JPS6167490A - Cyclic double-chain dna having pl promoter - Google Patents

Abstract

PURPOSE:The titled DNA containing a specific PL promoter operator, a region coding metapyrocatechase, a temperature-sensitive region, and a region coding ampicillin-resistance, and exhibiting metapyrocatechase. CONSTITUTION:A fragment containing cIts containing a temperature-sensitive gene region separated from pHT1 plasmid is linked and cyclized with a frag ment containing the sequence from the EcoRI terminal to the BamHI site and containing the region coding ampicillinresistance and the replication initiation point in Escherichia coli, containing HpaI site between said BamHI site and the BglII terminal, and containing the PL promoter operator region of lambda phage between the BglII terminal and the HpaI site. A plasmid PHT2 is constructed by this process. A C-230 fragment containing the region coding metapyrocatechase and separated from PYT3 plasmid is inserted into PHT2 to obtain PHT3A which is a cyclic double-chain DNA.

Description

[Detailed description of the invention]

The present invention relates to a circular double-stranded DNA having a PL promoter, and more particularly to a circular double-stranded DNA having a PL promoter and a DNA sequence encoding metavirocatecase. The present invention relates to a transformed microorganism containing this circular double-stranded DNA as a plasmid, and a method for producing metapyrocatechase using this microorganism. Metapyrocatechase was discovered in 1963 by Nozaki et al.
Zeitschrift) Volume 338, Page 582 (1
963)] by Pseudomonas albira (Pse
udomona sarvilla) mt-2 strain as colorless needle crystals. This enzyme has a molecular weight of 140,000 and a (Fe2/a)4 structure consisting of four identical subunits. The two irons essential for the expression of activity are easily oxidized to iron 311fi, but when oxidized, the enzymatic activity is lost. However, this enzyme is extremely stabilized in the presence of 10% acetone or ethanol, and its enzymatic activity is retained for several months [Nozaki et al., Journal of Biological Chemistry, T., Biological (lhe
migtr7), vol. 243, p. 2682 (1968)
). This enzyme was recently discovered in Pseudomonas putida (Pseudomonas putida).
It was analyzed using cloning technology that it was also encoded on the TOL plasmid of the TOL plasmid (monasputia) mt-2 strain, and its DNA sequence and primary amino acid structure were revealed [Nakai et al., Journal of Biological Chemistry (J , Biolog
icaICchemistry), Volume 258, 2923
(1983)). The reactivity of this enzyme has already been reported by Nozaki et al.
imica et Biophysica Acta)
, vol. 220, 213 Sada (1970)], and as shown in the general formula below, catechol derivatives are easily oxidized to give 2-oximuconic acid semialdehyde derivatives. kt=H, Me, UL% NIJ2 As described above, since this enzyme has relatively low substrate specificity, it is difficult to use as an important intermediate in the synthesis of useful organic compounds using various catechol derivatives as starting materials. can give. It is also said that this enzyme can be used in the food industry to prevent fruits from browning. Conventionally, this enzyme was obtained from Pseudomonas putida (P
seudomonas putida) mt-2
A method has been adopted in which the strain (ATCO23973) is cultured at 24°C for 140 hours. With this method, only about 2% of the total bacterial protein is obtained from metapyrocatechase [Nakai et al., Journal of Biological Chemistry (J. Biological Chemistry 7), 25
Volume 8, page 2916 (1983)]. In recent years, genetic recombination technology has been used to produce iK by expressing in microorganisms foreign direct genes encoding useful peptides and proteins such as enzymes, peptide hormones, antigens, and antibodies. Up to now, two methods are known for expressing foreign genes as proteins within microorganisms, such as Escherichia coli. One is to connect a foreign gene to a DNA sequence encoding a bacterial protein such as β-galactosidase to produce a fusion protein (hybrid protein method), and the other is to connect a foreign gene to a DNA sequence encoding a bacterial protein such as β-galactosidase to produce a fusion protein (hybrid protein method). This method (direct expression method) directly produces only the target protein by connecting the translation control region near the codon (ATG: methionine) to the hybrid SD sequence downstream of the promoter. Recently, it has been disclosed that a plasmid containing a gene encoding metapyrocatechase was expressed in Escherichia coli and Gram-positive bacteria such as Bacillus subtilis (Japanese Patent Application Laid-Open No. 180-1880
No. 499). However, the expression level there was 40 U in E. coli.
A. Protein, 3 U/in Bacillus lucieniformis
g・Protein content is variable. The present inventors have arrived at the present invention as a result of research on a plasmid system that can be used for direct expression of metapyrocatechase and its expression. That is, the present invention focuses on the PL promoter operator of lambda phage, the metavirocatecase-encoding region present in the downstream region under the control of the PL promoter operator, and the temperature control region present on the 5' upstream of the pL promoter operator and controlling its activity. A circular double-stranded DNA containing a susceptibility gene portion and capable of expressing metavirocatecase in E. coli is provided. The circular double-stranded DNA of the present invention preferably has a double-stranded DNA sequence containing a portion encoding ambiarin resistance downstream of the temperature-sensitive fusion gene portion,
Furthermore, the part encoding metavirocatecase is derived from the TOL plasmid of Pseudomonas putida mt-2 strain, and the part encoding ampicillin resistance is derived from pBR322.
Preferably, it is derived from a plasmid. The circular duplex 9g DNA of the present invention further preferably has:
The first DNA sequence of the PL promoter/operator, which controls transcription in the clockwise direction on the restriction enzyme cleavage map, is connected to the 5' upstream of this first double-stranded DNA sequence and is transcribed in the counterclockwise direction. a second DNA sequence containing a heat-sensitive gene portion whose 5' end ends in a coco RI restriction enzyme cleavage site, a portion encoding metapyrocatechase linked to the 3' end of the first DNA sequence; 3 of the third DNA sequence and a portion encoding ampicillin resistance that is transcribed counterclockwise starting from the above KCORI restriction enzyme cleavage site, and the 5' end thereof is 3 of the above third DNA sequence.
It consists of a fourth DNA sequence joined to the ' end. An example of such a circular double-stranded DNA is a plasmid having a restriction enzyme cleavage site as shown in FIG. The circular double-stranded DNA of the present invention can be constructed by cutting out an appropriate sequence from a vector containing the corresponding sequence and attaching it again. The PL 7” lomotor operator contained in the first DNA sequence of the circular duplex fi DNA of the present invention is
For example, the Hlnd derived from lambda phage
II-Ban HI restriction enzyme fragment. Such a fragment may be excised directly from lambda phage, or may be extracted from another suitable plasmid containing it (for example, pPmu-1amda: commercially available product). As such a plasmid, Id pKC30 planumid [Nature, No. 29]
2, p. 128 (July 9, 1981)]. The first DNA sequence of the cyclic double-stranded DNA of the present invention is the formula % formula % (hereinafter referred to as formula An example is a DNA sequence represented by a dylic acid unit or a deoxythymidylic acid unit. This sequence connects the PL promoter operator of lambda phage HlndIII-BaIII
It contains sequences corresponding to the Bgl IT-Hpa I fragment. K present in the second DNA sequence of the circular double-stranded DNA of the present invention L, Pt, Cl5sy K allele of lambda phage CI direct gene is exemplified as a temperature-sensitive gene portion that controls the activity of the 7' promoter operator. can do. This lovely gene: E'trD? J
The A fragment may also be extracted directly from lambda phage, but plasmids containing such fragments, such as pC!
