinant NDF-α2 induced changes in the distribution of cells from the low ratio peak (i.e., "nondifferentiated" cells with few neutral lipid vesicles) to the high ratio peak (i.e.,
"differentiated" with prominent neutral lipid vesicles). The data, summarized in Table 7, indicate that both E. coli-produced rHuNDF-α2 and CHO-produced rRtNDF-α2 stimulate accumulation of neutral lipids in BT-474 cells in a dose-dependent manner. This is reflected by an increase in the percentage of cells in the high-ratio peak. This effect was most pronounced at the highest concentration tested (100 ng/mL). Thus, treatment with either preparation of NDF-α2 can induce a
"differentiated" phenotype in BT-474 cells.
= = = = = == = = = = = = = = = = == = = = = = = = = = = == = = = = = = = = = = == = = = = = = = = = = == = = = = = = = = = = == = = = = = = = = = = == = = = = = = = = = = == = = = = TABLE 7. Accumulation of Neutral Lipids in BT-474 Mammary Carcinoma Cells = = = = = == = = = = = = = = = = == = = = = = = = = = = == = = = = = = = = = = == = = = = = = = = = = == = = = = = = = == = = = = = = = = = = == = = = = = = = = = = == = = = = = = =
LOW-RATIO PEAKb HIGH-RATIO PEAKc
[NDF-α2] Type of rNDFa Percent Percent- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
0 ng/mL E. coli/human 60.4 39 . 6
CHO/rat 63.2 36 . 8
4 ng/mL E. coli/human 45.6 54 . 5
CHO/rat 65.6 34 . 4
20 ng/mL E. coli/human 37.3 62 . 8
CHO/rat 30.2 69 . 0
100 ng/mL E. coli/human 20.1 79 . 9
CHO/rat 9.7 90 . 3
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - - - - - - - - - -
Cultures of BT-474 were treated for seven days with defined amounts of either E. col i-produced rlluNDF-α2 or CHO-produced rRtNDF-α2. Staining for lipids was as described in the Materials and Methods section. Data refer to either the fraction of cells (i.e., percent) residing in either tha low or high-ratio peak. The experiments were repeated and yielded similar results.
a " E. coli/human" refers to recombinant human met-NDF-α214-241 purified from E. coli ;
"CHO/rat" refers to recombinant rat NDF-α2 purified from CHO cell supernatents.
b Cells whose mean gold-to-red fluorescence ratio waa < 60.
c Cells whose mean gold-to-red fluorescence ratio waa > 60 and < 2000.
EXAMPLE 17
Stimulation of BT-474 Proliferation and Inhibition of
MDA-MB-468 Proliferation in the Presence of Recombinant
Human NDF-α2
The in vitro growth of cancer cells can be modified by numerous growth stimulatory and inhibitory factors (Aaronson, S.A., Science 254:1146-1153, 1991). Some of these, such as EGF (Imai, Y. et al., Cancer Res . 42:4394-4398, 1982), bFGF (Karey, K.P., and Sirbasku, D.A., Cancer Res . 48:4083-4092, 1988), IGF-I (Karey, K.P., and Sirbasku, D.A., Cancer Res . , above) and TGF-ßχ (Knabbe, C. et al.. Cell 48 :411-428, 1987), can regulate the proliferation of rodent and mammalian breast
carcinoma cells in culture. Responses can be dependent upon variables such as the target cell line, specific factor or factors, media and media supplements, cell density, and type of assay. In this experiment, we determined whether recombinant human met-NDF-α214-241 (rHuNDF-α2) stimulates, retards or has no affect on the in vitro growth of two human mammary cell lines: BT-474 cells, a ductal carcinoma line that overexpresses the neu/Her-2 receptor, and MDA-MB-468 cells, an
adenocarcinoma line which expresses the same receptor. rHuNDF-α2 was purified from E. coli and re-folded in an active configuration (Example 14).
I. Materials and Methods:
BT-474 (ATCC HTB 20) and MDA-MB-468 (ATCC HTB 132) cells were obtained from the American Type Culture Collection (Rockville, MD). Cells were
routinely cultured in "complete" media containing RPMI 1640 (GIBCO BRL, Gaithersburg, MD) supplemented with 10% heat-inactivated fetal bovine serum (Hyclone
Laboratories Inc., Logan, UT), 2 mM L-glutamine (GIBCO
BRL), and 10 μg/mL bovine insulin (Clonetics Corp., San Diego, CA). MDA-MB-468 were cultured in the same media without L-glutamine and insulin as supplements.
Proliferation was monitored using a modification of the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide "MTT" bioassay (Mosmann, T.,J. Immunol . Methods 65:55-63, 1983; Denizot, F., and Lang, R., J. Immunol . Methods 89: 271-277, 1986; Hansen, M.B. et al., J. Immunol . Methods 119: 203-210, 1989). Cells were seeded in 96-well flat-bottom microtiter dishes in 100 μL of cell-specific "complete" media at low density [i.e., 3,000 (BT-474) or 2,000 (MDA-MB-468) cells per well]. After incubation for twenty-four hours at 37°C in a humidified air/5% CO2 chamber, 100 μL aliquots with samples of rHuNDF-α2 were added which was followed by incubation of the plates using the
conditions described above. rHuNDF-α2 dilutions were prepared from a stock solution (rHuNDF-α214-241, 370 μg/mL) in diluent (1:1 mix of DMEM (GIBCO):F-12 (GIBCO) with 100 μM non-essential amino acids (GIBCO) and 2 mM L-glutamine (GIBCO)). All dilutions were tested in quadruplicate. Just prior to analysis, sample media were replaced with 50 μL per well phenol red-free RPMI 1640 (GIBCO) containing 1 mg/mL MTT (Sigma). Plates were then re-incubated for an additional three hours. The formazan precipitate was solubilized by adding
150 μL per well pre-heated (65°C) lysis buffer (50% dimethyl formamide (Fluka), 20% sodium dodecyl sulfate (Sigma)). Total absorbance (i.e., the result of subtracting background absorbance at 690 nm from
absorbance at 560nm) was determined for each well using a Vmax microplate reader (Molecular Devices Corp., Menlo Park, CA). Data were analyzed using Excel V3.0
(Microsoft) and plotted with CA-Cricket Graph VI.3.2 (Computer Associates, San Diego, CA).
II. Results and Discussion
Differentiation of mammary epithelial cells invokes two sometimes independent processes: the production of lipids and milk proteins (i.e.,
"functional" differentiation), and cessation of cell growth (i.e., "terminal" differentiation) (Stampfer, M.R., and Yasmen, P., CRC Press, above). Both can be regulated by a myriad of factors including steroid hormones, growth factors, and matrix components (Topper, Y.J., and Freeman, C.S., Physiol Rev, above). Native rat 44 kDa NDF, an EGF-related protein whose receptor is a member of the EGF receptor family, induces both terminal and functional differentiated phenotypes in AU-565, a human breast carcinoma cell line (Peles, E. et al., Cell, 1992, above).
In these experiments, the effects of rHuNDF-α2 on the in vitro proliferation of two mammary cell lines, BT-474 and MDA-MB-468, were studied. Both lines were treated with various concentrations of rHuNDF-α2 for periods of five, six, and seven days prior to analysis using the modified MTT assay described above in
Materials and Methods (Section I of this Example). Bar graphs depicting absorbance measurements (i.e., optical density) are presented in Figures 46 and 47. Since changes in absorbance are linearly proportional to cell number within a predetermined range, these data indicate that rHuNDF-α2 stimulates BT-474 proliferation, yet retards the growth of MDA-MB-468 cells in serum-containing media. Responses of each cell line were clearly both dose- and time-dependent. Increases in BT-474 growth of nearly 40% occurred by day 7 at doses greater than 25 ng/ml. Likewise, the most pronounced effects on MDA-MB-468 cells (i.e., > 80% inhibition) were also on day 7 at doses greater than 25 ng/mL.
Thus, as was the case for EGF, it appears that the effects of rHuNDF-α2 on proliferation are pleiotropic; some cells are stimulated to grow while a terminally differentiated (nonproliferating) phenotype is induced in others.
EXAMPLE 18
In Vitro Effects of Recombinant NDF-α2 on Colon
Epithelial Cells
To test the effect of NDF on mucosal epithelial cells, E. coli-expressed rat NDF-α214-241 was used in a crypt colony formation assay. Results show that this isoform stimulated the attachment of colon crypts on plates coated with rat collagen type IV.
Mouse colon crypts were prepared as described by Whitehead et al. ( In Vitro Cellular & Developmental Biology, Vol. 23, Number 6, Pages 436-442, 1987). Mice were sacrificed with a lethal dose of CO2, and large intestines were isolated. The large intestine was cut longitudinally, rinsed with buffer A(1x PBS containing 0.3 mg/ml of L-Glutamine, 100 units/ml of penicillin, 100 units/ml of streptomycin), and sliced into 0.5 cm pieces. These pieces were washed again with buffer A in a 50 ml conical tube several times. Clean tissue was washed with extraction buffer (0.5mM DTT, 2mM EDTA in buffer A) and incubated with 10 ml of extraction buffer for one hour. The extraction buffer was then
aspirated, and tissue was washed with Solution A.
Crypts were harvested by shaking the tissue in 5 ml of Solution A. Crypts were transferred to a 15-ml tube.
The crypts were distributed in collagen type IV coated 6-well plates (Collaborative Biomedical, Cambridge, MA) at a density of five hundred crypts per well. The medium (RPMI 1640, 0.3 mg/ml L-Glutamine,
100 units/ml penicillin, 100 units/ml streptomycin) contained either 1% or 10% fetal bovine serum (FBS). After twenty-four hours of incubation at 37°C, colonies of attached cells were stained with Gram Crystal Violet and counted. Formation of colonies was FBS dependent with 3-5 fold more colonies in medium containing 10% FBS than in media containing 1% FBS. The increase of colony formation was not due to the general increase of protein in the medium, since the same effect was not observed when BSA was used to substitute FBS.
To evaluate the effect of known protein factors in the assay, IL-1, IL-2, IL-3, IL-6, IL-8, PDGF, SCF, EGF, TGF-α, bFGF, aFGF, KGF, G-CSF, and NDF were comparison tested at 10 ng/ml in medium containing 1% FBS. Among these factors, only NDF gave results similar to the stimulation obtained with 10% FBS in this assay. The assay was then repeated with various
concentrations of NDF. Although the result shown in Figure 48 is from one experiment, similar results were obtained in two other experiments: stimulation started at a concentration of 2 ng/ml, and maximum stimulationwas achieved at 100 ng/ml. These results are consistent with the hypothesis that NDF stimulates the attachment of colonic crypts through binding to its receptor, and imply that NDF may play a role in the regulation of colon epithelial cell growth and development.
EXAMPLE 19
Use of Recombinant (Rat) NDF in Treatment of Human Colon Cancer by Modulating CEA Expression in Carcinoma Cells
I. Background
Colorectal carcinoma is among the most common malignancies in Western societies. The carcinogenic target is the colonic epithelium, which is a highly
proliferative organ that undergoes constant growth renewal and differentiation. Tumors are thought to arise from undifferentiated progenitor cells which harbor mutations that disrupt normal growth control mechanisms. Tumors begin as a small benign growth, or adenoma, which later acquire more aggressive growth characteristics and eventually metastasize. Current therapies include surgical resection and stringent chemotherapy in a combined modality prior to metastasis. Unfortunately, tumors that are not cured by surgery respond poorly to follow-up chemotherapy.
The HER-2/neu proto-oncogene encodes a type I transmembrane protein with intrinsic tyrosine kinase activity, which is generally believed to be a growth factor receptor that transmits mitogenic signals.
HER-2/neu is expressed in a variety of tissues including those enriched in epithelial surfaces such as breast, lung and the intestine. The HER-2 gene has previously been shown to be amplified in breast cancer, suggesting its role in tumorigenesis of epithelial cell types
(Slamon, D. et al. Science 244 : 707-713, 1989).
Identification of a ligand for the HER-2/neu receptor, Neu-Differentiation Factor (NDF), has made it possible to analyze the biological effects of the receptor-ligand interactions in HER-2/neu expressing cells (Peles et al., Ceil, 1992, above). Preliminary evidence suggests that NDF induces maturation of breast carcinoma cell lines in vitro. This suggests utility of
recombinant rat NDF in inducing maturation of carcinoma cells which express functional HER-2 receptors. II. Observations
From previous work it is shown that the HER-2 receptor is expressed on the colorectal carcinoma cell line LIM 1215. To determine the effects of recombinant
rat NDF on colonocyte growth and differentiation NDF treated LIM 1215 cells were analyzed using antibodies to carcinoembryonic antigen (CEA). CEA is a highly glycosylated membrane protein that functions as an intercellular adhesion molecule (Benchimol, S. et al., Cell 57: 327-334 , 1989). CEA is expressed at elevated levels in fully differentiated cells of the large intestine, suggesting its normal function is restricted to mature cell types. Modulation of CEA expression in undifferentiated colon carcinoma cell lines would indicate the ability to induce differentiation in those cell types. LIM 1215 cells were untreated, or treated with NDF, then stained with antibodies to the
extracellular domain of CEA. Recombinant rat NDF induces expression of CEA in LIM 1215 cells. This strongly suggests that recombinant rat NDF modulates the differentiation state of HER-2 positive colorectal carcinoma cells in vitro, and presumably in vivo. III. Materials and methods
The LIM 1215 colorectal carcinoma cell line was provided by Robert Whitehead (Ludwig Institute for Cancer Research, Melbourne) and maintained as adherent monolayer cultures in RPMI 1640 growth media containing 5% FBS, 1 μg/ml insulin, 10 μg/ml hydrocortisone and
10 μM α-thioglycerol as previously described (Whitehead, R.H. et al., J. Natl . Cancer Inst . 74:759-765, 1985). Exponentially growing cells were cultured in the presence or absence of 50 ng/ml recombinant rat NDF (CHO-derived NDF-α2,) for seventy-two hours in growth media containing 2% FBS. The cell cultures were washed with PBS, removed from the dish by scraping with a rubber policeman, then pelleted by centrifugation. The cell pellet was surrounded by O.C.T. embedding compound (#4583, Miles, Inc.) and flash-frozen in liquid
nitrogen. The embedded frozen pellets were sectioned using a cryostat fitted with a glass knife, and the frozen sections placed on glass slides. The sections were fixed in absolute acetone at -20°C for six minutes, then air dried. The fixed sections were rehydrated in PBS containing 0.3 μg/ml BSA (antibody diluent) then incubated with 1 μg/ml mouse monoclonal antibody to human CEA (MAB 425, Chemicon International, Inc.) in antibody diluent for one hour at 23°C. The slides were washed three times with antibody diluent, then incubated with 1 μg/ml FITC conjugated sheep anti-mouse IgG antibody (Amersham, Inc.) for approximately thirty minutes at room temperature. The slides were washed, and the area containing the stained section was covered with PBS solution containing 4% n-propyl galate (Sigma Chemical Co., # P-3130) and 90% glycerol to minimize fluorochrome bleaching. The slides were analyzed under a fluorescent microscope at UV 320 nm and photographed using Kodak Gold ASA 100 color film (Figure 49).
IV. Results and Discussion
In untreated LIM 1215 cells, basal level expression of CEA is detected in less than 5-10% of the cells. Treatment of these same cells with recombinant rat NDF induced a high level of CEA expression in 80-90% of the cells analyzed. The induced CEA molecule appears to be localized to the cell surface, suggesting that NDF treatment results in a functional increase in CEA activity.
CEA is detected at significant levels only at the apical portion of the colonic crypt (Benchimol, S. et al, Cell, 1989, above). Intestinal epithelial cells are known to differentiate as they migrate from the basal to apical crypt compartments (Cheng, H., and
Leblond, C . P . , Am . J. Anat . 141 : 537-562 , 1974 ) . The in
vivo pattern of CEA expression strongly suggests that in normal colon it is present only in fully differentiated or mature epithelium. Since colon carcinoma cells are generally believed to arise from undifferentiated basal crypt progenitor or stem cells, the resulting cell lines isolated from tumors are likely to be clonal,
transformed crypt progenitor cells. Modulation of surface CEA protein by exogenous recombinant rat NDF indicates that NDF is capable of regulating cell growth and differentiation control in HER-2 expressing colon carcinoma in vitro . NDF may therefore modulate CEA expression in normal and transformed colonic epithelial cells in vivo . This may prove to be useful in treatment of colon carcinoma by regulating carcinoma cell-cell and cell-matrix interactions by decreasing the mortality and morbidity associated with metastatic colon carcinoma.
EXAMPLE 20
Use of Recombinant Rat NDF in Treatment of Metastatic Colon Carcinoma by Modulating Expression of TIMP-2
I . Background
Tumor metastasis is a complex, multi-step process that begins when individual tumor cells leave the primary site of transformation by degrading their resident extracellular matrix. Matrix degradation is mediated by several classes of proteolytic enzymes, including members of the Zn+-dependent metalloproteinases, such as type IV collagenase and stromelysin (transin) (Liotta, L.A., et al., Cancer Research
51:5054s-5059s, 1991). The type IV collagenase and stromelysin genes are known to be induced following initiation of the growth response, and to be
constitutively secreted by proliferating cancer cells (Angel, P. et al, Mol . Cell . Biol . 7:2256-2266, 1987).
Since deregulation of matrix protease expression can enhance metastatic potential, an obvious anti-cancer therapy would be to block protease activity secreted by cancer cells. Naturally occurring inhibitors of metalloproteinases exist which regulate protease activities with tissues and body fluids. These include a family of proteins known as tissue inhibitors of metalloproteinase, or TIMPs. A novel member of this family has recently been isolated, known as TIMP-2
(Boone, T.C. et al, Proc. Natl . Acad. Sci . 87:2800-2804, 1990), and has been shown to inhibit both invasion and metastasis of tumor cells in an animal model system.
II. Observations
Previously, it has been shown that the HER-2 receptor is expressed by the metastatic ileoceacal carcinoma cell line LIM 1863. LIM 1863 cells were treated with recombinant rat NDF to determine its effects on TIMP-2 protein expression and found that NDF treatment dramatically increased TIMP-2 expression and secretion. Since NDF is believed to play a role in differentiation of epithelial cell types, NDF-induced expression of TIMP-2 may have a role in inducing
differentiation of LIM 1863 cells. Because TIMP-2 can inhibit metalloproteinases involved in tumor metastasis (DeClerck, Y.A. et al, Cancer Research 52:701-703, 1991), NDF treatment may alter the metastatic properties of LIM 1863 cell in vitro, and of other human colon cancers in vivo.
III. Materials and Methods
The LIM 1863 ileoceacal carcinoma cell line (Whitehead, R.H. et al, Cancer Research 47: 2704-2713 , 1987) was provided by Robert Whitehead (Ludwig Institute for Cancer Research, Melbourne) and maintained in
suspension cultures in RPMI 1640 growth media containing 5% FBS, 1 μg/ml insulin, 10 μg/ml hydrocortisone and 10 μM α-thioglycerol. Confluent cultures are
subcultured 1:5, then incubated for seventy-two hours. The culture was then harvested and the cells pelleted at 1 X g for five minutes. The supernatant was discarded, and the cell pellet was washed with ~50 ml of serum-free media (RPMI 1640 containing 0.03% BSA, 1 μg/ml insulin, 10 μg/ml hydrocortisone and 10 μM α-thioglycerol). The cells were re-pelleted, then resuspended in 500 ml of serum-free assay buffer. The cells were incubated for forty-eight hours in a 500 ml spinner culture flask at 50 rpm. The medium was removed at this time and replaced with 500 ml of fresh serum-free media, then allowed to incubate overnight. The cells were then harvested, counted, diluted to a concentration of about six hundred organoids per ml, then cultured in the presence or absence of 50 ng/ml of recombinant rat NDF (CHO-derived NDF-α2), for one hundred and twenty hours. After treatment, the cells were harvested and collected by centrifugation. The cell pellet was surrounded by optimum cooling temperature (OCT) embedding compound (#4583, Miles, Inc.) and flash-frozen in liquid
nitrogen. The embedded frozen pellets were sectioned using a cryostat fitted with a glass knife, and the frozen sections were placed on glass slides. The sections were fixed in absolute acetone at -20°C for six minutes, then air dried. The fixed sections were rehydrated in PBS containing 0.3 mg/ml BSA (antibody diluent), then incubated with a 1:300 dilution of rabbit polyclonal anti-TIMP-2 in antibody diluent for one hour at room temperature. The TIMP-2 antiserum, provided by Helen Hockmen (Amgen, Inc.), was raised against
bacterially expressed TIMP-2 protein (Boone, T.C.
et al., Proc. Natl . Acad. Sci . , 1990, above) . The
slides were washed three times with antibody diluent, then incubated with ~1 μg/ml phycoerythrin conjugated goat anti-rabbit IgG antibody (Cappel, Inc.) for
approximately thirty minutes at 23°C. The slides were washed, and the area containing the stained section was covered with PBS solution containing 4% n-propyl galate and 90% glycerol to minimize fluorochrome bleaching. The slides were analyzed under a fluorescent microscope at UV 320 nm and photographed using Kodak Gold ASA 100 color film.
IV. Results and Discussion
The LIM 1863 cell line spontaneously forms organoids in suspension consisting of a polarized layer of epithelial cells surrounding a central lumen
(Whitehead, R.H. et al., Cancer Research, 1987, above) In untreated LIM 1863 cells, basal levels of TIMP-2 expression is detected as both cell associated
(cytoplasmic) protein, and as a secreted protein
contained within the central lumen (Figure 50, panel A). In treated cells, there is a dramatic increase in the level of TIMP-2 protein, suggesting that NDF induces expression of the TIMP-2 gene (Figure 50, panel B). The induced TIMP-2 protein accumulated in the cytoplasmic and luminal compartments. In addition, there were significant amounts of TIMP-2 protein located at the basolateral edges of the organoid clumps and arranged in fiber-like structures (Figure 30, panel B). This result suggests that some of the TIMP-2 protein produced by LIM 1863 cells in response to NDF treatment is secreted and is therefore capable of interacting with tissue
metalloproteinases and modulating their activity.
Regulation of TIMP-2 expression in cells expressing HER-2 receptors implies that NDF treatment would result in the downstream modulation of
metalloproteinase activity in various compartments of the body. This may be useful in regulating tissue remodeling and repair in injured organs. More
significantly, regulating the expression of TIMP-2 in HER-2 receptor positive colon cancer cells by NDF treatment may inhibit their ability to metastasize and subsequently invade neighboring tissues. This effect may be difficult to mimic by the addition of even large amounts of recombinant TIMP-2 protein and may decrease the mortality and morbidity associated with metastatic colon carcinoma.
EXAMPLE 21
NDF-Stimulated Proliferation of Epithelium in an In Vivo
Wound Healing Model
In this Example, a modified rabbit ear partial thickness dermal wound model was used. The rabbit ear dermal ulcer model described by Mustoe, T., et al.
(J. Clin . Invest . , 87: 694-703, 1991) was modified to produce a wound through the cartilage, to the dermis on the back side of the ear, using a 6mm trephine. As a result, the wound heals by sprouting of epithelial elements beneath the cartilage and reepithelialization from wound borders. Contraction is not a variable during healing, permitting accurate quantitation of new tissues. Recombinant human met-NDF-α214-241 from E. coli was utilized as the therapeutic wound healing agent. A. Materials and Methods
Recombinant NDF-α2 or phosphate buffered saline vehicle alone was applied once on the day of surgery and the wounds (0.25 cm2) were covered with Tegaderm occlusive dressing (3M Company, St. Paul, MN). Prior to sacrifice five days later, each animal received
intravenous injections of BrdU (Aldrich Chemical
Company, Milwaukee, WI) in an amount of 50 mg per kg of body weight. Administration of BrdU was employed to better quantify the degree of basal keratinocyte
proliferation and the kinetics of basal cell migration toward the stratum spinosum and stratum corneum. After sacrifice, each wound was bisected, with one section being frozen in an OCT medium (Miles Inc., Elkhart, IN), and the second section being fixed in Omnifix (An-Con, Genetics, Inc., Melville, NY) and processed according to routine histological methods. Masson Trichrome, Oil Red 0, and immunohistochemical (IHC) stains were
performed on 3 μm-thick sections for each wound. B. Measurement of Reepithelialization
Measurements of the total wound gap and epithelial gap were made for each bisected wound using a calibrated stage micrometer. The amount of
reepithelialization for each wound was calculated by taking the difference of the total gap and the
epithelial gap. Data from two sections per wound were averaged. Differences in the amount of
reepthelialization were analyzed by a one-tailed, unpaired Student's t-test.
The area of epithelium produced in treated and untreated wounds was calculated from cross-sectional bisection for each dose group. A one-way ANOVA and Dunnett's t-test was run for each dose against the control group.
C. Measurement of Proliferation and Differentiation of Epithelial Cells
Paraffin-embedded 3 μm sections of tissue were stained using anti-BrdU (Dako Corp., Carpinteria, CA), Avidin-Biotin Complex (Vector Laboratories, Inc.,
Burlingame, CA) , and diaminobenzidine substrate (DAB; Sigma Chemical Co., St. Louis, MO). Sections were digested with 0.1% protease solution, followed by treatment with 2N HCl. Endogenous peroxidase was quenched by exposure to 3% hydrogen peroxide solution. Slides were blocked with a 10% solution of normal horse serum in phosphate buffered saline (PBS), then incubated with anti-BrdU diluted 1:400 in 1% bovine serum albumin. Following washing, sections were incubated with a secondary antibody, and subsequently incubated for twenty minutes with peroxidase-linked Avidin-Biotin Complex diluted 1:100 in 1% BSA. Slides were then exposed to DAB substrate (10 mg of DAB, 20 ml of PBS, 20 μl of 30% hydrogen peroxide) for ten minutes.
Sections were counterstained with hematoxylin.
D. Results and Discussion
An increase in new epithelium covering the wounds was observed for each concentration of recombinant NDF-α2 tested, in comparison with the control, indicating NDF-accelerated epithelial coverage of the wounds
(Figure 51). Increased epithelial area was also observed for all concentrations of recombinant NDF-α2 tested, indicating that NDF increased epithelial layer thickness and enhanced the integrity of epithelial coverage of the wounds (Figure 52). At doses of 5 μg per wound,
treatment with NDF-α2 resulted in an increase in the number and percentage of proliferating (BrdU positive) basal and suprabasal keratinocytes (Figure 53). These results indicate that NDF can directly stimulate basal and suprabasal keratinocytes to increase epithelial area and coverage of the treated wounds. The results also show that suprabasal cells, which are partially
differentiated in nature, also respond to NDF, suggesting
a role for NDF in the acceleration of differentiation in such cells.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANTS: Amgen Inc.
(ii) TITLE OF INVENTION: RECOMBINANT NEU DIFFERENTIATION FACTORS
(iii) NUMBER OF SEQUENCES: 127
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Amgen Inc.
