CA2065258A1 - Blood-clot-dissolving proteins, and their preparation from the leech hirudo medicinalis - Google Patents
Blood-clot-dissolving proteins, and their preparation from the leech hirudo medicinalisInfo
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- CA2065258A1 CA2065258A1 CA002065258A CA2065258A CA2065258A1 CA 2065258 A1 CA2065258 A1 CA 2065258A1 CA 002065258 A CA002065258 A CA 002065258A CA 2065258 A CA2065258 A CA 2065258A CA 2065258 A1 CA2065258 A1 CA 2065258A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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Abstract
O.Z. 0050/41274 Novel proteins and their preparation Abstract of the disclosure A protein which contains sequences 1 to 13 detailed in the sequence listing and cleaves glutamyl)-lysine linkages, and its muteins and natural variants with a comparable action.
Description
206~2~8 O.Z. 0050/41274 Novel proteins and their preparation Description The present invention relates to novel proteins and to their preparation.
A number of proteins, such as tPA, urokinase and streptokinase, which can dissolve blood clots have already been disclosed. Their action comprises de-polymerization, and thus solubilization, of the fibrin present in the blood clot by cleavage of peptide chains.
The dissolution of blood clots can al~o be effected by breaking ~ -glutamyl)-lysine linkages. Substances of this type have been isolated from Hirudo medicinalis.
They include destabilase (Biochemistry, USSR 50, 357 (1985)), which has a molecular weight of 12,300 Da (XII Congress of the International Society on Thrombosis and Haemostasis, Abstract 1727, (1989)).
The invention relates to a novel protein which contains the sequences 1-12 detailed in the ~equence listing and cleaves t - ( ~-glutamyl)-ly8ine linkages, and to its muteins and natural variants with a comparable action.
By muteins are meant proteins which are derived from the novel protein by exchange, deletion and/or addition of amino acids or peptides in the protein chain.
By natural variants are meant proteins which occur in blood-sucking animal species and have the same action with a similar amino-acid sequence.
The novel pxotein can be isolated from glandular secretion~ of the leech (Hirudo medicinalis) by chroma-tography on S-Sepharose9, concanavalin A and copper chelate chromatography columns. From 30 to 200 activity units of the protein can be isolated from the qlandular secretions of 500 g of leeches.
The novel protein is present in the glandular secretion3 in concentrations between 1 - 100 ~g/kg. In ' . ~ .
.
` 206~i25~
A number of proteins, such as tPA, urokinase and streptokinase, which can dissolve blood clots have already been disclosed. Their action comprises de-polymerization, and thus solubilization, of the fibrin present in the blood clot by cleavage of peptide chains.
The dissolution of blood clots can al~o be effected by breaking ~ -glutamyl)-lysine linkages. Substances of this type have been isolated from Hirudo medicinalis.
They include destabilase (Biochemistry, USSR 50, 357 (1985)), which has a molecular weight of 12,300 Da (XII Congress of the International Society on Thrombosis and Haemostasis, Abstract 1727, (1989)).
The invention relates to a novel protein which contains the sequences 1-12 detailed in the ~equence listing and cleaves t - ( ~-glutamyl)-ly8ine linkages, and to its muteins and natural variants with a comparable action.
By muteins are meant proteins which are derived from the novel protein by exchange, deletion and/or addition of amino acids or peptides in the protein chain.
By natural variants are meant proteins which occur in blood-sucking animal species and have the same action with a similar amino-acid sequence.
The novel pxotein can be isolated from glandular secretion~ of the leech (Hirudo medicinalis) by chroma-tography on S-Sepharose9, concanavalin A and copper chelate chromatography columns. From 30 to 200 activity units of the protein can be isolated from the qlandular secretions of 500 g of leeches.
The novel protein is present in the glandular secretion3 in concentrations between 1 - 100 ~g/kg. In ' . ~ .
.
` 206~i25~
- 2 - O.Z. 0050/41274 order to make the protein available in relatively large amounts for pharmaceutical purposes, it is possible to employ known genetic engineering method~ (cf.
Sambrook, T. et al.: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y., 1989). For this purpose it is necessary first to identify the genetic information for the novel protein and to isolate the corresponding nucleic acid. For this, the pure protein is reduced with dithiothreitol, then iodacetamide is added to derivatize the free S~ groups, and subsequently the protein treated in this way is cleaved into small pep-tides with cyanogen bromide or trypsin. The peptides are fractionated by reversed phase chromatography. The purified peptides are subsequently sequenced.
The available peptide sequences now permit, by synthesis of corresponding oligonucleotides, unambiguous identification of the gene from the genome or from appropriate cDNA banks by sequence-specific filter hybridization. The methods, which are necessary for this, for preparing degenerate oligonucleotide probes from protein sequence data, the isolation of mRNA and its transcription into cDNA and the preparation and the hybridization of cDNA banks with oligonucleotide probes are state of the art and described in the textbook ~Molecular Cloning" Sambrook et al (1989) chapter 11, 7 and 8).
Expression of the genetic information which has been obtained in this way for the protein can then be brought about in various host cells ~uch as eukaryotic cells, yeasts, Bacillus subtilis or E. coli by known methodR, and the protein can thus be obtained in rela-tively large amounts. The methods required for the expression of heterologous genes are also state of the art and are explained in Sambrook et al. (1989), chapter 16 and 17.
The muteins also, are preferably prepared by genetic engineering methods.