Q, V2 plasmid [C. Queen, Journal of Molecular and
Applied eGenetics (0, Q, ueen, Jo
urnal of Molecular and5'・
・A8I rCrCHacchoGTTTGACAGCTchohachir
CAr^rG88G8rl'CrTGCl'C8Arr
G DingTAGAGTAC88^CrG1'cGA^Ding^G
T81^C8IC [8^G88CG8GTTAAC8A
Ding [C8GC DingAI'GCGCCGACCAG^8CA
CC ding ding GCCG 8rCAGCCA 8AC (JCrCT
DingC^GTCGArACGCGGCrGGTcTTGT
GGAACGGCTAGTCGGTTTGCAGAGA
AG15〇8GGCC8CTG8CchoAGCG8rAAcchoTrc
cccAc8AcGG^8C88CrCTCATdingGC
TCCGGTGACTG8TCG(',TATIG888GGGGTGTTGCCTTGTTG868GT8A
CGAT(', GG8rC8rIC, GGTACTGT
GGGTTT8GIGQTTGIAAA8ACACCr
GAccG■8CCCT8GT88CCCATGACA
CCCA88TCACCMCATTTTTIGIGGAC
IGGCCT^rcccchoG8choC8GTchochoCchoTG
88 GGT 888 C [C^TCACCCCC^^GrC
I'GGCG8■8GGGACrAGTCA88GAA
CchoTCC＾choTTG8GT8GTGGGGGTTC8GACCGcho8TGC8G888rCACCTGGCr
C^8CAGCCl'GcTc8GGGrC88CGA
G^Hacchocho88TACG'rCTTTAGTGGG8CC
GAGTTGTCGGACGAGTCCCAGTTGC
ICrTAA ChohachiCArTCCGrcAGGAhachiAG
crrGGC DingTGGAGCCTGTT'GGrGc
GGrcAIGGAATGT^8GGC8GTCCII
TCG^8CCGAACCTCGGAC88CCACG
CC8 GTAC (JrTACCTTC88CCC8AGCC8G^8I'GC8GAADingCA(IGGCT
TTrTTGGrrGr88TGG^8G1'TGGA
GTTCGGTCTTACGTCTT8GI'G8CC
G88^^AACCAAC8GCTTACCCHacchoCT
CTCCGCArCACCTTTTGGTAAAGGTr
f': rAAGC DingT8GGrGCGA8TGGGT
8GAGAtff:GTAGTGGA^8CC8nTc
c88G8TTCG88rccAc8GAACAArcc
l':rGccrG88c8rGAG8AA8AAcA
GGGT8cICAI 8CIC8CI rTC'rr
GTAGGGAcGGAcTTGIACTCTTTTT
TGTCCCATGAGTA'rGAG-IG88CT
AAGrGAcGGcrGCATACTAACCGCT
TCATACArCTCGT8G8rTrcrcrG80TCACIGCCG8CGrAIG81TGGCGA
AGI^TGT8GAGCATCT88ΔG8G8GG
CG8rchoG88GGGCTAAArTCTchoC^8C
GCT product needle rGAG88rTTTTGC8ACCGC
TAACTTCCCCG8rTTAAGAAGrTGCG
ATTGAAACICrTAAA8 ACGIIGC 88-cho GCGGCG ding ■ ^ ding ^ 8 GC 8 TTTA 8-cho GC ^
rTG＾Ding GCC Haccho■88＾Ding 88＾GC＾C (JT
ACCCCGC88TIVTCG8^8^rTAcGT
8A (JACGGTA drunkenness TT^TITCGTCC88C
C, CCTGACTGCCCΔTCCCCC8TCTT
GTCdingGCGACAG8ITCCTGGG8GGTT
GCGGACTGACGGGGIΔGGGGFAG8Δ
CAGACGCJGICT88GGACCCTT'AA
GCC88G Ding Ding C8rrrrTCT Ding TT'rT
Ding C8 rAAA Ding TGcTTTAAGGCGACG
I'GCGA八TGGGTAGAGAGGCGTAGI
GG888CCArTTcc8AGHachoTCG88TC
CACCG chocC'rc88GC GCTCchochoGTG
rcho88rGGrcho [CchochoTTchoTG'l'GCrC
8f8CGTGC8GGAGITCGACG8G88C
AC88rrAcc888G8888AACACG8G
T8 IGCA [A88 CTATCACCGCAAGG
G eight eight eight eight [eight [C eight eight C eight CCGTGCGIGT
DingGACrΔ8rTT868TAGTGGCGTTCC
CTATTT8[8GATTGTGG(', 8CGC8C8^CTG84chochocho■8(',CI'CTGGCG
GTG8■^^rGGTTGc8rGrAcr Crystal GGA
GGTTGI 8 TG 8 Crystal 8 TGG 8 G 8 CCGCC 8 C
T8H8CCAACGr8(;^1G8[DingCCTCC
An example of a DNA sequence can be expressed as AAG81ΔCC...3°TAG (hereinafter referred to as formula (2). In the formula, A, G, C, and T have the same meanings as above). An example of the metavirocatecase-encoding portion present in the third DNA sequence of the circular double-stranded DNA of the present invention is that derived from Pseudomonas putida mt-2 TOL plus d. A DNA fragment containing such a gene part is Pseudomonas putida mt-j.
It can be prepared from the TOL plasmid of strain (ATOO23973). In addition, plasmids derived from the TOL plasmid, such as pTS11s (Nakazawa et al., Journal of Biological Chemistry (J, Biological
Chemietry), vol. 258, p. 2923 (19
83)), pYT 3 (Nakazawa et al., Journal of
Bacteriology (, T, BacteriolOgy
), Vol. 156, p. 487 (1983)). The third DNA sequence of the circular double-stranded DNA of the present invention is as follows: 5...