(B) STREET: Amgen Center
1840 Dehavilland Drive
(C) CITY: Thousand Oaks
(D) STATE: California
(E) COUNTRY: USA
(F) ZIP: 91320-1789
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy Disk
(B) COMPUTER: IBM PC Compatible
(C) OPERATING SYSTEM: MS-DOS
(D) SOFTWARE: Macintosh Microsoft Word Version 5.1a/Text only
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 08/066,384
(B) FILING DATE: 21-MAY-1993
(C) CLASSIFICATION: 435
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 07/877,431
(B) FILING DATE: 29-APR-1992
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu Cys
1 5 10 15
Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys Lys
20 25 30
Cys Gln Pro Gly Phe Thr Gly Ala Arg Cys
35 40
(3) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu Cys
1 5 10 15
Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys Lys
20 25 30
Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys
35 40
(4) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1063 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double stranded
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
G AATTCCGGGG GAG TGC TTC ATG GTG AAA GAC CTT TCA AAC CCC 44
Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro 1 5 10
TCG AGA TAC TTG TGC AAG TGC CAA CCT GGA TTC ACT GGA GCA AGA 89 Ser Arg Tyr Leu Cys Lys Cys Gln Pro Gly Phe Thr Gly Ala Arg
15 20 25
TGT ACT GAG AAT GTG CCC ATG AAA GTC CAA AAC CAA GAA AAG CAT 134 Cys Thr Glu Asn Val Pro Met Lys Val Gln Asn Gln Glu Lys His
30 35 40
CTT GGG ATT GAA TTT ATT GAG GCG GAG GAG CTG TAC CAG AAG AGA 179 Leu Gly Ile Glu Phe Ile Glu Ala Glu Glu Leu Tyr Gln Lys Arg
45 50 55
GTG CTG ACC ATA ACC GGC ATC TGC ATC GCC CTC CTT GTG GTC GGC 224 Val Leu Thr Ile Thr Gly Ile Cys Ile Ala Leu Leu Val Val Gly
60 65 70
ATC ATG TGT GTG GTG GCC TAC TGC AAA ACC AAG AAA CAG CGG AAA 269 Ile Met Cys Val Val Ala Tyr Cys Lys Thr Lys Lys Gln Arg Lys
75 80 85
AAG CTG CAT GAC CGT CTT CGG CAG AGC CTT CGG TCT GAA CGA AAC 314 Lys Leu His Asp Arg Leu Arg Gln Ser Leu Arg Ser Glu Arg Asn
90 95 100
AAT ATG ATG AAC ATT GCC AAT GGG CCT CAC CAT CCT AAC CCA CCC 359 Asn Met Met Asn Ile Ala Asn Gly Pro His His Pro Asn Pro Pro
105 110 115
CCC GAG AAT GTC CAG CTG GTG AAT CAA TAC GTA TCT AAA AAC GTC 404 Pro Glu Asn Val Gln Leu Val Asn Gln Tyr Val Ser Lys Asn Val
120 125 130
ATC TCC AGT GAG CAT ATT GTT GAG AGA GAA GCA GAG ACA TCC TTT 449 Ile Ser Ser Glu His Ile Val Glu Arg Glu Ala Glu Thr Ser Phe
135 140 145
TCC ACC AGT CAC TAT ACT TCC ACA GCC CAT CAC TCC ACT ACT GTC 494 Ser Thr Ser His Tyr Thr Ser Thr Ala His His Ser Thr Thr Val
150 155 160
ACC CAG ACT CCT AGC CAC AGC TGG AGC AAC GGA CAC ACT GAA AGC 539 Thr Gln Thr Pro Ser His Ser Trp Ser Asn Gly His Thr Glu Ser
165 170 175
ATC CTT TCC GAA AGC CAC TCT GTA ATC GTG ATG TCA TCC GTA GAA 584 Ile Leu Ser Glu Ser His Ser Val Ile Val Met Ser Ser Val Glu
180 185 190
AAC AGT AGG CAC AGC AGC CCA ACT GGG GGC CCA AGA GGA CGT CTT 629 Asn Ser Arg His Ser Ser Pro Thr Gly Gly Pro Arg Gly Arg Leu
195 200 205
AAT GGC ACA GGA GGC CCT CGT GAA TGT AAC AGC TTC CTC AGG CAT 674 Asn Gly Thr Gly Gly Pro Arg Glu Cys Asn Ser Phe Leu Arg His
210 215 220
GCC AGA GAA ACC CCT GAT TCC TAC CGA GAC TCT CCT CAT AGT GAA 719 Ala Arg Glu Thr Pro Asp Ser Tyr Arg Asp Ser Pro His Ser Glu
225 230 235
AGG TAT GTG TCA GCC ATG ACC ACC CCG GCT CGT ATG TCA CCT GTA 764 Arg Tyr Val Ser Ala Met Thr Thr Pro Ala Arg Met Ser Pro Val
240 245 250
GAT TTC CAC ACG CCA AGC TCC CCC AAA TCG CCC CCT TCG GAA ATG 809 Asp Phe His Thr Pro Ser Ser Pro Lys Ser Pro Pro Ser Glu Met
255 260 265
TCT CCA CCC GTG TCC AGC ATG ACG GTG TCC ATG CCT TCC ATG GCG 854 Ser Pro Pro Val Ser Ser Met Thr Val Ser Met Pro Ser Met Ala
270 275 280
GTC AGC CCC TTC ATG GAA GAA GAG AGA CCT CTA CTT CTC GTG ACA 899 Val Ser Pro Phe Met Glu Glu Glu Arg Pro Leu Leu Leu Val Thr
285 290 295
CCA CCA AGG CTG CGG GAG AAG AAG TTT GAC CAT CAC CCT CAG CAG 944 Pro Pro Arg Leu Arg Glu Lys Lys Phe Asp His His Pro Gln Gln
300 305 310
TTC AGC TCC TTC CAC CAC AAC CCC GCG CAT GAC AGT AAC AGC CTC 989 Phe Ser Ser Phe His His Asn Pro Ala His Asp Ser Asn Ser Leu
315 320 325
CCT GCT AGC CCC TTG AGG ATA GTG GAG GAT GAG GAG TAT GAA ACG 1034 Pro Ala Ser Pro Leu Arg Ile Val Glu Asp Glu Glu Tyr Glu Thr
330 335 340
ACC CAA GAG TAC GAG CCA GCC CGGAATTC 1063
Thr Gln Glu Tyr Glu Pro Ala
345
(5) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 348 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro 1 5 10
Ser Arg Tyr Leu Cys Lys Cys Gln Pro Gly Phe Thr Gly Ala Arg
15 20 25
Cys Thr Glu Asn Val Pro Met Lys Val Gln Asn Gln Glu Lys His
30 35 40
Leu Gly Ile Glu Phe Ile Glu Ala Glu Glu Leu Tyr Gln Lys Arg
45 50 55
Val Leu Thr Ile Thr Gly Ile Cys Ile Ala Leu Leu Val Val Gly
Ile Met Cys Val Val Ala Tyr Cys Lys Thr Lys Lys Gln Arg Lys 75 80 85
Lys Leu His Asp Arg Leu Arg Gln Ser Leu Arg Ser Glu Arg Asn
90 95 100
Asn Met Met Asn Ile Ala Asn Gly Pro His His Pro Asn Pro Pro
105 110 115
Pro Glu Asn Val Gln Leu Val Asn Gln Tyr Val Ser Lys Asn Val
120 125 130 Ile Ser Ser Glu His Ile Val Glu Arg Glu Ala Glu Thr Ser Phe
135 140 145
Ser Thr Ser His Tyr Thr Ser Thr Ala His His Ser Thr Thr Val
150 155 160
Thr Gln Thr Pro Ser His Ser Trp Ser Asn Gly His Thr Glu Ser
165 170 175 Ile Leu Ser Glu Ser His Ser Val Ile Val Met Ser Ser Val Glu
180 185 190
Asn Ser Arg His Ser Ser Pro Thr Gly Gly Pro Arg Gly Arg Leu
195 200 205
Asn Gly Thr Gly Gly Pro Arg Glu Cys Asn Ser Phe Leu Arg His
210 215 220
Ala Arg Glu Thr Pro Asp Ser Tyr Arg Asp Ser Pro His Ser Glu
225 230 235
Arg Tyr Val Ser Ala Met Thr Thr Pro Ala Arg Met Ser Pro Val
240 245 250
Asp Phe His Thr Pro Ser Ser Pro Lys Ser Pro Pro Ser Glu Met
255 260 265
Ser Pro Pro Val Ser Ser Met Thr Val Ser Met Pro Ser Met Ala
270 275 280
Val Ser Pro Phe Met Glu Glu Glu Arg Pro Leu Leu Leu Val Thr
285 290 295
Pro Pro Arg Leu Arg Glu Lys Lys Phe Asp His His Pro Gln Gln
300 305 310
Phe Ser Ser Phe His His Asn Pro Ala His Asp Ser Asn Ser Leu
315 320 325
Pro Ala Ser Pro Leu Arg Ile Val Glu Asp Glu Glu Tyr Glu Thr
330 335 340
Thr Gln Glu Tyr Glu Pro Ala
345
(6) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2335 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double stranded
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
GCGGCCGCTC GCTCTCCCCA TCGAGGGACA AACTTTTCCC AAACCCGATC 50
CGAGCCCTTG GACCAAACTC GCCTGCGCCG AGAGCCGTCC GCGTAGAGCG 100
CTCCGTCTCC GGCGAG ATG TCC GAG CGC AAA GAA GGC AGA GGC AAA 146
Met Ser Glu Arg Lys Glu Gly Arg Gly Lys
1 5 10
GGG AAG GGC AAG AAG AAG GAG CGA GGC TCC GGC AAG AAG CCG GAG 191 Gly Lys Gly Lys Lys Lys Glu Arg Gly Ser Gly Lys Lys Pro Glu
15 20 25
TCC GCG GCG GGC AGC CAG AGC CCA GCC TTG CCT CCC CGA TTG AAA 236 Ser Ala Ala Gly Ser Gln Ser Pro Ala Leu Pro Pro Arg Leu Lys
30 35 40
GAG ATG AAA AGC CAG GAA TCG GCT GCA GGT TCC AAA CTA GTC CTT 281 Glu Met Lys Ser Gln Glu Ser Ala Ala Gly Ser Lys Leu Val Leu
45 50 55
CGG TGT GAA ACC AGT TCT GAA TAC TCC TCT CTC AGA TTC AAG TGG 326 Arg Cys Glu Thr Ser Ser Glu Tyr Ser Ser Leu Arg Phe Lys Trp
60 65 70
TTC AAG AAT GGG AAT GAA TTG AAT CGA AAA AAC AAA CCA CAA AAT 371 Phe Lys Asn Gly Asn Glu Leu Asn Arg Lys Asn Lys Pro Gln Asn
75 80 85
ATC AAG ATA CAA AAA AAG CCA GGG AAG TCA GAA CTT CGC ATT AAC 416 Ile Lys Ile Gln Lys Lys Pro Gly Lys Ser Glu Leu Arg Ile Asn
90 95 100
AAA GCA TCA CTG GCT GAT TCT GGA GAG TAT ATG TGC AAA GTG ATC 461 Lys Ala Ser Leu Ala Asp Ser Gly Glu Tyr Met Cys Lys Val Ile
105 110 115
AGC AAA TTA GGA AAT GAC AGT GCC TCT GCC AAT ATC ACC ATC GTG 506 Ser Lys Leu Gly Asn Asp Ser Ala Ser Ala Asn Ile Thr Ile Val
120 125 130
GAA TCA AAC GAG ATC ATC ACT GGT ATG CCA GCC TCA ACT GAA GGA 551 Glu Ser Asn Glu Ile Ile Thr Gly Met Pro Ala Ser Thr Glu Gly
135 140 145
GCA TAT GTG TCT TCA GAG TCT CCC ATT AGA ATA TCA GTA TCC ACA 596 Ala Tyr Val Ser Ser Glu Ser Pro Ile Arg Ile Ser Val Ser Thr
150 155 160
GAA GGA GCA AAT ACT TCT TCA TCT ACA TCT ACA TCC ACC ACT GGG 641 Glu Gly Ala Asn Thr Ser Ser Ser Thr Ser Thr Ser Thr Thr Gly
165 170 175
ACA AGC CAT CTT GTA AAA TGT GCG GAG AAG GAG AAA ACT TTC TGT 686 Thr Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys
180 185 190
GTG AAT GGA GGG GAG TGC TTC ATG GTG AAA GAC CTT TCA AAC CCC 731 Val Asn Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro
195 200 205
TCG AGA TAC TTG TGC AAG TGC CAA CCT GGA TTC ACT GGA GCA AGA 776 Ser Arg Tyr Leu Cys Lys Cys Gln Pro Gly Phe Thr Gly Ala Arg
210 215 220
TGT ACT GAG AAT GTG CCC ATG AAA GTC CAA AAC CAA GAA AAG GCG 821 Cys Thr Glu Asn Val Pro Met Lys Val Gln Asn Gln Glu Lys Ala
225 230 235
GAG GAG CTG TAC CAG AAG AGA GTG CTG ACC ATA ACC GGC ATC TGC 866 Glu Glu Leu Tyr Gln Lys Arg Val Leu Thr Ile Thr Gly Ile Cys
240 245 250
ATC GCC CTC CTT GTG GTC GGC ATC ATG TGT GTG GTG GCC TAC TGC 911 Ile Ala Leu Leu Val Val Gly Ile Met Cys Val Val Ala Tyr Cys
255 260 265
AAA ACC AAG AAA CAG CGG AAA AAG CTG CAT GAC CGT CTT CGG CAG 956 Lys Thr Lys Lys Gln Arg Lys Lys Leu His Asp Arg Leu Arg Gln
270 275 280
AGC CTT CGG TCT GAA CGA AAC AAT ATG ATG AAC ATT GCC AAT GGG 1001 Ser Leu Arg Ser Glu Arg Asn Asn Met Met Asn Ile Ala Asn Gly
285 290 295
CCT CAC CAT CCT AAC CCA CCC CCC GAG AAT GTC CAG CTG GTG AAT 1046 Pro His His Pro Asn Pro Pro Pro Glu Asn Val Gln Leu Val Asn
300 305 310
CAA TAC GTA TCT AAA AAC GTC ATC TCC AGT GAG CAT ATT GTT GAG 1091 Gln Tyr Val Ser Lys Asn Val Ile Ser Ser Glu His Ile Val Glu
315 320 325
AGA GAA GCA GAG ACA TCC TTT TCC ACC AGT CAC TAT ACT TCC ACA 1136 Arg Glu Ala Glu Thr Ser Phe Ser Thr Ser His Tyr Thr Ser Thr
330 335 340
GCC CAT CAC TCC ACT ACT GTC ACC CAG ACT CCT AGC CAC AGC TGG 1181 Ala His His Ser Thr Thr Val Thr Gln Thr Pro Ser His Ser Trp
345 350 355
AGC AAC GGA CAC ACT GAA AGC ATC CTT TCC GAA AGC CAC TCT GTA 1226 Ser Asn Gly His Thr Glu Ser Ile Leu Ser Glu Ser His Ser Val
360 365 370
ATC GTG ATG TCA TCC GTA GAA AAC AGT AGG CAC AGC AGC CCA ACT 1271 Ile Val Met Ser Ser Val Glu Asn Ser Arg His Ser Ser Pro Thr
375 380 385
GGG GGC CCA AGA GGA CGT CTT AAT GGC ACA GGA GGC CCT CGT GAA 1316 Gly Gly Pro Arg Gly Arg Leu Asn Gly Thr Gly Gly Pro Arg Glu
390 395 400
TGT AAC AGC TTC CTC AGG CAT GCC AGA GAA ACC CCT GAT TCC TAC 1361 Cys Asn Ser Phe Leu Arg His Ala Arg Glu Thr Pro Asp Ser Tyr
405 410 415
CGA GAC TCT CCT CAT AGT GAA AGA CAT AAC CTT ATA GCT GAG CTA 1406 Arg Asp Ser Pro His Ser Glu Arg His Asn Leu Ile Ala Glu Leu
420 425 430
AGG AGA AAC AAG GCA CAC AGA TCC AAA TGC ATG CAG ATC CAG CTA 1451 Arg Arg Asn Lys Ala His Arg Ser Lys Cys Met Gln Ile Gln Leu
435 440 445
TCA GCA ACT CAT CTT AGA TCT TCT TCC ATT CCC CAT TTG GGC TTC 1496 Ser Ala Thr His Leu Arg Ser Ser Ser Ile Pro His Leu Gly Phe
450 455 460
ATT CTC TAA GACCCCTTGG CCTTTAGGAA GGTATGTGTC AGCCATGACC 1545 Ile Leu
ACCCCGGCTC GTATGTCACC TGTAGATTTC CACACGCCAA GCTCCCCCAA 1595
ATCGCCCCCT TCGGAAATGT CTCCACCCGT GTCCAGCATG ACGGTGTCCA 1645
TGCCTTCCAT GGCGGTCAGC CCCTTCATGG AAGAAGAGAG ACCTCTACTT 1695
CTCGTGACAC CACCAAGGCT GCGGGAGAAG AAGTTTGACC ATCACCCTCA 1745
GCAGTTCAGC TCCTTCCACC ACAACCCCGC GCATGACAGT AACAGCCTCC 1795
CTGCTAGCCC CTTGAGGATA GTGGAGGATG AGGAGTATGA AACGACCCAA 1845
GAGCACGAGC CAGCCCAAGA GCCTGTTAAG AAACTCGCCA ATAGCCGGCG 1895 GGCCAAAAGA ACCAAGCCCA ATGGCCACAT TGCTAACAGA TTGGAAGTGG 1945
ACAGCAACAC AAGCTCCCAG AGCAGTAACT CAGAGAGTGA AACAGAAGAT 1995
GAAAGAGTAG GTGAAGATAC GCCTTTCCTG GGCATACAGA ACCCCCTGGC 2045
AGCCAGTCTT GAGGCAACAC CTGCCTTCCG CCTGGCTGAC AGCAGGACTA 2095
ACCCAGCAGG CCGCTTCTCG ACACAGGAAG AAATCCAGGC CAGGCTGTCT 2145
AGTGTAATTG CTAACCAAGA CCCTATTGCT GTATAAAACC TAAATAAACA 2195
CATAGATTCA CCTGTAAAAC TTTATTTTAT ATAATAAAGT ATTCCACCTT 2245
AAATTAAACA ATTTATTTTA TTTTAGCAGT TCTGCAAAAA AAAAAAAAAA 2295
AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AGGGCGGCCG 2335
(7) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 462 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
Met Ser Glu Arg Lys Glu Gly Arg Gly Lys
1 5 10
Gly Lys Gly Lys Lys Lys Glu Arg Gly Ser Gly Lys Lys Pro Glu
15 20 25
Ser Ala Ala Gly Ser Gln Ser Pro Ala Leu Pro Pro Arg Leu Lys
30 35 40
Glu Met Lys Ser Gln Glu Ser Ala Ala Gly Ser Lys Leu Val Leu
45 50 55
Arg Cys Glu Thr Ser Ser Glu Tyr Ser Ser Leu Arg Phe Lys Trp
60 65 70
Phe Lys Asn Gly Asn Glu Leu Asn Arg Lys Asn Lys Pro Gln Asn
75 80 85
Ile Lys Ile Gln Lys Lys Pro Gly Lys Ser Glu Leu Arg Ile Asn 90 95 100
Lys Ala Ser Leu Ala Asp Ser Gly Glu Tyr Met Cys Lys Val Ile
105 110 115
Ser Lys Leu Gly Asn Asp Ser Ala Ser Ala Asn Ile Thr Ile Val
120 125 130
Glu Ser Asn Glu Ile Ile Thr Gly Met Pro Ala Ser Thr Glu Gly
135 140 145
Ala Tyr Val Ser Ser Glu Ser Pro Ile Arg Ile Ser Val Ser Thr
150 155 160
Glu Gly Ala Asn Thr Ser Ser Ser Thr Ser Thr Ser Thr Thr Gly
165 170 175
Thr Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys
180 185 190
Val Asn Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro
195 200 205
Ser Arg Tyr Leu Cys Lys Cys Gln Pro Gly Phe Thr Gly Ala Arg
210 215 220
Cys Thr Glu Asn Val Pro Met Lys Val Gln Asn Gln Glu Lys Ala
225 230 235
Glu Glu Leu Tyr Gln Lys Arg Val Leu Thr Ile Thr Gly Ile Cys
240 245 250 Ile Ala Leu Leu Val Val Gly Ile Met Cys Val Val Ala Tyr Cys
255 260 265
Lys Thr Lys Lys Gln Arg Lys Lys Leu His Asp Arg Leu Arg Gln
270 275 280
Ser Leu Arg Ser Glu Arg Asn Asn Met Met Asn Ile Ala Asn Gly
285 290 295
Pro His His Pro Asn Pro Pro Pro Glu Asn Val Gln Leu Val Asn
300 305 310 Gln Tyr Val Ser Lys Asn Val Ile Ser Ser Glu His Ile Val Glu
315 320 325
Arg Glu Ala Glu Thr Ser Phe Ser Thr Ser His Tyr Thr Ser Thr
330 335 340
Ala His His Ser Thr Thr Val Thr Gln Thr Pro Ser His Ser Trp
345 350 355
Ser Asn Gly His Thr Glu Ser Ile Leu Ser Glu Ser His Ser Val
360 365 370
Ile Val Met Ser Ser Val Glu Asn Ser Arg His Ser Ser Pro Thr
375 380 385
Gly Gly Pro Arg Gly Arg Leu Asn Gly Thr Gly Gly Pro Arg Glu
390 395 400
Cys Asn Ser Phe Leu Arg His Ala Arg Glu Thr Pro Asp Ser Tyr
405 410 415
Arg Asp Ser Pro His Ser Glu Arg His Asn Leu Ile Ala Glu Leu
420 425 430
Arg Arg Asn Lys Ala His Arg Ser Lys Cys Met Gln Ile Gln Leu
435 440 445
Ser Ala Thr His Leu Arg Ser Ser Ser Ile Pro His Leu Gly Phe
450 455 460
Ile Leu
(8) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1807 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double stranded
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
GCGGCCGCTC GCTCTCCCCA TCGAGGGACA AACTTTTCCC AAACCCGATC 50
CGAGCCCTTG GACCAAACTC GCCTGCGCCG AGAGCCGTCC GCGTAGAGCG 100
CTCCGTCTCC GGCGAG 116
ATG TCC GAG CGC AAA GAA GGC AGA GGC AAA GGG AAG GGC AAG AAG 161 Met Ser Glu Arg Lys Glu Gly Arg Gly Lys Gly Lys Gly Lys Lys
1 5 10 15
AAG GAG CGA GGC TCC GGC AAG AAG CCG GAG TCC GCG GCG GGC AGC 206 Lys Glu Arg Gly Ser Gly Lys Lys Pro Glu Ser Ala Ala Gly Ser
20 25 30
CAG AGC CCA GCC TTG CCT CCC CGA TTG AAA GAG ATG AAA AGC CAG 251 Gln Ser Pro Ala Leu Pro Pro Arg Leu Lys Glu Met Lys Ser Gln
35 40 45
GAA TCG GCT GCA GGT TCC AAA CTA GTC CTT CGG TGT GAA ACC AGT 296 Glu Ser Ala Ala Gly Ser Lys Leu Val Leu Arg Cys Glu Thr Ser
50 55 60
TCT GAA TAC TCC TCT CTC AGA TTC AAG TGG TTC AAG AAT GGG AAT 341 Ser Glu Tyr Ser Ser Leu Arg Phe Lys Trp Phe Lys Asn Gly Asn
65 70 75
GAA TTG AAT CGA AAA AAC AAA CCA CAA AAT ATC AAG ATA CAA AAA 386 Glu Leu Asn Arg Lys Asn Lys Pro Gln Asn Ile Lys Ile Gln Lys
80 85 90
AAG CCA GGG AAG TCA GAA CTT CGC ATT AAC AAA GCA TCA CTG GCT 431 Lys Pro Gly Lys Ser Glu Leu Arg Ile Asn Lys Ala Ser Leu Ala
95 100 105
GAT TCT GGA GAG TAT ATG TGC AAA GTG ATC AGC AAA TTA GGA AAT 476 Asp Ser Gly Glu Tyr Met Cys Lys Val Ile Ser Lys Leu Gly Asn
110 115 120
GAC AGT GCC TCT GCC AAT ATC ACC ATC GTG GAA TCA AAC GAG ATC 521 Asp Ser Ala Ser Ala Asn Ile Thr Ile Val Glu Ser Asn Glu Ile
125 130 135
ATC ACT GGT ATG CCA GCC TCA ACT GAA GGA GCA TAT GTG TCT TCA 566 Ile Thr Gly Met Pro Ala Ser Thr Glu Gly Ala Tyr Val Ser Ser
140 145 150
GAG TCT CCC ATT AGA ATA TCA GTA TCC ACA GAA GGA GCA AAT ACT 611 Glu Ser Pro Ile Arg Ile Ser Val Ser Thr Glu Gly Ala Asn Thr
155 160 165
TCT TCA TCT ACA TCT ACA TCC ACC ACT GGG ACA AGC CAT CTT GTA 656 Ser Ser Ser Thr Ser Thr Ser Thr Thr Gly Thr Ser His Leu Val
170 175 180
AAA TGT GCG GAG AAG GAG AAA ACT TTC TGT GTG AAT GGA GGG GAG 701 Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu
185 190 195
TGC TTC ATG GTG AAA GAC CTT TCA AAC CCC TCG AGA TAC TTG TGC 746 Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys
200 205 210
AAG TGC CAA CCT GGA TTC ACT GGA GCA AGA TGT ACT GAG AAT GTG 791 Lys Cys Gln Pro Gly Phe Thr Gly Ala Arg Cys Thr Glu Asn Val
215 220 225
CCC ATG AAA GTC CAA AAC CAA GAA AAG GCG GAG GAG CTG TAC CAG 836 Pro Met Lys Val Gln Asn Gln Glu Lys Ala Glu Glu Leu Tyr Gln
230 235 240
AAG AGA GTG CTG ACC ATA ACC GGC ATC TGC ATC GCC CTC CTT GTG 881 Lys Arg Val Leu Thr Ile Thr Gly Ile Cys Ile Ala Leu Leu Val
245 250 255
GTC GGC ATC ATG TGT GTG GTG GCC TAC TGC AAA ACC AAG AAA CAG 926 Val Gly Ile Met Cys Val Val Ala Tyr Cys Lys Thr Lys Lys Gln
260 265 270
CGG AAA AAG CTG CAT GAC CGT CTT CGG CAG AGC CTT CGG TCT GAA 971 Arg Lys Lys Leu His Asp Arg Leu Arg Gln Ser Leu Arg Ser Glu
275 280 285
CGA AAC AAT ATG ATG AAC ATT GCC AAT GGG CCT CAC CAT CCT AAC 1016 Arg Asn Asn Met Met Asn Ile Ala Asn Gly Pro His His Pro Asn
290 295 300
CCA CCC CCC GAG AAT GTC CAG CTG GTG AAT CAA TAC GTA TCT AAA 1061 Pro Pro Pro Glu Asn Val Gln Leu Val Asn Gln Tyr Val Ser Lys
305 310 315
AAC GTC ATC TCC AGT GAG CAT ATT GTT GAG AGA GAA GCA GAG ACA 1106 Asn Val Ile Ser Ser Glu His Ile Val Glu Arg Glu Ala Glu Thr
320 325 330
TCC TTT TCC ACC AGT CAC TAT ACT TCC ACA GCC CAT CAC TCC ACT 1151 Ser Phe Ser Thr Ser His Tyr Thr Ser Thr Ala His His Ser Thr
335 340 345
ACT GTC ACC CAG ACT CCT AGC CAC AGC TGG AGC AAC GGA CAC ACT 1196 Thr Val Thr Gln Thr Pro Ser His Ser Trp Ser Asn Gly His Thr
350 355 360
GAA AGC ATC CTT TCC GAA AGC CAC TCT GTA ATC GTG ATG TCA TCC 1241 Glu Ser Ile Leu Ser Glu Ser His Ser Val Ile Val Met Ser Ser
365 370 375
GTA GAA AAC AGT AGG CAC AGC AGC CCA ACT GGG GGC CCA AGA GGA 1286 Val Glu Asn Ser Arg His Ser Ser Pro Thr Gly Gly Pro Arg Gly
380 385 390
CGT CTT AAT GGC ACA GGA GGC CCT CGT GAA TGT AAC AGC TTC CTC 1331 Arg Leu Asn Gly Thr Gly Gly Pro Arg Glu Cys Asn Ser Phe Leu
395 400 405
AGG CAT GCC AGA GAA ACC CCT GAT TCC TAC CGA GAC TCT CCT CAT 1376 Arg His Ala Arg Glu Thr Pro Asp Ser Tyr Arg Asp Ser Pro His
410 415 420
AGT GAA AGA CAT AAC CTT ATA GCT GAG CTA AGG AGA AAC AAG GCA 1421 Ser Glu Arg His Asn Leu Ile Ala Glu Leu Arg Arg Asn Lys Ala
425 430 435
CAC AGA TCC AAA TGC ATG CAG ATC CAG CTA TCA GCA ACT CAT CTT 1466 His Arg Ser Lys Cys Met Gln Ile Gln Leu Ser Ala Thr His Leu
440 445 450
AGA TCT TCT TCC ATT CCC CAT TTG GGC TTC ATT CTC TAA 1505
Arg Ser Ser Ser Ile Pro His Leu Gly Phe Ile Leu
455 460
GACCCCTTGG CCTTTAGGAA GGTATGTGTC AGCCATGACC ACCCCGGCTC 1555
GTATGTCACC TGTAGATTTC CACACGCCAA GCTCCCCCAA ATCGCCCCCT 1605
TCGGAAATGT CTCCACCCGT GTCCAGCATG ACGGTGTCCA TGCCTTCCAT 1655
GGCGGTCAGC CCCTTCATGG AAGAAGAGAG ACCTCTACTT CTCGTGACAC 1705
CACCAAGGCT GCGGGAGAAG AAGTTTGACC ATCACCCTCA GCAGTTCAGC 1755
TCCTTCCACC ACAACCCCAC GCGCCCACGC GTCCGCGGAC GCGTGGGTCGAC 1807
(9) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 462 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 8 :
Met Ser Glu Arg Lys Glu Gly Arg Gly Lys Gly Lys Gly Lys Lys
1 5 10 15
Lys Glu Arg Gly Ser Gly Lys Lys Pro Glu Ser Ala Ala Gly Ser
20 25 30
Gln Ser Pro Ala Leu Pro Pro Arg Leu Lys Glu Met Lys Ser Gln
35 40 45
Glu Ser Ala Ala Gly Ser Lys Leu Val Leu Arg Cys Glu Thr Ser
50 55 60
Ser Glu Tyr Ser Ser Leu Arg Phe Lys Trp Phe Lys Asn Gly Asn 65 70 75
Glu Leu Asn Arg Lys Asn Lys Pro Gln Asn Ile Lys Ile Gln Lys
80 85 90
Lys Pro Gly Lys Ser Glu Leu Arg Ile Asn Lys Ala Ser Leu Ala
95 100 105
Asp Ser Gly Glu Tyr Met Cys Lys Val Ile Ser Lys Leu Gly Asn
110 115 120
Asp Ser Ala Ser Ala Asn Ile Thr Ile Val Glu Ser Asn Glu Ile
125 130 135 Ile Thr Gly Met Pro Ala Ser Thr Glu Gly Ala Tyr Val Ser Ser
140 145 150
Glu Ser Pro Ile Arg Ile Ser Val Ser Thr Glu Gly Ala Asn Thr
155 160 165
Ser Ser Ser Thr Ser Thr Ser Thr Thr Gly Thr Ser His Leu Val
170 175 180
Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu
185 190 195
Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys
200 205 210
Lys Cys Gln Pro Gly Phe Thr Gly Ala Arg Cys Thr Glu Asn Val
215 220 225
Pro Met Lys Val Gln Asn Gln Glu Lys Ala Glu Glu Leu Tyr Gln
230 235 240
Lys Arg Val Leu Thr Ile Thr Gly Ile Cys Ile Ala Leu Leu Val
245 250 255
Val Gly Ile Met Cys Val Val Ala Tyr Cys Lys Thr Lys Lys Gln
260 265 270
Arg Lys Lys Leu His Asp Arg Leu Arg Gln Ser Leu Arg Ser Glu
275 280 285
Arg Asn Asn Met Met Asn Ile Ala Asn Gly Pro His His Pro Asn
290 295 300
Pro Pro Pro Glu Asn Val Gln Leu Val Asn Gln Tyr Val Ser Lys
305 310' 315
Asn Val Ile Ser Ser Glu His Ile Val Glu Arg Glu Ala Glu Thr
320 325 330
Ser Phe Ser Thr Ser His Tyr Thr Ser Thr Ala His His Ser Thr
335 340 345
Thr Val Thr Gln Thr Pro Ser His Ser Trp Ser Asn Gly His Thr
350 355 360
Glu Ser Ile Leu Ser Glu Ser His Ser Val Ile Val Met Ser Ser
365 370 375
Val Glu Asn Ser Arg His Ser Ser Pro Thr Gly Gly Pro Arg Gly
380 385 390
Arg Leu Asn Gly Thr Gly Gly Pro Arg Glu Cys Asn Ser Phe Leu
395 400 405
Arg His Ala Arg Glu Thr Pro Asp Ser Tyr Arg Asp Ser Pro His
410 415 420
Ser Glu Arg His Asn Leu Ile Ala Glu Leu Arg Arg Asn Lys Ala
425 430 435
His Arg Ser Lys Cys Met Gln Ile Gln Leu Ser Ala Thr His Leu
440 445 450
Arg Ser Ser Ser Ile Pro His Leu Gly Phe Ile Leu
455 460
(10) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1651 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 20.. 817
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
GTCGACCCAC GCGTCCGGC TTC ATG GTG AAA GAC CTT TCA AAC CCC TCG AGA 52
Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg
1 5 10
TAC TTG TGC AAG TGC CAA CCT GGA TTC ACT GGA GCA AGA TGT ACT GAG 100 Tyr Leu Cys Lys Cys Gln Pro Gly Phe Thr Gly Ala Arg Cys Thr Glu
15 20 25
AAT GTG CCC ATG AAA GTC CAA AAC CAA GAA AAG GCG GAG GAG CTG TAC 148 Asn Val Pro Met Lys Val Gln Asn Gln Glu Lys Ala Glu Glu Leu Tyr
30 35 40
CAG AAG AGA GTG CTG ACC ATA ACC GGC ATC TGC ATC GCC CTC CTT GTG 196 Gln Lys Arg Val Leu Thr Ile Thr Gly Ile Cys Ile Ala Leu Leu Val
45 50 55
GTC GGC ATC ATG TGT GTG GTG GCC TAC TGC AAA ACC AAG AAA CAG CGG 244 Val Gly Ile Met Cys Val Val Ala Tyr Cys Lys Thr Lys Lys Gln Arg
60 65 70 75
AAA AAG CTG CAT GAC CGT CTT CGG CAG AGC CTT CGG TCT GAA CGA AAC 292 Lys Lys Leu His Asp Arg Leu Arg Gln Ser Leu Arg Ser Glu Arg Asn
80 85 90
AAT ATG ATG AAC ATT GCC AAT GGG CCT CAC CAT CCT AAC CCA CCC CCC 340 Asn Met Met Asn Ile Ala Asn Gly Pro His His Pro Asn Pro Pro Pro
95 100 105
GAG AAT GTC CAG CTG GTG AAT CAA TAC GTA TCT AAA AAC GTC ATC TCC 388 Glu Asn Val Gln Leu Val Asn Gln Tyr Val Ser Lys Asn Val Ile Ser
110 115 120
AGT GAG CAT ATT GTT GAG AGA GAA GCA GAG ACA TCC TTT TCC ACC AGT 436 Ser Glu His Ile Val Glu Arg Glu Ala Glu Thr Ser Phe Ser Thr Ser
125 130 135
CAC TAT ACT TCC ACA GCC CAT CAC TCC ACT ACT GTC ACC CAG ACT CCT 484 His Tyr Thr Ser Thr Ala His His Ser Thr Thr Val Thr Gln Thr Pro
140 145 150 155
AGC CAC AGC TGG AGC AAC GGA CAC ACT GAA AGC ATC CTT TCC GAA AGC 532 Ser His Ser Trp Ser Asn Gly His Thr Glu Ser Ile Leu Ser Glu Ser
160 165 170
CAC TCT GTA ATC GTG ATG TCA TCC GTA GAA AAC AGT AGG CAC AGC AGC 580 His Ser Val Ile Val Met Ser Ser Val Glu Asn Ser Arg His Ser Ser
175 180 185
CCA ACT GGG GGC CCA AGA GGA CGT CTT AAT GGC ACA GGA GGC CCT CGT 628 Pro Thr Gly Gly Pro Arg Gly Arg Leu Asn Gly Thr Gly Gly Pro Arg
190 195 200
GAA TGT AAC AGC TTC CTC AGG CAT GCC AGA GAA ACC CCT GAT TCC TAC 676 Glu Cys Asn Ser Phe Leu Arg His Ala Arg Glu Thr Pro Asp Ser Tyr
205 210 215
CGA GAC TCT CCT CAT AGT GAA AGA CAT AAC CTT ATA GCT GAG CTA AGG 724 Arg Asp Ser Pro His Ser Glu Arg His Asn Leu Ile Ala Glu Leu Arg
220 225 230 235
AGA AAC AAG GCA CAC AGA TCC AAA TGC ATG CAG ATC CAG CTA TCA GCA 772 Arg Asn Lys Ala His Arg Ser Lys Cys Met Gln Ile Gln Leu Ser Ala
240 245 250
ACT CAT CTT AGA TCT TCT TCC ATT CCC CAT TTG GGC TTC ATT CTC 817 Thr His Leu Arg Ser Ser Ser Ile Pro His Leu Gly Phe Ile Leu
255 260 265
TAAGACCCCT TGGCCTTTAG GAAGGTATGT GTCAGCCATG ACCACCCCGG CTCGTATGTC 877
ACCTGTAGAT TTCCACACGC CAAGCTCCCC CAAATCGCCC CCTTCGGAAA TGTCTCCACC 937
CGTGTCCAGC ATGACGGTGT CCATGCCTTC CATGGCGGTC AGCCCCTTCA TGGAAGAAGA 997
GAGACCTCTA CTTCTCGTGA CACCACCAAG GCTGCGGGAG AAGAAGTTTG ACCATCACCC 1057
TCAGCAGTTC AGCTCCTTCC ACCACAACCC CGCGCATGAC AGTAACAGCC TCCCTGCTAG 1117
CCCCTTGAGG ATAGTGGAGG ATGAGGAGTA TGAAACGACC CAAGAGCACG AGCCAGCCCA 1177
AGAGCCTGTT AAGAAACTCG CCAATAGCCG GCGGGCCAAA AGAACCAAGC CCAATGGCCA 1237
CATTGCTAAC AGATTGGAAG TGGACAGCAA CACAAGCTCC CAGAGCAGTA ACTCAGAGAG 1297
TGAAACAGAA GATGAAAGAG TAGGTGAAGA TACGCCTTTC CTGGGCATAC AGAACCCCCT 1357
GGCAGCCAGT CTTGAGGCAA CACCTGCCTT CCGCCTGGCT GACAGCAGGA CTAACCCAGC 1417
AGGCCGCTTC TCGACACAGG AAGAAATCCA GGCCAGGCTG TCTAGTGTAA TTGCTAACCA 1477
AGACCCTATT GCTGTATAAA ACCTAAATAA ACACATAGAT TCACCTGTAA AACTTTATTT 1537
TATATAATAA AGTATTCCAC CTTAAATTAA ACAATTTATT TTATTTTAGC AGTTCTGCAA 1597
AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAGGGCGG CCGC 1651
(11) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 266 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys Lys Cys
1 5 10 15
Gln Pro Gly Phe Thr Gly Ala Arg Cys Thr Glu Asn Val Pro Met Lys
20 25 30
Val Gln Asn Gln Glu Lys Ala Glu Glu Leu Tyr Gln Lys Arg Val Leu 35 40 45
Thr Ile Thr Gly Ile Cys Ile Ala Leu Leu Val Val Gly Ile Met Cys 50 55 60
Val Val Ala Tyr Cys Lys Thr Lys Lys Gln Arg Lys Lys Leu His Asp 65 70 75 80
Arg Leu Arg Gln Ser Leu Arg Ser Glu Arg Asn Asn Met Met Asn Ile
85 90 95
Ala Asn Gly Pro His His Pro Asn Pro Pro Pro Glu Asn Val Gln Leu
100 105 110
Val Asn Gln Tyr Val Ser Lys Asn Val Ile Ser Ser Glu His Ile Val
115 120 125