Sambrook, T. et al.: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, N.Y., 1989). For this purpose it is necessary first to identify the genetic information for the novel protein and to isolate the corresponding nucleic acid. For this, the pure protein is reduced with dithiothreitol, then iodacetamide is added to derivatize the free S~ groups, and subsequently the protein treated in this way is cleaved into small pep-tides with cyanogen bromide or trypsin. The peptides are fractionated by reversed phase chromatography. The purified peptides are subsequently sequenced.
The available peptide sequences now permit, by synthesis of corresponding oligonucleotides, unambiguous identification of the gene from the genome or from appropriate cDNA banks by sequence-specific filter hybridization. The methods, which are necessary for this, for preparing degenerate oligonucleotide probes from protein sequence data, the isolation of mRNA and its transcription into cDNA and the preparation and the hybridization of cDNA banks with oligonucleotide probes are state of the art and described in the textbook ~Molecular Cloning" Sambrook et al (1989) chapter 11, 7 and 8).
Expression of the genetic information which has been obtained in this way for the protein can then be brought about in various host cells ~uch as eukaryotic cells, yeasts, Bacillus subtilis or E. coli by known methodR, and the protein can thus be obtained in rela-tively large amounts. The methods required for the expression of heterologous genes are also state of the art and are explained in Sambrook et al. (1989), chapter 16 and 17.
The muteins also, are preferably prepared by genetic engineering methods.
- 3 - O.z. 0050/41274 When the novel protein i8 isolated from Hirudo medicinalis, it ic obtained in glyco~ylated form. The same applies to genetic engineering preparation when eukaryotic cells are used for the expression. If it is expressed in prokaryotic cells, it is obtained in non-glycosylated form. Both forms have the same action.
The novel protein is an isopeptidase and can, owing to its enzymatic activity, dissolve the fibrin clots (thrombus) crosslinked by activated factor XIII.
This crosslinking is produced by an isopeptide linkage between glutamate and lysine side chains. The cleavage i3 effected by the transfer of a y-glutamyl (sic) radical to another amino acid, to a dipeptide or by hydrolysis.
Hydrolysis of thiR isopeptide linkage abolishes the action of factor XIIIa. Thus, clots crosslinked (aged) by factor XIIIa are re~uvenated and thus either dissolved directly or dissolved in combination with tissue plas-minogen activators (e.g. tPA). It is thus possible to employ this enzyme in the treatment of thrombotic states (e.g. myocardial infarct, deep venous thrombosis and atherosclerosis) in acute therapy and in prevention.
Released tumor cells al~o anchor themselves in their target tissue via an 6-(~-glutamyl)-lysine linkage and then escape the immune system owing to envelopment in fibrin. The novel enzyme is thus also suitable for preventing the formation of meta~tases.
- Obtaining the novel protein Example l a) Obtaining the leech secretion 500 g of Hirudo medicinali~ were kept in portions of about 50 g at 4C in a cold room with a little water.
The animal~ were stimulated in 150 mN NaCl, 10 mM
arginine and 20 mM phosphate buffer ph ~sic) 7.4 at room temperature for 2 h. After 2 days, they were bathed in 150 mM NaCl, 20 mM phosphate buffer pH 7.4 and 1 %
pilocarpine for l h. During thi3 they secreted large amounts of mucus and an enzymatic activity which convert~
The novel protein is an isopeptidase and can, owing to its enzymatic activity, dissolve the fibrin clots (thrombus) crosslinked by activated factor XIII.
This crosslinking is produced by an isopeptide linkage between glutamate and lysine side chains. The cleavage i3 effected by the transfer of a y-glutamyl (sic) radical to another amino acid, to a dipeptide or by hydrolysis.
Hydrolysis of thiR isopeptide linkage abolishes the action of factor XIIIa. Thus, clots crosslinked (aged) by factor XIIIa are re~uvenated and thus either dissolved directly or dissolved in combination with tissue plas-minogen activators (e.g. tPA). It is thus possible to employ this enzyme in the treatment of thrombotic states (e.g. myocardial infarct, deep venous thrombosis and atherosclerosis) in acute therapy and in prevention.
Released tumor cells al~o anchor themselves in their target tissue via an 6-(~-glutamyl)-lysine linkage and then escape the immune system owing to envelopment in fibrin. The novel enzyme is thus also suitable for preventing the formation of meta~tases.
- Obtaining the novel protein Example l a) Obtaining the leech secretion 500 g of Hirudo medicinali~ were kept in portions of about 50 g at 4C in a cold room with a little water.
The animal~ were stimulated in 150 mN NaCl, 10 mM
arginine and 20 mM phosphate buffer ph ~sic) 7.4 at room temperature for 2 h. After 2 days, they were bathed in 150 mM NaCl, 20 mM phosphate buffer pH 7.4 and 1 %
pilocarpine for l h. During thi3 they secreted large amounts of mucus and an enzymatic activity which convert~
- 4 - O.Z. OOS0/41274 the color substrate ~-glutamyl para-nitroanilide. S00 g of leeches secreted from ~0 to 200 units of this enzymatic activity.
b) Isolation of the protein from the leech secretion 100 units (cf. a) were diluted with 5 1 of equilibration buffer ~20 mM sodium phosphate buffer pH 4.5, 10 mM NaCl, 0.01 % Tween0 80). The pH was cor-rected to 4.5. The final volume was about 6 1. The volume was loaded at a flow rate of 5 ml/minute onto 350 ml of S-Sepharose, washed with 4 column volumes of equi-libration buffer and developed with a salt gradient from 1 1 of 20 mM sodium phosphate buffer pH 4.5, 10 mM NaCl, 0.01 % Tween 80 to 1 1 of 20 mM sodium phosphate buffer pH 4.5, 500 mM NaCl, 0.01 % Tween~ 80. The enzymatic activity eluted at from 50 to 200 mM NaCl.