°・X'CAGIGCACGGCCCGGIrCCGGC
GGCGGCITCCGACCGGGrGGccI'G
1'C8AGrGTTTcc to c8AAccGAclT
To ACC81CG 80th AC1 1CGC^CCGG 8CAG rcAc 88 AGGCGTT GGCIGAA
CrGGrΔGCrC^rGA88GCGATGAAAC
To 888 GG To 888 TGC To CCGGCC 8 TG
Ding G to 8GC Ding G to Gl'Gr8C1'G to 8TACr
TGmCCAC8TTΔCGCI°GGCCCGGT8CACGTCGACGCACATG8CCTGTACT
CGTrccGGGAccTTGTG^1GcAGc1
'c8ACGACCCGGACTΔGCrCTACCT
GGCACTGC DingGGTCCCCGGCACAG8FA
G8CTTCCG to ACCGGCrlCACCr8T
TCAAAAGGG to CCACGATGCG to TCCG
ACrGcTCGGCCCGTACCT 8AATA
DingGGGTTTCAAGGrGAG to G1'GG
G to TGCTCTCCGGCA to CrGG GCGGG
Heding to CCCA ^GTTCC^ to C to CCTACTC
CTACGAG to GGCCGTTG to CCTCGCCC
300 to I'CrG to I'GGCAr to rGGcTG Ding GCC
GTTGAGCAGC1'ACCCGCAGGTG88CI'GTAGACTACCGT8TACCG8CAC
GGC^8CICGTCG^IGGGCGTCCACT
TGACTTG1'CA8C8CCGGCCGCGCA
CGCG88GGTCCGGGGGAGGCCCGI8GTGA8CG8e rTGTATGcAGAcAAGG
Eight to eight to eight TGGAA eight to rGGGGT Ding TGA
A To GACGGCIC ^ To C ■ To CGTCTG To T
To CC Ding T [to TG CCTTTCACCCC ^^C
TTAC DingGCrC8TCCCGAGGCA'l'G
GCCGCGCGATCTo GA GG Ding ATGGC
GGCrGTGCGT to GTT to GGGCrccGT to ccGGcGcGcIAG to CITTCCAT to CCG
CCG to CACGC^A to GCTGGTGCGGG
T^CATACCGCTGCTr^CGGC(',G
CTGG 8TACTG to 8C to aiccrICCAC
To G To GCC ^ To G ■ ^ GACC GGC Ding IGI'C
CACGACC'rGCrl'TATGGC8CGCCGICGCCC8GrTcchocAGrc1'G[G
ACCAAGGCCCACG8CTACCGIGCGC
G to AGCGGGrcA88 GAGTCAGACAG to GGITCCGGGTGCIGGTGGCCrrc8 rTCACCA to Ding CCGGAAAAAAGGCCGCC
r(J: ArC8r to IGrccI'TCACCGG8A to T88 to GGT8G CCTTT Ding TCCGG
CGGAGGrAGTAC to CAGGAACC 8 CT
CGAAACCDingGGGAAGACrrccncccG
ccccccAccrG to rcrccAGG'rGGA
To GC DingTTGGCCCTTCTGAACG^^GCG
CGGCGGCIGGA (J to GAGGTTGACCG
8CACArCrHacchocGAr^rCGGCCC88C
CCGCC8CGGCrcArrcAc8CTGGC
IGTGT8GAT8GCT8TAGCCGGGITG
GGCGGIGCCGG8filGAG'l'GGGC
AAG8CC8T to TA ding1'c'rTcGAccc
GTccGGrAAccGc88cG8AGrGITC
CGrTCTGGl'AGATGA8G8AGCTGG
GC8GGCCAr1'GGCGrTGCTTC8CA
AG to CGCCCCCTCTA TGTTG^rGGG
CCTGGTGTDINGTGGCCΔCIGGACCTG
rCCG8C to 8G DingGGGC8A to GCGArcT
I'I'TACCACG8C to GC8T to CTC88 to A8G C-cho-GG-cho-CGACCCGnCCGCT-8G
He^he^heGGTGCTGGCGTAAG 8G to TGC
Ding TT1'G1'GCAGATGA88G88^rcA
AGcArTTc Haccho [toGCGGrGCCrTCG DingCGGTAACACGTCTACTDingTCTDing■^GT
To TCGTAGTA^ICGCCACGGAAGCA
to GCCAGCGGTCGCCGITCG8C88GC
TGTI^CAG to CCGGGTTGCCGGTT
CAC1ACGGICIGCGTCCACG8GGCC
GGC8CGCGCC to AGGrCGAGCCG-Y
-^ to CCGIGcc to GACGCAGGrGc'1C
CGGCCG'1GCGCGGCTCC8GCIGCG
(koc-vir+c+koCGGG^■886^hehe GC
GAC1181icGTTTGGcTIT8GTGCT
C^TCTTrcG[:Glf;
^GACCrT8CAGrGTGGcdingGAAcho8C
CΔCA to ACΔGArTG 88 ATTT to GCr^
DingCrGGAATGICACGACCGACITΔTG
GTGnlGTC188CITTT^GCT8^GGA
to 88 IAG to CAA to GAAICT^TA8 CCG
G to ACCG 8^8 [to 8; to G1 cho ■^^TAT^
CCrAGT to TCGACCGTIG^TTA to GTC
rTT^] to AGGIC to GG^C ATAGGGAA
DingAATAr rGcTTrCCA DingTCCArcG
To GGA AAGrr Ding TGTrCCTT To rcccT
TATTATATAcGAA to GGT to GGTAGC
CC111TTC888^C8^GGCTCrTCTG
Chohachi AGGTrT to G to ArTACrGArCGCA
CTTl'ArcGTTrToGC l'cCGAG^ To GACATTCCHehehe CTT To AIG CTAGC
GrG to ^ to [to GC to ^ to CGT to G-r to ArG
CGTT Ding T (ITAGCTT 88 ArCG to 'rA
T^r (JGGCGCTGGC 88 rAGCA 81 to CGC 88 to AGA 8 TC hehe nl 8 to CGAA Δ
To T
G^DingTCrCrC to "■^^C^DinghachirTC to GGC
toTTGT^CGGTG1'GrAC1'CCn^TG
GCI8＾GAGAGT8ATTGI8TA8filc
cGGrCMrAGAcccGAArrTTcGICT
TCAGGTTGGGTCrAT'rGcIACT^[
8 ■ CA ding GGTrc1'crcc 8 to AGG ding [C^
rTAc Ding GAACACTCG Ding CCG 8G 88 ``Hehehehehe G[he CCAAGAGAGGTCTCC ^GT
A^TGACTTGTGAGCGGCTCITArT
GcIG[G...S(゛C to CC

[8G...:)゛(Hereinafter referred to as formula ■. In the formula, A%G, C and represent the same meanings as above, X represents G or AAT1'CCG, and X'
represents C which is the complementary base of X or HATTAAGGC; Y represents CGGAATT when x is G, t, att, 'Represents AA. ) is an example of a DNA sequence. This sequence contains the gene portion encoding metapyrocatechase, with both ends containing B, ttU or Eco RI.
The protruding end of the base pair fragment was repaired to make a complete duplex, and HlndIII-Ban containing the lambda phage PL promoter operator 3' downstream
Hpa [-BamH in the HI fragment (corresponds to the connection of fragments corresponding to the fragment. Fourth!7 of the circular two-lr strand DNA of the present invention) The portion of the DNA sequence encoding ampicillin resistance is pBR322. A considerable portion of plasmids can be exemplified. The fourth DNA sequence must contain a portion that will serve as the starting point for the reproduction when the circular double-stranded DNA of the present invention is introduced into E. coli as a plasmid. In this way, the NA sequence is the Ban HI t sequence in the counterclockwise direction on the restriction enzyme cleavage map from the 22R ■ restriction enzyme cleavage site of the pBR322 plasmid.
3987 bases for the -←) chain and 3987 bases for the (-) chain]. The circular double-stranded DNA of the present invention can be prepared from a plasmid containing such a corresponding sequence, for example, by the method shown in FIG. 2 as follows. pcQV2 plasmid with restriction enzyme C! Digest with /-a I, digest the protruding ends with 81 nuclease to make blunt ends, and circularize again with 'r4 DNA ligase to eliminate the Cta■ site to obtain the pHT1 plasmid. The method of Cohen et al. [Proceeding of National Academy of Sciences (Proc. Natl. Acad, 8ci), U.S.A.
, Vol. 69, p. 2110 (1972)': Transformation! +-, pHT1 plasmid is screened and amplified using ampicillin resistance as an indicator. The pHT1 plasmid is isolated from the resulting transformed bacterial cells. Between the Bam R1 and Kco Ri sites of this plasmid, there is a C (temperature-sensitive protein-encoding 'l') J crss'y crss'y which is a negative repressor of the PK and PL promoters of lambda phage. (hereinafter f″) is indicated as cItF3 in the explanation and in each figure. pHT1 plasmid with zcoRI and Foam
)! The cIts-enriched fragment is isolated by digestion with I. This fragment consists of the sequence represented by the formula (■). The ClaI site of the corresponding fragment of pCQ, V2: (was, CG
T. . The base pair (the one that existed between the 26th base pair and the 27th base pair in formula (■)) has been removed. Meanwhile, the pK030 plasmid was transformed with Kco RI and Bgt I.