Glu Arg Glu Ala Glu Thr Ser Phe Ser Thr Ser His Tyr Thr Ser Thr 130 135 140
Ala His His Ser Thr Thr Val Thr Gln Thr Pro Ser His Ser Trp Ser 145 150 155 160
Asn Gly His Thr Glu Ser Ile Leu Ser Glu Ser His Ser Val Ile Val
165 170 175
Met Ser Ser Val Glu Asn Ser Arg His Ser Ser Pro Thr Gly Gly Pro
180 185 190
Arg Gly Arg Leu Asn Gly Thr Gly Gly Pro Arg Glu Cys Asn Ser Phe
195 200 205
Leu Arg His Ala Arg Glu Thr Pro Asp Ser Tyr Arg Asp Ser Pro His 210 215 220
Ser Glu Arg His Asn Leu Ile Ala Glu Leu Arg Arg Asn Lys Ala His 225 230 235 240
Arg Ser Lys Cys Met Gln Ile Gln Leu Ser Ala Thr His Leu Arg Ser
245 250 255
Ser Ser Ile Pro His Leu Gly Phe Ile Leu
260 265
(12) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 946 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : unknown
(D) TOPOLOGY: unknown
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 20..394
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
GTCGACCCAC GCGTCCGGT GCC TCT GCC AAT ATC ACC ATC GTG GAA TCA AAC 52
Ala Ser Ala Asn Ile Thr Ile Val Glu Ser Asn
1 5 10
GAG ATC ATC ACT GGT ATG CCA GCC TCA ACT GAA GGA GCA TAT GTG TCT 100 Glu Ile Ile Thr Gly Met Pro Ala Ser Thr Glu Gly Ala Tyr Val Ser
15 20 25
TCA GAG TCT CCC ATT AGA ATA TCA GTA TCC ACA GAA GGA GCA AAT ACT 148 Ser Glu Ser Pro Ile Arg Ile Ser Val Ser Thr Glu Gly Ala Asn Thr
30 35 40
TCT TCA TCT ACA TCT ACA TCC ACC ACT GGG ACA AGC CAT CTT GTA AAA 196 Ser Ser Ser Thr Ser Thr Ser Thr Thr Gly Thr Ser His Leu Val Lys
45 50 55
TGT GCG GAG AAG GAG AAA ACT TTC TGT GTG AAT GGA GGG GAG TGC TTC 244 Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu Cys Phe
60 65 70 75
ATG GTG AAA GAC CTT TCA AAC CCC TCG AGA TAC TTG TGC AAG TGC CAA 292 Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys Lys Cys Gln
80 85 90
CCT GGA TTC ACT GGA GCA AGA TGT ACT GAG AAT GTG CCC ATG AAA GTC 340 Pro Gly Phe Thr Gly Ala Arg Cys Thr Glu Asn Val Pro Met Lys Val
95 100 105
CAA AAC CAA GAA AGT GCC CAA ATG AGT TTA CTG GTG ATC GCT GCC AAA 388 Gln Asn Gln Glu Ser Ala Gln Met Ser Leu Leu Val Ile Ala Ala Lys
110 115 120
ACT ACG TAATGGCCAG CTTCTACAGT ACGTCCACTC CCTTTCTGTC TCTGCCTGAA 444 Thr Thr
125
TAGGAGCATG CTCAGTTGGT GCTGCTTTCT TGTTGCTGCA TCTCCCCTCA GATTCCACCT 504
AGAGCTAGAT GTGTCTTACC AGATCTAATA TTGACTGCCT CTGCCTGTCG CATGAGAACA 564
TTAACAAAAG CAATTGTATT ACTTCCTCTG TTCGCGACTA GTTGGCTCTG AGATACTAAT 624
AGGTGTGTGA GGCTCCGGAT GTTTCTGGAA TTGATATTGA ATGATGTGAT ACAAATTGAT 684
AGTCAATATC AAGCAGTGAA ATATGATAAT AAAGGCATTT CAAAGTCTCA CTTTTATTGA 744
TAAAATAAAA ATCATTCTAC TGAACAGTCC ATCTTCTTTA TACAATGACC ACATCCTGAA 804
AAGGGTGTTG CTAAGCTGTA ACCGATATGC ACTTGAAATG ATGGTAAGTT AATTTTGATT 864
CAGAATGTGT TATTTGTCAC AAATAAACAT AATAAAAGGA AAAAAAAAAA AAAAAAAAAA 924
AAAAAAAAAA AAGGGCGGCC GC 946
(13) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 125 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:
Ala Ser Ala Asn Ile Thr Ile Val Glu Ser Asn Glu Ile Ile Thr Gly
1 5 10 15
Met Pro Ala Ser Thr Glu Gly Ala Tyr Val Ser Ser Glu Ser Pro Ile
20 25 30
Arg Ile Ser Val Ser Thr Glu Gly Ala Asn Thr Ser Ser Ser Thr Ser
35 40 45
Thr Ser Thr Thr Gly Thr Ser His Leu Val Lys Cys Ala Glu Lys Glu
50 55 60
Lys Thr Phe Cys Val Asn Gly Gly Glu Cys Phe Met Val Lys Asp Leu
65 70 75 80
Ser Asn Pro Ser Arg Tyr Leu Cys Lys Cys Gln Pro Gly Phe Thr Gly
85 90 95
Ala Arg Cys Thr Glu Asn Val Pro Met Lys Val Gln Asn Gln Glu Ser
100 105 110
Ala Gln Met Ser Leu Leu Val Ile Ala Ala Lys Thr Thr
115 120 125
(14) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1873 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
( ix) FEATURE :
(A) NAME/KEY: CDS
(B) LOCATION: 12..1664
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
GAATTCAAGC G TCA GAA CTT CGC ATT AAC AAA GCA TCA CTG .GCT GAT TCT 50
Ser Glu Leu Arg Ile Asn Lys Ala Ser Leu Ala Asp Ser
1 5 10
GGA GAG TAT ATG TGC AAA GTG ATC AGC AAA TTA GGA AAT GAC AGT GCC 98 Gly Glu Tyr Met Cys Lys Val Ile Ser Lys Leu Gly Asn Asp Ser Ala
15 20 25
TCT GCC AAT ATC ACC ATC GTG GAA TCA AAC GAG ATC ATC ACT GGT ATG 146 Ser Ala Asn Ile Thr Ile Val Glu Ser Asn Glu Ile Ile Thr Gly Met
30 35 40 45
CCA GCC TCA ACT GAA GGA GCA TAT GTG TCT TCA GAG TCT CCC ATT AGA 194 Pro Ala Ser Thr Glu Gly Ala Tyr Val Ser Ser Glu Ser Pro Ile Arg
50 55 60
ATA TCA GTA TCC ACA GAA GGA GCA AAT ACT TCT TCA TCT ACA TCT ACA 242 Ile Ser Val Ser Thr Glu Gly Ala Asn Thr Ser Ser Ser Thr Ser Thr
65 70 75
TCC ACC ACT GGG ACA AGC CAT CTT GTA AAA TGT GCG GAG AAG GAG AAA 290 Ser Thr Thr Gly Thr Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys
80 85 90
ACT TTC TGT GTG AAT GGA GGG GAG TGC TTC ATG GTG AAA GAC CTT TCA 338 Thr Phe Cys Val Asn Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser
95 100 105
AAC CCC TCG AGA TAC TTG TGC AAG TGC CCA AAT GAG TTT ACT GGT GAT 386 Asn Pro Ser Arg Tyr Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp
110 115 120 125
CGC TGC CAA AAC TAC GTA ATG GCC AGC TTC TAC AAG CAT CTT GGG ATT 434 Arg Cys Gln Asn Tyr Val Met Ala Ser Phe Tyr Lys His Leu Gly Ile
130 135 140
GAA TTT ATG GAG GCG GAG GAG CTG TAC CAG AAG AGA GTG CTG ACC ATA 482 Glu Phe Met Glu Ala Glu Glu Leu Tyr Gln Lys Arg Val Leu Thr Ile
145 150 155
ACC GGC ATC TGC ATC GCC CTC CTT GTG GTC GGC ATC ATG TGT GTG GTG 530 Thr Gly Ile Cys Ile Ala Leu Leu Val Val Gly Ile Met Cys Val Val
160 165 170
GCC TAC TGC AAA ACC AAG AAA CAG CGG AAA AAG CTG CAT GAC CGT CTT 578 Ala Tyr Cys Lys Thr Lys Lys Gln Arg Lys Lys Leu His Asp Arg Leu
175 180 185
CGG CAG AGC CTT CGG TCT GAA CGA AAC AAT ATG ATG AAC ATT GCC AAT 626 Arg Gln Ser Leu Arg Ser Glu Arg Asn Asn Met Met Asn Ile Ala Asn
190 195 200 205
GGG CCT CAC CAT CCT AAC CCA CCC CCC GAG AAT GTC CAG CTG GTG AAT 674 Gly Pro His His Pro Asn Pro Pro Pro Glu Asn Val Gln Leu Val Asn
210 215 220
CAA TAC GTA TCT AAA AAC GTC ATC TCC AGT GAG CAT ATT GTT GAG AGA 722 Gln Tyr Val Ser Lys Asn Val Ile Ser Ser Glu His Ile Val Glu Arg
225 230 235
GAA GCA GAG ACA TCC TTT TCC ACC AGT CAC TAT ACT TCC ACA GCC CAT 770 Glu Ala Glu Thr Ser Phe Ser Thr Ser His Tyr Thr Ser Thr Ala His
240 245 250
CAC TCC ACT ACT GTC ACC CAG ACT CCT AGC CAC AGC TGG AGC AAC GGA 818 His Ser Thr Thr Val Thr Gln Thr Pro Ser His Ser Trp Ser Asn Gly
255 260 265
CAC ACT GAA AGC ATC CTT TCC GAA AGC CAC TCT GTA ATC GTG ATG TCA 866 His Thr Glu Ser Ile Leu Ser Glu Ser His Ser Val Ile Val Met Ser
270 275 280 285
TCC GTA GAA AAC AGT AGG CAC AGC AGC CCA ACT GGG GGC CCA AGA GGA 914 Ser Val Glu Asn Ser Arg His Ser Ser Pro Thr Gly Gly Pro Arg Gly
290 295 300
CGT CTT AAT GGC ACA GGA GGC CCT CGT GAA TGT AAC AGC TTC CTC AGG 962 Arg Leu Asn Gly Thr Gly Gly Pro Arg Glu Cys Asn Ser Phe Leu Arg
305 310 315
CAT GCC AGA GAA ACC CCT GAT TTC TAC CGA GAC TCT CCT CAT AGT GAA 1010 His Ala Arg Glu Thr Pro Asp Phe Tyr Arg Asp Ser Pro His Ser Glu
320 325 330
AGG TAT GTG TCA GCC ATG ACC ACC CCG GCT CGT ATG TCA CCT GTA GAT 1058 Arg Tyr Val Ser Ala Met Thr Thr Pro Ala Arg Met Ser Pro Val Asp
335 340 345
TTC CAC ACG CCA AGC TCC CCC AAA TCG CCC CCT TCG GAA ATG TCT CCA 1106 Phe His Thr Pro Ser Ser Pro Lys Ser Pro Pro Ser Glu Met Ser Pro
350 355 360 365
CCC GTG TCC AGC ATG ACG GTG TCC ATG CCT TCC ATG GCG GTC AGC CCC 1154 Pro Val Ser Ser Met Thr Val Ser Met Pro Ser Met Ala Val Ser Pro
370 375 380
TTC ATG GAA GAA GAG AGA CCT CTA CTT CTC GTG ACA CCA CCA AGG CTG 1202 Phe Met Glu Glu Glu Arg Pro Leu Leu Leu Val Thr Pro Pro Arg Leu
385 390 395
CGG GAG AAG AAG TTT GAC CAT CAC CCT CAG CAG TTC AGC TCC TTC CAC 1250 Arg Glu Lys Lys Phe Asp His His Pro Gln Gln Phe Ser Ser Phe His
400 405 410
CAC AAC CCC GCG CAT GAC AGT AAC AGC CTC CCT GCT AGC CCC TTG AGG 1298 His Asn Pro Ala His Asp Ser Asn Ser Leu Pro Ala Ser Pro Leu Arg
415 420 425
ATA GTG GAG GAT GAG GAG TAT GAA ACG ACC CAA GAG TAC GAG CCA GCC 1346 Ile Val Glu Asp Glu Glu Tyr Glu Thr Thr Gln Glu Tyr Glu Pro Ala
430 435 440 445
CAA GAG CCT GTT AAG AAA CTC GCC AAT AGC CGG CGG GCC AAA AGA ACC 1394 Gln Glu Pro Val Lys Lys Leu Ala Asn Ser Arg Arg Ala Lys Arg Thr
450 455 460
AAG CCC AAT GGC CAC ATT GCT AAC AGA TTG GAA GTG GAC AGC AAC ACA 1442 Lys Pro Asn Gly His Ile Ala Asn Arg Leu Glu Val Asp Ser Asn Thr
465 470 475
AGC TCC CAG AGC AGT AAC TCA GAG AGT GAA ACA GAA GAT GAA AGA GTA 1490 Ser Ser Gln Ser Ser Asn Ser Glu Ser Glu Thr Glu Asp Glu Arg Val
480 485 490
GGT GAA GAT ACG CCT TTC CTG GGC ATA CAG AAC CCC CTG GCA GCC AGT 1538 Gly Glu Asp Thr Pro Phe Leu Gly Ile Gln Asn Pro Leu Ala Ala Ser
495 500 505
CTT GAG GCA ACA CCT GCC TTC CGC CTG GCT GAC AGC AGG ACT AAC CCA 1586 Leu Glu Ala Thr Pro Ala Phe Arg Leu Ala Asp Ser Arg Thr Asn Pro
510 515 520 525
GCA GGC CGC TTC TCG ACA CAG GAA GAA ATC CAG GCC AGG CTG TCT AGT 1634 Ala Gly Arg Phe Ser Thr Gln Glu Glu Ile Gln Ala Arg Leu Ser Ser
530 535 540
GTA ATT GCT AAC CAA GAC CCT ATT GCT GTA TAAAACCTAA ATAAACACAT 1684 Val Ile Ala Asn Gln Asp Pro Ile Ala Val
545 550
AGATTCACCT GTAAAACTTT ATTTTATATA ATAAAGTATT CCACCTTAAA TTAAACAATT 1744
TATTTTATTT TAGCAGTTCT GCAAATAGAA AACAGGAAAA AAACTTTTAT AAATTAAATA 1804
TATGTATGTA AAAATGTGTT ATGTGCCATA TGTAGCAATT TTTTACAGTA TTTCAAAAAA 1864
AAAAAGCTT 1873
(15) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 551 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
Ser Glu Leu Arg Ile Asn Lys Ala Ser Leu Ala Asp Ser Gly Glu Tyr 1 5 10 15
Met Cys Lys Val Ile Ser Lys Leu Gly Asn Asp Ser Ala Ser Ala Asn
20 25 30
Ile Thr Ile Val Glu Ser Asn Glu Ile Ile Thr Gly Met Pro Ala Ser
35 40 45
Thr Glu Gly Ala Tyr Val Ser Ser Glu Ser Pro Ile Arg Ile Ser Val 50 55 60
Ser Thr Glu Gly Ala Asn Thr Ser Ser Ser Thr Ser Thr Ser Thr Thr 65 70 75 80
Gly Thr Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys
85 90 95
Val Asn Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser
100 105 110
Arg Tyr Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln
115 120 125
Asn Tyr Val Met Ala Ser Phe Tyr Lys His Leu Gly Ile Glu Phe Met 130 135 140
Glu Ala Glu Glu Leu Tyr Gln Lys Arg Val Leu Thr Ile Thr Gly Ile 145 150 155 160
Cys Ile Ala Leu Leu Val Val Gly Ile Met Cys Val Val Ala Tyr Cys
165 170 175
Lys Thr Lys Lys Gln Arg Lys Lys Leu His Asp Arg Leu Arg Gln Ser
180 185 190
Leu Arg Ser Glu Arg Asn Asn Met Met Asn Ile Ala Asn Gly Pro His
195 200 205
His Pro Asn Pro Pro Pro Glu Asn Val Gln Leu Val Asn Gln Tyr Val 210 215 220
Ser Lys Asn Val Ile Ser Ser Glu His Ile Val Glu Arg Glu Ala Glu 225 230 235 240
Thr Ser Phe Ser Thr Ser His Tyr Thr Ser Thr Ala His His Ser Thr
245 250 255
Thr Val Thr Gln Thr Pro Ser His Ser Trp Ser Asn Gly His Thr Glu
260 265 270
Ser Ile Leu Ser Glu Ser His Ser Val Ile Val Met Ser Ser Val Glu
275 280 285
Asn Ser Arg His Ser Ser Pro Thr Gly Gly Pro Arg Gly Arg Leu Asn 290 295 300
Gly Thr Gly Gly Pro Arg Glu Cys Asn Ser Phe Leu Arg His Ala Arg 305 310 315 320
Glu Thr Pro Asp Phe Tyr Arg Asp Ser Pro His Ser Glu Arg Tyr Val
325 330 335
Ser Ala Met Thr Thr Pro Ala Arg Met Ser Pro Val Asp Phe His Thr
340 345 350
Pro Ser Ser Pro Lys Ser Pro Pro Ser Glu Met Ser Pro Pro Val Ser
355 360 365
Ser Met Thr Val Ser Met Pro Ser Met Ala Val Ser Pro Phe Met Glu 370 375 380
Glu Glu Arg Pro Leu Leu Leu Val Thr Pro Pro Arg Leu Arg Glu Lys 385 390 395 400
Lys Phe Asp His His Pro Gln Gln Phe Ser Ser Phe His His Asn Pro
405 410 415
Ala His Asp Ser Asn Ser Leu Pro Ala Ser Pro Leu Arg Ile Val Glu
420 425 430
Asp Glu Glu Tyr Glu Thr Thr Gln Glu Tyr Glu Pro Ala Gln Glu Pro
435 440 445
Val Lys Lys Leu Ala Asn Ser Arg Arg Ala Lys Arg Thr Lys Pro Asn 450 455 460
Gly His Ile Ala Asn Arg Leu Glu Val Asp Ser Asn Thr Ser Ser Gln 465 470 475 480
Ser Ser Asn Ser Glu Ser Glu Thr Glu Asp Glu Arg Val Gly Glu Asp
485 490 495
Thr Pro Phe Leu Gly Ile Gln Asn Pro Leu Ala Ala Ser Leu Glu Ala
500 505 510
Thr Pro Ala Phe Arg Leu Ala Asp Ser Arg Thr Asn Pro Ala Gly Arg
515 520 525
Phe Ser Thr Gln Glu Glu Ile Gln Ala Arg Leu Ser Ser Val Ile Ala
530 535 540
Asn Gln Asp Pro Ile Ala Val
545 550
(16) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 878 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 25..852
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
GTCGAAGGAA ATGACAGTGC CTCT GCC AAT ATC ACC ATC GTG GAA TCA AAC 51
Ala Asn Ile Thr Ile Val Glu Ser Asn.
1 5
GAG ATC ATC ACT GGT ATG CCA GCC TCA ACT GAA GGA GCA TAT GTG TCT 99 Glu Ile Ile Thr Gly Met Pro Ala Ser Thr Glu Gly Ala Tyr Val Ser
10 15 20 25
TCA GAG TCT CCC ATT AGA ATA TCA GTA TCC ACA GAA GGA GCA AAT ACT 147 Ser Glu Ser Pro Ile Arg Ile Ser Val Ser Thr Glu Gly Ala Asn Thr
30 35 40
TCT TCA TCT ACA TCT ACA TCC ACC ACT GGG ACA AGC CAT CTT GTA AAA 195 Ser Ser Ser Thr Ser Thr Ser Thr Thr Gly Thr Ser His Leu Val Lys
45 50 55
TGT GCG GAG AAG GAG AAA ACT TTC TGT GTG AAT GGA GGG GAG TGC TTC 243 Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu Cys Phe
60 65 70
ATG GTG AAA GAC CTT TCA AAC CCC TCG AGA TAC TTG TGC AAG TGC CCA 291 Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys Lys Cys Pro
75 80 85
AAT GAG TTT ACT GGT GAT CGC TGC CAA AAC TAC GTA ATG GCC AGC TTC 339 Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr Val Met Ala Ser Phe
90 95 100 105
TAC AAG GCG GAG GAG CTG TAC CAG AAG AGA GTG CTG ACC ATA ACC GGC 387 Tyr Lys Ala Glu Glu Leu Tyr Gln Lys Arg Val Leu Thr Ile Thr Gly
110 115 120
ATC TGC ATC GCC CTC CTT GTG GTC GGC ATC ATG TGT GTG GTG GCC TAC 435 Ile Cys Ile Ala Leu Leu Val Val Gly Ile Met Cys Val Val Ala Tyr
125 130 135
TGC AAA ACC AAG AAA CAG CGG AAA AAG CTG CAT GAC CGT CTT CGG CAG 483 Cys Lys Thr Lys Lys Gln Arg Lys Lys Leu His Asp Arg Leu Arg Gln
140 145 150
AGC CTT CGG TCT GAA CGA AAC AAT ATG ATG AAC ATT GCC AAT GGG CCT 531 Ser Leu Arg Ser Glu Arg Asn Asn Met Met Asn Ile Ala Asn Gly Pro
155 160 165
CAC CAT CCT AAC CCA CCC CCC GAG AAT GTC CAG CTG GTG AAT CAA TAC 579 His His Pro Asn Pro Pro Pro Glu Asn Val Gln Leu Val Asn Gln Tyr
170 175 180 185
GTA TCT AAA AAC GTC ATC TCC AGT GAG CAT ATT GTT GAG AGA GAA GCA 627 Val Ser Lys Asn Val Ile Ser Ser Glu His Ile Val Glu Arg Glu Ala
190 195 200
GAG ACA TCC TTT TCC ACC AGT CAC TAT ACT TCC ACA GCC CAT CAC TCC 675 Glu Thr Ser Phe Ser Thr Ser His Tyr Thr Ser Thr Ala His His Ser
205 210 215
ACT ACT GTC ACC CAG ACT CCT AGC CAC AGC TGG AGC AAC GGA CAC ACT 723 Thr Thr Val Thr Gln Thr Pro Ser His Ser Trp Ser Asn Gly His Thr
220 225 230
GAA AGC ATC CTT TCC GAA AGC CAC TCT GTA ATC GTG ATG TCA TCC GTA 771 Glu Ser Ile Leu Ser Glu Ser His Ser Val Ile Val Met Ser Ser Val
235 240 245
GAA AAC AGT AGG CAC AGC AGC CCA ACT GGG GGC CCA AGA GGA CGT CTT 819 Glu Asn Ser Arg His Ser Ser Pro Thr Gly Gly Pro Arg Gly Arg Leu
250 255 260 265
AAT GGC ACA GGA GGC CCT CGT GAA TGT AAC AGC TTCCTCAGGC ATGCCAGAGA 872 Asn Gly Thr Gly Gly Pro Arg Glu Cys Asn Ser
270 275
GGCCGC 878
(17) INFORMATION FOR SEQ ID NO: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 276 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
Ala Asn Ile Thr Ile Val Glu Ser Asn Glu Ile Ile Thr Gly Met Pro 1 5 10 15
Ala Ser Thr Glu Gly Ala Tyr Val Ser Ser Glu Ser Pro Ile Arg Ile
20 25 30
Ser Val Ser Thr Glu Gly Ala Asn Thr Ser Ser Ser Thr Ser Thr Ser
35 40 45
Thr Thr Gly Thr Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr 50 55 60
Phe Cys Val Asn Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn 65 70 75 80
Pro Ser Arg Tyr Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg
85 90 95
Cys Gln Asn Tyr Val Met Ala Ser Phe Tyr Lys Ala Glu Glu Leu Tyr
100 105 110
Gln Lys Arg Val Leu Thr Ile Thr Gly Ile Cys Ile Ala Leu Leu Val
115 120 125
Val Gly Ile Met Cys Val Val Ala Tyr Cys Lys Thr Lys Lys Gln Arg 130 135 140
Lys Lys Leu His Asp Arg Leu Arg Gln Ser Leu Arg Ser Glu Arg Asn 145 150 155 160
Asn Met Met Asn Ile Ala Asn Gly Pro His His Pro Asn Pro Pro Pro
165 170 175
Glu Asn Val Gln Leu Val Asn Gln Tyr Val Ser Lys Asn Val Ile Ser
180 185 190
Ser Glu His Ile Val Glu Arg Glu Ala Glu Thr Ser Phe Ser Thr Ser
195 200 205
His Tyr Thr Ser Thr Ala His His Ser Thr Thr Val Thr Gln Thr Pro 210 215 220
Ser His Ser Trp Ser Asn Gly His Thr Glu Ser Ile Leu Ser Glu Ser
225 230 235 240
His Ser Val Ile Val Met Ser Ser Val Glu Asn Ser Arg His Ser Ser
245 250 255
Pro Thr Gly Gly Pro Arg Gly Arg Leu Asn Gly Thr Gly Gly Pro Arg
260 265 270
Glu Cys Asn Ser
275
(18) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 906 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 43..531
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:
TCTAGATGCA TGCTCGAGCG GCCGCCAGTG TGCTCTAAAG AT CGA AAA AAC AAA 54
Arg Lys Asn Lys
1
CCA CAA AAT ATC AAG ATA CAA AAA AAG CCA GGG AAG TCA GAA CTT CGC 102 Pro Gln Asn Ile Lys Ile Gln Lys Lys Pro Gly Lys Ser Glu Leu Arg
5 10 15 20
ATT AAC AAA GCA TCA CTG GCT GAT TCT GGA GAG TAT ATG TGC AAA GTG 150 Ile Asn Lys Ala Ser Leu Ala Asp Ser Gly Glu Tyr Met Cys Lys Val
25 30 35
ATC AGC AAA TTA GGA AAT GAC AGT GCC TCT GCC AAT ATC ACC ATC GTG 198 Ile Ser Lys Leu Gly Asn Asp Ser Ala Ser Ala Asn Ile Thr Ile Val
40 45 50
GAA TCA AAC GAG ATC ATC ACT GGT ATG CCA GCC TCA ACT GAA GGA GCA 246 Glu Ser Asn Glu Ile Ile Thr Gly Met Pro Ala Ser Thr Glu Gly Ala
55 60 65
TAT GTG TCT TCA GAG TCT CCC ATT AGA ATA TCA GTA TCC ACA GAA GGA 294 Tyr Val Ser Ser Glu Ser Pro Ile Arg Ile Ser Val Ser Thr Glu Gly
70 75 80
GCA AAT ACT TCT TCA TCT ACA TCT ACA TCC ACC ACT GGG ACA AGC CAT 342 Ala Asn Thr Ser Ser Ser Thr Ser Thr Ser Thr Thr Gly Thr Ser His
85 90 95 100
CTT GTA AAA TGT GCG GAG AAG GAG AAA ACT TTC TGT GTG AAT GGA GGG 390 Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly
105 110 115
GAG TGC TTC ATG GTG AAA GAC CTT TCA AAC CCC TCG AGA TAC TTG TGC 438 Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys
120 125 130
AAG TGC CCA AAT GAG TTT ACT GGT GAT CGC TGC CAA AAC TAC GTA ATG 486 Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr Val Met
135 140 145
GCC AGC TTC TAC AGT ACG TCC ACT CCC TTT CTG TCT CTG CCT GAA 531
Ala Ser Phe Tyr Ser Thr Ser Thr Pro Phe Leu Ser Leu Pro Glu
150 155 160
TAGGAGCATG CTCAGTTGGT GCTGCTTTCT TGTTGCTGCA TCTCCCCTCA GATTCCACCT 591
AGAGCTAGAT GTGTCTTACC AGATCTAATA TTGACTGCCT CTGCCTGTCG CATGAGAACA 651
TTAACAAAAG CAATTGTATT ACTTCCTCTG TTCGCGACTA GTTGGCTCTG AGATACTAAT 711
AGGTGTGTGA GGCTCCGGAT GTTTCTGGAA TTGATATTGA ATGATGTGAT ACAAATTGAT 771
AGTCAATATC AAGCAGTGAA ATATGATAAT AAAGGCATTT CAAAGTCTCA AAAAAAAAAA 831
AAAAAAAAAA AAAAAAAAAA ACTTTAGAGC ACACTGGCGG CCGTTACTAG TGGATCCGAG 891
CTCGGTACCA AGCTT 906
(19) INFORMATION FOR SEQ ID NO: 18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 163 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:
Arg Lys Asn Lys Pro Gln Asn Ile Lys Ile Gln Lys Lys Pro Gly Lys
1 5 10 15
Ser Glu Leu Arg Ile Asn Lys Ala Ser Leu Ala Asp Ser Gly Glu Tyr
20 25 30
Met Cys Lys Val Ile Ser Lys Leu Gly Asn Asp Ser Ala Ser Ala Asn
35 40 45
Ile Thr Ile Val Glu Ser Asn Glu Ile Ile Thr Gly Met Pro Ala Ser
50 55 60
Thr Glu Gly Ala Tyr Val Ser Ser Glu Ser Pro Ile Arg Ile Ser Val
65 70 75 80
Ser Thr Glu Gly Ala Asn Thr Ser Ser Ser Thr Ser Thr Ser Thr Thr
85 90 95
Gly Thr Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys
100 105 110
Val Asn Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro Ser
115 120 125
Arg Tyr Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln
130 135 140
Asn Tyr Val Met Ala Ser Phe Tyr Ser Thr Ser Thr Pro Phe Leu Ser
145 150 155 160
Leu Pro Glu
(20) INFORMATION FOR SEQ ID NO: 19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2186 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double stranded
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
AGCTGCCGGG AGATGCGAGC GCAGACCGGA TTGTGATCAC CTTTCCCTCT TCGGGCTGTA 60
AGAGAGCGAG ACAAGCCACC GAAGCGAGGC CACTCCAGAG CCGGCAGCGG AGGGACCCGG 120
GACACTAGAG CAGCTCCGAG CCACTCCAGA CTGAGCGGAC GCTCCAGGTG ATCGAGTCCA 180
CGCTGCTTCC TGCAGGCGAC AGGCGACGCC TCCCGAGCAG CCCGGCCACT GGCTCTTCCC 240
CTCCTGGGAC AAACTTTTCT GCAAGCCCTT GGACCAAACT TGTCGCGCGT CACCGTCACC 300
CAACCGGGTC CGCGTAGAGC GCTCATCTTC GGCGAG ATG TCT GAG CGC AAA GAA 354
Met Ser Glu Arg Lys Glu
1 5
GGC AGA GGC AAG GGG AAG GGC AAG AAG AAG GAC CGG GGA TCC CGC GGG 402 Gly Arg Gly Lys Gly Lys Gly Lys Lys Lys Asp Arg Gly Ser Arg Gly
10 15 20
AAG CCC GGG CCC GCC GAG GGC GAC CCG AGC CCA GCA CTG CCT CCC AGA 450 Lys Pro Gly Pro Ala Glu Gly Asp Pro Ser Pro Ala Leu Pro Pro Arg
25 30 35
TTG AAA GAA ATG AAG AGC CAG GAG TCA GCT GCA GGC TCC AAG CTA GTG 498 Leu Lys Glu Met Lys Ser Gln Glu Ser Ala Ala Gly Ser Lys Leu Val
40 45 50
CTC CGG TGC GAA ACC AGC TCC GAG TAC TCC TCA CTC AGA TTC AAA TGG 546 Leu Arg Cys Glu Thr Ser Ser Glu Tyr Ser Ser Leu Arg Phe Lys Trp
55 60 65 70
TTC AAG AAT GGG AAC GAG CTG AAC CGC AAA AAT AAA CCA GAA AAC ATC 594 Phe Lys Asn Gly Asn Glu Leu Asn Arg Lys Asn Lys Pro Glu Asn Ile
75 80 85
AAG ATA CAG AAG AAG CCA GGG AAG TCA GAG CTT CGA ATT AAC AAA GCA 642 Lys Ile Gln Lys Lys Pro Gly Lys Ser Glu Leu Arg Ile Asn Lys Ala
90 95 100
TCC CTG GCT GAC TCT GGA GAG TAT ATG TGC AAA GTG ATC AGC AAG TTA 690 Ser Leu Ala Asp Ser Gly Glu Tyr Met Cys Lys Val Ile Ser Lys Leu
105 110 115
GGA AAT GAC AGT GCC TCT GCC AAC ATC ACC ATT GTT GAG TCA AAC GAG 738 Gly Asn Asp Ser Ala Ser Ala Asn Ile Thr Ile Val Glu Ser Asn Glu
120 125 130
TTC ATC ACT GGC ATG CCA GCC TCG ACT GAG ACA GCC TAT GTG TCC TCA 786 Phe Ile Thr Gly Met Pro Ala Ser Thr Glu Thr Ala Tyr Val Ser Ser
135 140 145 150
GAG TCT CCC ATT AGA ATC TCA GTT TCA ACA GAA GGC GCA AAC ACT TCT 834 Glu Ser Pro Ile Arg Ile Ser Val Ser Thr Glu Gly Ala Asn Thr Ser
155 160 165
TCA TCC ACA TCA ACA TCC ACG ACT GGG ACC AGC CAT CTC ATA AAG TGT 882 Ser Ser Thr Ser Thr Ser Thr Thr Gly Thr Ser His Leu Ile Lys Cys
170 175 180
GCG GAG AAG GAG AAA ACT TTC TGT GTG AAT GGG GGC GAG TGC TTC ACG 930 Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu Cys Phe Thr
185 190 195
GTG AAG GAC CTG TCA AAC CCG TCA AGA TAC TTG TGC AAG TGC CAA CCT 978 Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys Lys Cys Gln Pro
200 205 210
GGA TTC ACT GGA GCA AGA TGT ACT GAG AAT GTA CCC ATG AAA GTC CAA 1026 Gly Phe Thr Gly Ala Arg Cys Thr Glu Asn Val Pro Met Lys Val Gln
215 220 225 230
ACC CAA GAA AAA GCG GAG GAA CTC TAC CAG AAG AGG GTG CTG ACA ATT 1074 Thr Gln Glu Lys Ala Glu Glu Leu Tyr Gln Lys Arg Val Leu Thr Ile
235 240 245
ACT GGC ATC TGT ATC GCC CTG CTG GTG GTC GGC ATC ATG TGT GTG GTG 1122 Thr Gly Ile Cys Ile Ala Leu Leu Val Val Gly Ile Met Cys Val Val
250 255 260
GCC TAC TGC AAA ACC AAG AAG CAG CGG CAG AAG CTT CAT GAT CGG CTT 1170 Ala Tyr Cys Lys Thr Lys Lys Gln Arg Gln Lys Leu His Asp Arg Leu
265 270 275
CGG CAG AGT CTT CGG TCA GAA CGG AGC AAC CTG GTG AAC ATA GCG AAT 1218 Arg Gln Ser Leu Arg Ser Glu Arg Ser Asn Leu Val Asn Ile Ala Asn
280 285 290
GGG CCT CAC CAC CCA AAC CCA CCG CCA GAG AAC GTG CAG CTG GTG AAT 1266 Gly Pro His His Pro Asn Pro Pro Pro Glu Asn Val Gln Leu Val Asn
295 300 305 310
CAA TAC GTA TCT AAA AAC GTC ATC TCC AGT GAG CAT ATT GTT GAG AGA 1314 Gln Tyr Val Ser Lys Asn Val Ile Ser Ser Glu His Ile Val Glu Arg
315 320 325
GAA GTG GAG ACT TCC TTT TCC ACC AGT CAT TAC ACT TCC ACA GCC CAT 1362 Glu Val Glu Thr Ser Phe Ser Thr Ser His Tyr Thr Ser Thr Ala His
330 335 340
CAC TCC ACG ACT GTC ACC CAG ACT CCT AGT CAC AGC TGG AGT AAT GGG 1410 His Ser Thr Thr Val Thr Gln Thr Pro Ser His Ser Trp Ser Asn Gly
345 350 355
CAC ACG GAG AGC GTC ATT TCA GAA AGC AAC TCC GTA ATC ATG ATG TCT 1458 His Thr Glu Ser Val Ile Ser Glu Ser Asn Ser Val Ile Met Met Ser
360 365 370
TCG GTA GAG AAC AGC AGG CAC AGC AGT CCC GCC GGG GGC CCA CGA GGA 1506 Ser Val Glu Asn Ser Arg His Ser Ser Pro Ala Gly Gly Pro Arg Gly
375 380 385 390
CGT CTT CAT GGC CTG GGA GGC CCT CGT GAT AAC AGC TTC CTC AGG CAT 1554 Arg Leu His Gly Leu Gly Gly Pro Arg Asp Asn Ser Phe Leu Arg His
395 400 405
GCC AGA GAA ACC CCT GAC TCC TAC AGA GAC TCT CCT CAT AGC GAA AGG 1602 Ala Arg Glu Thr Pro Asp Ser Tyr Arg Asp Ser Pro His Ser Glu Arg
410 415 420
TAAAATGGAA GGGTAAAGCT ATCGTGGAGG AGAACCTCAT TCAGTGAGAG AATCCCATGA 1662
GCACCTGCGG TCTCTCCTAA GGAAACTCAT CCTTTCATAA GGGGCGTCAT CAATTCCCTG 1722
ACGCTCCTAG TTGATGAAGT CAACCTCTTA TTTGTCTGAA CTCCTTTCTC CTGAGCTTCT 1782
CCCCGCGTCC CAGTGGCTGA CAGGCAACCA ACTCCTAAAG AGCAGGAGTA ATGTAATGTG 1842
GAAGGCCCAG CCGACTTGGA GTTCTCAGAC CTGACCCTAA GGTCAGACTC ACTGGGGTCT 1902
TCCTTTCGTC AGGTGCACCA TTTTAAGGAC CCTCATCTAA TCCCGATCAG CCAGTTGTCC 1962
ATTCTCTACC TCGATGGTTG TTCTGCCCTC CTCTCCTCAT GCTCTAAGTA CCCAGCCTCT 2022
AGCCTCAGTT AAATCAAGTC GAGGGCTGGG ACACGGCTGA TGGTCATGGC GGAATGCCTC 2082
CTCGGGTTCC ACCACACTGT ATCCATTTAC GACATCCCTA GAAGTCATGG GTAAATAAAG 2142
TTGTTGTGTG ATGTGAAAAA AAAAAAAAAA AAAAAAAAAA AAAA 2186
(21) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 422 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
Met Ser Glu Arg Lys Glu
1 5
Gly Arg Gly Lys Gly Lys Gly Lys Lys Lys Asp Arg Gly Ser Arg Gly
10 15 20
Lys Pro Gly Pro Ala Glu Gly Asp Pro Ser Pro Ala Leu Pro Pro Arg
25 30 35
Leu Lys Glu Met Lys Ser Gln Glu Ser Ala Ala Gly Ser Lys Leu Val
40 45 50
Leu Arg Cys Glu Thr Ser Ser Glu Tyr Ser Ser Leu Arg Phe Lys Trp
55 60 65 70
Phe Lys Asn Gly Asn Glu Leu Asn Arg Lys Asn Lys Pro Glu Asn Ile
75 80 85
Lys Ile Gln Lys Lys Pro Gly Lys Ser Glu Leu Arg Ile Asn Lys Ala
90 95 100
Ser Leu Ala Asp Ser Gly Glu Tyr Met Cys Lys Val Ile Ser Lys Leu 105 110 115
Gly Asn Asp Ser Ala Ser Ala Asn Ile Thr Ile Val Glu Ser Asn Glu 120 125 130
Phe Ile Thr Gly Met Pro Ala Ser Thr Glu Thr Ala Tyr Val Ser Ser 135 140 145 150
Glu Ser Pro Ile Arg Ile Ser Val Ser Thr Glu Gly Ala Asn Thr Ser
155 160 165
Ser Ser Thr Ser Thr Ser Thr Thr Gly Thr Ser His Leu Ile Lys Cys
170 175 180
Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu Cys Phe Thr
185 190 195
Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys Lys Cys Gln Pro 200 205 210
Gly Phe Thr Gly Ala Arg Cys Thr Glu Asn Val Pro Met Lys Val Gln 215 220 225 230