The active fractions were collected. The buffer was ad~usted to 20 mM sodium acetate pH 7.0, lS0 mM NaCl, 1 mN MnCl2, 1 mM ~gCl2 and 1 mM CaCl2 by dilution. This solution was loaded onto a concanavalin A column (25 ml) and washed with 4 column volumes of the same buffer.
Elution was carried out with the same buffer with the addition of 10 mM mannose or 5 mM ~-methyl mannoside.
The active fractions were collected and diluted in a buffer 80 that a final concentration of 20 mM sodium phosphate pH 7.0, 500 m~ sodium chloride, 0.01 % Tween~
80, was reached. A copper chelate column ~2 ml volume) equilibrated in the same buffer was loaded and washed with lS column volumes of equilibration buffer followed by 15 column volumes of 20 mN succinic acid pH 7.0, 500 mM NaCl, 0.01 % TweenO 80. The column was then eluted with a gradient to 20 mN succinic acid pH 4.5, 500 mN
NaCl, 0.01 % Tween 80. The activity eluted at pH 6 to 5.4.
c) Purification of the protein by Mono S chromatography The required peak from the Cu chelate column was concentrated and the buffer was changed to 20 mN Na phosphate, 10 mM NaCl pH 5.0 (buffer A). This wa~
2 0 6 5 2 ~ 8 - S - o.z. 0050/41274 followed by loading onto a Mono S FPLC column (Pharmacia, Freiburg). After the unbound material had been eluted by wa~hing the column with 10 column volumes of buffer A, the column was eluted with a combined linear pH and salt gradient from 0 to 66 % buffer B (20 mM sodium phosphate, 500 mM NaCl, pH 8.5) in 85 min. Under these conditions the novel protein eluted from the column between 10 and 30 % buffer B.
The elution was followed by W brass (~ic) at 280 nm or measurement of the activity of aliquots of the fractions loaded on. The activity-containing fractions were combined, desalted and concentrated to dryness. The samples were stored at -20C until used further.
d) Purification of the protein by reversed phase (r)HPLC
The required fractions from the Cu chelate chromatography which had been concentrated to dryness were taken up in 0.1 % strength trifluoroacetic acid (TFA) solution and further purified on a C-4 reversed phase column (rP304, Biorad, Munich). Large and small subunits of the protein were separated in this step. The elution from the column was achieved by applying a linear acetonitrile gradient (in 0.1 ~ TFA) from 0 to 50 % in 90 min at a flow rate of 1 ml/min. The cours~ of the elution was followed by W measurement at 210 nm. Under these conditions, the protein chains assigned to the protein eluted between 35 to 45 % acetonitrile. The protein bands belonging to the protein were fractionated and, after removal of the solvent, sub~ected to a proteo-lytic digestion to generate peptides.e) Separation by rever~ed phase (r)HPLC o~ the destabilase peptides obtained after proteolytic fragmentation The peptide mixtures obtained after enzymatic digestion with trypsin (Sequence Grade, Boehringer Nannheim) or endoprotease Glu C (Boehringer Mannheim) were mixed with 0.1 % strength TFA and fractionated on 206~2~8 - 6 - O.Z. 0050/41274 a C-18 reversed phase HPLC column (rp 318, Biorad, Munich). This entailed a linear gradient from solution C
(0.1 ~ TFA in water) to solution D (0.1 % TFA in aceto-nitrile) being applied over a period of 2 h. Elution was followed by W measurement at 210 nm at a flow rate of 1 ml/min. The separated peptides were fractionated and the amino-acid sequence of individual peptides was determined in a peptide sequencer tApplied Biosystems).
The sequences indicated in the sequence listing were found.
Characterization of the protein After SDS polyacrylamide gel electrophoresis and subsequent silver staining, three bands are recognizable (low molecular weight marker from Pharmacia): one band with an apparent molecular weight of 57,000 Da and two bands at 30,000 Da. On determination of the molecular weight by molecular sieve chromatography (TSK G 3000, buffer: 20 mM Na2HPO4 0.1 M NaCl pH 7.0 flow rate:
0.7 ml/min), the protein has an apparent molecular weight of about 44,000 (+ 5000 Da) Da. The specific activity is about 580 U/mg. The following assay was used to determine the activity: 50 ~1 of buffer (5 mM tris pH 8.4, 0.01 %
Tween~ 80) 50 ~1 of color reagent (~-glutamyl) p-nitroanilide, 1 mg/ml) were incubated with 50 ~1 of sample at 37C for 60 min and then the increase in extinction at 405 nm was determined. 1 U is defined as enzymatic activity which converts 1 ~mol of ~-glutamyl para-nitroanilide in 1 min at 37C and causes thereby an increase in absorption at 405 nm.
The natural protein i8 a glycoprotein, which i8 demonstrated by its mannose-dependent elutien from the concanavalin A column.
Protease inhibitors such as cystatin C
(10 ~g/ml), aprotinin (100 ~g/ml) and benzamidine (10 mN) do not inhibit the activity. 100 ~m serine together with 100 ~m borate pH 8.4 inhibit about 50 % of the enzymatic activity. The enzyme i~ completely inhibited by addition 206~2~8 - 7 - O.Z. 0050/41274 of 1-5 mM HgCl2. Furthermore, reduced and oxidized gluta-thione inhibit the enzyme. Triton~ X-100 leaves the activity virtually unaffected and can protect from denaturation by SDS. The pH optimum is between pH 8 and S pH 9. However, the enzyme is still active up to pH 12.