Digest with I and separate the long fragments. This fragment has one Bam HI site, but there is a link from the Kco RI end to the Ba HI site.
m T (sequence at I site size is F of pBl 322
! Corresponding to the sequence at the Bam IT site in a counterclockwise direction on the curvature restriction map from the co R1 site. This contains a portion encoding ampicillin resistance and an origin of replication in E. coli. In addition, there is a lambda
Contains part of the phage's PL promoter/operator. The sequence between this Bgt II end and the Hpa site consists of the sequence represented by formula I. Also, the sequence between the Hpa T site and the Bam HI site
This corresponds to the sequence after A C(+) m :I following the 095th Y. Both DNA fragments thus obtained are adhered and circularized using T4DIJA ligase to construct plasmid pHT2. This plasmid is cloned and amplified in the same manner as pHT1. The pYT3 plasmid was digested with Bst fl restriction enzyme, and the part encoding metapyrocatechase was extracted with C-23.
The 0 fragment is isolated and the 3' overhanging ends are digested with 'r4 DNA polymerase to make blunt ends. This is the 1095th work.
(However, X is G, X'FiC
, Y is OteY' is G. The circular double-stranded DNA of the present invention, p[(T 3 A Construct a plasmid. Transform Escherichia coli with this plasmid, use ampicillin resistance as an indicator, and increase the culture temperature to 42° during the culture.
After increasing the concentration to about C, colonies that turn yellow with an aqueous catechol solution are collected, and this plasmid is cloned and amplified Q is performed. psLMK1 dulasmid was substituted for the 0-230 fragment from pYT a grasmid as shown in FIG.
C obtained by digesting with I! Both overhanging ends of the −230 fragment were dN
Fill with TP and add this to Hpa 1 of pHT 2 plasmid.
A pH73 plasmid may be prepared by inserting it into the site. The third DNA of the pHT3 plasmid thus prepared
The X in the sequence Q formula ■ is AATTOC! G, X'
is TTAAGGO%Y is cGGAATT SY' is GC
C! It is TTAA. Note that the psLMK1 plasmid can be prepared as shown in FIG. In the above operations, digestion with each restriction enzyme, ligation with 'r4 DNA ligase, screening using ampicillin resistance, and amplification of each plasmid can all be carried out using conventional methods. Further, for each 1) separation and purification of NA fragments, plasmids, etc., conventional techniques such as extraction with phenol, precipitation with ethanol, agarose gel electrophoresis and subsequent extraction techniques, and separation by liquid chromatography can be used. The present invention also includes the above-mentioned circular double-stranded I) NA as a plasmid to produce metaviral tecase.
The present invention provides a transformed microorganism belonging to the genus Escherichia (hereinafter referred to as the microorganism of the present invention). Among the microorganisms of the present invention, the most preferred is Escherichia coli, and a typical strain of microorganisms like this I7) is
It has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry on August 17, 2015, and has been assigned the following deposit number. The microorganism of the present invention has the above-mentioned circular double-stranded DNA of the present invention.
microorganisms belonging to the genus E. elicia, especially Escherichia
After transforming E. coli and culturing it in an appropriate medium, raising the culture temperature to about 42°C and continuing the culture, the colonies are easily screened by coloring them yellow with an aqueous catechol solution. be able to. If ampicillin-sensitive microorganisms are used, ampicillin resistance can also be used as an indicator, making screening easier. Transformation can be easily accomplished by conventional methods such as Cohen's method described above. The present invention further provides for culturing this transformed microorganism in a nutrient medium to accumulate metavirocatecase within the bacterial body,
The object of the present invention is to provide a method for producing metaviral tecase (hereinafter referred to as the method of the present invention), which involves collecting the metabirotecase. The nutrient medium used in the method of the invention is a conventional medium, for example L
B medium, LB agar medium, SB medium, etc. can be used; drugs such as ampicillin are added to these medium as necessary. In the method of the present invention, the microorganism of the present invention can be cultured by conventional methods such as submerged aerated culture on a solid medium. The culture conditions such as culture temperature and medium liquid properties are also the same as those of microorganisms before transformation, such as Escherichia coli HB 101, Escherichia coli W3110, and Nijieri 7ya coli csoo. The culture time is usually several hours to one day. During or after growth, the microorganism of the present invention can be grown by increasing the culture temperature from the usual 30°C to 42°C to inactivate the CI protein, which is a negative repressor of the pL promoter operator. P.L.
The promoter is activated, and the gene portion encoding metapyrocatechase under its control is transcribed strongly.
expressed. The grown microbial cells are collected by a conventional method, the microbial cells are destroyed if necessary in an aqueous suspension, and metabicocatechase is extracted and separated. The extraction separation method is used to extract soluble protein VC under conditions that do not substantially reduce the activity of metapyrocatechase; &
Any conventional method can be used, such as solvent precipitation, salting out, separation by liquid chromatography, etc. According to the method of the present invention, metapyrocatechase, which could only be obtained by conventional techniques at only about 2%, is increased to nearly 14% (towards a completely white tt in the bacterial extraction solution). be able to. Hereinafter, the present invention will be explained in more detail with reference to Examples. The compositions of the buffer solution, culture medium, etc. used in the persimmon example are as follows. XIO remission solution: A solution in which the (1) buffer for the restriction enzyme used was adjusted 10 times.TFj buffer: 10mM Trio-HCl 1mλ (FliDTA pH 7,6 or 8.0 TAA buffer; 50mM Trlg-acetic acid and 10% Aqueous solution containing acetone (pH 7.5) 0.1% bromophenol blue, 100 m M Na2
LB amp medium containing KDTA and 50 tiglycerin (manufactured by Bethesda Research Laboratories, Inc.): Na0t5 fl peptone (Bactotryptone, trademark) 52
Yeast extract 5? Ampicillin 25g water 1tL
B amp agar medium: SB medium containing the above LB amp t@ground and agar 15F: Peptone (trademark) 12
1 Yeast extract 241 Glycerin 5- Water 900-1
M potassium phosphate buffer (pH 7,6) 100rnt LB amp agar medium was used as the screening medium, and LB amp medium was used as the amplification culture medium. Example 1 (I) Preparation of pH 71 pCQv2 10 pL (7 μf) and Cja 110
pt (50 U), 5 μt of ×10 buffer, and 5 μt of B2020 were added to make a total volume of 50 μt, and the mixture was reacted at 37°C for 2 hours. The reaction solution was extracted with phenol, precipitated with ethanol, and the obtained O4a I fragment was purified with B202.
Dissolved in 0 pL. Add 5μtS of ×IO buffer to this solution.
Add 4 μL of B1 nuclease, 5 μt of 5% glycerin, and 6 μt of B2020 to make a total volume of 50 μt, and incubate at 37°C.
, the reaction was carried out for 2 minutes. After the reaction, phenol extraction and ethanol precipitation were performed, and the precipitate was dissolved in 20 μt of H2O. 20 μt of this solution, 5 μt of ×lQ buffer, 10 mM
ATP 5pt, 50mM DTT 5μt
1'r4 DNA ligase 1μt (2,5U), H2O1
4 μt was added to give a total f# of 50 μt, and the reaction was carried out at 15°C for 17 hours. Add 50mM IJaC! to this solution. t
Add 6μt and 4μt (20U), 37゛C12
The mixture was allowed to react for several hours, and then heat-treated at 70°C for 5 minutes to activate the enzyme. After cooling, pour this solution into the large intestine and apply it to Escherichia.
E. coli HB 101 was transformed according to Cohen's method, and colonies containing plasmid pHT1 were screened using ampicillin resistance as an indicator, and this plasmid was isolated. [■] Isolation of cItIll gene 1) HT 1 30 pt (10 py), ×10 buffer 5 pt, Bam T (I 10 μm (100 U)
) and 3 μt of H2O to make a total amount of 50 μt at 30°C.