Thr Gln Glu Lys Ala Glu Glu Leu Tyr Gln Lys Arg Val Leu Thr Ile
235 240 245
Thr Gly Ile Cys Ile Ala Leu Leu Val Val Gly Ile Met Cys Val Val
250 255 260
Ala Tyr Cys Lys Thr Lys Lys Gln Arg Gln Lys Leu His Asp Arg Leu
265 270 275
Arg Gln Ser Leu Arg Ser Glu Arg Ser Asn Leu Val Asn Ile Ala Asn
280 285 290
Gly Pro His His Pro Asn Pro Pro Pro Glu Asn Val Gln Leu Val Asn 295 300 305 310 Gln Tyr Val Ser Lys Asn Val Ile Ser Ser Glu His Ile Val Glu Arg
315 320 325
Glu Val Glu Thr Ser Phe Ser Thr Ser His Tyr Thr Ser Thr Ala His
330 335 340
His Ser Thr Thr Val Thr Gln Thr Pro Ser His Ser Trp Ser Asn Gly
345 350 355
His Thr Glu Ser Val Ile Ser Glu Ser Asn Ser Val Ile Met Met Ser 360 365 370
Ser Val Glu Asn Ser Arg His Ser Ser Pro Ala Gly Gly Pro Arg Gly 375 380 385 390
Arg Leu His Gly Leu Gly Gly Pro Arg Asp Asn Ser Phe Leu Arg His
395 400 405
Ala Arg Glu Thr Pro Asp Ser Tyr Arg Asp Ser Pro His Ser Glu Arg
410 415 4,20
(22) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1894 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double stranded
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
CCGT CACCCAACCG GGTCCGCGTA GAGCGCTCAT CTTCGGCGAG ATG TCT GAG 53
Met Ser Glu
1
CGC AAA GAA GGC AGA GGC AAG GGG AAG GGC AAG AAG AAG GAC CGG GGA 101 Arg Lys Glu Gly Arg Gly Lys Gly Lys Gly Lys Lys Lys Asp Arg Gly
5 10 15
TCC CGC GGG AAG CCC GGG CCC GCC GAG GGC GAC CCG AGC CCA GCA CTG 149 Ser Arg Gly Lys Pro Gly Pro Ala Glu Gly Asp Pro Ser Pro Ala Leu
20 25 30 35
CCT CCC AGA TTG AAA GAA ATG AAG AGC CAG GAG TCA GCT GCA GGC TCC 197 Pro Pro Arg Leu Lys Glu Met Lys Ser Gln Glu Ser Ala Ala Gly Ser
40 45 50
AAG CTA GTG CTC CGG TGC GAA ACC AGC TCC GAG TAC TCC TCA CTC AGA 245 Lys Leu Val Leu Arg Cys Glu Thr Ser Ser Glu Tyr Ser Ser Leu Arg
55 60 65
TTC AAA TGG TTC AAG AAT GGG AAC GAG CTG AAC CGC AAA AAT AAA CCA 293 Phe Lys Trp Phe Lys Asn Gly Asn Glu Leu Asn Arg Lys Asn Lys Pro
70 75 80
GAA AAC ATC AAG ATA CAG AAG AAG CCA GGG AAG TCA GAG CTT CGA ATT 341 Glu Asn Ile Lys Ile Gln Lys Lys Pro Gly Lys Ser Glu Leu Arg Ile
85 90 95
AAC AAA GCA TCC CTG GCT GAC TCT GGA GAG TAT ATG TGC AAA GTG ATC 389 Asn Lys Ala Ser Leu Ala Asp Ser Gly Glu Tyr Met Cys Lys Val Ile
100 105 110 115
AGC AAG TTA GGA AAT GAC AGT GCC TCT GCC AAC ATC ACC ATT GTT GAG 437 Ser Lys Leu Gly Asn Asp Ser Ala Ser Ala Asn Ile Thr Ile Val Glu
120 125 130
TCA AAC GAG TTC ATC ACT GGC ATG CCA GCC TCG ACT GAG ACA GCC TAT 485 Ser Asn Glu Phe Ile Thr Gly Met Pro Ala Ser Thr Glu Thr Ala Tyr
135 140 145
GTG TCC TCA GAG TCT CCC ATT AGA ATC TCA GTT TCA ACA GAA GGC GCA 533 Val Ser Ser Glu Ser Pro Ile Arg Ile Ser Val Ser Thr Glu Gly Ala
150 155 160
AAC ACT TCT TCA TCC ACA TCA ACA TCC ACG ACT GGG ACC AGC CAT CTC 581 Asn Thr Ser Ser Ser Thr Ser Thr Ser Thr Thr Gly Thr Ser His Leu
165 170 175
ATA AAG TGT GCG GAG AAG GAG AAA ACT TTC TGT GTG AAT GGG GGC GAG 629 Ile Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu
180 185 190 195
TGC TTC ACG GTG AAG GAC CTG TCA AAC CCG TCA AGA TAC TTG TGC AAG 677 Cys Phe Thr Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys Lys
200 205 210
TGC CAA CCT GGA TTC ACT GGA GCA AGA TGT ACT GAG AAT GTA CCC ATG 725 Cys Gln Pro Gly Phe Thr Gly Ala Arg Cys Thr Glu Asn Val Pro Met
215 220 225
AAA GTC CAA ACC CAA GAA AAA GCG GAG GAA CTC TAC CAG AAG AGG GTG 773 Lys Val Gln Thr Gln Glu Lys Ala Glu Glu Leu Tyr Gln Lys Arg Val
230 235 240
CTG ACA ATT ACT GGC ATC TGT ATC GCC CTG CTG GTG GTC GGC ATC ATG 821 Leu Thr Ile Thr Gly Ile Cys Ile Ala Leu Leu Val Val Gly Ile Met
245 250 255
TGT GTG GTG GCC TAC TGC AAA ACC AAG AAG CAG CGG CAG AAG CTT CAT 869 Cys Val Val Ala Tyr Cys Lys Thr Lys Lys Gln Arg Gln Lys Leu His
260 265 270 275
GAT CGG CTT CGG CAG AGT CTT CGG TCA GAA CGG AGC AAC CTG GTG AAC 917 Asp Arg Leu Arg Gln Ser Leu Arg Ser Glu Arg Ser Asn Leu Val Asn
280 285 290
ATA GCG AAT GGG CCT CAC CAC CCA AAC CCA CCG CCA GAG AAC GTG CAG 965 Ile Ala Asn Gly Pro His His Pro Asn Pro Pro Pro Glu Asn Val Gln
295 300 305
CTG GTG AAT CAA TAC GTA TCT AAA AAC GTC ATC TCC AGT GAG CAT ATT 1013 Leu Val Asn Gln Tyr Val Ser Lys Asn Val Ile Ser Ser Glu His Ile
310 315 320
GTT GAG AGA GAA GTG GAG ACT TCC TTT TCC ACC AGT CAT TAC ACT TCC 1061 Val Glu Arg Glu Val Glu Thr Ser Phe Ser Thr Ser His Tyr Thr Ser
325 330 335
ACA GCC CAT CAC TCC ACG ACT GTC ACC CAG ACT CCT AGT CAC AGC TGG 1109 Thr Ala His His Ser Thr Thr Val Thr Gln Thr Pro Ser His Ser Trp
340 345 350 355
AGT AAT GGG CAC ACG GAG AGC GTC ATT TCA GAA AGC AAC TCC GTA ATC 1157 Ser Asn Gly His Thr Glu Ser Val Ile Ser Glu Ser Asn Ser Val Ile
360 365 370
ATG ATG TCT TCG GTA GAG AAC AGC AGG CAC AGC AGT CCC GCC GGG GGC 1205 Met Met Ser Ser Val Glu Asn Ser Arg His Ser Ser Pro Ala Gly Gly
375 380 385
CCA CGA GGA CGT CTT CAT GGC CTG GGA GGC CCT CGT GAT AAC AGC TTC 1253 Pro Arg Gly Arg Leu His Gly Leu Gly Gly Pro Arg Asp Asn Ser Phe
390 395 400
CTC AGG CAT GCC AGA GAA ACC CCT GAC TCC TAC AGA GAC TCT CCT CAT 1301 Leu Arg His Ala Arg Glu Thr Pro Asp Ser Tyr Arg Asp Ser Pro His
405 410 415
AGC GAA AGG TAAAATGGAA GGGTAAAGCT ATCGTGGAGG AGAACCTCAT TCAGTGAGAG 1360
Ser Glu Arg
420
AATCCCATGA GCACCTGCGG TCTCTCCTAA GGAAACTCAT CCTTTCATAA GGGGCGTCAT 1420
CAATTCCCTG ACGCTCCTAG TTGATGAAGT CAACCTCTTA TTTGTCTGAA CTCCTTTCTC 1480
CTGAGCTTCT CCCCGCGTCC CAGTGGCTGA CAGGCAACCA ACTCCTAAAG AGCAGGAGTA 1540
ATGTAATGTG GAAGGCCCAG CCGACTTGGA GTTCTCAGAC CTGACCCTAA GGTCAGACTC 1600
ACTGGGGTCT TCCTTTCGTC AGGTGCACCA TTTTAAGGAC CCTCATCTAA TCCCGATCAG 1660
CCAGTTGTCC ATTCTCTACC TCGATGGTTG TTCTGCCCTC CTCTCCTCAT GCTCTAAGTA 1720
CCCAGCCTCT AGCCTCAGTT AAATCAAGTC GAGGGCTGGG ACACGGCTGA TGGTCATGGC 1780
GGAATGCCTC CTCGGGTTCC ACCACACTGT ATCCATTTAC GACATCCCTA GAAGTCATGG 1840
GTAAATAAAG TTGTTGTGTG ATGTGAAAAA AAAAAAAAAA AAAAAAAAAA AAAA 1894
(23) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 422 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
Met Ser Glu
1
Arg Lys Glu Gly Arg Gly Lys Gly Lys Gly Lys Lys Lys Asp Arg Gly 5 10 15
Ser Arg Gly Lys Pro Gly Pro Ala Glu Gly Asp Pro Ser Pro Ala Leu 20 25 30 35
Pro Pro Arg Leu Lys Glu Met Lys Ser Gln Glu Ser Ala Ala Gly Ser
40 45 50
Lys Leu Val Leu Arg Cys Glu Thr Ser Ser Glu Tyr Ser Ser Leu Arg
55 60 65
Phe Lys Trp Phe Lys Asn Gly Asn Glu Leu Asn Arg Lys Asn Lys Pro
70 75 80
Glu Asn Ile Lys Ile Gln Lys Lys Pro Gly Lys Ser Glu Leu Arg Ile 85 90 95
Asn Lys Ala Ser Leu Ala Asp Ser Gly Glu Tyr Met Cys Lys Val Ile 100 105 110 115
Ser Lys Leu Gly Asn Asp Ser Ala Ser Ala Asn Ile Thr Ile Val Glu
120 125 130
Ser Asn Glu Phe Ile Thr Gly Met Pro Ala Ser Thr Glu Thr Ala Tyr
135 140 145
Val Ser Ser Glu Ser Pro Ile Arg Ile Ser Val Ser Thr Glu Gly Ala
150 155 160
Asn Thr Ser Ser Ser Thr Ser Thr Ser Thr Thr Gly Thr Ser His Leu 165 170 175
Ile Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu 180 185 190 195
Cys Phe Thr Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys Lys
200 205 210
Cys Gln Pro Gly Phe Thr Gly Ala Arg Cys Thr Glu Asn Val Pro Met 215 220 225
Lys Val Gln Thr Gln Glu Lys Ala Glu Glu Leu Tyr Gln Lys Arg Val
230 235 240
Leu Thr Ile Thr Gly Ile Cys Ile Ala Leu Leu Val Val Gly Ile Met 245 250 255
Cys Val Val Ala Tyr Cys Lys Thr Lys Lys Gln Arg Gln Lys Leu His 260 265 270 275
Asp Arg Leu Arg Gln Ser Leu Arg Ser Glu Arg Ser Asn Leu Val Asn
280 285 290 Ile Ala Asn Gly Pro His His Pro Asn Pro Pro Pro Glu Asn Val Gln
295 300 305
Leu Val Asn Gln Tyr Val Ser Lys Asn Val Ile Ser Ser Glu His Ile
310 315 320
Val Glu Arg Glu Val Glu Thr Ser Phe Ser Thr Ser His Tyr Thr Ser 325 330 335
Thr Ala His His Ser Thr Thr Val Thr Gln Thr Pro Ser His Ser Trp 340 345 350 355
Ser Asn Gly His Thr Glu Ser Val Ile Ser Glu Ser Asn Ser Val Ile
360 365 370
Met Met Ser Ser Val Glu Asn Ser Arg His Ser Ser Pro Ala Gly Gly
375 380 385
Pro Arg Gly Arg Leu His Gly Leu Gly Gly Pro Arg Asp Asn Ser Phe
390 395 400
Leu Arg His Ala Arg Glu Thr Pro Asp Ser Tyr Arg Asp Ser Pro His 405 410 415
Ser Glu Arg
420
(24) INFORMATION FOR SEQ ID NO: 23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 64 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:
Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu Cys Phe 1 5 10 15
Thr Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys Lys Cys Gln
20 25 30
Pro Gly Phe Thr Gly Ala Arg Cys Thr Glu Asn Val Pro Met Lys Val
35 40 45
Gln Thr Gln Glu Lys Ala Glu Glu Leu Tyr Gln Lys Arg Val Leu Thr 50 55 60
(25) INFORMATION FOR SEQ ID NO: 24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
Cys Pro Asp Ser His Thr Gln Tyr Cys Phe His Gly Thr Cys Arg Phe 1 5 10 15
Leu Val Gln Glu Glu Lys Pro Ala Cys Val Cys His Ser Gly Tyr Val
20 25 30
Gly Val Arg Cys Glu His Ala Asp Leu Leu Ala Val Val Ala Ala Ser
35 40 45
Gln Lys Lys Gln Ala Ile Thr Ala Ala Val Val Val
50 55 60
(26) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY : unknown
(xi) SEQUENCE DESCRIPTION : SEQ ID NO : 25 :
Cys Asn Ala Glu Phe Gln Asn Phe Cys Ile His Gly Glu Cys Lys Tyr 1 5 10 15 Ile Glu His Leu Glu Ala Val Thr Cys Asn Cys Gln Gln Glu Tyr Phe
20 25 30
Gly Glu Arg Cys Gly Glu Lys Ser Met Lys Thr His Ser Met Ile Asp 35 40 45
Ser Ser Leu Ser Lys Ile Ala Leu Leu Ala Ile Ala
50 55 60
(27) INFORMATION FOR SEQ ID NO: 26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 61 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:26:
Cys Pro Ser Ser Tyr Asp Gly Tyr Cys Leu Asn Gly Gly Val Cys Met 1 5 10 15
His Ile Glu Ser Leu Asp Ser Tyr Thr Cys Asn Cys Val Ile Gly Tyr
20 25 30
Ser Gly Asp Arg Cys Gln Thr Arg Asp Leu Arg Trp Trp Glu Leu Arg
35 40 45
His Ala Gly Tyr Gly Gln Lys His Asp Ile Met Val Val
50 55 60
(28) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:
Cys Leu Arg Lys Tyr Lys Asp Phe Cys Ile His Gly Glu Cys Lys Tyr 1 5 10 15
Val Lys Glu Leu Arg Ala Pro Ser Cys Ile Cys His Pro Gly Tyr His
20 25 30
Gly Glu Arg Cys His Gly Leu Ser Leu Pro Val Glu Asn Arg Val Tyr
35 40 45
Thr Tyr Asp His Thr Thr Ile Leu Ala Val Val Ala
50 55 60
(29) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
Cys Ala Ala Lys Phe Gln Asn Phe Cys Ile His Gly Glu Cys Arg Tyr 1 5 10 15 Ile Glu Asn Leu Glu Val Val Thr Cys His Cys His Gln Asp Tyr Phe
20 25 30
Gly Glu Arg Cys Gly Glu Lys Thr Met Lys Thr Gln Lys Lys Asp Asp
35 40 45
Ser Asp Leu Ser Lys Ile Ala Leu Ala Ala Ile Ile
50 55 60
(30) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
Cys Gly Pro Glu Gly Asp Gly Tyr Cys Leu His Gly Asp Cys Ile His 1 5 10 15
Ala Arg Asp Ile Asp Gly Met Tyr Cys Arg Cys Ser His Gly Tyr Thr
20 25 30
Gly Ile Arg Cys Gln His Val Val Leu Val Asp Tyr Gln Arg Ser Glu
35 40 45
Asn Pro Asn Thr Thr Thr Ser Tyr Ile Pro Ser Pro
50 55 60
(31) INFORMATION FOR SEQ ID NO: 30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:
Cys Asn His Asp Tyr Glu Asn Tyr Cys Leu Asn Asn Gly Thr Cys Phe 1 5 10 15
Thr Ile Ala Leu Asp Asn Val Ser Ile Thr Pro Phe Cys Val Cys Arg
20 25 30
Ile Asn Tyr Glu Gly Ser Arg Cys Gln Phe Ile Asn Leu Val Thr Tyr
35 40 45
(32) INFORMATION FOR SEQ ID NO: 31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 49 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:
Cys Asn Asp Asp Tyr Lys Asn Tyr Cys Leu Asn Asn Gly Thr Cys Phe 1 5 10 15
Thr Val Ala Leu Asn Asn Val Ser Leu Asn Pro Phe Cys Ala Cys His
20 25 30
Ile Asn Tyr Val Gly Ser Arg Cys Gln Phe Ile Asn Leu Ile Thr Ile
35 40 45
Lys
(33) INFORMATION FOR SEQ ID NO: 32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 65 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
Leu Val Leu Arg Cys Glu Thr Ser Ser Glu Tyr Ser Ser Leu Arg Phe 1 5 10 15
Arg Trp Phe Lys Asn Gly Asn Glu Leu Asn Arg Lys Asn Asn Lys Pro
20 25 30
Glu Asn Ile Lys Ile Gln Lys Lys Pro Gly Lys Ser Glu Leu Arg Ile
35 40 45
Asn Lys Ala Ser Leu Ala Asp Ser Gly Glu Tyr Met Cys Lys Val Ile
50 55 60
Ser
65
(34) INFORMATION FOR SEQ ID NO: 33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 65 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:
Val Thr Leu Thr Cys Glu Ala Ser Gly Asp Pro Ile Pro Ser Ile Thr
1 5 10 15
Trp Arg Thr Ser Thr Arg Asn Ile Ser Ser Glu Glu Gln Asp Leu Asp
20 25 30
Gly His Met Val Val Arg Ser His Ala Arg Val Ser Ser Leu Thr Leu
35 40 45
Lys Ser Ile Gln Tyr Arg Asp Ala Gly Glu Tyr Met Cys Thr Ala Ser
50 55 60
Asn
65
(35) INFORMATION FOR SEQ ID NO: 34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1098 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 17..709
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
GTCGACCCAC GCGTCC GGG AAG GGC AAG AAG AAG GAC CGG GGA TCC CGC 49
Gly Lys Gly Lys Lys Lys Asp Arg Gly Ser Arg
1 5 10
GGG AAG CCC GGG CCC GCC GAG GGC GAC CCG AGC CCA GCA CTG CCT CCC 97 Gly Lys Pro Gly Pro Ala Glu Gly Asp Pro Ser Pro Ala Leu Pro Pro
15 20 25
AGA TTG AAA GAA ATG AAG AGC CAG GAG TCA GCT GCA GGC TCC AAG CTA 145 Arg Leu Lys Glu Met Lys Ser Gln Glu Ser Ala Ala Gly Ser Lys Leu
30 35 40
GTG CTC CGG TGC GAA ACC AGC TCC GAG TAC TCC TCA CTC AGA TTC AAA 193 Val Leu Arg Cys Glu Thr Ser Ser Glu Tyr Ser Ser Leu Arg Phe Lys
45 50 55
TGG TTC AAG AAT GGG AAC GAG CTG AAC CGC AAA AAT AAA CCA GAA AAC 241 Trp Phe Lys Asn Gly Asn Glu Leu Asn Arg Lys Asn Lys Pro Glu Asn
60 65 70 75
ATC AAG ATA CAG AAG AAG CCA GGG AAG TCA GAG CTT CGA ATT AAC AAA 289 Ile Lys Ile Gln Lys Lys Pro Gly Lys Ser Glu Leu Arg Ile Asn Lys
80 85 90
GCA TCC CTG GCT GAC TCT GGA GAG TAT ATG TGC AAA GTG ATC AGC AAG 337 Ala Ser Leu Ala Asp Ser Gly Glu Tyr Met Cys Lys Val Ile Ser Lys
95 100 105
TTA GGA AAT GAC AGT GCC TCT GCC AAC ATC ACC ATT GTT GAG TCA AAC 385 Leu Gly Asn Asp Ser Ala Ser Ala Asn Ile Thr Ile Val Glu Ser Asn
110 115 120
GAG TTC ATC ACT GGC ATG CCA GCC TCG ACT GAG ACA GCC TAT GTG TCC 433 Glu Phe Ile Thr Gly Met Pro Ala Ser Thr Glu Thr Ala Tyr Val Ser
125 130 135
TCA GAG TCT CCC ATT AGA ATC TCA GTT TCA ACA GAA GGC GCA AAC ACT 481 Ser Glu Ser Pro Ile Arg Ile Ser Val Ser Thr Glu Gly Ala Asn Thr
140 145 150 155
TCT TCA TCC ACA TCA ACA TCC ACG ACT GGG ACC AGC CAT CTC ATA AAG 529 Ser Ser Ser Thr Ser Thr Ser Thr Thr Gly Thr Ser His Leu Ile Lys
160 165 170
TGT GCG GAG AAG GAG AAA ACT TTC TGT GTG AAT GGG GGC GAG TGC TTC 577 Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu Cys Phe
175 180 185
ACG GTG AAG GAC CTG TCA AAC CCG TCA AGA TAC TTG TGC AAG TGC CCA 625 Thr Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys Lys Cys Pro
190 195 200
AAT GAG TTT ACT GGT GAT CGT TGC CAA AAC TAC GTA ATG GCC AGC TTC 673 Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr Val Met Ala Ser Phe
205 210 215
TAC AGT ACG TCC ACT CCC TTT CTG TCT CTG CCT GAG TAGGAGCATG 719
Tyr Ser Thr Ser Thr Pro Phe Leu Ser Leu Pro Glu
220 225 230
CTCAGTCGAT GCTGCTTTCT TGTTGCTACA TCTCCCCTCA GAGTCACCTA GAGCCTAGAA 779
CCTTCTACCA GGTCTAATAT TGACTGCCTC TGCCTGTCGC ATGAGAACAT TAACAAGGCC 839
GCTGTGTTAC TTCTCTGTTT GTGACTAGTC GGCTCAGAGT TACTAAATAG GTGTGTGAGG 899
CCCCAGAGGT TTCTGAAAAT GGTCCCGCAG AAGGTGATGC AAATCGATAG TCAACATGCC 959
AAGAGTGAAA TAATAATAAT AATAATAATA ATAATAATAA TAATAATAAA GGCGTTTCAA 1019
ACTCGCACTT GTATTGGTAA AATAAAATCA TTCCAACAAG TAAAAAAAAA AAAAAAAAAA 1079
AAAAAAAAAG GGCGCCCGC 1098
(36) INFORMATION FOR SEQ ID NO: 35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 231 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:
Gly Lys Gly Lys Lys Lys Asp Arg Gly Ser Arg Gly Lys Pro Gly Pro
1 5 10 15
Ala Glu Gly Asp Pro Ser Pro Ala Leu Pro Pro Arg Leu Lys Glu Met
20 25 30
Lys Ser Gln Glu Ser Ala Ala Gly Ser Lys Leu Val Leu Arg Cys Glu
35 40 45
Thr Ser Ser Glu Tyr Ser Ser Leu Arg Phe Lys Trp Phe Lys Asn Gly
50 55 60
Asn Glu Leu Asn Arg Lys Asn Lys Pro Glu Asn Ile Lys Ile Gln Lys
65 70 75 80
Lys Pro Gly Lys Ser Glu Leu Arg Ile Asn Lys Ala Ser Leu Ala Asp
85 90 95
Ser Gly Glu Tyr Met Cys Lys Val Ile Ser Lys Leu Gly Asn Asp Ser
100 105 110
Ala Ser Ala Asn Ile Thr Ile Val Glu Ser Asn Glu Phe Ile Thr Gly
115 120 125
Met Pro Ala Ser Thr Glu Thr Ala Tyr Val Ser Ser Glu Ser Pro Ile
130 135 140
Arg Ile Ser Val Ser Thr Glu Gly Ala Asn Thr Ser Ser Ser Thr Ser
145 150 155 160
Thr Ser Thr Thr Gly Thr Ser His Leu Ile Lys Cys Ala Glu Lys Glu
165 170 175
Lys Thr Phe Cys Val Asn Gly Gly Glu Cys Phe Thr Val Lys Asp Leu
180 185 190
Ser Asn Pro Ser Arg Tyr Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly
195 200 205
Asp Arg Cys Gln Asn Tyr Val Met Ala Ser Phe Tyr Ser Thr Ser Thr
210 215 220
Pro Phe Leu Ser Leu Pro Glu
225 230
(37) INFORMATION FOR SEQ ID NO: 36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2914 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 345..1727
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:
GCGGCCGCAG CTGCCGGGAG ATGCGAGCGC AGACCGGATT GTGATCACCT TTCCCTCTTC 60
GGGCTGTAAG AGAGCGAGAC AAGCCACCGA AGCGAGGCCA CTCCAGAGCC GGCAGCGGAG 120
GGACCCGGGA CACTAGAGCA GCTCCGAGCC ACTCCAGACT GAGCGGACGC TCCAGGTGAT 180
CGAGTCCACG CTGCTTCCTG CAGGCGACAG GCGACGCCTC CCGAGCAGCC CGGCCACTGG 240
CTCTTCCCCT CCTGGGACAA ACTTTTCTGC AAGCCCTTGG ACCAAACTTG TCGCGCGTCA 300
CCGTCACCCA ACCGGGTCCG CGTAGAGCGC TCATCTTCGG CGAG ATG TCT GAG CGC 356
Met Ser Glu Arg
1
AAA GAA GGC AGA GGC AAG GGG AAG GGC AAG AAG AAG GAC CGG GGA TCC 404 Lys Glu Gly Arg Gly Lys Gly Lys Gly Lys Lys Lys Asp Arg Gly Ser
5 10 15 20
CGC GGG AAG CCC GGG CCC GCC GAG GGC GAC CCG AGC CCA GCA CTG CCT 452 Arg Gly Lys Pro Gly Pro Ala Glu Gly Asp Pro Ser Pro Ala Leu Pro
25 30 35
CCC AGA TTG AAA GAA ATG AAG AGC CAG GAG TCA GCT GCA GGC TCC AAG 500 Pro Arg Leu Lys Glu Met Lys Ser Gln Glu Ser Ala Ala Gly Ser Lys
40 45 50
CTA GTG CTC CGG TGC GAA ACC AGC TCC GAG TAC TCC TCA CTC AGA TTC 548 Leu Val Leu Arg Cys Glu Thr Ser Ser Glu Tyr Ser Ser Leu Arg Phe
55 60 65
AAA TGG TTC AAG AAT GGG AAC GAG CTG AAC CGC AAA AAT AAA CCA GAA 596 Lys Trp Phe Lys Asn Gly Asn Glu Leu Asn Arg Lys Asn Lys Pro Glu
70 75 80
AAC ATC AAG ATA CAG AAG AAG CCA GGG AAG TCA GAG CTT CGA ATT AAC 644 Asn Ile Lys Ile Gln Lys Lys Pro Gly Lys Ser Glu Leu Arg Ile Asn
85 90 95 100
AAA GCA TCC CTG GCT GAC TCT GGA GAG TAT ATG TGC AAA GTG ATC AGC 692 Lys Ala Ser Leu Ala Asp Ser Gly Glu Tyr Met Cys Lys Val Ile Ser
105 110 115
AAG TTA GGA AAT GAC AGT GCC TCT GCC AAC ATC ACC ATT GTT GAG TCA 740 Lys Leu Gly Asn Asp Ser Ala Ser Ala Asn Ile Thr Ile Val Glu Ser
120 125 130
AAC GAG TTC ATC ACT GGC ATG CCA GCC TCG ACT GAG ACA GCC TAT GTG 788 Asn Glu Phe Ile Thr Gly Met Pro Ala Ser Thr Glu Thr Ala Tyr Val
135 140 145
TCC TCA GAG TCT CCC ATT AGA ATC TCA GTT TCA ACA GAA GGC GCA AAC 836 Ser Ser Glu Ser Pro Ile Arg Ile Ser Val Ser Thr Glu Gly Ala Asn
150 155 160
ACT TCT TCA TCC ACA TCA ACA TCC ACG ACT GGG ACC AGC CAT CTC ATA 884 Thr Ser Ser Ser Thr Ser Thr Ser Thr Thr Gly Thr Ser His Leu Ile
165 170 175 180
AAG TGT GCG GAG AAG GAG AAA ACT TTC TGT GTG AAT GGG GGC GAG TGC 932 Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu Cys
185 190 195
TTC ACG GTG AAG GAC CTG TCA AAC CCG TCA AGA TAC TTG TGC AAG TGC 980 Phe Thr Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys Lys Cys
200 205 210
CAA CCT GGA TTC ACT GGA GCA AGA TGT ACT GAG AAT GTA CCC ATG AAA 1028 Gln Pro Gly Phe Thr Gly Ala Arg Cys Thr Glu Asn Val Pro Met Lys
215 220 225
GTC CAA ACC CAA GAA AAA GCG GAG GAA CTC TAC CAG AAG AGG GTG CTG 1076 Val Gln Thr Gln Glu Lys Ala Glu Glu Leu Tyr Gln Lys Arg Val Leu
230 235 240
ACA ATT ACT GGC ATC TGT ATC GCC CTG CTG GTG GTC GGC ATC ATG TGT 1124 Thr Ile Thr Gly Ile Cys Ile Ala Leu Leu Val Val Gly Ile Met Cys
245 250 255 260
GTG GTG GCC TAC TGC AAA ACC AAG AAG CAG CGG CAG AAG CTT CAT GAT 1172 Val Val Ala Tyr Cys Lys Thr Lys Lys Gln Arg Gln Lys Leu His Asp
265 270 275
CGG CTT CGG CAG AGT CTT CGG TCA GAA CGG AGC AAC CTG GTG AAC ATA 1220 Arg Leu Arg Gln Ser Leu Arg Ser Glu Arg Ser Asn Leu Val Asn Ile
280 285 290
GCG AAT GGG CCT CAC CAC CCA AAC CCA CCG CCA GAG AAC GTG CAG CTG 1268 Ala Asn Gly Pro His His Pro Asn Pro Pro Pro Glu Asn Val Gln Leu
295 300 305
GTG AAT CAA TAC GTA TCT AAA AAC GTC ATC TCC AGT GAG CAT ATT GTT 1316 Val Asn Gln Tyr Val Ser Lys Asn Val Ile Ser Ser Glu His Ile Val
310 315 320
GAG AGA GAA GTG GAG ACT TCC TTT TCC ACC AGT CAT TAC ACT TCC ACA 1364 Glu Arg Glu Val Glu Thr Ser Phe Ser Thr Ser His Tyr Thr Ser Thr
325 330 335 340
GCC CAT CAC TCC ACG ACT GTC ACC CAG ACT CCT AGT CAC AGC TGG AGT 1412 Ala His His Ser Thr Thr Val Thr Gln Thr Pro Ser His Ser Trp Ser
345 350 355
AAT GGG CAC ACG GAG AGC GTC ATT TCA GAA AGC AAC TCC GTA ATC ATG 1460 Asn Gly His Thr Glu Ser Val Ile Ser Glu Ser Asn Ser Val Ile Met
360 365 370
ATG TCT TCG GTA GAG AAC AGC AGG CAC AGC AGT CCC GCC GGG GGC CCA 1508 Met Ser Ser Val Glu Asn Ser Arg His Ser Ser Pro Ala Gly Gly Pro
375 380 385
CGA GGA CGT CTT CAT GGC CTG GGA GGC CCT CGT GAT AAC AGC TTC CTC 1556 Arg Gly Arg Leu His Gly Leu Gly Gly Pro Arg Asp Asn Ser Phe Leu
390 395 400
AGG CAT GCC AGA GAA ACC CCT GAC TCC TAC AGA GAC TCT CCT CAT AGC 1604 Arg His Ala Arg Glu Thr Pro Asp Ser Tyr Arg Asp Ser Pro His Ser
405 410 415 420
GAA AGA CAT AAC CTT ATA GCT GAG CTA AGG AGA AAC AAG GCT TAC AGA 1652 Glu Arg His Asn Leu Ile Ala Glu Leu Arg Arg Asn Lys Ala Tyr Arg
425 430 435
TCC AAA TGC ATG CAG ATC CAG CTG TCA GCA ACT CAT CTT AGA CCC TCT 1700 Ser Lys Cys Met Gln Ile Gln Leu Ser Ala Thr His Leu Arg Pro Ser
440 445 450
TCC ATT ACC CAT TTG GGC TTC ATT CTC TAAGACCCCT TGGCCTTTAG 1747 Ser Ile Thr His Leu Gly Phe Ile Leu
455 460
GAAGGTATGT ATCAGCCATG ACCACCCCGG CTCGTATGTC ACCTGTAGAT TTCCACACGC 1807
CAAGCTCCCC TAAATCGCCC CCTTCGGAAA TGTCTCCACC CGTGTCCAGC ATGACGGTGT 1867
CCATGCCCTC TGTGGCAGTC AGCCCCTTTG TGGAAGAAGA GAGGCCTCTG CTGCTTGTGA 1927
CGCCACCAAG GCTACGGGAG AAGAAATATG ATCATCACCC CCAGCAACTC AACTCCTTTC 1987
ATCACAACCC TGCACATCAG AGTACCAGCC TCCCCCCTAG CCCACTGAGG ATAGTGGAGG 2047
ATGAGGAGTA CGAGACGACC CAGGAGTATG AGTCAGTTCA AGAGCCCGTT AAGAAAGTCA 2107
CCAATAGCCG GCGGGCCAAA AGAACCAAGC CCAATGGCCA CATTGCCAAT AGGTTGGAAA 2167
TGGACAGCAA CACAAGTTCT GTGAGCAGTA ACTCAGAAAG TGAGACAGAA GACGAAAGAG 2227
TAGGTGAAGA CACACCATTC CTGGGCATAC AGAACCCCCT GGCAGCCAGC CTTGAGGTGG 2287
CCCCTGCCTT CCGTCTGGCT GAGAGCAGGA CTAACCCAGC AGGCCGCTTC TCCACACAGG 2347
AGGAATTACA GGCCAGGCTG TCTAGTGTAA TCGCTAACCA AGACCCTATT GCTGTATAAA 2407
ACCTAAATAA ACACATAGAT TCACCTGTAA AACTTTATTT TATATAATAA AGTATTTCAC 2467
CTTAAATTAA ACAATTTATT TTATTTTAGC AGTTCTGCAA ATAGAAAACA GGAAGAAAAA 2527
AAAACTTTTA TAAATTAAAT ATATGTATGT AAAAATGTGT TATGTGCCAT ATGTAGCAAT 2587
TTTTTACAGT ATTTCAAAAA CGAGAAAGAT ATCAATGGTG CCTTTATGTT CTGTTATGTC 2647
GAGAGCAAGT TTTATAAAGT TATGGTGATT TCTTTTTCAC AGTATTTCAG CAAAACCTCC 2707
CATATATTCA GTTTCTGCTG GCTTTTTGTG CATTGCATTA TGATGTTGAC TGGATGTATG 2767
GTTTGCAAGG CTAGCAGCTC GCTCGTGTTC TCTCTCTCTC TCTCTCTCTC TCTCTCTCTG 2827
TCTCTCTCTC TGTCTCTCTC TCTCTCTCTC TCTCTCTCTC TCTCTCTCTC TCGTGATCCT 2887
CCTGCCTCTG CGGACGCGTG GGTCGAC 2914
(38) INFORMATION FOR SEQ ID NO: 37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 461 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:
Met Ser Glu Arg Lys Glu Gly Arg Gly Lys Gly Lys Gly Lys Lys Lys 1 5 10 15
Asp Arg Gly Ser Arg Gly Lys Pro Gly Pro Ala Glu Gly Asp Pro Ser
20 25 30
Pro Ala Leu Pro Pro Arg Leu Lys Glu Met Lys Ser Gln Glu Ser Ala
35 40 45
Ala Gly Ser Lys Leu Val Leu Arg Cys Glu Thr Ser Ser Glu Tyr Ser
50 55 60
Ser Leu Arg Phe Lys Trp Phe Lys Asn Gly Asn Glu Leu Asn Arg Lys 65 70 75 80
Asn Lys Pro Glu Asn Ile Lys Ile Gln Lys Lys Pro Gly Lys Ser Glu
85 90 95
Leu Arg Ile Asn Lys Ala Ser Leu Ala Asp Ser Gly Glu Tyr Met Cys
100 105 110
Lys Val Ile Ser Lys Leu Gly Asn Asp Ser Ala Ser Ala Asn Ile Thr
115 120 125
Ile Val Glu Ser Asn Glu Phe Ile Thr Gly Met Pro Ala Ser Thr Glu 130 135 140
Thr Ala Tyr Val Ser Ser Glu Ser Pro Ile Arg Ile Ser Val Ser Thr 145 150 155 160
Glu Gly Ala Asn Thr Ser Ser Ser Thr Ser Thr Ser Thr Thr Gly Thr
165 170 175
Ser His Leu Ile Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn
180 185 190
Gly Gly Glu Cys Phe Thr Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr
195 200 205
Leu Cys Lys Cys Gln Pro Gly Phe Thr Gly Ala Arg Cys Thr Glu Asn 210 215 220
Val Pro Met Lys Val Gln Thr Gln Glu Lys Ala Glu Glu Leu Tyr Gln 225 230 235 240
Lys Arg Val Leu Thr Ile Thr Gly Ile Cys Ile Ala Leu Leu Val Val 245 250 255
Gly Ile Met Cys Val Val Ala Tyr Cys Lys Thr Lys Lys Gln Arg Gln
260 265 270
Lys Leu His Asp Arg Leu Arg Gln Ser Leu Arg Ser Glu Arg Ser Asn
275 280 285
Leu Val Asn Ile Ala Asn Gly Pro His His Pro Asn Pro Pro Pro Glu 290 295 300
Asn Val Gln Leu Val Asn Gln Tyr Val Ser Lys Asn Val Ile Ser Ser 305 310 315 320
Glu His Ile Val Glu Arg Glu Val Glu Thr Ser Phe Ser Thr Ser His
325 330 335
Tyr Thr Ser Thr Ala His His Ser Thr Thr Val Thr Gln Thr Pro Ser
340 345 350
His Ser Trp Ser Asn Gly His Thr Glu Ser Val Ile Ser Glu Ser Asn
355 360 365
Ser Val Ile Met Met Ser Ser Val Glu Asn Ser Arg His Ser Ser Pro 370 375 380
Ala Gly Gly Pro Arg Gly Arg Leu His Gly Leu Gly Gly Pro Arg Asp 385 390 395 400
Asn Ser Phe Leu Arg His Ala Arg Glu Thr Pro Asp Ser Tyr Arg Asp
405 410 415
Ser Pro His Ser Glu Arg His Asn Leu Ile Ala Glu Leu Arg Arg Asn
420 425 430
Lys Ala Tyr Arg Ser Lys Cys Met Gln Ile Gln Leu Ser Ala Thr His
435 440 445
Leu Arg Pro Ser Ser Ile Thr His Leu Gly Phe Ile Leu
450 455 460
(39) INFORMATION FOR SEQ ID NO: 38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2531 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
( ix) FEATURE :
(A) NAME/KEY: CDS
(B) LOCATION: 345..1727
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:
GCGGCCGCAG CTGCCGGGAG ATGCGAGCGC AGACCGGATT GTGATCACCT TTCCCTCTTC 60
GGGCTGTAAG AGAGCGAGAC AAGCCACCGA AGCGAGGCCA CTCCAGAGCC GGCAGCGGAG 120
GGACCCGGGA CACTAGAGCA GCTCCGAGCC ACTCCAGACT GAGCGGACGC TCCAGGTGAT 180
CGAGTCCACG CTGCTTCCTG CAGGCGACAG GCGACGCCTC CCGAGCAGCC CGGCCACTGG 240
CTCTTCCCCT CCTGGGACAA ACTTTTCTGC AAGCCCTTGG ACCAAACTTG TCGCGCGTCA 300
CCGTCACCCA ACCGGGTCCG CGTAGAGCGC TCATCTTCGG CGAG ATG TCT GAG CGC 356
Met Ser Glu Arg
1