Beyond this non-specific hydrolysis of the substrate is already dominant.
Detection of the fibrin-dissolving action 100 ~1 of bovine fibrinogen (Niles no. 82-0222-4;
contains factor XIII) in a concentration of 4 mg/ml in TBS (= 10 mM tris/HCl pH 7.5, 150 mM NaCl) are mixed with 10 ~1 of bovine thrombin (sigma no. T6634, 5 U/ml in TBS
with 25 mM calcium acetate) and incubated in Eppendorf reaction tubes at 37C for 2 to 5 h. This results in stable, factor XIIIa-crosslinked fibrin clots.
The latter are washed once with 10 mM sodium phosphate pH 7.5, 150 mM NaCl and subsequently incubated with 200 ~1 of salivary secretion of Hirudo medicinalis (37C, 20 h). After addition of 1 ml of 5 % strenqth monochloroacetic acid, the amount of liberated protein in the supernatant i8 determined (OD 280 nm).
Example 2 1. Isolation of m-RNA
~ RNA wa~ obtained from whole leeches after dis-ruption in guanidinium thiocyanate and subsequent centri-fugation through a CsCl cushion. The portion of the RNA
containing at the 3' end a polyA (polyA+) was isolated by oligo(dT) affinity chromatography. Both process steps were carried out in aecordanee with Sambrook et al ~et al (sie) (1989) ~'MoIeeular Cloning" 2nd edition, CSH
Press, page 7.19-7.22 and 7.26-7.29.
2. Preparation of DNA probes The starting point for the cloning of cDNA
fragments by means of the polymerase chain reaetion (PCR, for details of this technique, see Sambrook et al, chapter 14) was the peptide sequence Gly Ly~ Asp Ile Ile Lys Lys Tyr 20652~8 - 8 - O.Z. 0050/41274 on the basis of the known genetic code, this amino-acid ~equence can be transcribed into a nucleic acid sequence.
Because of the known degeneracy of the genetic code, several nucleic acids can be inserted in make (sic) positions. It emerges from this that there are the following 576 possibilities for the sequence of the mRNA
strand:
b) Isolation of the protein from the leech secretion 100 units (cf. a) were diluted with 5 1 of equilibration buffer ~20 mM sodium phosphate buffer pH 4.5, 10 mM NaCl, 0.01 % Tween0 80). The pH was cor-rected to 4.5. The final volume was about 6 1. The volume was loaded at a flow rate of 5 ml/minute onto 350 ml of S-Sepharose, washed with 4 column volumes of equi-libration buffer and developed with a salt gradient from 1 1 of 20 mM sodium phosphate buffer pH 4.5, 10 mM NaCl, 0.01 % Tween 80 to 1 1 of 20 mM sodium phosphate buffer pH 4.5, 500 mM NaCl, 0.01 % Tween~ 80. The enzymatic activity eluted at from 50 to 200 mM NaCl.
The active fractions were collected. The buffer was ad~usted to 20 mM sodium acetate pH 7.0, lS0 mM NaCl, 1 mN MnCl2, 1 mM ~gCl2 and 1 mM CaCl2 by dilution. This solution was loaded onto a concanavalin A column (25 ml) and washed with 4 column volumes of the same buffer.
Elution was carried out with the same buffer with the addition of 10 mM mannose or 5 mM ~-methyl mannoside.
The active fractions were collected and diluted in a buffer 80 that a final concentration of 20 mM sodium phosphate pH 7.0, 500 m~ sodium chloride, 0.01 % Tween~
80, was reached. A copper chelate column ~2 ml volume) equilibrated in the same buffer was loaded and washed with lS column volumes of equilibration buffer followed by 15 column volumes of 20 mN succinic acid pH 7.0, 500 mM NaCl, 0.01 % TweenO 80. The column was then eluted with a gradient to 20 mN succinic acid pH 4.5, 500 mN
NaCl, 0.01 % Tween 80. The activity eluted at pH 6 to 5.4.
c) Purification of the protein by Mono S chromatography The required peak from the Cu chelate column was concentrated and the buffer was changed to 20 mN Na phosphate, 10 mM NaCl pH 5.0 (buffer A). This wa~
2 0 6 5 2 ~ 8 - S - o.z. 0050/41274 followed by loading onto a Mono S FPLC column (Pharmacia, Freiburg). After the unbound material had been eluted by wa~hing the column with 10 column volumes of buffer A, the column was eluted with a combined linear pH and salt gradient from 0 to 66 % buffer B (20 mM sodium phosphate, 500 mM NaCl, pH 8.5) in 85 min. Under these conditions the novel protein eluted from the column between 10 and 30 % buffer B.
The elution was followed by W brass (~ic) at 280 nm or measurement of the activity of aliquots of the fractions loaded on. The activity-containing fractions were combined, desalted and concentrated to dryness. The samples were stored at -20C until used further.