The reaction was carried out for 3 hours, and after the reaction was heated at 70°C for 5 minutes. After cooling, ×10 buffer $10μt, μyoRI 15μt (
100 U) and 25 μt of H2O were added to bring the total amount to 100 μt, and the mixture was reacted at 37°C for 2 hours. After the reaction, it was heated at 70°C for 5 minutes and allowed to cool. 30μt of this bath solution and 5μt of stop solution
t was added, placed on a 0.7% agarose gel for separation, and subjected to electric method M (50 mA, 30 minutes). A defined 400'bp fragment of the citθ gene was excised and purified.
(Dissolved in 2020 μt.
μt was added to make a total volume of 60 pt, and the reaction was carried out at 37°C for 90 minutes. After the reaction, phenol extraction and ethanol precipitation were performed, and the precipitate was dissolved in 20 μt of H2O. 1 μt of this solution, 5 μt of the solution prepared in (Il,), 2 μt of XIO buffer 150 mM DTT 2 Pt, 10 mM
ATP 2111. T4 DNA ligase 1 μt (2
, 8U) was added with 7μt of H2O to make a total amount of 20μt, and 1
The reaction was carried out at 5°C for 16 hours. 10 μL of this solution K
90 μL of H2O was added, and E. coli and Escherichia coli HB 101 were transformed in the same manner as for pE71. Colonies containing plasmid pHT2 were screened using ampicillin resistance as an indicator, and this plasmid was isolated. [■] Metapyrocatechase microgene preparation plasmid DN
A psLMK 1 15μt (33μ?) x 10
Yuuei Q, 5 PL, Kco RI 3011t (
180U) was added and digested at 37°C for 3 hours. After the reaction, the reaction solution was heated at 70°C for 5 minutes. After cooling, 8 μt of stop solution was added to the reaction solution, and 0.0 μt of stop solution was added to the reaction solution.
Place it on a 7% agarose gel and perform electrophoresis (150 mAS
2.5 hours). A 1 to 2 Kbp DNA fragment containing the metaviral tecase gene was excised, purified according to a conventional method, and dissolved in 20 μt of H2O. Dissolve 18 μt (2 μ?) of this solution in 5 μt of ×10 buffer and add 2 pl of 2 mM dNTP and t H2O2 in a box.
4 μt 1 followed by DNA polymerase ■ Klenow fragment 1
μt (5 U) was added and heated at 25° C. for 30 minutes to perform a plant-end reaction. After the reaction, phenol extraction and chloroform extraction were performed, and after ethanol precipitation, the precipitate was dissolved in 10 pt of H2O. (Creation of V′1pH73 [:Il[] plasmid pH723μt (5
, 4μf), XIO buffer 511tSHpa 110
pt (60U), added H2O32111, total A1
After 3 hours of reaction at 37°C, phenol extraction and ethanol precipitation were performed, and the precipitate was heated with 20 μt of H2O.
VC was dissolved. 15 μt (4 μ?) of this solution, 8 μt (x μF) of the DNA fragment solution obtained in [■], 5 pt of ×10 buffer, 50 mM DTT 5 /It, 1
0 mM ATP 5 11t, 'r4 DNA
Add ligase 15μz (42U) to 12”C18
After reacting for an hour, phenol extraction and ethanol precipitation were performed, and the precipitate was dissolved in 1 (2020 μt). To this solution, 3 μt of ×10 buffer, 9 pL of H2, Hp
a I 8111 (48U) was added, and the approved amount was 40 μt.
After the reaction was carried out at 37°C for 13 hours, the temperature was increased to 70°C.
Heat treated for minutes. After cooling, Escherichia coli was transformed into E. coli (B 101) in the same manner as in the case of pH 1, and the culture temperature was raised from 30°C to 42°C to increase ampicillin resistance. Plasmid pHT3 using the yellow color of the body as an indicator.
We screened strains with Furthermore, the obtained bacterial cells were cultured in an amplification medium suspension according to a conventional method, and a plasmid pH3 of about 1 to r was obtained by preparing 4"H. The DNA of this plasmid was sequenced according to the Maxam-Gilbert method. The pL promoter controls transcription clockwise on the restriction enzyme cleavage map.
a first DNA sequence containing an operator, a temperature-sensitive gene portion that is connected to the 5' upstream of this first double-stranded DNA sequence and is transcribed in a counterclockwise direction; a second DNA sequence ending in the RI restriction enzyme cleavage site, a third 1) HA (X = AAT
TCCG;
17) It was confirmed that it is a plasmid, which consists of a fourth DNA sequence joined to the 3' end of the sequence. [Reference] The preparation of pEtLMK 1 is as follows: [■]
I followed the instructions. [■] Preparation of pSLK110 ■ Preparation of pBR322Ban HI-Pvu II fragment Plasmid pnR322 (Pesseda Laboratories, Inc. [) 10 pt (9
pf ) with restriction enzyme Pvu U ] 0 μt (40 U)
, 100 mM Tris-HCt, 70
mMgct2.600 mM NaCl and 7Q
10 μt of a pH 7.5 buffer consisting of mM β-mercaptoethanol and 70 pt of water were added to make a total of 100 μt, and the mixture was reacted at 37° C. for 90 minutes. The reaction solution was subjected to phenol extraction and ethanol precipitation, and the obtained Pvu TI
The cut piece was soaked in 20μLK61% water, and the restricted surface was
HI 8μ, (80U), 100mM Tris
-HCl, 70mM MgC72, 1000m
8 μt of a pH 8.0 buffer consisting of M NaCL and 70 mM β-mercaptoethanol and 44 μl of water were added to give a total weight of 80 μt, and the reaction was carried out at 30° C. for 3 hours. The reaction solution was extracted with phenol and precipitated with ethanol, and the precipitate was dissolved in 50 μtK of water. 7) 10 µt of stop solution was added to 40 µt of DNA solution, and the mixture was subjected to 0.7 thiagarose gel electrophoresis for separation. 2. Cut out the 7 Kbfl band and perform BamHI-PV
The u II fragment was prepared using the hydroxyapatite method [H. F. Tabak et al. Nuclei, c Ac1dq Re8earch
) 5, 2321 (1978)],
It was dissolved in 10 μl of ethanol precipitation water. Preparation of double-digested fragment with K. plasmid pKB2527μz (10pf) and restriction enzymes Pvu IT 6 fil (24U), Bgt I
t4μt(32U), 100mM Tris-)T
e3゜70mM M,nir,012.600mM
10 pt of a pH 7.5 buffer consisting of NaCt and 70 mM β-mercaptoethanol and 73 μt of water were added, the total volume was 100 pl, the mixture was closed, and the reaction was carried out at 37° C. for 90 minutes. After phenol extraction of the reaction solution and ethanol precipitation, D
The NA fragment was dissolved in 50 μtVC of water. ■ Construction of psLK 11 Bam HI-PvuTI of plasmid pBR322
5 μt of fragment solution and Pvu of plasmid pKB 252
[-10 pt of BgtII digest (7i7'r4 DNA
Ligase Q, 5 pt (1,25U), 500 m
MTrln-HCl, 100 mM M(zcl-2
, 200rnM dithiothreitol and lmM AT
9 μl of PI (7, 8) buffer containing P and 5 μt of water were added to make a total volume of 30 μt, and the mixture was reacted at 15°C for 22 hours.
7) As a result of agarose gel Ia electrophoresis, circular DNA other than the raw material was observed. The resulting circular DNA was
The method of Cohen et al.
U,S,A,, (Cohen.!Eet, al,, Proc, Natl, Ac
ad, 8ci, U, S, A,) 69, 2110-2
114 (1972)), and the recombinant plasmid p8LK was transformed with ampicillin resistance as a target.