AAA GAA GGC AGA GGC AAG GGG AAG GGC AAG AAG AAG GAC CGG GGA TCC 404 Lys Glu Gly Arg Gly Lys Gly Lys Gly Lys Lys Lys Asp Arg Gly Ser
5 10 15 20
CGC GGG AAG CCC GGG CCC GCC GAG GGC GAC CCG AGC CCA GCA CTG CCT 452 Arg Gly Lys Pro Gly Pro Ala Glu Gly Asp Pro Ser Pro Ala Leu Pro
25 30 35
CCC AGA TTG AAA GAA ATG AAG AGC CAG GAG TCA GCT GCA GGC TCC AAG 500 Pro Arg Leu Lys Glu Met Lys Ser Gln Glu Ser Ala Ala Gly Ser Lys
40 45 50
CTA GTG CTC CGG TGC GAA ACC AGC TCC GAG TAC TCC TCA CTC AGA TTC 548 Leu Val Leu Arg Cys Glu Thr Ser Ser Glu Tyr Ser Ser Leu Arg Phe
55 60 65
AAA TGG TTC AAG AAT GGG AAC GAG CTG AAC CGC AAA AAT AAA CCA GAA 596 Lys Trp Phe Lys Asn Gly Asn Glu Leu Asn Arg Lys Asn Lys Pro Glu
70 75 80
AAC ATC AAG ATA CAG AAG AAG CCA GGG AAG TCA GAG CTT CGA ATT AAC 644 Asn Ile Lys Ile Gln Lys Lys Pro Gly Lys Ser Glu Leu Arg Ile Asn
85 90 95 100
AAA GCA TCC CTG GCT GAC TCT GGA GAG TAT ATG TGC AAA GTG ATC AGC 692 Lys Ala Ser Leu Ala Asp Ser Gly Glu Tyr Met Cys Lys Val Ile Ser
105 110 115
AAG TTA GGA AAT GAC AGT GCC TCT GCC AAC ATC ACC ATT GTT GAG TCA 740 Lys Leu Gly Asn Asp Ser Ala Ser Ala Asn Ile Thr Ile Val Glu Ser
120 125 130
AAC GAG TTC ATC ACT GGC ATG CCA GCC TCG ACT GAG ACA GCC TAT GTG 788 Asn Glu Phe Ile Thr Gly Met Pro Ala Ser Thr Glu Thr Ala Tyr Val
135 140 145
TCC TCA GAG TCT CCC ATT AGA ATC TCA GTT TCA ACA GAA GGC GCA AAC 836 Ser Ser Glu Ser Pro Ile Arg Ile Ser Val Ser Thr Glu Gly Ala Asn
150 155 160
ACT TCT TCA TCC ACA TCA ACA TCC ACG ACT GGG ACC AGC CAT CTC ATA 884 Thr Ser Ser Ser Thr Ser Thr Ser Thr Thr Gly Thr Ser His Leu Ile
165 170 175 180
AAG TGT GCG GAG AAG GAG AAA ACT TTC TGT GTG AAT GGG GGC GAG TGC 932 Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu Cys
185 190 195
TTC ACG GTG AAG GAC CTG TCA AAC CCG TCA AGA TAC TTG TGC AAG TGC 980 Phe Thr Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys Lys Cys
200 205 210
CAA CCT GGA TTC ACT GGA GCA AGA TGT ACT GAG AAT GTA CCC ATG AAA 1028 Gln Pro Gly Phe Thr Gly Ala Arg Cys Thr Glu Asn Val Pro Met Lys
215 220 225
GTC CAA ACC CAA GAA AAA GCG GAG GAA CTC TAC CAG AAG AGG GTG CTG 1076 Val Gln Thr Gln Glu Lys Ala Glu Glu Leu Tyr Gln Lys Arg Val Leu
230 235 240
ACA ATT ACT GGC ATC TGT ATC GCC CTG CTG GTG GTC GGC ATC ATG TGT 1124 Thr Ile Thr Gly Ile Cys Ile Ala Leu Leu Val Val Gly Ile Met Cys
245 250 255 260
GTG GTG GCC TAC TGC AAA ACC AAG AAG CAG CGG CAG AAG CTT CAT GAT 1172 Val Val Ala Tyr Cys Lys Thr Lys Lys Gln Arg Gln Lys Leu His Asp
265 270 275
CGG CTT CGG CAG AGT CTT CGG TCA GAA CGG AGC AAC CTG GTG AAC ATA 1220 Arg Leu Arg Gln Ser Leu Arg Ser Glu Arg Ser Asn Leu Val Asn Ile
280 285 290
GCG AAT GGG CCT CAC CAC CCA AAC CCA CCG CCA GAG AAC GTG CAG CTG 1268 Ala Asn Gly Pro His His Pro Asn Pro Pro Pro Glu Asn Val Gln Leu
295 300 305
GTG AAT CAA TAC GTA TCT AAA AAC GTC ATC TCC AGT GAG CAT ATT GTT 1316 Val Asn Gln Tyr Val Ser Lys Asn Val Ile Ser Ser Glu His Ile Val
310 315 320
GAG AGA GAA GTG GAG ACT TCC TTT TCC ACC AGT CAT TAC ACT TCC ACA 1364 Glu Arg Glu Val Glu Thr Ser Phe Ser Thr Ser His Tyr Thr Ser Thr
325 330 335 340
GCC CAT CAC TCC ACG ACT GTC ACC CAG ACT CCT AGT CAC AGC TGG AGT 1412 Ala His His Ser Thr Thr Val Thr Gln Thr Pro Ser His Ser Trp Ser
345 350 355
AAT GGG CAC ACG GAG AGC GTC ATT TCA GAA AGC AAC TCC GTA ATC ATG 1460 Asn Gly His Thr Glu Ser Val Ile Ser Glu Ser Asn Ser Val Ile Met
360 365 370
ATG TCT TCG GTA GAG AAC AGC AGG CAC AGC AGT CCC GCC GGG GGC CCA 1508 Met Ser Ser Val Glu Asn Ser Arg His Ser Ser Pro Ala Gly Gly Pro
375 380 385
CGA GGA CGT CTT CAT GGC CTG GGA GGC CCT CGT GAT AAC AGC TTC CTC 1556 Arg Gly Arg Leu His Gly Leu Gly Gly Pro Arg Asp Asn Ser Phe Leu
390 395 400
AGG CAT GCC AGA GAA ACC CCT GAC TCC TAC AGA GAC TCT CCT CAT AGC 1604 Arg His Ala Arg Glu Thr Pro Asp Ser Tyr Arg Asp Ser Pro His Ser
405 410 415 420
GAA AGA CAT AAC CTT ATA GCT GAG CTA AGG AGA AAC AAG GCT TAC AGA 1652 Glu Arg His Asn Leu Ile Ala Glu Leu Arg Arg Asn Lys Ala Tyr Arg
425 430 435
TCC AAA TGC ATG CAG ATC CAG CTG TCA GCA ACT CAT CTT AGA CCC TCT 1700 Ser Lys Cys Met Gln Ile Gln Leu Ser Ala Thr His Leu Arg Pro Ser
440 445 450
TCC ATT ACC CAT TTG GGC TTC ATT CTC TAAGACCCCT TGGCCTTTAG 1747
Ser Ile Thr His Leu Gly Phe Ile Leu
455 460
GAAGGTATGT ATCAGCCATG ACCACCCCGG CTCGTATGTC ACCTGTAGAT TTCCACACGC 1807
CAAGCTCCCC TAAATCGCCC CCTTCGGAAA TGTCTCCACC CGTGTCCAGC ATGACGGTGT 1867
CCATGCCCTC TGTGGCAGTC AGCCCCTTTG TGGAAGAAGA GAGGCCTCTG CTGCTTGTGA 1927
CGCCACCAAG GCTACGGGAG AAGAAATATG ATCATCACCC CCAGCAACTC AACTCCTTTC 1987
ATCACAACCC TGCACATCAG AGTACCAGCC TCCCCCCTAG CCCACTGAGG ATAGTGGAGG 2047
ATGAGGAGTA CGAGACGACC CAGGAGTATG AGTCAGTTCA AGAGCCCGTT AAGAAAGTCA 2107
CCAATAGCCG GCGGGCCAAA AGAACCAAGC CCAATGGCCA CATTGCCAAT AGGTTGGAAA 2167
TGGACAGCAA CACAAGTTCT GTGAGCAGTA ACTCAGAAAG TGAGACAGAA GACGAAAGAG 2227
TAGGTGAAGA CACACCATTC CTGGGCATAC AGAACCCCCT GGCAGCCAGC CTTGAGGTGG 2287
CCCCTGCCTT CCGTCTGGCT GAGAGCAGGA CTAACCCAGC AGGCCGCTTC TCCACACAGG 2347
AGGAATTACA GGCCAGGCTG TCTAGTGTAA TCGCTAACCA AGACCCTATT GCTGTATAAA 2407
ACCTAAATAA ACACATAGAT TCACCTGTAA AACTTTATTT TATATAATAA AGTATTTCAC 2467
CTTAAAAAAA AAAAAAAAGG ACGGCCGCTC GCGATCTAGA ACTAGTCCGG ACGCGTGGGT 2527
CGAC 2531
(40) INFORMATION FOR SEQ ID NO: 39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 461 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:
Met Ser Glu Arg Lys Glu Gly Arg Gly Lys Gly Lys Gly Lys Lys Lys
1 5 10 15
Asp Arg Gly Ser Arg Gly Lys Pro Gly Pro Ala Glu Gly Asp Pro Ser
20 25 30
Pro Ala Leu Pro Pro Arg Leu Lys Glu Met Lys Ser Gln Glu Ser Ala
35 40 45
Ala Gly Ser Lys Leu Val Leu Arg Cys Glu Thr Ser Ser Glu Tyr Ser
50 55 60
Ser Leu Arg Phe Lys Trp Phe Lys Asn Gly Asn Glu Leu Asn Arg Lys
65 70 75 80
Asn Lys Pro Glu Asn Ile Lys Ile Gln Lys Lys Pro Gly Lys Ser Glu
85 90 95
Leu Arg Ile Asn Lys Ala Ser Leu Ala Asp Ser Gly Glu Tyr Met Cys
100 105 110
Lys Val Ile Ser Lys Leu Gly Asn Asp Ser Ala Ser Ala Asn Ile Thr
115 120 125
Ile Val Glu Ser Asn Glu Phe Ile Thr Gly Met Pro Ala Ser Thr Glu
130 135 140
Thr Ala Tyr Val Ser Ser Glu Ser Pro Ile Arg Ile Ser Val Ser Thr
145 150 155 160
Glu Gly Ala Asn Thr Ser Ser Ser Thr Ser Thr Ser Thr Thr Gly Thr
165 170 175
Ser His Leu Ile Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn 180 185 190
Gly Gly Glu Cys Phe Thr Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr
195 200 205
Leu Cys Lys Cys Gln Pro Gly Phe Thr Gly Ala Arg Cys Thr Glu Asn 210 215 220
Val Pro Met Lys Val Gln Thr Gln Glu Lys Ala Glu Glu Leu Tyr Gln 225 230 235 240
Lys Arg Val Leu Thr Ile Thr Gly Ile Cys Ile Ala Leu Leu Val Val
245 250 255
Gly Ile Met Cys Val Val Ala Tyr Cys Lys Thr Lys Lys Gln Arg Gln
260 265 270
Lys Leu His Asp Arg Leu Arg Gln Ser Leu Arg Ser Glu Arg Ser Asn
275 280 285
Leu Val Asn Ile Ala Asn Gly Pro His His Pro Asn Pro Pro Pro Glu 290 295 300
Asn Val Gln Leu Val Asn Gln Tyr Val Ser Lys Asn Val Ile Ser Ser 305 310 315 320
Glu His Ile Val Glu Arg Glu Val Glu Thr Ser Phe Ser Thr Ser His
325 330 335
Tyr Thr Ser Thr Ala His His Ser Thr Thr Val Thr Gln Thr Pro Ser
340 345 350
His Ser Trp Ser Asn Gly His Thr Glu Ser Val Ile Ser Glu Ser Asn
355 360 365
Ser Val Ile Met Met Ser Ser Val Glu Asn Ser Arg His Ser Ser Pro 370 375 380
Ala Gly Gly Pro Arg Gly Arg Leu His Gly Leu Gly Gly Pro Arg Asp 385 390 395 400
Asn Ser Phe Leu Arg His Ala Arg Glu Thr Pro Asp Ser Tyr Arg Asp
405 410 415
Ser Pro His Ser Glu Arg His Asn Leu Ile Ala Glu Leu Arg Arg Asn
420 425 430
Lys Ala Tyr Arg Ser Lys Cys Met Gln Ile Gln Leu Ser Ala Thr His
435 440 445
Leu Arg Pro Ser Ser Ile Thr His Leu Gly Phe Ile Leu
450 455 460
(41) INFORMATION FOR SEQ ID NO: 40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3344 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 345..2252
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:
GCGGCCGCAG CTGCCGGGAG ATGCGAGCGC AGACCGGATT GTGATCACCT TTCCCTCTTC 60
GGGCTGTAAG AGAGCGAGAC AAGCCACCGA AGCGAGGCCA CTCCAGAGCC GGCAGCGGAG 120
GGACCCGGGA CACTAGAGCA GCTCCGAGCC ACTCCAGACT GAGCGGACGC TCCAGGTGAT 180
CGAGTCCACG CTGCTTCCTG CAGGCGACAG GCGACGCCTC CCGAGCAGCC CGGCCACTGG 240
CTCTTCCCCT CCTGGGACAA ACTTTTCTGC AAGCCCTTGG ACCAAACTTG TCGCGCGTCA 300
CCGTCACCCA ACCGGGTCCG CGTAGAGCGC TCATCTTCGG CGAG ATG TCT GAG CGC 356
Met Ser Glu Arg
1
AAA GAA GGC AGA GGC AAG GGG AAG GGC AAG AAG AAG GAC CGG GGA TCC 404 Lys Glu Gly Arg Gly Lys Gly Lys Gly Lys Lys Lys Asp Arg Gly Ser
5 10 15 20
CGC GGG AAG CCC GGG CCC GCC GAG GGC GAC CCG AGC CCA GCA CTG CCT 452 Arg Gly Lys Pro Gly Pro Ala Glu Gly Asp Pro Ser Pro Ala Leu Pro
25 30 35
CCC AGA TTG AAA GAA ATG AAG AGC CAG GAG TCA GCT GCA GGC TCC AAG 500 Pro Arg Leu Lys Glu Met Lys Ser Gln Glu Ser Ala Ala Gly Ser Lys
40 45 50
CTA GTG CTC CGG TGC GAA ACC AGC TCC GAG TAC TCC TCA CTC AGA TTC 548 Leu Val Leu Arg Cys Glu Thr Ser Ser Glu Tyr Ser Ser Leu Arg Phe
55 60 65
AAA TGG TTC AAG AAT GGG AAC GAG CTG AAC CGC AAA AAT AAA CCA GAA 596 Lys Trp Phe Lys Asn Gly Asn Glu Leu Asn Arg Lys Asn Lys Pro Glu
70 75 80
AAC ATC AAG ATA CAG AAG AAG CCA GGG AAG TCA GAG CTT CGA ATT AAC 644 Asn Ile Lys Ile Gln Lys Lys Pro Gly Lys Ser Glu Leu Arg Ile Asn
85 90 95 100
AAA GCA TCC CTG GCT GAC TCT GGA GAG TAT ATG TGC AAA GTG ATC AGC 692 Lys Ala Ser Leu Ala Asp Ser Gly Glu Tyr Met Cys Lys Val Ile Ser
105 110 115
AAG TTA GGA AAT GAC AGT GCC TCT GCC AAC ATC ACC ATT GTT GAG TCA 740 Lys Leu Gly Asn Asp Ser Ala Ser Ala Asn Ile Thr Ile Val Glu Ser
120 125 130
AAC GAG TTC ATC ACT GGC ATG CCA GCC TCG ACT GAG ACA GCC TAT GTG 788 Asn Glu Phe Ile Thr Gly Met Pro Ala Ser Thr Glu Thr Ala Tyr Val
135 140 145
TCC TCA GAG TCT CCC ATT AGA ATC TCA GTT TCA ACA GAA GGC GCA AAC 836 Ser Ser Glu Ser Pro Ile Arg Ile Ser Val Ser Thr Glu Gly Ala Asn
150 155 160
ACT TCT TCA TCC ACA TCA ACA TCC ACG ACT GGG ACC AGC CAT CTC ATA 884 Thr Ser Ser Ser Thr Ser Thr Ser Thr Thr Gly Thr Ser His Leu Ile
165 170 175 180
AAG TGT GCG GAG AAG GAG AAA ACT TTC TGT GTG AAT GGG GGC GAG TGC 932 Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu Cys
185 190 195
TTC ACG GTG AAG GAC CTG TCA AAC CCG TCA AGA TAC TTG TGC AAG TGC 980 Phe Thr Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys Lys Cys
200 205 210
CCA AAT GAG TTT ACT GGT GAT CGT TGC CAA AAC TAC GTA ATG GCC AGC 1028 Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr Val Met Ala Ser
215 220 225
TTC TAC AAA GCG GAG GAA CTC TAC CAG AAG AGG GTG CTG ACA ATT ACT 1076 Phe Tyr Lys Ala Glu Glu Leu Tyr Gln Lys Arg Val Leu Thr Ile Thr
230 235 240
GGC ATC TGT ATC GCC CTG CTG GTG GTC GGC ATC ATG TGT GTG GTG GCC 1124 Gly Ile Cys Ile Ala Leu Leu Val Val Gly Ile Met Cys Val Val Ala
245 250 255 260
TAC TGC AAA ACC AAG AAG CAG CGG CAG AAG CTT CAT GAT CGG CTT CGG 1172 Tyr Cys Lys Thr Lys Lys Gln Arg Gln Lys Leu His Asp Arg Leu Arg
265 270 275
CAG AGT CTT CGG TCA GAA CGG AGC AAC CTG GTG AAC ATA GCG AAT GGG 1220 Gln Ser Leu Arg Ser Glu Arg Ser Asn Leu Val Asn Ile Ala Asn Gly
280 285 290
CCT CAC CAC CCA AAC CCA CCG CCA GAG AAC GTG CAG CTG GTG AAT CAA 1268 Pro His His Pro Asn Pro Pro Pro Glu Asn Val Gln Leu Val Asn Gln
295 300 305
TAC GTA TCT AAA AAC GTC ATC TCC AGT GAG CAT ATT GTT GAG AGA GAA 1316 Tyr Val Ser Lys Asn Val Ile Ser Ser Glu His Ile Val Glu Arg Glu
310 315 320
GTG GAG ACT TCC TTT TCC ACC AGT CAT TAC ACT TCC ACA GCC CAT CAC 1364 Val Glu Thr Ser Phe Ser Thr Ser His Tyr Thr Ser Thr Ala His His
325 330 335 340
TCC ACG ACT GTC ACC CAG ACT CCT AGT CAC AGC TGG AGT AAT GGG CAC 1412 Ser Thr Thr Val Thr Gln Thr Pro Ser His Ser Trp Ser Asn Gly His
345 350 355
ACG GAG AGC GTC ATT TCA GAA AGC AAC TCC GTA ATC ATG ATG TCT TCG 1460 Thr Glu Ser Val Ile Ser Glu Ser Asn Ser Val Ile Met Met Ser Ser
360 365 370
GTA GAG AAC AGC AGG CAC AGC AGT CCC GCC GGG GGC CCA CGA GGA CGT 1508 Val Glu Asn Ser Arg His Ser Ser Pro Ala Gly Gly Pro Arg Gly Arg
375 380 385
CTT CAT GGC CTG GGA GGC CCT CGT GAT AAC AGC TTC CTC AGG CAT GCC 1556 Leu His Gly Leu Gly Gly Pro Arg Asp Asn Ser Phe Leu Arg His Ala
390 395 400
AGA GAA ACC CCT GAC TCC TAC AGA GAC TCT CCT CAT AGC GAA AGG TAT 1604 Arg Glu Thr Pro Asp Ser Tyr Arg Asp Ser Pro His Ser Glu Arg Tyr
405 410 415 420
GTA TCA GCC ATG ACC ACC CCG GCT CGT ATG TCA CCT GTA GAT TTC CAC 1652 Val Ser Ala Met Thr Thr Pro Ala Arg Met Ser Pro Val Asp Phe His
425 430 435
ACG CCA AGC TCC CCT AAA TCG CCC CCT TCG GAA ATG TCT CCA CCC GTG 1700 Thr Pro Ser Ser Pro Lys Ser Pro Pro Ser Glu Met Ser Pro Pro Val
440 445 450
TCC AGC ATG ACG GTG TCC ATG CCC TCT GTG GCA GTC AGC CCC TTT GTG 1748 Ser Ser Met Thr Val Ser Met Pro Ser Val Ala Val Ser Pro Phe Val
455 460 465
GAA GAA GAG AGG CCT CTG CTG CTT GTG ACG CCA CCA AGG CTA CGG GAG 1796 Glu Glu Glu Arg Pro Leu Leu Leu Val Thr Pro Pro Arg Leu Arg Glu
470 475 480
AAG AAA TAT GAT CAT CAC CCC CAG CAA CTC AAC TCC TTT CAT CAC AAC 1844 Lys Lys Tyr Asp His His Pro Gln Gln Leu Asn Ser Phe His His Asn
485 490 495 500
CCT GCA CAT CAG AGT ACC AGC CTC CCC CCT AGC CCA CTG AGG ATA GTG 1892 Pro Ala His Gln Ser Thr Ser Leu Pro Pro Ser Pro Leu Arg Ile Val
505 510 515
GAG GAT GAG GAG TAC GAG ACG ACC CAG GAG TAT GAG TCA GTT CAA GAG 1940 Glu Asp Glu Glu Tyr Glu Thr Thr Gln Glu Tyr Glu Ser Val Gln Glu
520 525 530
CCC GTT AAG AAA GTC ACC AAT AGC CGG CGG GCC AAA AGA ACC AAG CCC 1988 Pro Val Lys Lys Val Thr Asn Ser Arg Arg Ala Lys Arg Thr Lys Pro
535 540 545
AAT GGC CAC ATT GCC AAT AGG TTG GAA ATG GAC AGC AAC ACA AGT TCT 2036 Asn Gly His Ile Ala Asn Arg Leu Glu Met Asp Ser Asn Thr Ser Ser
550 555 560
GTG AGC AGT AAC TCA GAA AGT GAG ACA GAA GAC GAA AGA GTA GGT GAA 2084 Val Ser Ser Asn Ser Glu Ser Glu Thr Glu Asp Glu Arg Val Gly Glu
565 570 575 580
GAC ACA CCA TTC CTG GGC ATA CAG AAC CCC CTG GCA GCC AGC CTT GAG 2132 Asp Thr Pro Phe Leu Gly Ile Gln Asn Pro Leu Ala Ala Ser Leu Glu
585 590 595
GTG GCC CCT GCC TTC CGT CTG GCT GAG AGC AGG ACT AAC CCA GCA GGC 2180 Val Ala Pro Ala Phe Arg Leu Ala Glu Ser Arg Thr Asn Pro Ala Gly
600 605 610
CGC TTC TCC ACA CAG GAG GAA TTA CAG GCC AGG CTG TCT AGT GTA ATC 2228 Arg Phe Ser Thr Gln Glu Glu Leu Gln Ala Arg Leu Ser Ser Val Ile
615 620 625
GCT AAC CAA GAC CCT ATT GCT GTA TAAAACCTAA ATAAACACAT AGATTCACCT 2282 Ala Asn Gln Asp Pro Ile Ala Val
630 635
GTAAAACTTT ATTTTATATA ATAAAGTATT TCACCTTAAA TTAAACAATT TATTTTATTT 2342
TAGCAGTTCT GCAAATAGAA AACAGGAAGA AAAAAAAACT TTTATAAATT AAATATATGT 2402
ATGTAAAAAT GTGTTATGTG CCATATGTAG CAATTTTTTT ACAGTATTTC AAAAACGAGA 2462
AAGATATCAA TGGTGCCTTT ATGTTCTGTT ATGTCGAGAG CAAGTTTTAT AAAGTTATGG 2522
TGATTTCTTT TTCACAGTAT TTCAGCAAAA CCTCCCATAT ATTCAGTTTC TGCTGGCTTT 2582
TTGTGCATTG CATTATGATG TTGACTGGAT GTATGGTTTG CAAGGCTAGC AGCTCGCTCG 2642
TGTTCTCTCT CTCTCTCTCT CTCTCTCTCT CTCTGTCTCT CTCTCTGTCT CTCTCTCTCT 2702
CTCTCTCTCT CTCTCTCTCT CTCTCTCTCT CTCTCTCTCT CTGTCTCTCT CTCTGCTTCC 2762
CGTAGCTCCC AACCAGTACT GTCTTGGACT GGCACATCCA TCCAAATACC TTTCTACTTT 2822
GTATGAAGTT TTCTTTGCTT TCCCAATATG AAATGAGTTC TCTCTACTCT GTCAGCCAAA 2882
GGTTTGCTTC ACTGGACTCT GAGATAATAG TAGACCCAGC AGCATGCTAC TATTACGTAT 2942
AGCAGGAAAC TGCACCAAGT AATGTCCAAT AATAGGAAGA AAGTAATACT GTGATTTAAA 3002
AAAAAAAACA AACTATATTA TTAATCAGAA GACAGCTTGC TCTTGGTAAA AGGAGCTACC 3062
ATTGACTCTA ATTTTGACTT TTTAGTTATT GTTCTTGACA AAGAGTAACA GCTTCAAGTA 3122
CAGCCTAGAA AAAAAAATGG GTTCTGGCCT GCTATCAGGA TAAATCTATC GACGTAGATA 3182
GATTCAACTC AGTTTCACTT TCTGTCTTGG GGGAAATGAT CCAGCCACTC ATATGACGAC 3242
CAACCAACCA CAGGTGCCTC TGCTCCCTGT AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 3302
AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAGGGCGGCC GC 3344
(42) INFORMATION FOR SEQ ID NO: 41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 636 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:
Met Ser Glu Arg Lys Glu Gly Arg Gly Lys Gly Lys Gly Lys Lys Lys
1 5 10 15
Asp Arg Gly Ser Arg Gly Lys Pro Gly Pro Ala Glu Gly Asp Pro Ser
20 25 30
Pro Ala Leu Pro Pro Arg Leu Lys Glu Met Lys Ser Gln Glu Ser Ala
35 40 45
Ala Gly Ser Lys Leu Val Leu Arg Cys Glu Thr Ser Ser Glu Tyr Ser
50 55 60
Ser Leu Arg Phe Lys Trp Phe Lys Asn Gly Asn Glu Leu Asn Arg Lys
65 70 75 80
Asn Lys Pro Glu Asn Ile Lys Ile Gln Lys Lys Pro Gly Lys Ser Glu
85 90 95
Leu Arg Ile Asn Lys Ala Ser Leu Ala Asp Ser Gly Glu Tyr Met Cys 100 105 110
Lys Val Ile Ser Lys Leu Gly Asn Asp Ser Ala Ser Ala Asn Ile Thr
115 120 125
Ile Val Glu Ser Asn Glu Phe Ile Thr Gly Met Pro Ala Ser Thr Glu 130 135 140
Thr Ala Tyr Val Ser Ser Glu Ser Pro Ile Arg Ile Ser Val Ser Thr 145 150 155 160
Glu Gly Ala Asn Thr Ser Ser Ser Thr Ser Thr Ser Thr Thr Gly Thr
165 170 175
Ser His Leu Ile Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn
180 185 190
Gly Gly Glu Cys Phe Thr Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr
195 200 205
Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr 210 215 220
Val Met Ala Ser Phe Tyr Lys Ala Glu Glu Leu Tyr Gln Lys Arg Val 225 230 235 240
Leu Thr Ile Thr Gly Ile Cys Ile Ala Leu Leu Val Val Gly Ile Met
245 250 255
Cys Val Val Ala Tyr Cys Lys Thr Lys Lys Gln Arg Gln Lys Leu His
260 265 270
Asp Arg Leu Arg Gln Ser Leu Arg Ser Glu Arg Ser Asn Leu Val Asn
275 280 285
Ile Ala Asn Gly Pro His His Pro Asn Pro Pro Pro Glu Asn Val Gln 290 295 300
Leu Val Asn Gln Tyr Val Ser Lys Asn Val Ile Ser Ser Glu His Ile 305 310 315 320
Val Glu Arg Glu Val Glu Thr Ser Phe Ser Thr Ser His Tyr Thr Ser
325 330 335
Thr Ala His His Ser Thr Thr Val Thr Gln Thr Pro Ser His Ser Trp
340 345 350
Ser Asn Gly His Thr Glu Ser Val Ile Ser Glu Ser Asn Ser Val Ile
355 360 365
Met Met Ser Ser Val Glu Asn Ser Arg His Ser Ser Pro Ala Gly Gly 370 375 380
Pro Arg Gly Arg Leu His Gly Leu Gly Gly Pro Arg Asp Asn Ser Phe 385 390 395 400
Leu Arg His Ala Arg Glu Thr Pro Asp Ser Tyr Arg Asp Ser Pro His
405 410 415
Ser Glu Arg Tyr Val Ser Ala Met Thr Thr Pro Ala Arg Met Ser Pro
420 425 430
Val Asp Phe His Thr Pro Ser Ser Pro Lys Ser Pro Pro Ser Glu Met
435 440 445
Ser Pro Pro Val Ser Ser Met Thr Val Ser Met Pro Ser Val Ala Val 450 455 460
Ser Pro Phe Val Glu Glu Glu Arg Pro Leu Leu Leu Val Thr Pro Pro 465 470 475 480
Arg Leu Arg Glu Lys Lys Tyr Asp His His Pro Gln Gln Leu Asn Ser
485 490 495
Phe His His Asn Pro Ala His Gln Ser Thr Ser Leu Pro Pro Ser Pro
500 505 510
Leu Arg Ile Val Glu Asp Glu Glu Tyr Glu Thr Thr Gln Glu Tyr Glu
515 520 525
Ser Val Gln Glu Pro Val Lys Lys Val Thr Asn Ser Arg Arg Ala Lys 530 535 540
Arg Thr Lys Pro Asn Gly His Ile Ala Asn Arg Leu Glu Met Asp Ser 545 550 555 560
Asn Thr Ser Ser Val Ser Ser Asn Ser Glu Ser Glu Thr Glu Asp Glu
565 570 575
Arg Val Gly Glu Asp Thr Pro Phe Leu Gly Ile Gln Asn Pro Leu Ala
580 585 590
Ala Ser Leu Glu Val Ala Pro Ala Phe Arg Leu Ala Glu Ser Arg Thr
595 600 605
Asn Pro Ala Gly Arg Phe Ser Thr Gln Glu Glu Leu Gln Ala Arg Leu 610 615 620
Ser Ser Val Ile Ala Asn Gln Asp Pro Ile Ala Val
625 630 635
(43) INFORMATION FOR SEQ ID NO: 42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2356 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 345..2261
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:
GCGGCCGCAG CTGCCGGGAG ATGCGAGCGC AGACCGGATT GTGATCACCT TTCCCTCTTC 60
GGGCTGTAAG AGAGCGAGAC AAGCCACCGA AGCGAGGCCA CTCCAGAGCC GGCAGCGGAG 120
GGACCCGGGA CACTAGAGCA GCTCCGAGCC ACTCCAGACT GAGCGGACGC TCCAGGTGAT 180
CGAGTCCACG CTGCTTCCTG CAGGCGACAG GCGACGCCTC CCGAGCAGCC CGGCCACTGG 240
CTCTTCCCCT CCTGGGACAA ACTTTTCTGC AAGCCCTTGG ACCAAACTTG TCGCGCGTCA 300
CCGTCACCCA ACCGGGTCCG CGTAGAGCGC TCATCTTCGG CGAG ATG TCT GAG CGC 356
Met Ser Glu Arg
1
AAA GAA GGC AGA GGC AAG GGG AAG GGC AAG AAG AAG GAC CGG GGA TCC 404 Lys Glu Gly Arg Gly Lys Gly Lys Gly Lys Lys Lys Asp Arg Gly Ser
5 10 15 20
CGC GGG AAG CCC GGG CCC GCC GAG GGC GAC CCG AGC CCA GCA CTG CCT 452 Arg Gly Lys Pro Gly Pro Ala Glu Gly Asp Pro Ser Pro Ala Leu Pro
25 30 35
CCC AGA TTG AAA GAA ATG AAG AGC CAG GAG TCA GCT GCA GGC TCC AAG 500 Pro Arg Leu Lys Glu Met Lys Ser Gln Glu Ser Ala Ala Gly Ser Lys
40 45 50
CTA GTG CTC CGG TGC GAA ACC AGC TCC GAG TAC TCC TCA CTC AGA TTC 548 Leu Val Leu Arg Cys Glu Thr Ser Ser Glu Tyr Ser Ser Leu Arg Phe
55 60 65
AAA TGG TTC AAG AAT GGG AAC GAG CTG AAC CGC AAA AAT AAA CCA GAA 596 Lys Trp Phe Lys Asn Gly Asn Glu Leu Asn Arg Lys Asn Lys Pro Glu
70 75 80
AAC ATC AAG ATA CAG AAG AAG CCA GGG AAG TCA GAG CTT CGA ATT AAC 644 Asn Ile Lys Ile Gln Lys Lys Pro Gly Lys Ser Glu Leu Arg Ile Asn
85 90 95 100
AAA GCA TCC CTG GCT GAC TCT GGA GAG TAT ATG TGC AAA GTG ATC AGC 692 Lys Ala Ser Leu Ala Asp Ser Gly Glu Tyr Met Cys Lys Val Ile Ser
105 110 115
AAG TTA GGA AAT GAC AGT GCC TCT GCC AAC ATC ACC ATT GTT GAG TCA 740 Lys Leu Gly Asn Asp Ser Ala Ser Ala Asn Ile Thr Ile Val Glu Ser
120 125 130
AAC GAG TTC ATC ACT GGC ATG CCA GCC TCG ACT GAG ACA GCC TAT GTG 788 Asn Glu Phe Ile Thr Gly Met Pro Ala Ser Thr Glu Thr Ala Tyr Val
135 140 145
TCC TCA GAG TCT CCC ATT AGA ATC TCA GTT TCA ACA GAA GGC GCA AAC 836 Ser Ser Glu Ser Pro Ile Arg Ile Ser Val Ser Thr Glu Gly Ala Asn
150 155 160
ACT TCT TCA TCC ACA TCA ACA TCC ACG ACT GGG ACC AGC CAT CTC ATA 884 Thr Ser Ser Ser Thr Ser Thr Ser Thr Thr Gly Thr Ser His Leu Ile
165 170 175 180
AAG TGT GCG GAG AAG GAG AAA ACT TTC TGT GTG AAT GGG GGC GAG TGC 932 Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu Cys
185 190 195
TTC ACG GTG AAG GAC CTG TCA AAC CCG TCA AGA TAC TTG TGC AAG TGC 980 Phe Thr Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys Lys Cys
200 205 210
CAA CCT GGA TTC ACT GGA GCA AGA TGT ACT GAG AAT GTA CCC ATG AAA 1028 Gln Pro Gly Phe Thr Gly Ala Arg Cys Thr Glu Asn Val Pro Met Lys
215 220 225
GTC CAA ACC CAA GAA AAA GCG GAG GAA CTC TAC CAG AAG AGG GTG CTG 1076 Val Gln Thr Gln Glu Lys Ala Glu Glu Leu Tyr Gln Lys Arg Val Leu
230 235 240
ACA ATT ACT GGC ATC TGT ATC GCC CTG CTG GTG GTC GGC ATC ATG TGT 1124 Thr Ile Thr Gly Ile Cys Ile Ala Leu Leu Val Val Gly Ile Met Cys
245 250 255 260
GTG GTG GCC TAC TGC AAA ACC AAG AAG CAG CGG CAG AAG CTT CAT GAT 1172 Val Val Ala Tyr Cys Lys Thr Lys Lys Gln Arg Gln Lys Leu His Asp
265 270 275
CGG CTT CGG CAG AGT CTT CGG TCA GAA CGG AGC AAC CTG GTG AAC ATA 1220 Arg Leu Arg Gln Ser Leu Arg Ser Glu Arg Ser Asn Leu Val Asn Ile
280 285 290
GCG AAT GGG CCT CAC CAC CCA AAC CCA CCG CCA GAG AAC GTG CAG CTG 1268 Ala Asn Gly Pro His His Pro Asn Pro Pro Pro Glu Asn Val Gln Leu
295 300 305
GTG AAT CAA TAC GTA TCT AAA AAC GTC ATC TCC AGT GAG CAT ATT GTT 1316 Val Asn Gln Tyr Val Ser Lys Asn Val Ile Ser Ser Glu His Ile Val
310 315 320
GAG AGA GAA GTG GAG ACT TCC TTT TCC ACC AGT CAT TAC ACT TCC ACA 1364 Glu Arg Glu Val Glu Thr Ser Phe Ser Thr Ser His Tyr Thr Ser Thr
325 330 335 340
GCC CAT CAC TCC ACG ACT GTC ACC CAG ACT CCT AGT CAC AGC TGG AGT 1412 Ala His His Ser Thr Thr Val Thr Gln Thr Pro Ser His Ser Trp Ser
345 350 355
AAT GGG CAC ACG GAG AGC GTC ATT TCA GAA AGC AAC TCC GTA ATC ATG 1460 Asn Gly His Thr Glu Ser Val Ile Ser Glu Ser Asn Ser Val Ile Met
360 365 370
ATG TCT TCG GTA GAG AAC AGC AGG CAC AGC AGT CCC GCC GGG GGC CCA 1508 Met Ser Ser Val Glu Asn Ser Arg His Ser Ser Pro Ala Gly Gly Pro
375 380 385
CGA GGA CGT CTT CAT GGC CTG GGA GGC CCT CGT GAT AAC AGC TTC CTC 1556 Arg Gly Arg Leu His Gly Leu Gly Gly Pro Arg Asp Asn Ser Phe Leu
390 395 400
AGG CAT GCC AGA GAA ACC CCT GAC TCC TAC AGA GAC TCT CCT CAT AGC 1604 Arg His Ala Arg Glu Thr Pro Asp Ser Tyr Arg Asp Ser Pro His Ser
405 410 415 420
GAA AGG TAT GTA TCA GCC ATG ACC ACC CCG GCT CGT ATG TCA CCT GTA 1652 Glu Arg Tyr Val Ser Ala Met Thr Thr Pro Ala Arg Met Ser Pro Val
425 430 435
GAT TTC CAC ACG CCA AGC TCC CCT AAA TCG CCC CCT TCG GAA ATG TCT 1700 Asp Phe His Thr Pro Ser Ser Pro Lys Ser Pro Pro Ser Glu Met Ser
440 445 450
CCA CCC GTG TCC AGC ATG ACG GTG TCC ATG CCC TCT GTG GCA GTC AGC 1748 Pro Pro Val Ser Ser Met Thr Val Ser Met Pro Ser Val Ala Val Ser
455 460 465
CCC TTT GTG GAA GAA GAG AGG CCT CTG CTG CTT GTG ACG CCA CCA AGG 1796 Pro Phe Val Glu Glu Glu Arg Pro Leu Leu Leu Val Thr Pro Pro Arg
470 475 480
CTA CGG GAG AAG AAA TAT GAT CAT CAC CCC CAG CAA CTC AAC TCC TTT 1844 Leu Arg Glu Lys Lys Tyr Asp His His Pro Gln Gln Leu Asn Ser Phe
485 490 495 500
CAT CAC AAC CCT GCA CAT CAG AGT ACC AGC CTC CCC CCT AGC CCA CTG 1892 His His Asn Pro Ala His Gln Ser Thr Ser Leu Pro Pro Ser Pro Leu
505 510 515
AGG ATA GTG GAG GAT GAG GAG TAC GAG ACG ACC CAG GAG TAT GAG TCA 1940 Arg Ile Val Glu Asp Glu Glu Tyr Glu Thr Thr Gln Glu Tyr Glu Ser
520 525 530
GTT CAA GAG CCC GTT AAG AAA GTC ACC AAT AGC CGG CGG GCC AAA AGA 1988 Val Gln Glu Pro Val Lys Lys Val Thr Asn Ser Arg Arg Ala Lys Arg
535 540 545
ACC AAG CCC AAT GGC CAC ATT GCC AAT AGG TTG GAA ATG GAC AGC AAC 2036 Thr Lys Pro Asn Gly His Ile Ala Asn Arg Leu Glu Met Asp Ser Asn
550 555 560
ACA AGT TCT GTG AGC AGT AAC TCA GAA AGT GAG ACA GAA GAC GAA AGA 2084 Thr Ser Ser Val Ser Ser Asn Ser Glu Ser Glu Thr Glu Asp Glu Arg
565 570 575 580
GTA GGT GAA GAC ACA CCA TTC CTG GGC ATA CAG AAC CCC CTG GCA GCC 2132 Val Gly Glu Asp Thr Pro Phe Leu Gly Ile Gln Asn Pro Leu Ala Ala
585 590 595
AGC CTT GAG GTG GCC CCT GCC TTC CGT CTG GCT GAG AGC AGG ACT AAC 2180 Ser Leu Glu Val Ala Pro Ala Phe Arg Leu Ala Glu Ser Arg Thr Asn
600 605 610
CCA GCA GGC CGC TTC TCC ACA CAG GAG GAA TTA CAG GCC AGG CTG TCT 2228 Pro Ala Gly Arg Phe Ser Thr Gln Glu Glu Leu Gln Ala Arg Leu Ser
615 620 625
AGT GTA ATC GCT AAC CAA GAC CCT ATT GCT GTA TAAAACCTAA ATAAACACAT 2281 Ser Val Ile Ala Asn Gln Asp Pro Ile Ala Val
630 635
AGATTCACCT GTAAAACTTT ATTTTATATA ATAAAGTATT TCACCTTAAA AAAAAAAAAA 2341
AAAAAAGGGG GCCGC 2356
(44) INFORMATION FOR SEQ ID NO: 43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 639 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:
Met Ser Glu Arg Lys Glu Gly Arg Gly Lys Gly Lys Gly Lys Lys Lys
1 5 10 15
Asp Arg Gly Ser Arg Gly Lys Pro Gly Pro Ala Glu Gly Asp Pro Ser 20 25 30
Pro Ala Leu Pro Pro Arg Leu Lys Glu Met Lys Ser Gln Glu Ser Ala
35 40 45