d) Purification of the protein by reversed phase (r)HPLC
The required fractions from the Cu chelate chromatography which had been concentrated to dryness were taken up in 0.1 % strength trifluoroacetic acid (TFA) solution and further purified on a C-4 reversed phase column (rP304, Biorad, Munich). Large and small subunits of the protein were separated in this step. The elution from the column was achieved by applying a linear acetonitrile gradient (in 0.1 ~ TFA) from 0 to 50 % in 90 min at a flow rate of 1 ml/min. The cours~ of the elution was followed by W measurement at 210 nm. Under these conditions, the protein chains assigned to the protein eluted between 35 to 45 % acetonitrile. The protein bands belonging to the protein were fractionated and, after removal of the solvent, sub~ected to a proteo-lytic digestion to generate peptides.e) Separation by rever~ed phase (r)HPLC o~ the destabilase peptides obtained after proteolytic fragmentation The peptide mixtures obtained after enzymatic digestion with trypsin (Sequence Grade, Boehringer Nannheim) or endoprotease Glu C (Boehringer Mannheim) were mixed with 0.1 % strength TFA and fractionated on 206~2~8 - 6 - O.Z. 0050/41274 a C-18 reversed phase HPLC column (rp 318, Biorad, Munich). This entailed a linear gradient from solution C
(0.1 ~ TFA in water) to solution D (0.1 % TFA in aceto-nitrile) being applied over a period of 2 h. Elution was followed by W measurement at 210 nm at a flow rate of 1 ml/min. The separated peptides were fractionated and the amino-acid sequence of individual peptides was determined in a peptide sequencer tApplied Biosystems).
The sequences indicated in the sequence listing were found.
Characterization of the protein After SDS polyacrylamide gel electrophoresis and subsequent silver staining, three bands are recognizable (low molecular weight marker from Pharmacia): one band with an apparent molecular weight of 57,000 Da and two bands at 30,000 Da. On determination of the molecular weight by molecular sieve chromatography (TSK G 3000, buffer: 20 mM Na2HPO4 0.1 M NaCl pH 7.0 flow rate:
0.7 ml/min), the protein has an apparent molecular weight of about 44,000 (+ 5000 Da) Da. The specific activity is about 580 U/mg. The following assay was used to determine the activity: 50 ~1 of buffer (5 mM tris pH 8.4, 0.01 %
Tween~ 80) 50 ~1 of color reagent (~-glutamyl) p-nitroanilide, 1 mg/ml) were incubated with 50 ~1 of sample at 37C for 60 min and then the increase in extinction at 405 nm was determined. 1 U is defined as enzymatic activity which converts 1 ~mol of ~-glutamyl para-nitroanilide in 1 min at 37C and causes thereby an increase in absorption at 405 nm.
The natural protein i8 a glycoprotein, which i8 demonstrated by its mannose-dependent elutien from the concanavalin A column.
Protease inhibitors such as cystatin C
(10 ~g/ml), aprotinin (100 ~g/ml) and benzamidine (10 mN) do not inhibit the activity. 100 ~m serine together with 100 ~m borate pH 8.4 inhibit about 50 % of the enzymatic activity. The enzyme i~ completely inhibited by addition 206~2~8 - 7 - O.Z. 0050/41274 of 1-5 mM HgCl2. Furthermore, reduced and oxidized gluta-thione inhibit the enzyme. Triton~ X-100 leaves the activity virtually unaffected and can protect from denaturation by SDS. The pH optimum is between pH 8 and S pH 9. However, the enzyme is still active up to pH 12.
Beyond this non-specific hydrolysis of the substrate is already dominant.
Detection of the fibrin-dissolving action 100 ~1 of bovine fibrinogen (Niles no. 82-0222-4;
contains factor XIII) in a concentration of 4 mg/ml in TBS (= 10 mM tris/HCl pH 7.5, 150 mM NaCl) are mixed with 10 ~1 of bovine thrombin (sigma no. T6634, 5 U/ml in TBS
with 25 mM calcium acetate) and incubated in Eppendorf reaction tubes at 37C for 2 to 5 h. This results in stable, factor XIIIa-crosslinked fibrin clots.
The latter are washed once with 10 mM sodium phosphate pH 7.5, 150 mM NaCl and subsequently incubated with 200 ~1 of salivary secretion of Hirudo medicinalis (37C, 20 h). After addition of 1 ml of 5 % strenqth monochloroacetic acid, the amount of liberated protein in the supernatant i8 determined (OD 280 nm).
Example 2 1. Isolation of m-RNA
~ RNA wa~ obtained from whole leeches after dis-ruption in guanidinium thiocyanate and subsequent centri-fugation through a CsCl cushion. The portion of the RNA
containing at the 3' end a polyA (polyA+) was isolated by oligo(dT) affinity chromatography. Both process steps were carried out in aecordanee with Sambrook et al ~et al (sie) (1989) ~'MoIeeular Cloning" 2nd edition, CSH
Press, page 7.19-7.22 and 7.26-7.29.
2. Preparation of DNA probes The starting point for the cloning of cDNA
fragments by means of the polymerase chain reaetion (PCR, for details of this technique, see Sambrook et al, chapter 14) was the peptide sequence Gly Ly~ Asp Ile Ile Lys Lys Tyr 20652~8 - 8 - O.Z. 0050/41274 on the basis of the known genetic code, this amino-acid ~equence can be transcribed into a nucleic acid sequence.
Because of the known degeneracy of the genetic code, several nucleic acids can be inserted in make (sic) positions. It emerges from this that there are the following 576 possibilities for the sequence of the mRNA
strand:
5'GGNAARGAYATHATHAARAARTA3' In order to reduce the complexity, 4 groups each of 144 oligonucleotides were synthesized (316 A-D).
Likewise, 4 groups each of 144 oligonucleotides (316 rev A-D) from the complementary strand (cDNA strand) were synthesized.
The 2nd peptide sequence u~ed was the peptide Ile Val Gln Glu Ile Gln Ser Glu After transcription into the nucleic acid code, the following 1728 pos3ibilities emerged for the mRNA strand:
5~ATHGTNCARGARATHCARTCNGA3' 5'ATHGTNCARGARATHCARAGYGA3' This sequence was synthesized in 12 groups each of 144 oligonucleotides (317 A-L). Likewise, 12 groups each of 144 oliqonucleotides (317 rev A-L) from the complementary strand ~cDNA strand) were synthesized.