11 was subjected to rapid plasmid DNA isolation (Rap
idP'lasmid DNA l5olation
> Law [D, S. HO: tmqs, Analytical Biochemistry (
,Analytical Biochemistry
), 127.428 (1982)]. Gchleif et al., Practical Meth
oas 1nlvlolscalar Bioloq
y), Springel-7 Eller, New York Incorporated, published in 1980, 106 pages]
The plasmid psLK I1 was isolated according to the procedure described above. [IT-, +7) Preparation of 8LK 21 thus F4
% plasmid psLK 110, 5/11 (0
,5μf) KKco RI 1μt (6U),
1000 mM Tris, -Hot, 70+
nM NaCA2.5 Q OncM NaC6 and 7
pH 7 containing 0 mM β-mercaptoethanol, 2 µt of 5 buffers, 16 t of water, 5111 t) D (amount of 2
The temperature was set to 0 pt, and the skin was exposed to 37°C for 1 hour. Reaction solution 1μ
t (equivalent to 25 ng of DNA) and 0.5 μt of Ta DNA ligase (1,40). SL) OmM Tris-HCl, , 10
0 mM MgC42,2Q OmM dithiothreitol 1 roh (ATP-rich pH 7,8 buffer 5
Add 33.5 μt of μt1 water to make a total volume of 50 ttL,
The reaction was carried out at 15°C overnight. The reaction solution was transformed into E. coli HB 101 according to the method described above, and ampicillin resistance was used as a rapid plasmid DNA 'S-Piit'.
A strain carrying the plasmid 13SDK21 with one Eco)1 site was screened using the method (supra). This strain was amplified using conventional methods, the plasmid was purified, and a cleavage map was created using restriction enzymes.
During the sequencing of plasmid pKR252-derived resequencing, proceed upstream from the Bco RI cleavage site of the plasmid pBR322-derived sequence! 7) Bco RI
It was confirmed that a 2.5 Kb plasmid deleted at the cleavage site could be used. [10] Sst T to the immediate release plasmid PYT350μt (69μf) of Metavirocate sword - Seiyuko (C230)
f 10 pL (70U'), 100 mM Tr
is-HCl, 70 mbt MgG! , 10 μl of pH 7, 5 buffer solution containing 2 and 600 mM NaCA f, and 30 μt of water were added to make a total of ¥100 μt.
``C4 time reaction princess''. Add stop solution 10-tx to reaction solution
Add t to 07% agarose gel for separation! Electrophoresis was performed. Cut out the 1 and 2 Kb bands contained in the metapyrolysis force - 7; transfer gene 17') and send the fragment to DN4 ]] 1 CLUT engineering I'-d column
After isolation using water 4.
Dissolved at 0 μt. Add 5 μt of this sample solution to 1 μt of T4 DNA polymerase (2,50), 2:nM +1NT
P(N=A, G, c, T) 1#lt, 330mMT
rio-%tL 660 mM potassium acetate, 11
00rn: fy nM? Gunenium [:Mg(OA
O) pH 7 containing 2F and 5 mM dithiothreitol
, Add 11 μL of water to 2 μ, 37'
The reaction was carried out for C5 minutes. Reaction solution D was subjected to phenol extraction and ethanol precipitation.
T1 love liquid (pH 7,6) 20 μl K solution - Inebriated. ko'>fBB100 pl-,s Oo rrhp
ATrj, o-HCl, ]OOmM NaC
A2.200mM dithiothreitol and lmM
(pH 7, 8 to read ATP) 1 μt of mild liquid, T4 D
Pico RT-phosphorylated pL, which had been previously phosphorylated at the 5' end, was added to pL and 20 μm of Pico RT-phosphorylated poma was prepared. This solution was converted into 22"Cf 6 hr 1 ij 1 reaction (
It's r. After stopping the reaction by adding 0.25 M KCT) (volume: 92 μt), phenol extrusion and ethanol precipitation were performed, and the warped product was subjected to TFX (pH 8,0) 90 p
t Km understood. Next, add 3.3 μ/μ of RI to this solution.
(20U), 1000 m＞ノI Tris
-HCl, 70mM M2Ot
2. 50 mM NaCl and 70 n>M β-mercaptoethanol were added to the mixture, and a buffer solution of pH 7.5 was added and allowed to react for 37 days. The phenol extract of the reaction solution was applied to a Sepharose 0L-4B column. The DNA fraction was collected and precipitated with ethanol, then TE (pH 7,6) 5μ/
-K (?+Understood. [lV] Preparation of pEILMK 1 [■I] Plasmid psLK 21 5μ (11μ?) RI5μt (30[7), 1000
mM Tris-HOI, 70 mM MgC
l2. Add 5 μt of pH 7.5 buffer containing 500 mM NaCl and 70 mM β-mercaptoethanol and 35 μt of water to make a total @50 μt, and incubate at 37°C for 2 hours 30
Allowed to react for minutes. Furthermore, 50μt of stop solution was added with Yoso RIIμt (6U) and reacted at 37°C for 1 hour.
was added and placed on a 0.7 thiagarose gel for separation and electrophoresis was performed. After extracting the DNA 1fIr fragment corresponding to 2,5 Kb Vclil according to the method using (rlT"l 't'), it was precipitated in ethanol, and the product
0') 100 pt-v was dissolved. This solution 50
calf digestive phosphatase 5 pt (60U
), 500 mM Tris-HCL, 10
inM MgCl2, lmM ZnC62 and 10
Add 6 μt of pH 9.0 buffer containing mM spermidine and react at 37°C for 30 minutes.
After adding 1 pl of DTA fii solution to float the reaction, phenol extraction was performed three times and ethanol precipitation was performed once, and the reaction product was TI! f (pH 8,0) was dissolved in 10 μt. Add 2 μl of this solution and 5 μl of the metavirocatecase gene DNA fragment solution prepared with [:lI[] to 0.5 μt of 74 DNA ligase (-1,4[7), 500 mM
Tris-HOt, 100 mM MgC22,
200mM dithiothret (Toll and ITIM AT
1 ttL of pH 7.8 buffer containing P and 1.5 liters of water
- was added, and the reaction was carried out overnight at 12°C. The reaction solution was mixed with Escherichia coli 11B 10 according to the method described above.
Plasmid 1 was transformed into plasmid 1, into which a metaviral tecase enzyme was inserted, with ampicillin resistance and the fact that the bacterial cells turned dark color when sprayed with a 5-ticatecol aqueous solution.
) A bacterial strain harboring SLMKl was screened. The resulting bacterial cells were then cultured in an amplification culture solution according to a conventional method and purified to obtain about 4 ml of plasmid psLMK1. The DNA of this plasmid was sequenced according to the Maxam-Gilbert method and was found to be pBR322.
The first two strands consisting of 2297 bases (-) (complementary (+) strand 2293 bases) correspond to the Pvu TI cleavage site counterclockwise from the gco RI cleavage site on the Grasmid restriction enzyme cleavage map. heavy@DNA sequence and the gco RI cleaved end of this sequence followed by the 20 Kco
A double-stranded DNA sequence, which can be represented by formula (1), terminating in the RI cleavage terminus, followed by a restriction enzyme Pvu TT cleavage N-position of the first double-stranded sequence, which terminates in the formula (2).