Ala Gly Ser Lys Leu Val Leu Arg Cys Glu Thr Ser Ser Glu Tyr Ser 50 55 60
Ser Leu Arg Phe Lys Trp Phe Lys Asn Gly Asn Glu Leu Asn Arg Lys 65 70 75 80
Asn Lys Pro Glu Asn Ile Lys Ile Gln Lys Lys Pro Gly Lys Ser Glu
85 90 95
Leu Arg Ile Asn Lys Ala Ser Leu Ala Asp Ser Gly Glu Tyr Met Cys
100 105 110
Lys Val Ile Ser Lys Leu Gly Asn Asp Ser Ala Ser Ala Asn Ile Thr
115 120 125
Ile Val Glu Ser Asn Glu Phe Ile Thr Gly Met Pro Ala Ser Thr Glu 130 135 140
Thr Ala Tyr Val Ser Ser Glu Ser Pro Ile Arg Ile Ser Val Ser Thr 145 150 155 160
Glu Gly Ala Asn Thr Ser Ser Ser Thr Ser Thr Ser Thr Thr Gly Thr
165 170 175
Ser His Leu Ile Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn
180 185 190
Gly Gly Glu Cys Phe Thr Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr
195 200 205
Leu Cys Lys Cys Gln Pro Gly Phe Thr Gly Ala Arg Cys Thr Glu Asn 210 215 220
Val Pro Met Lys Val Gln Thr Gln Glu Lys Ala Glu Glu Leu Tyr Gln 225 230 235 240
Lys Arg Val Leu Thr Ile Thr Gly Ile Cys Ile Ala Leu Leu Val Val
245 250 255
Gly Ile Met Cys Val Val Ala Tyr Cys Lys Thr Lys Lys Gln Arg Gln
260 265 270
Lys Leu His Asp Arg Leu Arg Gln Ser Leu Arg Ser Glu Arg Ser Asn
275 280 285
Leu Val Asn Ile Ala Asn Gly Pro His His Pro Asn Pro Pro Pro Glu 290 295 300
Asn Val Gln Leu Val Asn Gln Tyr Val Ser Lys Asn Val Ile Ser Ser 305 310 315 320
Glu His Ile Val Glu Arg Glu Val Glu Thr Ser Phe Ser Thr Ser His
325 330 335
Tyr Thr Ser Thr Ala His His Ser Thr Thr Val Thr Gln Thr Pro Ser
340 345 350
His Ser Trp Ser Asn Gly His Thr Glu Ser Val Ile Ser Glu Ser Asn
355 360 365
Ser Val Ile Met Met Ser Ser Val Glu Asn Ser Arg His Ser Ser Pro 370 375 380
Ala Gly Gly Pro Arg Gly Arg Leu His Gly Leu Gly Gly Pro Arg Asp 385 390 395 400
Asn Ser Phe Leu Arg His Ala Arg Glu Thr Pro Asp Ser Tyr Arg Asp
405 410 415
Ser Pro His Ser Glu Arg Tyr Val Ser Ala Met Thr Thr Pro Ala Arg
420 425 430
Met Ser Pro Val Asp Phe His Thr Pro Ser Ser Pro Lys Ser Pro Pro
435 440 445
Ser Glu Met Ser Pro Pro Val Ser Ser Met Thr Val Ser Met Pro Ser 450 455 460
Val Ala Val Ser Pro Phe Val Glu Glu Glu Arg Pro Leu Leu Leu Val 465 470 475 480
Thr Pro Pro Arg Leu Arg Glu Lys Lys Tyr Asp His His Pro Gln Gln
485 490 495
Leu Asn Ser Phe His His Asn Pro Ala His Gln Ser Thr Ser Leu Pro
500 505 510
Pro Ser Pro Leu Arg Ile Val Glu Asp Glu Glu Tyr Glu Thr Thr Gln
515 520 525
Glu Tyr Glu Ser Val Gln Glu Pro Val Lys Lys Val Thr Asn Ser Arg 530 535 540
Arg Ala Lys Arg Thr Lys Pro Asn Gly His Ile Ala Asn Arg Leu Glu 545 550 555 560
Met Asp Ser Asn Thr Ser Ser Val Ser Ser Asn Ser Glu Ser Glu Thr
565 570 575
Glu Asp Glu Arg Val Gly Glu Asp Thr Pro Phe Leu Gly Ile Gln Asn
580 585 590
Pro Leu Ala Ala Ser Leu Glu Val Ala Pro Ala Phe Arg Leu Ala Glu
595 600 605
Ser Arg Thr Asn Pro Ala Gly Arg Phe Ser Thr Gln Glu Glu Leu Gln
610 615 620
Ala Arg Leu Ser Ser Val Ile Ala Asn Gln Asp Pro Ile Ala Val
625 630 635
(45) INFORMATION FOR SEQ ID NO: 44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1261 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 345..1238
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:
GCGGCCGCAG CTGCCGGGAG ATGCGAGCGC AGACCGGATT GTGATCACCT TTCCCTCTTC 60
GGGCTGTAAG AGAGCGAGAC AAGCCACCGA AGCGAGGCCA CTCCAGAGCC GGCAGCGGAG 120
GGACCCGGGA CACTAGAGCA GCTCCGAGCC ACTCCAGACT GAGCGGACGC TCCAGGTGAT 180
CGAGTCCACG CTGCTTCCTG CAGGCGACAG GCGACGCCTC CCGAGCAGCC CGGCCACTGG 240
CTCTTCCCCT CCTGGGACAA ACTTTTCTGC AAGCCCTTGG ACCAAACTTG TCGCGCGTCA 300
CCGTCACCCA ACCGGGTCCG CGTAGAGCGC TCATCTTCGG CGAG ATG TCT GAG CGC 356
Met Ser Glu Arg
1
AAA GAA GGC AGA GGC AAG GGG AAG GGC AAG AAG AAG GAC CGG GGA TCC 404 Lys Glu Gly Arg Gly Lys Gly Lys Gly Lys Lys Lys Asp Arg Gly Ser
5 10 15 20
CGC GGG AAG CCC GGG CCC GCC GAG GGC GAC CCG AGC CCA GCA CTG CCT 452 Arg Gly Lys Pro Gly Pro Ala Glu Gly Asp Pro Ser Pro Ala Leu Pro
25 30 35
CCC AGA TTG AAA GAA ATG AAG AGC CAG GAG TCA GCT GCA GGC TCC AAG 500 Pro Arg Leu Lys Glu Met Lys Ser Gln Glu Ser Ala Ala Gly Ser Lys
40 45 50
CTA GTG CTC CGG TGC GAA ACC AGC TCC GAG TAC TCC TCA CTC AGA TTC 548 Leu Val Leu Arg Cys Glu Thr Ser Ser Glu Tyr Ser Ser Leu Arg Phe
55 60 65
AAA TGG TTC AAG AAT GGG AAC GAG CTG AAC CGC AAA AAT AAA CCA GAA 596 Lys Trp Phe Lys Asn Gly Asn Glu Leu Asn Arg Lys Asn Lys Pro Glu
70 75 80
AAC ATC AAG ATA CAG AAG AAG CCA GGG AAG TCA GAG CTT CGA ATT AAC 644 Asn Ile Lys Ile Gln Lys Lys Pro Gly Lys Ser Glu Leu Arg Ile Asn
85 90 95 100
AAA GCA TCC CTG GCT GAC TCT GGA GAG TAT ATG TGC AAA GTG ATC AGC 692 Lys Ala Ser Leu Ala Asp Ser Gly Glu Tyr Met Cys Lys Val Ile Ser
105 110 115
AAG TTA GGA AAT GAC AGT GCC TCT GCC AAC ATC ACC ATT GTT GAG TCA 740 Lys Leu Gly Asn Asp Ser Ala Ser Ala Asn Ile Thr Ile Val Glu Ser
120 125 130
AAC GAG TTC ATC ACT GGC ATG CCA GCC TCG ACT GAG ACA GCC TAT GTG 788 Asn Glu Phe Ile Thr Gly Met Pro Ala Ser Thr Glu Thr Ala Tyr Val
135 140 145
TCC TCA GAG TCT CCC ATT AGA ATC TCA GTT TCA ACA GAA GGC GCA AAC 836 Ser Ser Glu Ser Pro Ile Arg Ile Ser Val Ser Thr Glu Gly Ala Asn
150 155 160
ACT TCT TCA TCC ACA TCA ACA TCC ACG ACT GGG ACC AGC CAT CTC ATA 884 Thr Ser Ser Ser Thr Ser Thr Ser Thr Thr Gly Thr Ser His Leu Ile
165 170 175 180
AAG TGT GCG GAG AAG GAG AAA ACT TTC TGT GTG AAT GGG GGC GAG TGC 932 Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu Cys
185 190 195
TTC ACG GTG AAG GAC CTG TCA AAC CCG TCA AGA TAC TTG TGC AAG TGC 980 Phe Thr Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys Lys Cys
200 205 210
CCA AAT GAG TTT ACT GGT GAT CGT TGC CAA AAC TAC GTA ATG GCC AGC 1028 Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr Val Met Ala Ser
215 220 225
TTC TAC AAA GCG GAG GAA CTC TAC CAG AAG AGG GTG CTG ACA ATT ACT 1076 Phe Tyr Lys Ala Glu Glu Leu Tyr Gln Lys Arg Val Leu Thr Ile Thr
230 235 240
GGC ATC TGT ATC GCC CTG CTG GTG GTC GGC ATC ATG TGT GTG GTG GCC 1124 Gly Ile Cys Ile Ala Leu Leu Val Val Gly Ile Met Cys Val Val Ala
245 250 255 260
TAC TGC AAA ACC AAG AAG CAG CGG CAG AAG CTT CAT GAT CGG CTT CGG 1172 Tyr Cys Lys Thr Lys Lys Gln Arg Gln Lys Leu His Asp Arg Leu Arg
265 270 275
CAG AGT CTT CGG TCA GAA CGG AGC AAC CTG GTG AAC ATA GCG AAT GGG 1220 Gln Ser Leu Arg Ser Glu Arg Ser Asn Leu Val Asn Ile Ala Asn Gly
280 285 290
CCT CAC CAC CCA AAC CCA CGCGTCCGGA CGCGTGGGTC GAC 1261
Pro His His Pro Asn Pro
295
(46) INFORMATION FOR SEQ ID NO: 45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 298 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:
Met Ser Glu Arg Lys Glu Gly Arg Gly Lys Gly Lys Gly Lys Lys Lys
1 5 10 15
Asp Arg Gly Ser Arg Gly Lys Pro Gly Pro Ala Glu Gly Asp Pro Ser
20 25 30
Pro Ala Leu Pro Pro Arg Leu Lys Glu Met Lys Ser Gln Glu Ser Ala
35 40 45
Ala Gly Ser Lys Leu Val Leu Arg Cys Glu Thr Ser Ser Glu Tyr Ser
50 55 60
Ser Leu Arg Phe Lys Trp Phe Lys Asn Gly Asn Glu Leu Asn Arg Lys
65 70 75 80
Asn Lys Pro Glu Asn Ile Lys Ile Gln Lys Lys Pro Gly Lys Ser Glu
85 90 95
Leu Arg Ile Asn Lys Ala Ser Leu Ala Asp Ser Gly Glu Tyr Met Cys 100 105 110
Lys Val Ile Ser Lys Leu Gly Asn Asp Ser Ala Ser Ala Asn Ile Thr
115 120 125
Ile Val Glu Ser Asn Glu Phe Ile Thr Gly Met Pro Ala Ser Thr Glu 130 135 140
Thr Ala Tyr Val Ser Ser Glu Ser Pro Ile Arg Ile Ser Val Ser Thr 145 150 155 160
Glu Gly Ala Asn Thr Ser Ser Ser Thr Ser Thr Ser Thr Thr Gly Thr
165 170 175
Ser His Leu Ile Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn
180 185 190
Gly Gly Glu Cys Phe Thr Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr
195 200 205
Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr 210 215 220
Val Met Ala Ser Phe Tyr Lys Ala Glu Glu Leu Tyr Gln Lys Arg Val 225 230 235 240
Leu Thr Ile Thr Gly Ile Cys Ile Ala Leu Leu Val Val Gly Ile Met
245 250 255
Cys Val Val Ala Tyr Cys Lys Thr Lys Lys Gln Arg Gln Lys Leu His
260 265 270
Asp Arg Leu Arg Gln Ser Leu Arg Ser Glu Arg Ser Asn Leu Val Asn
275 280 285
Ile Ala Asn Gly Pro His His Pro Asn Pro
290 295
(47) INFORMATION FOR SEQ ID NO: 46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2743 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : unknown
(D) TOPOLOGY: unknown
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 345..2252
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:
GCGGCCGCAG CTGCCGGGAG ATGCGAGCGC AGACCGGATT GTGATCACCT TTCCCTCTTC 60
GGGCTGTAAG AGAGCGAGAC AAGCCACCGA AGCGAGGCCA CTCCAGAGCC GGCAGCGGAG 120
GGACCCGGGA CACTAGAGCA GCTCCGAGCC ACTCCAGACT GAGCGGACGC TCCAGGTGAT 180
CGAGTCCACG CTGCTTCCTG CAGGCGACAG GCGACGCCTC CCGAGCAGCC CGGCCACTGG 240
CTCTTCCCCT CCTGGGACAA ACTTTTCTGC AAGCCCTTGG ACCAAACTTG TCGCGCGTCA 300
CCGTCACCCA ACCGGGTCCG CGTAGAGCGC TCATCTTCGG CGAG ATG TCT GAG CGC 356
Met Ser Glu Arg
1
AAA GAA GGC AGA GGC AAG GGG AAG GGC AAG AAG AAG GAC CGG GGA TCC 404 Lys Glu Gly Arg Gly Lys Gly Lys Gly Lys Lys Lys Asp Arg Gly Ser
5 10 15 20
CGC GGG AAG CCC GGG CCC GCC GAG GGC GAC CCG AGC CCA GCA CTG CCT 452 Arg Gly Lys Pro Gly Pro Ala Glu Gly Asp Pro Ser Pro Ala Leu Pro
25 30 35
CCC AGA TTG AAA GAA ATG AAG AGC CAG GAG TCA GCT GCA GGC TCC AAG 500 Pro Arg Leu Lys Glu Met Lys Ser Gln Glu Ser Ala Ala Gly Ser Lys
40 45 50
CTA GTG CTC CGG TGC GAA ACC AGC TCC GAG TAC TCC TCA CTC AGA TTC 548 Leu Val Leu Arg Cys Glu Thr Ser Ser Glu Tyr Ser Ser Leu Arg Phe
55 60 65
AAA TGG TTC AAG AAT GGG AAC GAG CTG AAC CGC AAA AAT AAA CCA GAA 596 Lys Trp Phe Lys Asn Gly Asn Glu Leu Asn Arg Lys Asn Lys Pro Glu
70 75 80
AAC ATC AAG ATA CAG AAG AAG CCA GGG AAG TCA GAG CTT CGA ATT AAC 644 Asn Ile Lys Ile Gln Lys Lys Pro Gly Lys Ser Glu Leu Arg Ile Asn
85 90 95 100
AAA GCA TCC CTG GCT GAC TCT GGA GAG TAT ATG TGC AAA GTG ATC AGC 692 Lys Ala Ser Leu Ala Asp Ser Gly Glu Tyr Met Cys Lys Val Ile Ser
105 110 115
AAG TTA GGA AAT GAC AGT GCC TCT GCC AAC ATC ACC ATT GTT GAG TCA 740 Lys Leu Gly Asn Asp Ser Ala Ser Ala Asn Ile Thr Ile Val Glu Ser
120 125 130
AAC GAG TTC ATC ACT GGC ATG CCA GCC TCG ACT GAG ACA GCC TAT GTG 788 Asn Glu Phe Ile Thr Gly Met Pro Ala Ser Thr Glu Thr Ala Tyr Val
135 140 145
TCC TCA GAG TCT CCC ATT AGA ATC TCA GTT TCA ACA GAA GGC GCA AAC 836 Ser Ser Glu Ser Pro Ile Arg Ile Ser Val Ser Thr Glu Gly Ala Asn
150 155 160
ACT TCT TCA TCC ACA TCA ACA TCC ACG ACT GGG ACC AGC CAT CTC ATA 884 Thr Ser Ser Ser Thr Ser Thr Ser Thr Thr Gly Thr Ser His Leu Ile
165 170 175 180
AAG TGT GCG GAG AAG GAG AAA ACT TTC TGT GTG AAT GGG GGC GAG TGC 932 Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu Cys
185 190 195
TTC ACG GTG AAG GAC CTG TCA AAC CCG TCA AGA TAC TTG TGC AAG TGC 980 Phe Thr Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys Lys Cys
200 205 210
CCA AAT GAG TTT ACT GGT GAT CGT TGC CAA AAC TAC GTA ATG GCC AGC 1028 Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr Val Met Ala Ser
215 220 225
TTC TAC AAA GCG GAG GAA CTC TAC CAG AAG AGG GTG CTG ACA ATT ACT 1076 Phe Tyr Lys Ala Glu Glu Leu Tyr Gln Lys Arg Val Leu Thr Ile Thr
230 235 240
GGC ATC TGT ATC GCC CTG CTG GTG GTC GGC ATC ATG TGT GTG GTG GCC 1124 Gly Ile Cys Ile Ala Leu Leu Val Val Gly Ile Met Cys Val Val Ala
245 250 255 260
TAC TGC AAA ACC AAG AAG CAG CGG CAG AAG CTT CAT GAT CGG CTT CGG 1172 Tyr Cys Lys Thr Lys Lys Gln Arg Gln Lys Leu His Asp Arg Leu Arg
265 270 275
CAG AGT CTT CGG TCA GAA CGG AGC AAC CTG GTG AAC ATA GCG AAT GGG 1220 Gln Ser Leu Arg Ser Glu Arg Ser Asn Leu Val Asn Ile Ala Asn Gly
280 285 290
CCT CAC CAC CCA AAC CCA CCG CCA GAG AAC GTG CAG CTG GTG AAT CAA 1268 Pro His His Pro Asn Pro Pro Pro Glu Asn Val Gln Leu Val Asn Gln
295 300 305
TAC GTA TCT AAA AAC GTC ATC TCC AGT GAG CAT ATT GTT GAG AGA GAA 1316 Tyr Val Ser Lys Asn Val Ile Ser Ser Glu His Ile Val Glu Arg Glu
310 315 320
GTG GAG ACT TCC TTT TCC ACC AGT CAT TAC ACT TCC ACA GCC CAT CAC 1364 Val Glu Thr Ser Phe Ser Thr Ser His Tyr Thr Ser Thr Ala His His
325 330 335 340
TCC ACG ACT GTC ACC CAG ACT CCT AGT CAC AGC TGG AGT AAT GGG CAC 1412 Ser Thr Thr Val Thr Gln Thr Pro Ser His Ser Trp Ser Asn Gly His
345 350 355
ACG GAG AGC GTC ATT TCA GAA AGC AAC TCC GTA ATC ATG ATG TCT TCG 1460 Thr Glu Ser Val Ile Ser Glu Ser Asn Ser Val Ile Met Met Ser Ser
360 365 370
GTA GAG AAC AGC AGG CAC AGC AGT CCC GCC GGG GGC CCA CGA GGA CGT 1508 Val Glu Asn Ser Arg His Ser Ser Pro Ala Gly Gly Pro Arg Gly Arg
375 380 385
CTT CAT GGC CTG GGA GGC CCT CGT GAT AAC AGC TTC CTC AGG CAT GCC 1556 Leu His Gly Leu Gly Gly Pro Arg Asp Asn Ser Phe Leu Arg His Ala
390 395 400
AGA GAA ACC CCT GAC TCC TAC AGA GAC TCT CCT CAT AGC GAA AGG TAT 1604 Arg Glu Thr Pro Asp Ser Tyr Arg Asp Ser Pro His Ser Glu Arg Tyr
405 410 415 420
GTA TCA GCC ATG ACC ACC CCG GCT CGT ATG TCA CCT GTA GAT TTC CAC 1652 Val Ser Ala Met Thr Thr Pro Ala Arg Met Ser Pro Val Asp Phe His
425 430 435
ACG CCA AGC TCC CCT AAA TCG CCC GCT TCG GAA ATG TCT CCA CCC GTG 1700 Thr Pro Ser Ser Pro Lys Ser Pro Ala Ser Glu Met Ser Pro Pro Val
440 445 450
TCC AGC ATG ACG GTG TCC ATG CCC TCT GTG GCA GTC AGC CCC TTT GTG 1748 Ser Ser Met Thr Val Ser Met Pro Ser Val Ala Val Ser Pro Phe Val
455 460 465
GAA GAA GAG AGG CCT CTG CTG CTT GTG ACG CCA CCA AGG CTA CGG GAG 1796 Glu Glu Glu Arg Pro Leu Leu Leu Val Thr Pro Pro Arg Leu Arg Glu
470 475 480
AAG AAA TAT GAT CAT CAC CCC CAG CAA CTC AAC TCC TTT CAT CAC AAC 1844 Lys Lys Tyr Asp His His Pro Gln Gln Leu Asn Ser Phe His His Asn
485 490 495 500
CCT GCA CAT CAG AGT ACC AGC CTC CCC CCT AGC CCA CTG AGG ATA GTG 1892 Pro Ala His Gln Ser Thr Ser Leu Pro Pro Ser Pro Leu Arg Ile Val
505 510 515
GAG GAT GAG GAG TAC GAG ACG ACC CAG GAG TAT GAG TCA GTT CAA GAG 1940 Glu Asp Glu Glu Tyr Glu Thr Thr Gln Glu Tyr Glu Ser Val Gln Glu
520 525 530
CCC GTT AAG AAA GTC ACC AAT AGC CGG CGG GCC AAA AGA ACC AAG CCC 1988 Pro Val Lys Lys Val Thr Asn Ser Arg Arg Ala Lys Arg Thr Lys Pro
535 540 545
AAT GGC CAC ATT GCC AAT AGG TTG GAA ATG GAC AGC AAC ACA AGT TCT 2036 Asn Gly His Ile Ala Asn Arg Leu Glu Met Asp Ser Asn Thr Ser Ser
550 555 560
GTG AGC AGT AAC TCA GAA AGT GAG ACA GAA GAC GAA AGA GTA GGT GAA 2084 Val Ser Ser Asn Ser Glu Ser Glu Thr Glu Asp Glu Arg Val Gly Glu
565 570 575 580
GAC ACA CCA TTC CTG GGC ATA CAG AAC CCC CTG GCA GCC AGC CTT GAG 2132 Asp Thr Pro Phe Leu Gly Ile Gln Asn Pro Leu Ala Ala Ser Leu Glu
585 590 595
GTG GCC CCT GCC TTC CGT CTG GCT GAG AGC AGG ACT AAC CCA GCA GGC 2180 Val Ala Pro Ala Phe Arg Leu Ala Glu Ser Arg Thr Asn Pro Ala Gly
600 605 610
CGC TTC TCC ACA CAG GAG GAA TTA CAG GCC AGG CTG TCT AGT GTA ATC 2228 Arg Phe Ser Thr Gln Glu Glu Leu Gln Ala Arg Leu Ser Ser Val Ile
615 620 625
GCT AAC CAA GAC CCT ATT GCT GTA TAAAACCTAA ATAAACACAT AGATTCACCT 2282 Ala Asn Gln Asp Pro Ile Ala Val
630 635
GTAAAACTTT ATTTTATATA ATAAAGTATT TCACCTTAAA TTAAACAATT TATTTTATTT 2342
TAGCAGTTCT GCAAATAGAA AACAGGAAGA AAAAAAAACT TTTATAAATT AAATATATGT 2402
ATGTAAAAAT GTGTTATGTG CCATATGTAG CAATTTTTTA CAGTATTTCA AAAACGAGAA 2462
AGATATCAAT GGTGCCTTTA TGTTCTGTTA TGTCGAGAGC AAGTTTTATA AAGTTATGGT 2522
GATTTCTTTT TCACAGTATT TCAGCAAAAC CTCCCATATA TTCAGTTTCT GCTGGCTTTT 2582
TGTGCATTGC ATTATGATGT TGACTGGATG TATGGTTTGC AAGGCTAGCA GCTCGCTCGT 2642
GTTCTCTCTC TCTCTCTCTC TCTCTCTCTC TCTGTCTCTC TCTCTGTCTC TCTCTCTCTC 2702
TCTCTCTCTC TCTCTCTCTC TCTCCGGACG CGTGGGTCGA C 2743
(48) INFORMATION FOR SEQ ID NO: 47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 636 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:
Met Ser Glu Arg Lys Glu Gly Arg Gly Lys Gly Lys Gly Lys Lys Lys
1 5 10 15
Asp Arg Gly Ser Arg Gly Lys Pro Gly Pro Ala Glu Gly Asp Pro Ser 20 25 30
Pro Ala Leu Pro Pro Arg Leu Lys Glu Met Lys Ser Gln Glu Ser Ala
35 40 45
Ala Gly Ser Lys Leu Val Leu Arg Cys Glu Thr Ser Ser Glu Tyr Ser 50 55 60
Ser Leu Arg Phe Lys Trp Phe Lys Asn Gly Asn Glu Leu Asn Arg Lys 65 70 75 80
Asn Lys Pro Glu Asn Ile Lys Ile Gln Lys Lys Pro Gly Lys Ser Glu
85 90 95
Leu Arg Ile Asn Lys Ala Ser Leu Ala Asp Ser Gly Glu Tyr Met Cys
100 105 110
Lys Val Ile Ser Lys Leu Gly Asn Asp Ser Ala Ser Ala Asn Ile Thr
115 120 125
Ile Val Glu Ser Asn Glu Phe Ile Thr Gly Met Pro Ala Ser Thr Glu 130 135 140
Thr Ala Tyr Val Ser Ser Glu Ser Pro Ile Arg Ile Ser Val Ser Thr 145 150 155 160
Glu Gly Ala Asn Thr Ser Ser Ser Thr Ser Thr Ser Thr Thr Gly Thr
165 170 175
Ser His Leu Ile Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn
180 185 190
Gly Gly Glu Cys Phe Thr Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr
195 200 205
Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr 210 215 220
Val Met Ala Ser Phe Tyr Lys Ala Glu Glu Leu Tyr Gln Lys Arg Val 225 230 235 240
Leu Thr Ile Thr Gly Ile Cys Ile Ala Leu Leu Val Val Gly Ile Met
245 250 255
Cys Val Val Ala Tyr Cys Lys Thr Lys Lys Gln Arg Gln Lys Leu His
260 265 270
Asp Arg Leu Arg Gln Ser Leu Arg Ser Glu Arg Ser Asn Leu Val Asn
275 280 285
Ile Ala Asn Gly Pro His His Pro Asn Pro Pro Pro Glu Asn Val Gln 290 295 300
Leu Val Asn Gln Tyr Val Ser Lys Asn Val Ile Ser Ser Glu His Ile 305 310 315 320
Val Glu Arg Glu Val Glu Thr Ser Phe Ser Thr Ser His Tyr Thr Ser
325 330 335
Thr Ala His His Ser Thr Thr Val Thr Gln Thr Pro Ser His Ser Trp
340 345 350
Ser Asn Gly His Thr Glu Ser Val Ile Ser Glu Ser Asn Ser Val Ile
355 360 365
Met Met Ser Ser Val Glu Asn Ser Arg His Ser Ser Pro Ala Gly Gly 370 375 380
Pro Arg Gly Arg Leu His Gly Leu Gly Gly Pro Arg Asp Asn Ser Phe 385 390 395 400
Leu Arg His Ala Arg Glu Thr Pro Asp Ser Tyr Arg Asp Ser Pro His
405 410 415
Ser Glu Arg Tyr Val Ser Ala Met Thr Thr Pro Ala Arg Met Ser Pro
420 425 430
Val Asp Phe His Thr Pro Ser Ser Pro Lys Ser Pro Ala Ser Glu Met
435 440 445
Ser Pro Pro Val Ser Ser Met Thr Val Ser Met Pro Ser Val Ala Val 450 455 460
Ser Pro Phe Val Glu Glu Glu Arg Pro Leu Leu Leu Val Thr Pro Pro 465 470 475 480
Arg Leu Arg Glu Lys Lys Tyr Asp His His Pro Gln Gln Leu Asn Ser
485 490 495
Phe His His Asn Pro Ala His Gln Ser Thr Ser Leu Pro Pro Ser Pro
500 505 510
Leu Arg Ile Val Glu Asp Glu Glu Tyr Glu Thr Thr Gln Glu Tyr Glu
515 520 525
Ser Val Gln Glu Pro Val Lys Lys Val Thr Asn Ser Arg Arg Ala Lys 530 535 540
Arg Thr Lys Pro Asn Gly His Ile Ala Asn Arg Leu Glu Met Asp Ser 545 550 555 560
Asn Thr Ser Ser Val Ser Ser Asn Ser Glu Ser Glu Thr Glu Asp Glu
565 570 575
Arg Val Gly Glu Asp Thr Pro Phe Leu Gly Ile Gln Asn Pro Leu Ala
580 585 590
Ala Ser Leu Glu Val Ala Pro Ala Phe Arg Leu Ala Glu Ser Arg Thr
595 600 605
Asn Pro Ala Gly Arg Phe Ser Thr Gln Glu Glu Leu Gln Ala Arg Leu
610 615 620
Ser Ser Val Ile Ala Asn Gln Asp Pro Ile Ala Val
625 630 635
(49) INFORMATION FOR SEQ ID NO: 48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2430 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 345..2330
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:
GCGGCCGCAG CTGCCGGGAG ATGCGAGCGC AGACCGGATT GTGATCACCT TTCCCTCTTC 60
GGGCTGTAAG AGAGCGAGAC AAGCCACCGA AGCGAGGCCA CTCCAGAGCC GGCAGCGGAG 120
GGACCCGGGA CACTAGAGCA GCTCCGAGCC ACTCCAGACT GAGCGGACGC TCCAGGTGAT 180
CGAGTCCACG CTGCTTCCTG CAGGCGACAG GCGACGCCTC CCGAGCAGCC CGGCCACTGG 240
CTCTTCCCCT CCTGGGACAA ACTTTTCTGC AAGCCCTTGG ACCAAACTTG TCGCGCGTCA 300
CCGTCACCCA ACCGGGTCCG CGTAGAGCGC TCATCTTCGG CGAG ATG TCT GAG CGC 356
Met Ser Glu Arg
1
AAA GAA GGC AGA GGC AAG GGG AAG GGC AAG AAG AAG GAC CGG GGA TCC 404 Lys Glu Gly Arg Gly Lys Gly Lys Gly Lys Lys Lys Asp Arg Gly Ser
5 10 15 20
CGC GGG AAG CCC GGG CCC GCC GAG GGC GAC CCG AGC CCA GCA CTG CCT 452 Arg Gly Lys Pro Gly Pro Ala Glu Gly Asp Pro Ser Pro Ala Leu Pro
25 30 35
CCC AGA TTG AAA GAA ATG AAG AGC CAG GAG TCA GCT GCA GGC TCC AAG 500 Pro Arg Leu Lys Glu Met Lys Ser Gln Glu Ser Ala Ala Gly Ser Lys
40 45 50
CTA GTG CTC CGG TGC GAA ACC AGC TCC GAG TAC TCC TCA CTC AGA TTC 548 Leu Val Leu Arg Cys Glu Thr Ser Ser Glu Tyr Ser Ser Leu Arg Phe
55 60 65
AAA TGG TTC AAG AAT GGG AAC GAG CTG AAC CGC AAA AAT AAA CCA GAA 596 Lys Trp Phe Lys Asn Gly Asn Glu Leu Asn Arg Lys Asn Lys Pro Glu
70 75 80
AAC ATC AAG ATA CAG AAG AAG CCA GGG AAG TCA GAG CTT CGA ATT AAC 644 Asn Ile Lys Ile Gln Lys Lys Pro Gly Lys Ser Glu Leu Arg Ile Asn
85 90 95 100
AAA GCA TCC CTG GCT GAC TCT GGA GAG TAT ATG TGC AAA GTG ATC AGC 692 Lys Ala Ser Leu Ala Asp Ser Gly Glu Tyr Met Cys Lys Val Ile Ser
105 110 115
AAG TTA GGA AAT GAC AGT GCC TCT GCC AAC ATC ACC ATT GTT GAG TCA 740 Lys Leu Gly Asn Asp Ser Ala Ser Ala Asn Ile Thr Ile Val Glu Ser
120 125 130
AAC GAG TTC ATC ACT GGC ATG CCA GCC TCG ACT GAG ACA GCC TAT GTG 788 Asn Glu Phe Ile Thr Gly Met Pro Ala Ser Thr Glu Thr Ala Tyr Val
135 140 145
TCC TCA GAG TCT CCC ATT AGA ATC TCA GTT TCA ACA GAA GGC GCA AAC 836 Ser Ser Glu Ser Pro Ile Arg Ile Ser Val Ser Thr Glu Gly Ala Asn
150 155 160
ACT TCT TCA TCC ACA TCA ACA TCC ACG ACT GGG ACC AGC CAT CTC ATA 884 Thr Ser Ser Ser Thr Ser Thr Ser Thr Thr Gly Thr Ser His Leu Ile
165 170 175 180
AAG TGT GCG GAG AAG GAG AAA ACT TTC TGT GTG AAT GGG GGC GAG TGC 932 Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu Cys
185 190 195
TTC ACG GTG AAG GAC CTG TCA AAC CCG TCA AGA TAC TTG TGC AAG TGC 980 Phe Thr Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys Lys Cys
200 205 210
CCA AAT GAG TTT ACT GGT GAT CGT TGC CAA AAC TAC GTA ATG GCC AGC 1028 Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr Val Met Ala Ser
215 220 225
TTC TAC ATG ACT TCT AGG AGG AAA AGG CAA GAA ACA GAG AAG CCT CTA 1076 Phe Tyr Met Thr Ser Arg Arg Lys Arg Gln Glu Thr Glu Lys Pro Leu
230 235 240
GAA AGA AAA TTG GAT CAT AGC CTT GTG AAA GAA TCG AAA GCG GAG GAA 1124 Glu Arg Lys Leu Asp His Ser Leu Val Lys Glu Ser Lys Ala Glu Glu
245 250 255 260
CTC TAC CAG AAG AGG GTG CTG ACA ATT ACT GGC ATC TGT ATC GCC CTG 1172 Leu Tyr Gln Lys Arg Val Leu Thr Ile Thr Gly Ile Cys Ile Ala Leu
265 270 275
CTG GTG GTC GGC ATC ATG TGT GTG GTG GCC TAC TGC AAA ACC AAG AAG 1220 Leu Val Val Gly Ile Met Cys Val Val Ala Tyr Cys Lys Thr Lys Lys
280 285 290
CAG CGG CAG AAG CTT CAT GAT CGG CTT CGG CAG AGT CTT CGG TCA GAA 1268 Gln Arg Gln Lys Leu His Asp Arg Leu Arg Gln Ser Leu Arg Ser Glu
295 300 305
CGG AGC AAC CTG GTG AAC ATA GCG AAT GGG CCT CAC CAC CCA AAC CCA 1316 Arg Ser Asn Leu Val Asn Ile Ala Asn Gly Pro His His Pro Asn Pro
310 315 320
CCG CCA GAG AAC GTG CAG CTG GTG AAT CAA TAC GTA TCT AAA AAC GTC 1364 Pro Pro Glu Asn Val Gln Leu Val Asn Gln Tyr Val Ser Lys Asn Val
325 330 335 340
ATC TCC AGT GAG CAT ATT GTT GAG AGA GAA GTG GAG ACT TCC TTT TCC 1412 Ile Ser Ser Glu His Ile Val Glu Arg Glu Val Glu Thr Ser Phe Ser
345 350 355
ACC AGT CAT TAC ACT TCC ACA GCC CAT CAC TCC ACG ACT GTC ACC CAG 1460 Thr Ser His Tyr Thr Ser Thr Ala His His Ser Thr Thr Val Thr Gln
360 365 370
ACT CCT AGT CAC AGC TGG AGT AAT GGG CAC ACG GAG AGC GTC ATT TCA 1508 Thr Pro Ser His Ser Trp Ser Asn Gly His Thr Glu Ser Val Ile Ser
375 380 385
GAA AGC AAC TCC GTA ATC ATG ATG TCT TCG GTA GAG AAC AGC AGG CAC 1556 Glu Ser Asn Ser Val Ile Met Met Ser Ser Val Glu Asn Ser Arg His
390 395 400
AGC AGT CCC GCC GGG GGC CCA CGA GGA CGT CTT CAT GGC CTG GGA GGC 1604 Ser Ser Pro Ala Gly Gly Pro Arg Gly Arg Leu His Gly Leu Gly Gly
405 410 415 420
CCT CGT GAT AAC AGC TTC CTC AGG CAT GCC AGA GAA ACC CCT GAC TCC 1652 Pro Arg Asp Asn Ser Phe Leu Arg His Ala Arg Glu Thr Pro Asp Ser
425 430 435
TAC AGA GAC TCT CCT CAT AGC GAA AGG TAT GTA TCA GCC ATG ACC ACC 1700 Tyr Arg Asp Ser Pro His Ser Glu Arg Tyr Val Ser Ala Met Thr Thr
440 445 450
CCG GCT CGT ATG TCA CCT GTA GAT TTC CAC ACG CCA AGC TCC CCT AAA 1748 Pro Ala Arg Met Ser Pro Val Asp Phe His Thr Pro Ser Ser Pro Lys
455 460 465
TCG CCC CCT TCG GAA ATG TCT CCA CCC GTG TCC AGC ATG ACG GTG TCC 1796 Ser Pro Pro Ser Glu Met Ser Pro Pro Val Ser Ser Met Thr Val Ser
470 475 480
ATG CCC TCT GTG GCA GTC AGC CCC TTT GTG GAA GAA GAG AGG CCT CTG 1844 Met Pro Ser Val Ala Val Ser Pro Phe Val Glu Glu Glu Arg Pro Leu
485 490 495 500
CTG CTT GTG ACG CCA CCA AGG CTA CGG GAG AAG AAA TAT GAT CAT CAC 1892 Leu Leu Val Thr Pro Pro Arg Leu Arg Glu Lys Lys Tyr Asp His His
505 510 515
CCC CAG CAA CTC AAC TCC TTT CAT CAC AAC CCT GCA CAT CAG AGT ACC 1940 Pro Gln Gln Leu Asn Ser Phe His His Asn Pro Ala His Gln Ser Thr
520 525 530
AGC CTC CCC CCT AGC CCA CTG AGG ATA GTG GAG GAT GAG GAG TAC GAG 1988 Ser Leu Pro Pro Ser Pro Leu Arg Ile Val Glu Asp Glu Glu Tyr Glu
535 540 545
ACG ACC CAG GAG TAT GAG TCA GTT CAA GAG CCC GTT AAG AAA GTC ACC 2036 Thr Thr Gln Glu Tyr Glu Ser Val Gln Glu Pro Val Lys Lys Val Thr
550 555 560
AAT AGC CGG CGG GCC AAA AGA ACC AAG CCC AAT GGC CAC ATT GCC AAT 2084 Asn Ser Arg Arg Ala Lys Arg Thr Lys Pro Asn Gly His Ile Ala Asn
565 570 575 580
AGG TTG GAA ATG GAC AGC AAC ACA AGT TCT GTG AGC AGT AAC TCA GAA 2132 Arg Leu Glu Met Asp Ser Asn Thr Ser Ser Val Ser Ser Asn Ser Glu
585 590 595
AGT GAG ACA GAA GAC GAA AGA GTA GGT GAA GAC ACA CCA TTC CTG GGC 2180 Ser Glu Thr Glu Asp Glu Arg Val Gly Glu Asp Thr Pro Phe Leu Gly
600 605 610
ATA CAG AAC CCC CTG GCA GCC AGC CTT GAG GTG GCC CCT GCC TTC CGT 2228 Ile Gln Asn Pro Leu Ala Ala Ser Leu Glu Val Ala Pro Ala Phe Arg
615 620 625
CTG GCT GAG AGC AGG ACT AAC CCA GCA GGC CGC TTC TCC ACA CAG GAG 2276 Leu Ala Glu Ser Arg Thr Asn Pro Ala Gly Arg Phe Ser Thr Gln Glu
630 635 640
GAA TTA CAG GCC AGG CTG TCT AGT GTA ATC GCT AAC CAA GAC CCT ATT 2324 Glu Leu Gln Ala Arg Leu Ser Ser Val Ile Ala Asn Gln Asp Pro Ile
645 650 655 660
GCT GTA TAAAACCTAA ATAAACACAT AGATTCACCT GTAAAACTTT ATTTTATATA 2380 Ala Val
ATAAAGTATT TCACCTTAAA TTAAAAAAAA AAAAAAAAAA GGACGGCCGC 2430
(50) INFORMATION FOR SEQ ID NO: 49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 662 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:
Met Ser Glu Arg Lys Glu Gly Arg Gly Lys Gly Lys Gly Lys Lys Lys
1 5 10 15
Asp Arg Gly Ser Arg Gly Lys Pro Gly Pro Ala Glu Gly Asp Pro Ser
20 25 30
Pro Ala Leu Pro Pro Arg Leu Lys Glu Met Lys Ser Gln Glu Ser Ala
35 40 45
Ala Gly Ser Lys Leu Val Leu Arg Cys Glu Thr Ser Ser Glu Tyr Ser
50 55 60
Ser Leu Arg Phe Lys Trp Phe Lys Asn Gly Asn Glu Leu Asn Arg Lys
65 70 75 80