The syntheses were carried out with an Applied Biosystems type 360A DNA synthesizer. The oligo-nucleotides were, after removal of the protective groups, purified by gel electrophoresis on an acrylamide/urea gel.
3. Preparation of cDNA
12 x 3 ~g of polyA+ RNA were transcribed into single-stranded cDNA with the aid of a commercially available cDNA synthesis kit (Boehringer Mannheim, order no. 10 13 882) with oligo(dT)l5 as starter. The synthe~is was stopped after 1 hour by heating at 95C (5 min) and low molecular weight constituents were removed on a "cDNA
Spun Column" (Pharmacia no. 27-5099).
. . .
206~2~8 - 9 - O.Z. 0050/41274 4. PCR reaction The method for the polymerase chain reaction (PCR) is described in Sambrook et al, chapter 14. The method permits the multiplication of DNA segments.
Necessary for this is, besides the DNA (in the present case the single-stranded cDNA), a set of complementary oligonucleotides. For this, one oligonucleotide must be complementary to the mRNA strand and the other complemen-tary to the cDNA strand. The PCR reaction then permits the segment between the two oligonucleotides to be multiplied and cloned. A total of 96 PCR reaction~ was carried out, in each case combining the oligonucleotide group~ 316 and 317 rev or 316 rev with 317. 1/10 of the cDNA mixture (0.3 ~g of RNA) was employed for each reaction. The PCR reaction was carried out in an ap-paratus supplied by Perkin-Elmer (DNA-Thermo Cycler) with 40 cycles each of 2 min at 95C, 2 min at 50C and 2 min at 72C.
The oligonucleotide mixture 316B
5'GGCAARGAYATHATNAARAARTA3' combined with 317 revI
5~TCACTTTGDATYTCYTGNACDAT3' yielded a DNA fragment 590 bp in size. It was possible to isolate this fragment from a preparative agarose gel and, after attachment of 5~-phosphate residues (~kinasing~
clone it into the SmaI cleavage site of the cloning vector puc 18. The clone was called Dest-pcr 1. It was possible to replicate the DNA of the chimeric plasmid in E. coli (CMK 603) and determine the DNA sequence of the pas~enger by the Sanger method (sequence listingt sequence 13). The methods nece~ary for this are ex-plained in detail in Sambrook et al (1989).
The DNA sequence and the protein sequence belong-ing thereto is sequence 13. The sequence starts with a portion of peptide 11 and terminates with a portion of peptide 1. The sequence from nucleotide 307 to 333 codes for peptide 9.
- 10 - O.Z. 0050/41274 Sequence listing Sequence 1:
Ile Val Gln Glu lle Gln Ser Glu Gly Gly Ile Ile Thr Glu Glu ASp Leu Ala Asn Sequence 2:
Phe Val Thr Gly Ala Ser Gly Gly Ser Arg Sequence 3:
Leu Leu Gly Phe Glu Val Thr Ala Gtu Lys Sequence 4 Pro Met Ser Gly Met Val Pro Thr Leu lle Asp ASp Lys Sequence 5 Val Gly Val Cys Val Tyr Lys S
Sequence 6 Phe Val Tyr Thr Gly Thr Val Ala Glu Arg Lys Sequence 7 Leu Asn Tyr ~la Pro Ala Sequence 8 Ile Ile Glu Ala Pho Sor s Sequence 9 Leu Pho Gln Pro Thr Ile Asp Leu Leu S tO
Sequence 10 Leu Gln Leu Gly Asp Gln Asn Pho Thr Xaa V~l Thr ASp Leu llo Ser .
, 20652~8 - 11 - o.Z~ 0050/41274 Sequence 1 1 Thr Val Ala Cys Ser Lys Ile Gly Lys Asp Il~ lle Lys Lys Tyr Sequence 12 Xab Phe Xac Phe Al a Tyr Al a Gty Pro .
`
206S2a 8 Sequence 13 ATA ATC AAG AAG TAT GGA TCG GTC GTG GAT TCT GcG ATT GCC TCA ATG 48 Ile Ile Ly~ Ly~ Tyr Gly Ser Val Val A~p S-r Ala I1Q Ala S~r MQt Leu Cy~ Val Gly Va1 Val S~r Ala H1- sQr A~n Gly Il- Gly Gly Gly H1~ Val Ala Ile I1- Tyr Ala Ly~ Pro A~n Asn Gly Thr Lys Ly~ Glu L-u Val Thr L-u Il- Ala Arg Glu Arg Ala Pro Ly- Ly- Ala A-n Gln Thr ~ t Ph- Ala S-r Arg S-r S-r ~ u Ala Gly Cly L-u Ala Ala Gly ATT CCT GGA GAA TTA AAG GGA TAT TAC GAA GCT TGG AAA AGA m GGA 288 Il- Pro Gly Glu L u Ly- Gly Tyr Tyr Glu Ala Trp Ly- Arg Ph- Gly Trp L-u Pro Trp LY- a-p L U Ph- Gln Pro Thr Il- Asp L-u L-u Glu lOO 105 110 A n Gly Tyr Val Il- Glu Ly- Ala L u Ala S-r Tbr L-u Asn Ly~ Glu llS 120 125 Il- Val Ala S-r L u Val ~ u A n T~r S-r ~u Val Ly- S-r S-r L-U
L-u Arg ~y- T~r Tyr Tyr Pro A n Cly Cln Ala ~u Arg Ala Gly A p lSO lS5 160 16S
ACT TTC M A GAC CAA AAC T~G GCC GCC ACT TTT AAA AAC ATA GCT CAA 528 T~r L u ~y A p Cln Ly- L u Al~ Ala Thr Ph- Ly- Ly- Il- Ala Gln 170 17~ 180 ACT CCA CAC aGT Trr TAC AGC GCT CAT CTr GCA AAA ACG ATC GT$ CAC 576 S-r Pro ~U S-r Ph - Tyr g-r Gly A p ~u Al~ Ly- Thr rl- V~1 Gln 185 190 19~
GAA ATC CAA ACT GA Sgo G1U Il- Gln S-r
Likewise, 4 groups each of 144 oligonucleotides (316 rev A-D) from the complementary strand (cDNA strand) were synthesized.