It was confirmed that the plasmid was a 3.7 Kb plasmid consisting of the third double-stranded DNA represented by -/-. Example 2 Using the plasmid pHT3f obtained in Example 1, E. coli IJ C600 and W3110 were transformed and screened in the same manner as in Example 1 for Escherichia coli HB 101, and the transformed strain Escherichia coli 0600 was obtained. /pHT3, w3110/pH
I got T3. Escherichia Cori HBIOI/1)H obtained in this way
T3.0600/pHT3, W3]10/pHT3 to L
The cells were cultured overnight at 30°C in Bamp medium 5me, and then at 42°C for 3 hours. 1 m of the resulting culture solution
Place e in an Ebbendorf tube and set it at 4"C for 120'
The bacterial cells were separated from the Beret R medium by centrifugation for 00010 minutes. Bacterial cell pellet K, sampling solution (3M urea and 0.08M dithiothreitol, 1% SDS) 20
μt was added and heat treated at 95° C. for 5 minutes. After removing insoluble matter by centrifugation, 5DS-polyacrylamide gradient gel (M Take and Rosenberg et al., Nature Vol. 292,
128 (1981)) was loaded with the above sample solution (4 μl), and 50v1 night electrophoresis was performed. After swimming Sl,
Stain the gel with solution (0.195 phosphorus blue - 10% vinegar)
u-254 Ingropanoru water) for 1.5 hours at F2.
6fI-C11 color, then decolorizing solution (10% vinegar IIm!
-25% inopropanol-water, 2 hours; 10% acetic acid-
It was soaked in 10 acetate isopropano-A-water, 2 hours: 5-methanol-7 thiacetic acid-water, 2 hours). The obtained gel pattern is shown in No. 51 ¥1. The number on the left side indicates the molecular weight of the protein used as a marker. Lanes A and A' are untransformed Escherichia coli HBOI and HE I Qx/pH.
The pattern of T3 is shown. Lane B, rtic6oo and a 600/paT3, lane c, c' is W3110
, W3110/1) shows the pattern of HT3. In both cases, a thick band that is not observed in bacteria that has not been transformed to a molecular weight of about 35,000 is observed in bacteria that retain pHT3. On the other hand, as with each sample, EIDg-
After polyacrylamide toge 2 dientoglu electricity > mausoleum and ribs, nitrocellulose gg i: transfer the protein,
r3 antibody staining (internal history, cell engineering, vol. 2, 1061
Page r1983)) was carried out. To prevent non-specific absorption of the nitrocellulose membrane to which proteins were transferred from the 5DR-polyacrylamide gel, 3 T/TEG was added.
(2gm, +riTris -HC Otsupi (7, 5, 5
00m), 4) obtained from a rabbit that was crushed in NaC6) and stimulated with metabilocatecase. The cells were soaked for 2 hours in 11 days of serum, washed with TBEI solution, and then crushed in horseradish peroxidase-labeled sheep's G (anti-rabbit IgG) solution for 1 hour. Washed well. After that, coloring solution (20mM Tr
is-110' pH 7, 5,500mM NaC/,
,, 0.06% 4-chloro-1-naphthol,
0.015=H2O2) for 5 minutes to develop color. The pattern is shown in No. 61. Lane numbers are the same as in FIG. In both strains, a protein band that specifically binds to the metavirocatecase antiserum is seen in the sample of the bacteria transformed at pH 73. This corresponds to a molecular weight of approximately 35,000, indicating that the protein that was present only in dead bacteria transformed at pH 73 shown in FIG. 5 was metapyrocatechase. Example 3 [Measurement of metabilocatecase activity] 1.0 - of the above-mentioned Escherichia coli W3110/1) HT3 culture solution was placed in an Etzpendorf tube and incubated at 4°C.
Centrifuge at 12,000G for 10 minutes at C, add TAA buffer (pH 7,5) 1.0- to the resulting bacterial pellet, and perform ultrasonication 5 times for 10 seconds each while cooling on ice to destroy the bacteria. did. The treatment solution was heated to 12,000G at 4°C for 10
The mixture was centrifuged for a minute to remove insoluble matter and obtain a supernatant. Regarding this solution, Nozaki's method [Nozaki, Methods in Enzymol
oBy) 17A, 522-525, Academic
Press, published in 1976]. Metapyrocatechase catalyzes the reaction of oxidizing catechol to α-human aximconic β-aldehyde. The product has a spectrum in the ultraviolet region with an absorption maximum at 375 nm. Therefore, the activity of metapyrocatechase can be determined by measuring the rate of increase in 0D375. Optical path 1
- In a cuvette, add 2.6-10.5M potassium phosphate buffer (pH 7.5) of pure water, 0.3 m, 0.001 M
0.1 - of an aqueous catechol solution was added and mixed. The bacterial cell extraction supernatant was diluted 100 times with TAA buffer, 5 to 100 pt of the diluted solution was added, and the mixture was thoroughly mixed, and the change in OD3-5 over time was measured at 24°C. Since the molecular extinction coefficient of the product under the measurement conditions used is 4.4 x 104 M-1, the amount of catechol oxidized per unit time can be calculated from the slope of the straight line plotting the change over time. The unit of enzyme activity, IU, is defined as the amount of enzyme that oxidizes 1 μmot of catechol per minute at 24°C. Therefore, IU corresponds to an increase in 0D37S of 14.7 per minute. . Specific activity was expressed as activity per ~protein. Protein measurement is carried out using the Lowry method (0, H, Lowr
y et al., J. Biol, Ohem, 193.265
(1951)]. In addition, literature values [Nakai et al., Biochemica et Biophyθi
ca Acta) Volume 220, 213 (1970)]
Accordingly, the specific activity of metapyrocatechase was 320 U/
The amount was calculated assuming +q. The results are shown in Table 1. Table 1: Metapyrocatechase activity in supernatant after bacterial cell destruction (υ shoulder) 20.0 Protein concentration (drops) 0.45 Specific activity (
U/#) 44.4 t (T) of metavirocatecase relative to total protein 13.9 From these results, it is estimated that about 139aF of metavirocatecase is present in the culture solution lt. Ta. Example 4 Escherichia coli c 6, a convertible stock obtained in Actual Example 2
00/pHT 3 was cultured and a bacterial cell extract was prepared in the same manner as in Example 2, and the production of metavirocatecase was confirmed by antigen-antibody reaction. A bacterial cell extract was further prepared in the same manner as in Example 3, and the metapyrocatechase activity and protein amount were measured. The results obtained are shown in Table 2. Table 2 Metapyrocatechase activity (137mg) 14.
7 Protein concentration (Vml) 0.48 Specific activity (U/P) 30.6 Metapyrocatechase f# (%) relative to total protein 9゛6

[Brief explanation of drawings]

FIG. 1 is a diagram showing a restriction map of an example of a circular double-stranded DNA having a PL promoter of the present invention, and FIG. ! ! FIG. 4 is a diagram illustrating an example of lIm, and FIG.
FIG. 5 is a diagram of the electrophoresis pattern of E. coli protein containing metapyrocatechase produced by the method of the present invention.
The figure shows the electrophoretic pattern of enzyme staining method 1 between metavirocatecase and metapyrocatechase antibodies produced by the method of the great invention. ■ Eco Shakuko ■ Part 2!! ! Figure 3 珈 2 J-11 Figure 4 Wa □ C!! v A A'F5B-
・:,' ,1. ,,,+ , , , :::'
;,I :; ,: ,,Z,,",:,:' ,
+L,,y,,”,;S-”'-'
, I3", ・C11- ・/Cl+1/N1)-:
I Ta-do! Jt1. -2-1-9GEHD
(ZZZZ2)CO2 );1-2',,he-,・0,・2°],,'rN'ba・,ξ-;)゛2゛-tonotapiro

Claims (1)

[Scope of Claims] (1) The PL promoter operator of lambda phage and the metapyrocatechase-encoding region that is present in the downstream region under its control, and the part that is present on the 5' upstream of the PL promoter operator and that A circular double-stranded DNA containing a temperature-sensitive gene portion that controls activity and capable of expressing metapyrocatechase in E. coli. (2) The circular double-stranded DNA according to claim (1), which has a double-stranded DNA sequence containing a portion encoding ampicillin resistance downstream of the temperature-sensitive gene portion. (3) The part encoding metapyrocatechase is derived from the TOL plasmid of Pseudomonas putida mt-2 strain, and the part encoding ampicillin resistance is derived from pBR322.