Asn Lys Pro Glu Asn Ile Lys Ile Gln Lys Lys Pro Gly Lys Ser Glu
85 90 95
Leu Arg Ile Asn Lys Ala Ser Leu Ala Asp Ser Gly Glu Tyr Met Cys
100 105 110
Lys Val Ile Ser Lys Leu Gly Asn Asp Ser Ala Ser Ala Asn Ile Thr
115 120 125
Ile Val Glu Ser Asn Glu Phe Ile Thr Gly Met Pro Ala Ser Thr Glu
130 135 140
Thr Ala Tyr Val Ser Ser Glu Ser Pro Ile Arg Ile Ser Val Ser Thr
145 150 155 160
Glu Gly Ala Asn Thr Ser Ser Ser Thr Ser Thr Ser Thr Thr Gly Thr
165 170 175
Ser His Leu Ile Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn
180 185 190
Gly Gly Glu Cys Phe Thr Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr
195 200 205
Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr 210 215 220
Val Met Ala Ser Phe Tyr Met Thr Ser Arg Arg Lys Arg Gln Glu Thr 225 230 235 240
Glu Lys Pro Leu Glu Arg Lys Leu Asp His Ser Leu Val Lys Glu Ser
245 250 255
Lys Ala Glu Glu Leu Tyr Gln Lys Arg Val Leu Thr Ile Thr Gly Ile
260 265 270
Cys Ile Ala Leu Leu Val Val Gly Ile Met Cys Val Val Ala Tyr Cys
275 280 285
Lys Thr Lys Lys Gln Arg Gln Lys Leu His Asp Arg Leu Arg Gln Ser 290 295 300
Leu Arg Ser Glu Arg Ser Asn Leu Val Asn Ile Ala Asn Gly Pro His 305 310 315 320
His Pro Asn Pro Pro Pro Glu Asn Val Gln Leu Val Asn Gln Tyr Val
325 330 335
Ser Lys Asn Val Ile Ser Ser Glu His Ile Val Glu Arg Glu Val Glu
340 345 350
Thr Ser Phe Ser Thr Ser His Tyr Thr Ser Thr Ala His His Ser Thr
355 360 365
Thr Val Thr Gln Thr Pro Ser His Ser Trp Ser Asn Gly His Thr Glu 370 375 380
Ser Val Ile Ser Glu Ser Asn Ser Val Ile Met Met Ser Ser Val Glu 385 390 395 400
Asn Ser Arg His Ser Ser Pro Ala Gly Gly Pro Arg Gly Arg Leu His
405 410 415
Gly Leu Gly Gly Pro Arg Asp Asn Ser Phe Leu Arg His Ala Arg Glu
420 425 430
Thr Pro Asp Ser Tyr Arg Asp Ser Pro His Ser Glu Arg Tyr Val Ser
435 440 445
Ala Met Thr Thr Pro Ala Arg Met Ser Pro Val Asp Phe His Thr Pro 450 455 460
Ser Ser Pro Lys Ser Pro Pro Ser Glu Met Ser Pro Pro Val Ser Ser 465 470 475 480
Met Thr Val Ser Met Pro Ser Val Ala Val Ser Pro Phe Val Glu Glu
485 490 495
Glu Arg Pro Leu Leu Leu Val Thr Pro Pro Arg Leu Arg Glu Lys Lys
500 505 510
Tyr Asp His His Pro Gln Gln Leu Asn Ser Phe His His Asn Pro Ala
515 520 525
His Gln Ser Thr Ser Leu Pro Pro Ser Pro Leu Arg Ile Val Glu Asp
530 535 540
Glu Glu Tyr Glu Thr Thr Gln Glu Tyr Glu Ser Val Gln Glu Pro Val
545 550 555 560
Lys Lys Val Thr Asn Ser Arg Arg Ala Lys Arg Thr Lys Pro Asn Gly
565 570 575
His Ile Ala Asn Arg Leu Glu Met Asp Ser Asn Thr Ser Ser Val Ser
580 585 590
Ser Asn Ser Glu Ser Glu Thr Glu Asp Glu Arg Val Gly Glu Asp Thr
595 600 605
Pro Phe Leu Gly Ile Gln Asn Pro Leu Ala Ala Ser Leu Glu Val Ala
610 615 620
Pro Ala Phe Arg Leu Ala Glu Ser Arg Thr Asn Pro Ala Gly Arg Phe
625 630 635 640
Ser Thr Gln Glu Glu Leu Gln Ala Arg Leu Ser Ser Val Ile Ala Asn
645 650 655
Gln Asp Pro Ile Ala Val
660
(51) INFORMATION FOR SEQ ID NO: 50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3161 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 345..2261
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:
GCGGCCGCAG CTGCCGGGAG ATGCGAGCGC AGACCGGATT GTGATCACCT TTCCCTCTTC 60
GGGCTGTAAG AGAGCGAGAC AAGCCACCGA AGCGAGGCCA CTCCAGAGCC GGCAGCGGAG 120
GGACCCGGGA CACTAGAGCA GCTCCGAGCC ACTCCAGACT GAGCGGACGC TCCAGGTGAT 180
CGAGTCCACG CTGCTTCCTG CAGGCGACAG GCGACGCCTC CCGAGCAGCC CGGCCACTGG 240
CTCTTCCCCT CCTGGGACAA ACTTTTCTGC AAGCCCTTGG ACCAAACTTG TCGCGCGTCA 300
CCGTCACCCA ACCGGGTCCG CGTAGAGCGC TCATCTTCGG CGAG ATG TCT GAG CGC 356
Met Ser Glu Arg
1
AAA GAA GGC AGA GGC AAG GGG AAG GGC AAG AAG AAG GAC CGG GGA TCC 404 Lys Glu Gly Arg Gly Lys Gly Lys Gly Lys Lys Lys Asp Arg Gly Ser
5 10 15 20
CGC GGG AAG CCC GGG CCC GCC GAG GGC GAC CCG AGC CCA GCA CTG CCT 452 Arg Gly Lys Pro Gly Pro Ala Glu Gly Asp Pro Ser Pro Ala Leu Pro
25 30 35
CCC AGA TTG AAA GAA ATG AAG AGC CAG GAG TCA GCT GCA GGC TCC AAG 500 Pro Arg Leu Lys Glu Met Lys Ser Gln Glu Ser Ala Ala Gly Ser Lys
40 45 50
CTA GTG CTC CGG TGC GAA ACC AGC TCC GAG TAC TCC TCA CTC AGA TTC 548 Leu Val Leu Arg Cys Glu Thr Ser Ser Glu Tyr Ser Ser Leu Arg Phe
55 60 65
AAA TGG TTC AAG AAT GGG AAC GAG CTG AAC CGC AAA AAT AAA CCA GAA 596 Lys Trp Phe Lys Asn Gly Asn Glu Leu Asn Arg Lys Asn Lys Pro Glu
70 75 80
AAC ATC AAG ATA CAG AAG AAG CCA GGG AAG TCA GAG CTT CGA ATT AAC 644 Asn lie Lys Ile Gln Lys Lys Pro Gly Lys Ser Glu Leu Arg Ile Asn
85 90 95 100
AAA GCA TCC CTG GCT GAC TCT GGA GAG TAT ATG TGC AAA GTG ATC AGC 692 Lys Ala Ser Leu Ala Asp Ser Gly Glu Tyr Met Cys Lys Val Ile Ser
105 110 115
AAG TTA GGA AAT GAC AGT GCC TCT GCC AAC ATC ACC ATT GTT GAG TCA 740 Lys Leu Gly Asn Asp Ser Ala Ser Ala Asn Ile Thr Ile Val Glu Ser
120 125 130
AAC GAG TTC ATC ACT GGC ATG CCA GCC TCG ACT GAG ACA GCC TAT GTG 788 Asn Glu Phe Ile Thr Gly Met Pro Ala Ser Thr Glu Thr Ala Tyr Val
135 140 145
TCC TCA GAG TCT CCC ATT AGA ATC TCA GTT TCA ACA GAA GGC GCA AAC 836 Ser Ser Glu Ser Pro Ile Arg Ile Ser Val Ser Thr Glu Gly Ala Asn
150 155 160
ACT TCT TCA TCC ACA TCA ACA TCC ACG ACT GGG ACC AGC CAT CTC ATA 884 Thr Ser Ser Ser Thr Ser Thr Ser Thr Thr Gly Thr Ser His Leu Ile
165 170 175 180
AAG TGT GCG GAG AAG GAG AAA ACT TTC TGT GTG AAT GGG GGC GAG TGC 932 Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu Cys
185 190 195
TTC ACG GTG AAG GAC CTG TCA AAC CCG TCA AGA TAC TTG TGC AAG TGC 980 Phe Thr Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr Leu Cys Lys Cys
200 205 210
CAA CCT GGA TTC ACT GGA GCA AGA TGT ACT GAG AAT GTA CCC ATG AAA 1028 Gln Pro Gly Phe Thr Gly Ala Arg Cys Thr Glu Asn Val Pro Met Lys
215 220 225
GTC CAA ACC CAA GAA AAA GCG GAG GAA CTC TAC CAG AAG AGG GTG CTG 1076 Val Gln Thr Gln Glu Lys Ala Glu Glu Leu Tyr Gln Lys Arg Val Leu
230 235 240
ACA ATT ACT GGC ATC TGT ATC GCC CTG CTG GTG GTC GGC ATC ATG TGT 1124 Thr Ile Thr Gly Ile Cys Ile Ala Leu Leu Val Val Gly Ile Met Cys
245 250 255 260
GTG GTG GCC TAC TGC AAA ACC AAG AAG CAG CGG CAG AAG CTT CAT GAT 1172 Val Val Ala Tyr Cys Lys Thr Lys Lys Gln Arg Gln Lys Leu His Asp
265 270 275
CGG CTT CGG CAG AGT CTT CGG TCA GAA CGG AGC AAC CTG GTG AAC ATA 1220 Arg Leu Arg Gln Ser Leu Arg Ser Glu Arg Ser Asn Leu Val Asn Ile
280 285 290
GCG AAT GGG CCT CAC CAC CCA AAC CCA CCG CCA GAG AAC GTG CAG CTG 1268 Ala Asn Gly Pro His His Pro Asn Pro Pro Pro Glu Asn Val Gln Leu
295 300 305
GTG AAT CAA TAC GTA TCT AAA AAC GTC ATC TCC AGT GAG CAT ATT GTT 1316 Val Asn Gln Tyr Val Ser Lys Asn Val Ile Ser Ser Glu His Ile Val
310 315 320
GAG AGA GAA GTG GAG ACT TCC TTT TCC ACC AGT CAT TAC ACT TCC ACA 1364 Glu Arg Glu Val Glu Thr Ser Phe Ser Thr Ser His Tyr Thr Ser Thr
325 330 335 340
GCC CAT CAC TCC ACG ACT GTC ACC CAG ACT CCT AGT CAC AGC TGG AGT 1412 Ala His His Ser Thr Thr Val Thr Gln Thr Pro Ser His Ser Trp Ser
345 350 355
AAT GGG CAC ACG GAG AGC GTC ATT TCA GAA AGC AAC TCC GTA ATC ATG 1460 Asn Gly His Thr Glu Ser Val Ile Ser Glu Ser Asn Ser Val Ile Met
360 365 370
ATG TCT TCG GTA GAG AAC AGC AGG CAC AGC AGT CCC GCC GGG GGC CCA 1508
Met Ser Ser Val Glu Asn Ser Arg His Ser Ser Pro Ala Gly Gly Pro
375 380 385
CGA GGA CGT CTT CAT GGC CTG GGA GGC CCT CGT GAT AAC AGC TTC CTC 1556
Arg Gly Arg Leu His Gly Leu Gly Gly Pro Arg Asp Asn Ser Phe Leu
390 395 400
AGG CAT GCC AGA GAA ACC CCT GAC TCC TAC AGA GAC TCT CCT CAT AGC 1604
Arg His Ala Arg Glu Thr Pro Asp Ser Tyr Arg Asp Ser Pro His Ser
405 410 415 420
GAA AGG TAT GTA TCA GCC ATG ACC ACC CCG GCT CGT ATG TCA CCT GTA 1652
Glu Arg Tyr Val Ser Ala Met Thr Thr Pro Ala Arg Met Ser Pro Val
425 430 435
GAT TTC CAC ACG CCA AGC TCC CCT AAA TCG CCC CCT TCG GAA ATG TCT 1700
Asp Phe His Thr Pro Ser Ser Pro Lys Ser Pro Pro Ser Glu Met Ser
440 445 450
CCA CCC GTG TCC AGC ATG ACG GTG TCC ATG CCC TCT GTG GCA GTC AGC 1748
Pro Pro Val Ser Ser Met Thr Val Ser Met Pro Ser Val Ala Val Ser
455 460 465
CCC TTT GTG GAA GAA GAG AGG CCT CTG CTG CTT GTG ACG CCA CCA AGG 1796
Pro Phe Val Glu Glu Glu Arg Pro Leu Leu Leu Val Thr Pro Pro Arg
470 475 480
CTA CGG GAG AAG AAA TAT GAT CAT CAC CCC CAG CAA CTC AAC TCC TTT 1844
Leu Arg Glu Lys Lys Tyr Asp His His Pro Gln Gln Leu Asn Ser Phe
485 490 495 500
CAT CAC AAC CCT GCA CAT CAG AGT ACC AGC CTC CCC CCT AGC CCA CTG 1892
His His Asn Pro Ala His Gln Ser Thr Ser Leu Pro Pro Ser Pro Leu
505 510 515
AGG ATA GTG GAG GAT GAG GAG TAC GAG ACG ACC CAG GAG TAT GAG TCA 1940
Arg Ile Val Glu Asp Glu Glu Tyr Glu Thr Thr Gln Glu Tyr Glu Ser
520 525 530
GTT CAA GAG CCC GTT AAG AAA GTC ACC AAT AGC CGG CGG GCC AAA AGA 1988
Val Gln Glu Pro Val Lys Lys Val Thr Asn Ser Arg Arg Ala Lys Arg
535 540 545
ACC AAG CCC AAT GGC CAC ATT GCC AAT AGG TTG GAA ATG GAC AGC AAC 2036
Thr Lys Pro Asn Gly His Ile Ala Asn Arg Leu Glu Met Asp Ser Asn
550 555 560
ACA AGT TCT GTG AGC AGT AAC TCA GAA AGT GAG ACA GAA GAC GAA AGA 2084
Thr Ser Ser Val Ser Ser Asn Ser Glu Ser Glu Thr Glu Asp Glu Arg
565 570 575 580
GTA GGT GAA GAC ACA CCA TTC CTG GGC ATA CAG AAC CCC CTG GCA GCC 2132 Val Gly Glu Asp Thr Pro Phe Leu Gly Ile Gln Asn Pro Leu Ala Ala
585 590 595
AGC CTT GAG GTG GCC CCT GCC TTC CGT CTG GCT GAG AGC AGG ACT AAC 2180 Ser Leu Glu Val Ala Pro Ala Phe Arg Leu Ala Glu Ser Arg Thr Asn
600 605 610
CCA GCA GGC CGC TTC TCC ACA CAG GAG GAA TTA CAG GCC AGG CTG TCT 2228 Pro Ala Gly Arg Phe Ser Thr Gln Glu Glu Leu Gln Ala Arg Leu Ser
615 620 625
AGT GTA ATC GCT AAC CAA GAC CCT ATT GCT GTA TAAAACCTAA ATAAACACAT 2281 Ser Val Ile Ala Asn Gln Asp Pro Ile Ala Val
630 635
AGATTCACCT GTAAAACTTT ATTTTATATA ATAAAGTATT TCACCTTAAA TTAAACAATT 2341
TATTTTATTT TAGCAGTTCT GCAAATAGAA AACAGGAAGA AAAAAAAACT TTTATAAATT 2401
AAATATATGT ATGTAAAAAT GTGTTATGTG CCATATGTAG CAATTTTTTA CAGTATTTCA 2461
AAAACGAGAA AGATATCAAT GGTGCCTTTA TGTTCTGTTA TGTCGAGAGC AAGTTTTATA 2521
AAGTTATGGT GATTTCTTTT TCACAGTATT TCAGCAAAAC CTCCCATATA TTCAGTTTCT 2581
GCTGGCTTTT TGTGCATTGC ATTATGATGT TGACTGGATG TATGGTTTGC AAGGCTAGCA 2641
GCTCGCTCGT GTTCTCTCTC TCTCTCTCTC TCTCTCTCTC TCTGTCTCTC TCTCTGTCTC 2701
TCTCTCTCTC TCTCTCTCTC TCTCTCTCTC TCTCTCTCTC TCTCTCTCTC TGTCTCTCTC 2761
TCTGCTTCCC GTAGCTCCCA ACCAGTACTG TCTTGGACTG GCACATCCAT CCAAATACCT 2821
TTCTACTTTG TATGAAGTTT TCTTTGCTTT CCCAATATGA AATGAGTTCT CTCTACTCTG 2881
TCAGCCAAAG GTTTGCTTCA CTGGACTCTG AGATAATAGT AGACCCAGCA GCATGCTACT 2941
ATTACGTATA GCAGGAAACT GCACCAAGTA ATGTCCAATA ATAGGAAGAA AGTAATACTG 3001
TGATTTAAAA AAAAAAACAA ACTATATTAT TAATCAGAAG ACAGCTTGCT CTTGGTAAAA 3061
GGAGCTACCA TTGACTCTAA TTTTGACTTT TTAGTTATTG TTCTTGACAA AGAGTAACAG 3121
CTTCAAGTAC AGCCTAAAAA AAAAAAAAGG GCGGCCGCCC 3161
(52) INFORMATION FOR SEQ ID NO: 51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 639 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:
Met Ser Glu Arg Lys Glu Gly Arg Gly Lys Gly Lys Gly Lys Lys Lys 1 5 10 15
Asp Arg Gly Ser Arg Gly Lys Pro Gly Pro Ala Glu Gly Asp Pro Ser
20 25 30
Pro Ala Leu Pro Pro Arg Leu Lys Glu Met Lys Ser Gln Glu Ser Ala
35 40 45
Ala Gly Ser Lys Leu Val Leu Arg Cys Glu Thr Ser Ser Glu Tyr Ser 50 55 60
Ser Leu Arg Phe Lys Trp Phe Lys Asn Gly Asn Glu Leu Asn Arg Lys 65 70 75 80
Asn Lys Pro Glu Asn Ile Lys Ile Gln Lys Lys Pro Gly Lys Ser Glu
85 90 95
Leu Arg Ile Asn Lys Ala Ser Leu Ala Asp Ser Gly Glu Tyr Met Cys
100 105 110
Lys Val Ile Ser Lys Leu Gly Asn Asp Ser Ala Ser Ala Asn Ile Thr
115 120 125
Ile Val Glu Ser Asn Glu Phe Ile Thr Gly Met Pro Ala Ser Thr Glu 130 135 140
Thr Ala Tyr Val Ser Ser Glu Ser Pro Ile Arg Ile Ser Val Ser Thr 145 150 155 160
Glu Gly Ala Asn Thr Ser Ser Ser Thr Ser Thr Ser Thr Thr Gly Thr
165 170 175
Ser His Leu Ile Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val Asn
180 185 190
Gly Gly Glu Cys Phe Thr Val Lys Asp Leu Ser Asn Pro Ser Arg Tyr
195 200 205
Leu Cys Lys Cys Gln Pro Gly Phe Thr Gly Ala Arg Cys Thr Glu Asn 210 215 220
Val Pro Met Lys Val Gln Thr Gln Glu Lys Ala Glu Glu Leu Tyr Gln
225 230 235 240
Lys Arg Val Leu Thr Ile Thr Gly Ile Cys Ile Ala Leu Leu Val Val
245 250 255
Gly Ile Met Cys Val Val Ala Tyr Cys Lys Thr Lys Lys Gln Arg Gln
260 265 270
Lys Leu His Asp Arg Leu Arg Gln Ser Leu Arg Ser Glu Arg Ser Asn
275 280 285
Leu Val Asn Ile Ala Asn Gly Pro His His Pro Asn Pro Pro Pro Glu 290 295 300
Asn Val Gln Leu Val Asn Gln Tyr Val Ser Lys Asn Val Ile Ser Ser 305 310 315 320
Glu His Ile Val Glu Arg Glu Val Glu Thr Ser Phe Ser Thr Ser His
325 330 335
Tyr Thr Ser Thr Ala His His Ser Thr Thr Val Thr Gln Thr Pro Ser
340 345 350
His Ser Trp Ser Asn Gly His Thr Glu Ser Val Ile Ser Glu Ser Asn
355 360 365
Ser Val Ile Met Met Ser Ser Val Glu Asn Ser Arg His Ser Ser Pro 370 375 380
Ala Gly Gly Pro Arg Gly Arg Leu His Gly Leu Gly Gly Pro Arg Asp 385 390 395 400
Asn Ser Phe Leu Arg His Ala Arg Glu Thr Pro Asp Ser Tyr Arg Asp
405 410 415
Ser Pro His Ser Glu Arg Tyr Val Ser Ala Met Thr Thr Pro Ala Arg
420 425 430
Met Ser Pro Val Asp Phe His Thr Pro Ser Ser Pro Lys Ser Pro Pro
435 440 445
Ser Glu Met Ser Pro Pro Val Ser Ser Met Thr Val Ser Met Pro Ser 450 455 460
Val Ala Val Ser Pro Phe Val Glu Glu Glu Arg Pro Leu Leu Leu Val 465 470 475 480
Thr Pro Pro Arg Leu Arg Glu Lys Lys Tyr Asp His His Pro Gln Gln
485 490 495
Leu Asn Ser Phe His His Asn Pro Ala His Gln Ser Thr Ser Leu Pro 500 505 510
Pro Ser Pro Leu Arg Ile Val Glu Asp Glu Glu Tyr Glu Thr Thr Gln
515 520 525
Glu Tyr Glu Ser Val Gln Glu Pro Val Lys Lys Val Thr Asn Ser Arg 530 535 540
Arg Ala Lys Arg Thr Lys Pro Asn Gly His Ile Ala Asn Arg Leu Glu 545 550 555 560
Met Asp Ser Asn Thr Ser Ser Val Ser Ser Asn Ser Glu Ser Glu Thr
565 570 575
Glu Asp Glu Arg Val Gly Glu Asp Thr Pro Phe Leu Gly Ile Gln Asn
580 585 590
Pro Leu Ala Ala Ser Leu Glu Val Ala Pro Ala Phe Arg Leu Ala Glu
595 600 605
Ser Arg Thr Asn Pro Ala Gly Arg Phe Ser Thr Gln Glu Glu Leu Gln 610 615 620
Ala Arg Leu Ser Ser Val Ile Ala Asn Gln Asp Pro Ile Ala Val 625 630 635
(53) INFORMATION FOR SEQ ID NO: 52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:
Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr Val Met Ala 1 5 10 15
Ser Phe Tyr Met Thr Ser Arg Arg Lys Arg Gln Glu Thr Glu Lys
20 25 30
Pro Leu Glu Arg Lys Leu Asp His Ser Leu Val Lys Glu Ser Lys
35 40 45
(54) INFORMATION FOR SEQ ID NO: 53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:
Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr Val Met Ala
1 5 10 15
Ser Phe Tyr Lys His Leu Gly Ile Glu Phe Met Glu
20 25
(55) INFORMATION FOR SEQ ID NO: 54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:
His Asn Leu Ile Ala Glu Leu Arg Arg Asn Lys Ala Tyr Arg Ser
1 5 10 15
Lys Cys Met Gln Ile Gln Leu Ser Ala Thr His Leu Arg Pro Ser
20 25 30
Ser Ile Thr His Leu Gly Phe Ile Leu
35
(56) INFORMATION FOR SEQ ID NO: 55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 159 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 27..128
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:
TCTAGATGAA GGACCTGTCA AACCCG TCA AGA TAC TTG TGC AAG TGC CCA AAT 53
Ser Arg Tyr Leu Cys Lys Cys Pro Asn
1 5
GAG TTT ACT GGT GAT CGT TGC CAA AAC TAC GTA ATG GCC AGC TTC TAC 101 Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr Val Met Ala Ser Phe Tyr
10 15 20 25
AAG TAT CTT GGG ATT GAA TTT ATG GAA GCGGAGGAAC TCTACCAGAA 148
Lys Tyr Leu Gly Ile Glu Phe Met Glu
30
GGGATCCCGC G 159
(57) INFORMATION FOR SEQ ID NO:56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:
Ser Arg Tyr Leu Cys Lys Cys Pro Asn Glu Phe Thr Gly Asp Arg Cys
1 5 10 15 Gln Asn Tyr Val Met Ala Ser Phe Tyr Lys Tyr Leu Gly Ile Glu Phe
20 25 30
Met Glu
(58) INFORMATION FOR SEQ ID NO: 57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:
His Leu Gly Ile Glu Phe Ile Glu
1 5
(59) INFORMATION FOR SEQ ID NO: 58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:
Ser Ala Gln Met Ser Leu Leu Val Ile Ala Ala Lys Thr Thr
1 5 10
(60) INFORMATION FOR SEQ ID NO: 59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:
Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr Val Met Ala Ser 1 5 10 15
Phe Tyr Lys His Leu Gly Ile Glu Phe Met Glu
20 25
(61) INFORMATION FOR SEQ ID NO: 60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60:
Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr Val Met Ala Ser 1 5 10 15
Phe Tyr Lys
(62) INFORMATION FOR SEQ ID NO: 61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61:
Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr Val Met Ala Ser 1 5 10 15
Phe Tyr Ser Thr Ser Thr Pro Phe Leu Ser Leu Pro Glu
20 25
(63) INFORMATION FOR SEQ ID NO: 62:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 111 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62:
Tyr Val Ser Ala Met Thr Thr Pro Ala Arg Met Ser Pro Val Asp Phe 1 5 10 15
His Thr Pro Ser Ser Pro Lys Ser Pro Pro Ser Glu Met Ser Pro Pro
20 25 30
Val Ser Ser Met Thr Val Ser Met Pro Ser Met Ala Val Ser Pro Phe
35 40 45
Met Glu Glu Glu Arg Pro Leu Leu Leu Val Thr Pro Pro Arg Leu Arg 50 55 60
Glu Lys Lys Phe Asp His His Pro Gln Gln Phe Ser Ser Phe His His 65 70 75 80
Asn Pro Ala His Asp Ser Asn Ser Leu Pro Ala Ser Pro Leu Arg Ile
85 90 95
Val Glu Asp Glu Glu Tyr Glu Thr Thr Gln Glu Tyr Glu Pro Ala
100 105 110
(64) INFORMATION FOR SEQ ID NO: 63:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 70 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63:
Phe Tyr Val Ser Ala Met Thr Thr Pro Ala Arg Met Ser Pro Val Asp 1 5 10 15
Phe His Thr Pro Ser Ser Pro Lys Ser Pro Pro Ser Glu Met Ser Pro
20 25 30
Pro Val Ser Ser Met Thr Val Ser Met Pro Ser Met Ala Val Ser Pro
35 40 45
Phe Met Glu Glu Glu Arg Pro Leu Leu Leu Val Thr Pro Pro Arg Leu 50 55 60
Arg Glu Lys Lys Phe Asp
65 70
(65) INFORMATION FOR SEQ ID NO: 64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 148 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64:
His His Pro Gln Gln Phe Ser Ser Phe His His Asn Pro Ala His Asp 1 5 10 15
Ser Asn Ser Leu Pro Ala Ser Pro Leu Arg Ile Val Glu Asp Glu Glu
20 25 30
Tyr Glu Thr Thr Gln Glu Tyr Glu Pro Ala Gln Glu Pro Val Lys Lys
35 40 45
Leu Ala Asn Ser Arg Arg Ala Lys Arg Thr Lys Pro Asn Gly His Ile 50 55 60
Ala Asn Arg Leu Glu Val Asp Ser Asn Thr Ser Ser Gln Ser Ser Asn 65 70 75 80
Ser Glu Ser Glu Thr Glu Asp Glu Arg Val Gly Glu Asp Thr Pro Phe
85 90 95
Leu Gly Ile Gln Asn Pro Leu Ala Ala Ser Leu Glu Ala Thr Pro Ala
100 105 110
Phe Arg Leu Ala Asp Ser Arg Thr Asn Pro Ala Gly Arg Phe Ser Thr
115 120 125
Gln Glu Glu Ile Gln Ala Arg Leu Ser Ser Val Ile Ala Asn Gln Asp 130 135 140
Pro Ile Ala Val
145
(66) INFORMATION FOR SEQ ID NO: 65:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65:
Trp Phe Lys
1
(67) INFORMATION FOR SEQ ID NO: 66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66:
Leu Val Leu Arg
1
(68) INFORMATION FOR SEQ ID NO: 67:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67:
Gly Lys Pro Gly Pro Ala Glu Gly Asp Pro Ser Pro Ala Leu Pro 1 5 10 15
Pro Arg
(69) INFORMATION FOR SEQ ID NO: 68:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 68:
Cys Glu Thr Ser Ser Glu Tyr Ser Ser Leu Arg
1 5 10
(70) INFORMATION FOR SEQ ID NO: 69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69:
Ala Ser Leu Ala Asp Ser Gly Glu Tyr Met Cys Lys
1 5 10
(71) INFORMATION FOR SEQ ID NO: 70:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:
Leu Gly Asn Asp Ser Ala Ser Ala Asn Ile Thr Ile Val Glu Ser 1 5 10 15
Asn Glu Phe Ile Ile Gly Met Pro Ala
20
(72) INFORMATION FOR SEQ ID NO: 71:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:71:
Arg Gly Ser Arg Gly Lys Pro Gly Pro Ala Glu Gly Asp Pro Ser 1 5 10 15
Pro Ala Leu Pro Pro
20
(73) INFORMATION FOR SEQ ID NO: 72:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:
Gly Glu Tyr Met Cys Lys
1 5
(74) INFORMATION FOR SEQ ID NO: 73:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 60 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : single stranded
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73:
ATAGGGAAGG GCGGGGGAAG GRTCNCCYTC NGCAGGGCCG GGCTTGCCTC 50
TGGAGCCTCT 60
(75) INFORMATION FOR SEQ ID NO:74:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74:
YTTRCACATR TAYTCNCC 18
(76) INFORMATION FOR SEQ ID NO: 75:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75:
GCGTCTAGAT GAAGGACCTG TCAAACCC 28
(77) INFORMATION FOR SEQ ID NO: 76:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76:
GCGGGATCCC TTCTGGTAGA GTTCCTCC 28
(78) INFORMATION FOR SEQ ID NO: 77:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:
TGAAGGACCT GTCAAACCC 19
(79) INFORMATION FOR SEQ ID NO: 78:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:
AGGAAATGAC AGTGCCTCT 19
(80) INFORMATION FOR SEQ ID NO: 79:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 79:
TCTCTGGCAT GCCTGAGG 18
(81) INFORMATION FOR SEQ ID NO: 80:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 80:
CGGTCTAGAA GCTTCCACCA TGTCTGAGCG CAAAGAA 37
(82) INFORMATION FOR SEQ ID NO: 81:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 81:
GCCGTCGACC TATTACCTTT CGCTATGAGG 30
(83) INFORMATION FOR SEQ ID NO: 82:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 82:
GCTCTAGAGG CTTCTCTGTT TCTTGCC 27
(84) INFORMATION FOR SEQ ID NO: 83:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 83:
GCTCTAGAAA GAAAATTGGA TCATAGCCTT GTG 33
(85) INFORMATION FOR SEQ ID NO: 84:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 84:
GCCGTCGACC TATTATACAG CAATAGGGTC TTG 33
(86) INFORMATION FOR SEQ ID NO: 85:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 85:
CGGTCTAGAA GCTTCCACCA TGTCCGAGCG CAAAGAA 37
(87) INFORMATION FOR SEQ ID NO: 86:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 86:
GCCGTCGACC TATTAGAGAA TGAAGCCCAA 30
(88) INFORMATION FOR SEQ ID NO: 87:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 39 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 87:
GCCGAAGACG GTCATGAAGC TTCTGCCGCT GTTTCTTGG 39
(89) INFORMATION FOR SEQ ID NO: 88:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 88:
CCTTTCAAAC CCCTCGAGAT ACTTGTGCAA GTG 33
(90) INFORMATION FOR SEQ ID NO: 89:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 89:
Lys Glu Gly Arg Gly Lys Gly Lys Gly Lys Lys Lys Asp Arg Gly
1 5 10 15
Ser Arg Gly
(91) INFORMATION FOR SEQ ID NO: 90:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 141 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double stranded
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 90:
CTAATTCCGC TCTCACCTAC CAAACAATGC CCCCCTGCAA AAAATAAATT 50
CATATAAAAA ACATACAGAT AACCATCTGC GGTGATAAAT TATCTCTGGC 100
GGTGTTGACA TAAATACCAC TGGCGGTGAT ACTGAGCACA T 141
(92) INFORMATION FOR SEQ ID NO: 91:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 147 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double stranded
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 91:
CGATGTGCTC AGTATCACCG CCAGTGGTAT TTATGTCAAC ACCGCCAGAG 50
ATAATTTATC ACCGCAGATG GTTATCTGTA TGTTTTTTAT ATGAATTTAT 100
TTTTTGCAGG GGGGCATTGT TTGGTAGGTG AGAGCGGAAT TAGACGT 147
(93) INFORMATION FOR SEQ ID NO: 92:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 55 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double stranded
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 92:
CGATTTGATT CTAGAAGGAG GAATAACATA TGGTTAACGC GTTGGAATTC 50
GGTAC 55
(94) INFORMATION FOR SEQ ID NO: 93:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 49 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double stranded
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 93:
CGAATTCCAA CGCGTTAACC ATATCTTATT CCTCCTTCTA GAATCAAAT 49
(95) INFORMATION FOR SEQ ID NO: 94:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double stranded
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 94:
GAGCTCACTA GTGTCGACCT GCAG 24
(96) INFORMATION FOR SEQ ID NO: 95:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double stranded
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 95:
CTGCAGGTCG ACACTAGTGA GCTC 24
(97) INFORMATION FOR SEQ ID NO: 96:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 96:
CACTGGCGGT GATAATGATC 20
(98) INFORMATION FOR SEQ ID NO: 97:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 49 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 97:
TTTGATCTAG AAGGAGGAAT AACATATGAA GAAGAAGGAG CGAGGCTCC 49
(99) INFORMATION FOR SEQ ID NO: 98:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 98:
CCGTTAGGAT CCTTACTTCT GGTACAGCTC CTCCGC 36
(100) INFORMATION FOR SEQ ID NO: 99:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 99:
AACACACTCG AGTTACTTCT GGTAGAGTTC CTCCGC 36
(101) INFORMATION FOR SEQ ID NO: 100:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 100:
TATGAAAAAA AAAGAACGTG GTTCTGGTAA AAAACCGGAA TCCGC 45
(102) INFORMATION FOR SEQ ID NO: 101:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 41 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 101:
GGATTCCGGT TTTTTACCAG AACCACGTTC TTTTTTTTTC A 41
(103) INFORMATION FOR SEQ ID NO: 102:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 48 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 102:
ACCACATCTA GAAGGAGGAA TAACATATGA GCCATCTTGT AAAATGTG 48
(104) INFORMATION FOR SEQ ID NO: 103:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 103:
ACCACAGGAT CCTTACTTCT GGTACAGCTC C 31
(105) INFORMATION FOR SEQ ID NO: 104:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 104:
ACACAAGGAT CCTTACTTCT GGTACAGCTC 30
(106) INFORMATION FOR SEQ ID NO: 105:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 50 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : single stranded
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 105:
ACACAATCTA GAAGGAGGAA TAACATATGT CTCATCTTGT AAAATGTGCT 50
(107) INFORMATION FOR SEQ ID NO:106:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 106:
AGGAAATGAC AGTGCCTCTG C 21
(108) INFORMATION FOR SEQ ID NO: 107:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 107:
TATGGGGATC CATTACTTCT GGTACAGCTC CTCCG 35
(109) INFORMATION FOR SEQ ID NO: 108:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 111 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 108:
TGATTCTAGA AGGAGGAATA ACATATGAAR AARACYGCDA TYGCDGTWGC 50
DCTGGCDCTG GCDGGYTTYG CDACYGTWGC DCAGGCDAGC CATCTTGTAA 100
AATGTGCGGA G 111
(110) INFORMATION FOR SEQ ID NO: 109:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 109:
AGTATCTCGA GGGGTTTGAA AGG 23
(111) INFORMATION FOR SEQ ID NO: 110:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 46 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 110:
ACAAACTCTA GAAGGAGGAA TAACATATGT CTGAACGTAA AGAAGG 46
(112) INFORMATION FOR SEQ ID NO: 111:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:111:
GTCTGAACGT AAAGAAGGTC GTGGTAAAGG GAAGGGCAAG AA 42
(113) INFORMATION FOR SEQ ID NO: 112:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 112:
ACAAACGGAT CCTTACTTCT GGTACAGCTC C 31
(114) INFORMATION FOR SEQ ID NO: 113:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 113:
CAAACCCCTC GAGATACTTG TG 22
(115) INFORMATION FOR SEQ ID NO: 114:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single stranded
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 114:
AGAAGGGATC CATTACGTAG TTTTGGCAGC GATCAC 36
(116) INFORMATION FOR SEQ ID NO: 115:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double stranded
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 115:
AAGAAGAAGG AGCGAGGCTC CGGCAAGAAG CCGGAG 36
(117) INFORMATION FOR SEQ ID NO: 116:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double stranded
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:116:
AAAAAAAAAG AACGTGGTTC TGGTAAAAAA CCGGAA 36
(118) INFORMATION FOR SEQ ID NO: 117:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double stranded
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 117:
TCCGAGCGCA AAGAAGGCAG AGGC 24
(119) INFORMATION FOR SEQ ID NO: 118:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double stranded
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 118:
TCTGAACGTA AAGAAGGTCG TGGT 24
(120) INFORMATION FOR SEQ ID NO: 119:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 63 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double stranded
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 119:
ATGAAGAAGA CCGCGATTGC AATTGCGGTA GCGCTGGCGG GTTTTGCGAC 50
CGTTGCGCAG GCG 63
(121) INFORMATION FOR SEQ ID NO: 120:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 120:
Met Lys Lys Lys Asp Arg Gly Ser Arg Gly Lys Pro Gly Pro Ala 1 5 10 15
Glu Gly Asp Pro Ser
20
(122) INFORMATION FOR SEQ ID NO: 121:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 121:
Met Ser Glu Arg Lys Glu Gly Arg Gly Lys Gly Lys Gly Lys Lys 1 5 10 15
Lys Glu Arg Gly Ser Gly Lys Lys Pro Glu
20 25
(123) INFORMATION FOR SEQ ID NO: 122:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 122:
Met Lys Lys Lys Glu Arg Gly Ser Gly Lys
1 5 10
(124) INFORMATION FOR SEQ ID NO: 123:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 123:
Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys Val 1 5 10 15
(125) INFORMATION FOR SEQ ID NO: 124:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 124:
Met Lys Lys Lys Glu Arg Gly Ser Gly Lys Lys Pro Glu Ser Ala 1 5 10 15
Ala Gly Ser Gln Ser Pro Ala Leu Pro Pro
20 25
(126) INFORMATION FOR SEQ ID NO: 125:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 125:
Met Ser His Leu Val Lys Cys Ala Glu Lys Glu Lys Thr Phe Cys 1 5 10 15
(127) INFORMATION FOR SEQ ID NO: 126:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 126:
Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr Val Met Ala 1 5 10 15
Ser Phe Tyr Lys
(128) INFORMATION FOR SEQ ID NO: 127:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: unknown
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:127:
Pro Asn Glu Phe Thr Gly Asp Arg Cys Gln Asn Tyr Val Met Ala 1 5 10 15
Ser Phe Tyr Ser Thr Ser Thr Pro Phe Leu Ser Leu Pro Glu
20 25
Claims
1. A non-naturally-occurring polypeptide which possesses neu receptor stimulatory activity and comprises an amino acid sequence that includes one of the following two sequences:
CysAlaGluLysGluLysThrPheCysValAsnGlyGlyGluCysPheMetVal LysAspLeuSerAsnProSerArgTyrLeuCysLysCysGlnProGlyPheThr (α) GlyAlaArgCys [SEQ ID NO: 1] CysAlaGluLysGluLysThrPheCysValAsnGlyGlyGluCysPheMetVal
LysAspLeuSerAsnProSerArgTyrLeuCysLysCysProAsnGluPheThr (ß ) GlyAspArgCys [SEQ ID NO: 2] or an analog thereof.
2. A polypeptide according to Claim 1 which is a product of procaryotic or eucaryotic expression of an exogenous DNA sequence.
3. A polypeptide according to Claim 2 which is a product of non-human mammalian cell expression.
4. A polypeptide according to Claim 3 in which the non-human mammalian cell is a Chinese hamster ovary cell.
5. A polypeptide according to Claim 2 which is a product of bacterial cell expression.
6. A polypeptide according to Claim 5 in which the bacterial cell is an E. coli cell.
7. A polypeptide according to Claim 2 which is a product of yeast cell expression.
8. A polypeptide according to Claim 2 which is a product of an exogenous DNA sequence which is a
manufactured DNA sequence.
9. A polypeptide according to Claim 2 which is the expression product of an exogenous DNA sequence which is a complementary DNA sequence.
10. A polypeptide according to Claim 1 which has all or a portion of the amino acid sequence of Figure 31, 32, 34, 35, 36, or 37 [SEQ ID NOS: 6, 8, 10, 12, 14, or 16, respectively].
11. A polypeptide according to Claim 1 which has the amino acid sequence of Figure 32 [SEQ ID NO: 8] from amino acid position 1 to amino acid position 241.
12. A polypeptide according to claim 1 comprising the amino acid sequence of Figure 32 [SEQ ID NO: 8] from amino acid position 14 to amino acid position 241.
13. A polypeptide according to Claim 12 which further includes a methionine residue at the amino terminus.
14. A polypeptide according to Claim 1 which has the amino acid sequence of proNDF-α1a in Figure 38 from amino acid position 14 to amino acid position 249.
15. A polypeptide according to Claim 1 which has the amino acid sequence of Figure 32 [SEQ ID NO: 8] from amino acid position 177 to amino acid position 241.
16. A polypeptide according to Claim 15 which further includes a methionine residue at the amino terminus.
17. A polypeptide according to Claim 1 which has the amino acid sequence of proNDF-ßla in Figure 38 from amino acid position 14 to amino acid position 246.
18. A polypeptide according to Claim 17 which further includes a methionine residue at the amino terminus.
19. A polypeptide according to Claim 1 which has the amino acid sequence of Figure 35 [SEQ ID NO: 14] from amino acid position 177 to amino acid position 246.
20. A polypeptide according to Claim 19 which further includes a methionine residue at the amino terminus.
21. A polypeptide according to Claim 1 which has the amino acid sequence of proNDF-α3 in Figure 38 from amino acid position 14 to amino acid position 247.
22. A polypeptide according to Claim 21 which further includes a methionine residue at the amino terminus.
23. A polypeptide according to Claim 1 which is the expression product of a DNA sequence encoding, in addition to one of the two amino acid sequences specified
in Claim 1, the cytoplasmic domain of a rat neu
differentiation factor.
24. An isolated DNA molecule comprising a nucleic acid sequence encoding a polypeptide which
possess neu receptor stimulatory activity and comprises an amino acid sequence that includes one of the following two sequences: CysAlaGluLysGluLysThrPheCysValAsnGlyGlyGluCysPheMetVal
LysAspLeuSerAsnProSerArgTyrLeuCysLysCysGlnProGlyPheThr (α)
GlyAlaArgCys [SEQ ID NO: 1]
CysAlaGluLysGluLysThrPheCysValAsnGlyGlyGluCysPheMetVal
LysAspLeuSerAsnProSerArgTyrLeuCysLysCysProAsnGluPheThr (ß )
GlyAspArgCys [SEQ ID NO: 2] or an analog thereof.
25. A DNA molecule according to Claim 24 which comprises a DNA sequence selected from among the following:
(a) the DNA sequences set out in Figures 31, 32, 34, 35, 36, and 37 [SEQ ID NOS: 5, 7, 11, 13, or 15, respectively], or their complementary strands;
(b) DNA sequences which hybridize to the DNA sequences defined in (a) or fragments thereof; and
(c) DNA sequences which, but for the degeneracy of the genetic code, would hybridize to the DNA sequences defined in (a) and (b).
26. A recombinant DNA molecule according to Claim 24 which is deposited with the ATCC under
accession number 69302.
27. A recombinant DNA molecule according to Claim 24 which is deposited with the ATCC under accession number 69304.
28. A recombinant DNA molecule having the nucleotide sequence shown in Figure 30 [SEQ ID NO: 3].
29. A recombinant DNA molecule according to Claim 28 which is deposited with the ATCC under accession number 69305.
30. A recombinant DNA molecule according to Claim 24 which is deposited with the ATCC under accession number 69306.
31. A recombinant DNA molecule according to Claim 24 which is deposited with the ATCC under accession number 69307.
32. A recombinant DNA molecule according to Claim 24 which is deposited with the ATCC under accession number 69308.
33. A complementary DNA molecule according to
Claim 24.
34. A manufactured DNA molecule according to Claim 24.
35. A DNA molecule according to Claim 24 which includes one or more codons preferred for
expression in E. coli cells.
36. A DNA molecule according to Claim 24 which also encodes the cytoplasmic domain of a rat neu differentiation factor.
37. A biologically functional plasmid or viral DNA vector which includes a DNA molecule according to Claim 24.
38. A procaryotic or eucaryotic host cell stably transformed or transfected with a DNA vector according to Claim 37 in a manner allowing expression of the polypeptide encoded by the DNA molecule.
39. A host cell according to Claim 38 which is a non-human mammalian cell.
40. A host cell according to Claim 39 which is a Chinese hamster ovary cell.
41. A host cell according to Claim 38 which is an E. coli cell.
42. A host cell according to Claim 38 which is a yeast cell.
43. A polypeptide product of the expression in a procaryotic or eucaryotic host cell of a DNA
molecule according to Claim 24.
44. A process for the production of a
biologically active, non-naturally occurring polypeptide, comprising:
growing, under suitable nutrient conditions, procaryotic or eucaryotic host cells transformed or
transfected with a DNA molecule according to Claim 24, and
isolating the desired polypeptide product of the expression in a biologically active form.
45. An antibody specifically generated by immunization with a polypeptide according to Claim 1.
46. An antibody according to Claim 45 which is a monoclonal antibody.
47. A biologically active composition comprising the polypeptide of Claim 1 covalently attached to a water soluble polymer.
48. A composition according to Claim 47, in which the water soluble polymer is from the group
consisting of polyethylene glycol, polypropylene glycol, and copolymers of polyethylene glycol and polypropylene glycol.
49. A method of modulating cellular proliferation and differentiation, comprising contacting the cells with an effective amount of the polypeptide of Claim 1.
50. A method of enhancing repair and regeneration, of human tissues that express the neu receptor, comprising contacting the tissue with an effective amount of a polypeptide according to Claim 1.
51. A method of treating a dermal wound in a human, comprising administering to the human a
therapeutically effective amount of a polypeptide according to Claim 1.
52. A method of treating tumors derived from epithelial tissue of the breast, stomach, lung, pancreas, colon, kidney, prostate or bladder in a human, comprising administering to the human a therapeutically effective amount of a polypeptide according to Claim 1.
53. A method according to Claim 52 in which a polypeptide according to Claim 1 is used in conjunction with one or more anti-cancer chemotherapeutic agents.
54. A method of treating gastrointestinal disease in a human, comprising administering to the human a therapeutically effective amount of a polypeptide according to Claim 1.
55. A method of treating Barrett's esophagus in a human, comprising administering to the human a therapeutically effective amount of a polypeptide
according to Claim 1.
56. A method of promoting reepithelialization in the human gastrointestinal, respiratory, reproductive or urinary tract, comprising administering to a human a therapeutically effective amount of a polypeptide according to Claim 1.
57. A method of treating cystic kidney disease or non-cystic end stage kidney disease in a human, comprising administering to the human a
therapeutically effective amount of a polypeptide according to Claim 1.
58. A method of treating inflammatory bowel disease in a human, comprising administering to the human
a therapeutically effective amount of a polypeptide according to Claim 1.
59. A pharmaceutical composition comprising a therapeutically effective amount of a polypeptide according to Claim 1 and a pharmaceutically acceptable diluent, adjuvant or carrier.
60. An assay for detecting the level of naturally-occurring neu differentiation factor in cells or biological fluids from a human subject, comprising contacting an antibody according to Claim 45 with a sample of the cells or biological fluids to be tested under conditions appropriate for binding of the antibody to naturally-occurring neu differentiation factor, and
determining the level of antibody-antigen reaction as indicative of the amount of naturally-occurring neu differentiation factor in the sample.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU70429/94A AU7042994A (en) | 1993-05-21 | 1994-05-23 | Recombinant (neu) differentiation factors |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US6638493A | 1993-05-21 | 1993-05-21 | |
US08/066,384 | 1993-05-21 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO1994028133A1 true WO1994028133A1 (en) | 1994-12-08 |
WO1994028133A9 WO1994028133A9 (en) | 1995-02-16 |
Family
ID=22069174
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1994/005769 WO1994028133A1 (en) | 1993-05-21 | 1994-05-23 | Recombinant neu differentiation factors |
Country Status (2)
Country | Link |
---|---|
AU (1) | AU7042994A (en) |
WO (1) | WO1994028133A1 (en) |
Cited By (20)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5670342A (en) * | 1995-04-06 | 1997-09-23 | Amgen Inc. | NDF peptides |
US5814308A (en) * | 1996-03-26 | 1998-09-29 | Zhang; Ke | Methods for the treatment of gastrointestinal tract disorders |
WO1998056804A1 (en) * | 1997-06-13 | 1998-12-17 | Human Genome Sciences, Inc. | 86 human secreted proteins |
WO1999014323A1 (en) * | 1997-09-17 | 1999-03-25 | Urs Eppenberger | Heregulin-gamma |
US5912326A (en) * | 1995-09-08 | 1999-06-15 | President And Fellows Of Harvard College | Cerebellum-derived growth factors |
US6017886A (en) * | 1998-08-05 | 2000-01-25 | Amgen Inc. | Use of NDF peptide as growth factor for sensory epithelium |
US6080845A (en) * | 1998-08-05 | 2000-06-27 | Amgen Inc. | Monoclonal antibody against utricular epithelium |
US6096873A (en) * | 1996-07-12 | 2000-08-01 | Genentech, Inc. | Gamma-heregulin |
US6136558A (en) * | 1997-02-10 | 2000-10-24 | Genentech, Inc. | Heregulin variants |
WO2001064877A2 (en) * | 2000-02-28 | 2001-09-07 | Decode Genetics Ehf | Human schizophrenia gene |
WO2001064876A2 (en) * | 2000-02-28 | 2001-09-07 | Decode Genetics Ehf | Human schizophrenia gene |
US6387638B1 (en) | 1997-02-10 | 2002-05-14 | Genentech, Inc. | Heregulin variants |
US7153828B2 (en) | 1998-02-04 | 2006-12-26 | Genentech, Inc. | Use of heregulin as a growth factor |
WO2010053472A1 (en) | 2008-11-04 | 2010-05-14 | Novocell, Inc. | Stem cell aggregate suspension compositions and methods for differentiation thereof |
WO2011147981A3 (en) * | 2010-05-28 | 2012-02-02 | Mind-Nrg Sa | Neuregulin isoforms, neuregulin polypeptides and uses thereof |
EP2420565A1 (en) | 2006-02-23 | 2012-02-22 | ViaCyte, Inc. | Compositions and methods useful for culturing differentiable cells |
EP2730649A1 (en) | 2012-11-08 | 2014-05-14 | Viacyte, Inc. | Scalable primate pluripotent stem cell aggregate suspension culture and differentiation thereof |
WO2015158743A1 (en) * | 2014-04-15 | 2015-10-22 | Universiteit Antwerpen | Treatment of nephropathy |
US9828635B2 (en) | 2011-10-06 | 2017-11-28 | Aveo Pharmaceuticals, Inc. | Predicting tumor response to anti-ERBB3 antibodies |
US20180237540A1 (en) * | 2010-05-21 | 2018-08-23 | Merrimack Pharmaceuticals, Inc. | Bi-Specific Fusion Proteins |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0505148A1 (en) * | 1991-03-17 | 1992-09-23 | Yeda Research And Development Company Limited | Stimulating factor of the neu receptor |
WO1992020798A1 (en) * | 1991-05-24 | 1992-11-26 | Genentech, Inc. | HEREGULINS (HRGs), BINDING PROTEINS OF P185?erb2¿ |
EP0583050A2 (en) * | 1992-04-29 | 1994-02-16 | Amgen Inc. | Recombinant stimulating factor of the neu receptor |
-
1994
- 1994-05-23 WO PCT/US1994/005769 patent/WO1994028133A1/en active Application Filing
- 1994-05-23 AU AU70429/94A patent/AU7042994A/en not_active Abandoned
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0505148A1 (en) * | 1991-03-17 | 1992-09-23 | Yeda Research And Development Company Limited | Stimulating factor of the neu receptor |
WO1992020798A1 (en) * | 1991-05-24 | 1992-11-26 | Genentech, Inc. | HEREGULINS (HRGs), BINDING PROTEINS OF P185?erb2¿ |
EP0583050A2 (en) * | 1992-04-29 | 1994-02-16 | Amgen Inc. | Recombinant stimulating factor of the neu receptor |
Non-Patent Citations (3)
Title |
---|
D. WEN ET AL.: "Neu differentiation factor: A transmembrane glycoprotein containing an EGF domain and an immunglobulin homology unit", CELL, vol. 69, no. 3, 1 May 1992 (1992-05-01), CELL PRESS, CAMBRIDGE, MA,US;, pages 559 - 572 * |
E. PELES ET AL.: "Isolation of the Neu/Her-2 stimulatory ligand: A 44kd glycoprotein that induces differentiation of mammary tumor cells", CELL, vol. 69, no. 1, 3 April 1992 (1992-04-03), CELL PRESS, CAMBRIDGE, MA,US;, pages 205 - 216 * |
W.E. HOLMES ET AL.: "Identification of heregulin, a specific activator of p185erbB2", SCIENCE, vol. 256, 22 May 1992 (1992-05-22), AAAS, WASHINGTON, DC, US;, pages 1205 - 1210 * |
Cited By (38)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5670342A (en) * | 1995-04-06 | 1997-09-23 | Amgen Inc. | NDF peptides |
US5686415A (en) * | 1995-04-06 | 1997-11-11 | Amgen Inc. | Method for the treatment of colon epithelial cells in vivo |
US5849705A (en) * | 1995-04-06 | 1998-12-15 | Amgen Inc. | Method for the treatment of Schwann cells in vivo |
US5929032A (en) * | 1995-04-06 | 1999-07-27 | Amgen Inc. | Method for treating Schwann and colon cells in vitro |
US5912326A (en) * | 1995-09-08 | 1999-06-15 | President And Fellows Of Harvard College | Cerebellum-derived growth factors |
US5814308A (en) * | 1996-03-26 | 1998-09-29 | Zhang; Ke | Methods for the treatment of gastrointestinal tract disorders |
US6916624B2 (en) | 1996-07-12 | 2005-07-12 | Genentech, Inc. | Antibodies that bind gamma-heregulin |
US6096873A (en) * | 1996-07-12 | 2000-08-01 | Genentech, Inc. | Gamma-heregulin |
US7585673B2 (en) | 1996-07-12 | 2009-09-08 | Genentech, Inc. | γ-heregulin |
US7563583B2 (en) | 1997-02-10 | 2009-07-21 | Genentech, Inc. | Heregulin variants |
US6136558A (en) * | 1997-02-10 | 2000-10-24 | Genentech, Inc. | Heregulin variants |
US7063961B2 (en) | 1997-02-10 | 2006-06-20 | Genentech, Inc. | Heregulin variants |
US6387638B1 (en) | 1997-02-10 | 2002-05-14 | Genentech, Inc. | Heregulin variants |
WO1998056804A1 (en) * | 1997-06-13 | 1998-12-17 | Human Genome Sciences, Inc. | 86 human secreted proteins |
WO1999014323A1 (en) * | 1997-09-17 | 1999-03-25 | Urs Eppenberger | Heregulin-gamma |
US7153828B2 (en) | 1998-02-04 | 2006-12-26 | Genentech, Inc. | Use of heregulin as a growth factor |
US6080845A (en) * | 1998-08-05 | 2000-06-27 | Amgen Inc. | Monoclonal antibody against utricular epithelium |
US6017886A (en) * | 1998-08-05 | 2000-01-25 | Amgen Inc. | Use of NDF peptide as growth factor for sensory epithelium |
WO2001064876A3 (en) * | 2000-02-28 | 2002-04-18 | Decode Genetics Ehf | Human schizophrenia gene |
WO2001064876A2 (en) * | 2000-02-28 | 2001-09-07 | Decode Genetics Ehf | Human schizophrenia gene |
WO2001064877A2 (en) * | 2000-02-28 | 2001-09-07 | Decode Genetics Ehf | Human schizophrenia gene |
WO2001064877A3 (en) * | 2000-02-28 | 2002-05-30 | Decode Genetics Ehf | Human schizophrenia gene |
EP2420565A1 (en) | 2006-02-23 | 2012-02-22 | ViaCyte, Inc. | Compositions and methods useful for culturing differentiable cells |
US8658352B2 (en) | 2006-02-23 | 2014-02-25 | Viacyte, Inc. | Compositions and methods useful for culturing differentiable cells |
WO2010053472A1 (en) | 2008-11-04 | 2010-05-14 | Novocell, Inc. | Stem cell aggregate suspension compositions and methods for differentiation thereof |
EP3550012A1 (en) | 2008-11-04 | 2019-10-09 | ViaCyte, Inc. | Stem cell aggregate suspension compositions and methods for differentiation thereof |
US10988547B2 (en) | 2010-05-21 | 2021-04-27 | Silver Creek Pharmaceuticals, Inc. | Bi-specific fusion proteins |
US20180237540A1 (en) * | 2010-05-21 | 2018-08-23 | Merrimack Pharmaceuticals, Inc. | Bi-Specific Fusion Proteins |
US10858450B2 (en) * | 2010-05-21 | 2020-12-08 | Silver Creek Pharmaceuticals, Inc. | Bi-specific fusion proteins |
US11673970B2 (en) | 2010-05-21 | 2023-06-13 | Silver Creek Pharmaceuticals, Inc. | Bi-specific fusion proteins |
US11814443B2 (en) | 2010-05-21 | 2023-11-14 | Silver Creek Pharmaceuticals, Inc. | Bi-specific fusion proteins |
WO2011147981A3 (en) * | 2010-05-28 | 2012-02-02 | Mind-Nrg Sa | Neuregulin isoforms, neuregulin polypeptides and uses thereof |
JP2013529911A (en) * | 2010-05-28 | 2013-07-25 | マインド−エヌアールジー ソシエテ アノニム | Neuregulin isoform, neuregulin polypeptide and methods of use thereof |
CN103118698A (en) * | 2010-05-28 | 2013-05-22 | 意识-Nrg公司 | Neuregulin isoforms, neuregulin polypeptides and uses thereof |
US9828635B2 (en) | 2011-10-06 | 2017-11-28 | Aveo Pharmaceuticals, Inc. | Predicting tumor response to anti-ERBB3 antibodies |
EP2730649A1 (en) | 2012-11-08 | 2014-05-14 | Viacyte, Inc. | Scalable primate pluripotent stem cell aggregate suspension culture and differentiation thereof |
EP4053264A1 (en) | 2012-11-08 | 2022-09-07 | ViaCyte, Inc. | Scalable primate pluripotent stem cell aggregate suspension culture and differentiation thereof |
WO2015158743A1 (en) * | 2014-04-15 | 2015-10-22 | Universiteit Antwerpen | Treatment of nephropathy |
Also Published As
Publication number | Publication date |
---|---|
AU7042994A (en) | 1994-12-20 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO1994028133A1 (en) | Recombinant neu differentiation factors | |
WO1994028133A9 (en) | Recombinant neu differentiation factors | |
Wen et al. | Structural and functional aspects of the multiplicity of Neu differentiation factors | |
EP0814827B1 (en) | Vascular endothelial growth factor-b | |
EP0583050A2 (en) | Recombinant stimulating factor of the neu receptor | |
EP0842273B1 (en) | Receptor ligand vegf-c | |
TOYODA et al. | Distribution of mRNA for human epiregulin, a differentially expressed member of the epidermal growth factor family | |
CA2267670C (en) | Method for preparing human stem cell factor polypeptide | |
KR100306244B1 (en) | Composition for treating muscle disease and disorders | |
EP1241258A2 (en) | Stem cell factor | |
GB2214185A (en) | Proteins and methods for their production | |
US5115096A (en) | Amphiregulin: a bifunctional growth modulating glycoprotein | |
CA2249666C (en) | Macrophage stimulating protein for the treatment of gastrointestinal disorders | |
WO1999032515A2 (en) | Angiopoietin homolog, dna encoding it, and method of making it | |
WO1999040193A1 (en) | Angiopoietin homolog zapo3, dna encoding it, and method of making it | |
AU2003261493B2 (en) | Stem Cell Factor | |
AU2003201371B2 (en) | Receptor Ligand VEGF-C | |
Wu | Tumorigenesis of A431 human epidermoid carcinoma cell | |
NO179453B (en) | Method of Preparation of Therapeutically Active Amphiregulin, Host Cell Producing Protein, Recombinant Vector Contained in the Host Cell, and Nucleotide Sequence Encoding Proteins with Amphiregulin Activity | |
SK97999A3 (en) | Dna sequence, a polypeptide having hematopoietic biological property, the use thereof and pharmaceutical composition | |
SK98099A3 (en) | Dna sequence, a polypeptide having hematopoietic biological property, the use thereof and pharmaceutical composition | |
DD296691A5 (en) | PROCESS FOR THE PREPARATION OF ANTIBODIES AGAINST AN EPITOPE OF AMPHIREGULIN | |
AU1007200A (en) | Receptor ligand VEGF-C |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AU CA JP |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE |
|
COP | Corrected version of pamphlet |
Free format text: PAGES 1/86-86/86,DRAWINGS,REPLACED BY NEW PAGES 1/88-88/88;DUE TO LATE TRANSMITTAL BY THE RECEIVINGOFFICE |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
122 | Ep: pct application non-entry in european phase | ||
NENP | Non-entry into the national phase |
Ref country code: CA |