The 2nd peptide sequence u~ed was the peptide Ile Val Gln Glu Ile Gln Ser Glu After transcription into the nucleic acid code, the following 1728 pos3ibilities emerged for the mRNA strand:
5~ATHGTNCARGARATHCARTCNGA3' 5'ATHGTNCARGARATHCARAGYGA3' This sequence was synthesized in 12 groups each of 144 oligonucleotides (317 A-L). Likewise, 12 groups each of 144 oliqonucleotides (317 rev A-L) from the complementary strand ~cDNA strand) were synthesized.
The syntheses were carried out with an Applied Biosystems type 360A DNA synthesizer. The oligo-nucleotides were, after removal of the protective groups, purified by gel electrophoresis on an acrylamide/urea gel.
3. Preparation of cDNA
12 x 3 ~g of polyA+ RNA were transcribed into single-stranded cDNA with the aid of a commercially available cDNA synthesis kit (Boehringer Mannheim, order no. 10 13 882) with oligo(dT)l5 as starter. The synthe~is was stopped after 1 hour by heating at 95C (5 min) and low molecular weight constituents were removed on a "cDNA
Spun Column" (Pharmacia no. 27-5099).
. . .
206~2~8 - 9 - O.Z. 0050/41274 4. PCR reaction The method for the polymerase chain reaction (PCR) is described in Sambrook et al, chapter 14. The method permits the multiplication of DNA segments.
Necessary for this is, besides the DNA (in the present case the single-stranded cDNA), a set of complementary oligonucleotides. For this, one oligonucleotide must be complementary to the mRNA strand and the other complemen-tary to the cDNA strand. The PCR reaction then permits the segment between the two oligonucleotides to be multiplied and cloned. A total of 96 PCR reaction~ was carried out, in each case combining the oligonucleotide group~ 316 and 317 rev or 316 rev with 317. 1/10 of the cDNA mixture (0.3 ~g of RNA) was employed for each reaction. The PCR reaction was carried out in an ap-paratus supplied by Perkin-Elmer (DNA-Thermo Cycler) with 40 cycles each of 2 min at 95C, 2 min at 50C and 2 min at 72C.
The oligonucleotide mixture 316B
5'GGCAARGAYATHATNAARAARTA3' combined with 317 revI
5~TCACTTTGDATYTCYTGNACDAT3' yielded a DNA fragment 590 bp in size. It was possible to isolate this fragment from a preparative agarose gel and, after attachment of 5~-phosphate residues (~kinasing~
clone it into the SmaI cleavage site of the cloning vector puc 18. The clone was called Dest-pcr 1. It was possible to replicate the DNA of the chimeric plasmid in E. coli (CMK 603) and determine the DNA sequence of the pas~enger by the Sanger method (sequence listingt sequence 13). The methods nece~ary for this are ex-plained in detail in Sambrook et al (1989).
The DNA sequence and the protein sequence belong-ing thereto is sequence 13. The sequence starts with a portion of peptide 11 and terminates with a portion of peptide 1. The sequence from nucleotide 307 to 333 codes for peptide 9.
- 10 - O.Z. 0050/41274 Sequence listing Sequence 1:
Ile Val Gln Glu lle Gln Ser Glu Gly Gly Ile Ile Thr Glu Glu ASp Leu Ala Asn Sequence 2:
Phe Val Thr Gly Ala Ser Gly Gly Ser Arg Sequence 3:
Leu Leu Gly Phe Glu Val Thr Ala Gtu Lys Sequence 4 Pro Met Ser Gly Met Val Pro Thr Leu lle Asp ASp Lys Sequence 5 Val Gly Val Cys Val Tyr Lys S
Sequence 6 Phe Val Tyr Thr Gly Thr Val Ala Glu Arg Lys Sequence 7 Leu Asn Tyr ~la Pro Ala Sequence 8 Ile Ile Glu Ala Pho Sor s Sequence 9 Leu Pho Gln Pro Thr Ile Asp Leu Leu S tO
Sequence 10 Leu Gln Leu Gly Asp Gln Asn Pho Thr Xaa V~l Thr ASp Leu llo Ser .
, 20652~8 - 11 - o.Z~ 0050/41274 Sequence 1 1 Thr Val Ala Cys Ser Lys Ile Gly Lys Asp Il~ lle Lys Lys Tyr Sequence 12 Xab Phe Xac Phe Al a Tyr Al a Gty Pro .