The circular double-stranded DNA according to claim (2), which is derived from a plasmid. (4) A first DNA sequence containing the PL promoter/operator that controls transcription in the clockwise direction on the restriction enzyme cleavage map; a second DNA sequence containing the temperature-sensitive gene portion to be transcribed and ending with an EcoRI restriction enzyme cleavage site at its 5'end;
A third DNA sequence containing a portion encoding metapyrocatechase linked to the 3' end of the NA sequence and a portion encoding ampicillin resistance starting from the EcoRI restriction enzyme cleavage site and transcribed counterclockwise. , a fourth DNA sequence whose 5' end is linked to the 3' end of the third DNA sequence, according to any one of claims (1) to (3). double stranded DNA. (5) The circular double-stranded DNA according to claim (4), wherein the first double-stranded DNA sequence consists of base pairs represented by the formula [there is a gene sequence]. (6) The cyclic duplex according to claim (4) or (5), wherein the second double-stranded DNA sequence consists of base pairs represented by the formula [there is a gene sequence]. Stranded DNA. (7) The third double-stranded DNA sequence has the formula [there is a gene sequence] (X represents G or AATTCCG, X' represents C or TTAAGGC, which is the complementary base of X, and Y represents when When C is AATTCCG, it represents CGGAATT, and Y' is the complementary base of Y, G or GCCTT.
Represents AA. ) The circular double-stranded DNA according to any one of claims (4) to (6), which consists of base pairs represented by: (8) The fourth DNA sequence is a base pair corresponding to the sequence from the EcoRI restriction enzyme site of the pBR322 plasmid to \Bam\HI in the counterclockwise direction on the restriction enzyme cleavage map. ) to (7). (9) The PL promoter operator of lambda phage and the metapyrocatechase-encoding part that exists in the downstream region under its control, and the temperature sensitivity that exists on the 5' upstream of the PL promoter operator and controls its activity. A double strand D containing a gene portion and a portion encoding ampicillin resistance downstream of the temperature-sensitive gene portion.
A transformed microorganism belonging to the genus Escherichia that contains NA as a plasmid and is capable of producing metapyrocatechase. (10) The microorganism according to claim (9), wherein the microorganism is Escherichia coli. (11) A patent claim in which the metapyrocatechase-encoding portion of the circular double-stranded DNA contained as a plasmid is derived from the TOL plasmid of Pseudomonas putida mt-2 strain, and the ampicillin resistance-encoding portion is derived from the pBR322 plasmid. Range (9) or (10)
Microorganisms listed in section. (12) Circular double-stranded DNA contained as a plasmid has a PL that controls transcription in the clockwise direction on its restriction enzyme cleavage map.
A first DNA sequence containing a promoter operator, a temperature-sensitive gene portion that is connected to the 5' upstream of this first double-stranded DNA sequence and is transcribed in a counterclockwise direction, and whose 5' end is ¥ A second DNA sequence ending in the Eco\RI restriction enzyme cleavage site, the first D above.
A third DNA sequence containing a portion encoding metapyrocatechase linked to the 3' end of the NA sequence and encoding ampicillin resistance starting from the \Eco\RI restriction enzyme cleavage site and transcribed counterclockwise. Claims (9) to (11), wherein the fourth DNA sequence comprises a fourth DNA sequence whose 5' end is linked to the 3' end of the third DNA sequence. Microorganisms described in any of the sections. (13) Claim (12) states that the first double-stranded DNA sequence of the circular double-stranded DNA contained as a plasmid consists of base pairs represented by the formula [There is a gene sequence] microorganisms. Claim (12) or (13), wherein the second double-stranded DNA sequence of the circular double-stranded DNA contained as a plasmid consists of base pairs represented by the formula: [There is a gene sequence] Microorganisms listed in section. (15) The third double-stranded DNA sequence of the circular double-stranded DNA contained as a plasmid has the following gene sequence: Y represents C when X is G, CGGAATT when X is AATTCCG, and Y' is G or GCCTT which is the complementary base of Y.
Represents AA. ) The microorganism according to any one of claims (12) to (14), which is composed of base pairs represented by the following. (16) The fourth double-stranded DNA sequence of the double-stranded DNA contained in the plasmid is
The base pairs of claims (12) to (15) are base pairs corresponding to the sequence from the ¥Eco¥RI restriction enzyme site to ¥Bam¥HI in a counterclockwise direction on the restriction enzyme cleavage map. Microorganisms described in any of the sections. (17) The PL promoter operator of lambda phage and the metapyrocatechase-encoding part that exists in the downstream region under its control, and the temperature that exists on the 5' upstream of the PL promoter operator and controls its activity. A metapyrocatechase-producing bacterium belonging to the genus Escherichia, which has been transformed with a plasmid containing a sensitivity gene part and a part encoding ampicillin resistance downstream of the temperature-sensitive gene part, is cultured in a nutrient medium to produce metapyrocatechase in the bacterial body. A method for producing metapyrocatechase, which comprises accumulating case and collecting the same. (18) Claim No. 1, wherein the metapyrocatechase-producing bacterium obtained by transformation is Escherichia coli (
17) The manufacturing method described in item 17). (19) The transformed plasmid has a PL promoter that controls transcription in the clockwise direction on its restriction enzyme cleavage map.
A first DNA sequence containing an operator, connected to the 5' side upstream of this first double-stranded DNA sequence, and containing a temperature-sensitive gene portion that is transcribed in a counterclockwise direction, the 5' end of which is ¥Eco¥ a second DNA sequence ending in an RI restriction enzyme cleavage site, a third DNA sequence containing a portion encoding metapyrocatechase linked to the 3' end of the first DNA sequence and ¥Eco¥
starting from the RI restriction enzyme cleavage site and containing a portion encoding ampicillin resistance that is transcribed in a counterclockwise direction;
The manufacturing method according to claims (17) and (18), comprising a fourth DNA sequence whose 5' end is linked to the 3' end of the third DNA sequence. (20) First double-stranded DNA of transformed plasmid
The manufacturing method according to claim (19), wherein the sequence consists of base pairs represented by the formula: [There is a gene sequence]. (21) Second double-stranded DNA of transformed plasmid
The manufacturing method according to claim (19) or (20), wherein the sequence consists of base pairs represented by the formula: [There is a gene sequence]. (22) Third double-stranded DNA of transformed plasmid
The A sequence has the formula [There is a gene sequence] (X represents G or AATTCCG, X' represents C or TTAAGGC, which is the complementary base of X, Y represents X when X is G, and CGGAATT when X is AATTCCG) , and Y' is G or GCCTT, which is the complementary base of Y.
Represents AA. ) The manufacturing method according to any one of claims (19) to (21). (23) Fourth double-stranded DNA of transformed plasmid
The A sequence is ¥Eco¥ of pBR322 plasmid.
Claims (19) to (22) are base pairs corresponding to the sequence from the RI restriction enzyme site to \Bam\HI in a counterclockwise direction on the restriction enzyme cleavage map.
) The manufacturing method described in any of the following paragraphs. (24) The manufacturing method according to any one of claims (19) to (23), wherein the culture is performed by adding a gene expression inducer to the medium. (25) The production method according to claim (24), wherein the inducer is isopropylthiogalactoside.