`
206S2a 8 Sequence 13 ATA ATC AAG AAG TAT GGA TCG GTC GTG GAT TCT GcG ATT GCC TCA ATG 48 Ile Ile Ly~ Ly~ Tyr Gly Ser Val Val A~p S-r Ala I1Q Ala S~r MQt Leu Cy~ Val Gly Va1 Val S~r Ala H1- sQr A~n Gly Il- Gly Gly Gly H1~ Val Ala Ile I1- Tyr Ala Ly~ Pro A~n Asn Gly Thr Lys Ly~ Glu L-u Val Thr L-u Il- Ala Arg Glu Arg Ala Pro Ly- Ly- Ala A-n Gln Thr ~ t Ph- Ala S-r Arg S-r S-r ~ u Ala Gly Cly L-u Ala Ala Gly ATT CCT GGA GAA TTA AAG GGA TAT TAC GAA GCT TGG AAA AGA m GGA 288 Il- Pro Gly Glu L u Ly- Gly Tyr Tyr Glu Ala Trp Ly- Arg Ph- Gly Trp L-u Pro Trp LY- a-p L U Ph- Gln Pro Thr Il- Asp L-u L-u Glu lOO 105 110 A n Gly Tyr Val Il- Glu Ly- Ala L u Ala S-r Tbr L-u Asn Ly~ Glu llS 120 125 Il- Val Ala S-r L u Val ~ u A n T~r S-r ~u Val Ly- S-r S-r L-U
L-u Arg ~y- T~r Tyr Tyr Pro A n Cly Cln Ala ~u Arg Ala Gly A p lSO lS5 160 16S
ACT TTC M A GAC CAA AAC T~G GCC GCC ACT TTT AAA AAC ATA GCT CAA 528 T~r L u ~y A p Cln Ly- L u Al~ Ala Thr Ph- Ly- Ly- Il- Ala Gln 170 17~ 180 ACT CCA CAC aGT Trr TAC AGC GCT CAT CTr GCA AAA ACG ATC GT$ CAC 576 S-r Pro ~U S-r Ph - Tyr g-r Gly A p ~u Al~ Ly- Thr rl- V~1 Gln 185 190 19~
GAA ATC CAA ACT GA Sgo G1U Il- Gln S-r
Claims (4)
1. A protein which contains sequences 1 to 13 detailed in the sequence listing and cleaves .epsilon.-(.gamma.-glutamyl)-lysine linkages, and its muteins and natural variants with a comparable action.
2. A process for preparing the proteins as claimed in claim 1, which comprises preparing them by known genetic engineering processes.
3. DNA coding for a protein as claimed in claim 1.
4. A protein as claimed in claim 1 for use for controlling diseases.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE3939801A DE3939801A1 (en) | 1989-12-01 | 1989-12-01 | NEW PROTEINS AND THEIR PRODUCTION |
| DEP3939801.3 | 1989-12-01 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2065258A1 true CA2065258A1 (en) | 1991-06-02 |
Family
ID=6394621
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002065258A Abandoned CA2065258A1 (en) | 1989-12-01 | 1990-11-22 | Blood-clot-dissolving proteins, and their preparation from the leech hirudo medicinalis |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP0502876B1 (en) |
| AT (1) | ATE119571T1 (en) |
| CA (1) | CA2065258A1 (en) |
| DE (2) | DE3939801A1 (en) |
| ES (1) | ES2069098T3 (en) |
| WO (1) | WO1991008233A1 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU659432B2 (en) * | 1991-03-08 | 1995-05-18 | Novartis Ag | A method for the inhibition or prevention of tumor cell metastasis with hirudin |
| RU2112528C1 (en) * | 1994-12-23 | 1998-06-10 | Институт биоорганической химии им.М.М.Шемякина и Ю.А.Овчинникова РАН | Method of preparing enzyme destabilase showing fibrinolytic, thrombolytic, endo- and exo-gamma)-lys-isopeptidase and amidolytic activity |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4588587A (en) * | 1983-03-01 | 1986-05-13 | Pennsylvania Hospital | Method of treatment to inhibit metastasis |
| PT79519B (en) * | 1983-11-21 | 1986-12-11 | Ciba Geigy Ag | Process for preparing protease inhibitors based on egline compounds |
| DE3438296A1 (en) * | 1984-04-18 | 1985-11-07 | Hoechst Ag, 6230 Frankfurt | NEW POLYPEPTIDES WITH A BLOOD-CLOTHING EFFECT, METHOD FOR THE PRODUCTION OR THEIR RECOVERY, THEIR USE AND THE CONTAINERS THEREOF |
| DE3689525D1 (en) * | 1985-07-17 | 1994-02-24 | Hoechst Ag | New polypeptides with an anticoagulant effect, processes for their preparation or extraction, their use and agents containing them. |
-
1989
- 1989-12-01 DE DE3939801A patent/DE3939801A1/en not_active Withdrawn
-
1990
- 1990-11-22 DE DE59008680T patent/DE59008680D1/en not_active Expired - Fee Related
- 1990-11-22 EP EP90917037A patent/EP0502876B1/en not_active Expired - Lifetime
- 1990-11-22 ES ES90917037T patent/ES2069098T3/en not_active Expired - Lifetime
- 1990-11-22 AT AT90917037T patent/ATE119571T1/en not_active IP Right Cessation
- 1990-11-22 CA CA002065258A patent/CA2065258A1/en not_active Abandoned
- 1990-11-22 WO PCT/EP1990/001996 patent/WO1991008233A1/en active IP Right Grant
Also Published As
| Publication number | Publication date |
|---|---|
| ATE119571T1 (en) | 1995-03-15 |
| EP0502876B1 (en) | 1995-03-08 |
| EP0502876A1 (en) | 1992-09-16 |
| DE3939801A1 (en) | 1991-06-06 |
| DE59008680D1 (en) | 1995-04-13 |
| WO1991008233A1 (en) | 1991-06-13 |
| ES2069098T3 (en) | 1995-05-01 |
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Legal Events
| Date | Code | Title | Description |
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| FZDE | Discontinued |