EP0338634A2
Expired / Lapsed
EP
⏱️ 0.0 years remaining
Viper venom polypeptides and genetic expression
Assignee: Merck and Co Inc
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Filing: 1989-04-17
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Granted: —
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Expires: 2009-04-17
Abstract
Abstract A platelet aggregation inhibiting polypeptide having the following amino acid sequence: X-Cys-R-R-R-Arg-Gly-Asp-R-R-R-R-R-Cys-Y where X is H or at least one amino acid; Y is OH or at least one amino acid; and each R, either the same or different, is any amino acid. Genetically engineered genes for the polypeptides are described and expressed as a fusion protein using the pJC264 expression vector in E. coli . Synthetic polypeptides and methods of synthesis are also described.
Inventors
- 👤 Paul A. Friedman
- 👤 Mark A. Polokoff
- 👤 Robert J. Gould
- 👤 Gerard H. Bencen
- 👤 John W. Jacobs
- 👤 Victor M. Garsky
- 👤 Zhong-Ru Gan
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-28 01:26
CPC Classifications
C; C07; C07K; C07K14/00; C07K14/435; C07K14/745; A; A61; A61P; A61P7/00; A61P7/02; C07K14/195; C07K14/24; C07K14/245; C07K14/46; A61K; A61K38/00; C07K2319/00; C07K2319/01; C07K2319/02; C07K2319/70; C07K2319/74
Claims Analysis
Total Claims: 29
Claim 1
Independent Claim
40 words
A polypeptide having the following amino acid sequence: X-Cys-R-R-R-Arg-Gly-Asp-R-R-R-R-R-Cys-Y wherein X is H or at least one amino acid; Y is OH or at least one amino acid; and each R, either the same or different, is any amino acid.
Claim 2
Dependent Claim
40 words
A polypeptide having the following amino acid sequence: X-Cys-R-R-R-Arg-Gly-Asp-R-R-R-R-R-Cys-Y wherein X is H or at least one amino acid; Y is OH or at least one amino acid; and each R, either the same or different, is any amino acid.
Claim 3
Dependent Claim
41 words
A polypeptide according to Claim 1, having the following amino acid sequence: H₂N- Ch)-Cys-R-R-R-Arg-Gly-Asp-R-R-R-R-R-Cys-(Cx)-H wherein Ch represents at least one amino acid; Cx represents at least one amino acid; and each R, either the same or different, represents an amino acid.
Claim 4
Dependent Claim
16 words
A substantially pure polypeptide of Claim 1 called "Echistatin" having the following amino acid sequence: Glu-Cys-Glu-Ser-Gly-Pro-Cys-Cys-Arg-Asn-Cys-Lys-Phe-Leu-Lys-Glu-Gly-Thr-Ile-Cys-Lys-Arg-Ala-Arg-Gly-Asp-Asp-Met-Asp-Asp-Tyr-Cys-Asn-Gly-Lys-Thr-Cys-Asp-Cys-Pro-Arg-Asn-Pro-His-Lys-Gly-Pro-Ala-Thr
Claim 5
Dependent Claim
28 words
A substantially pure polypeptide of Claim 1 called "Agkistrostatin" having the following amino acid sequence: Val-Gln-Pro-Ala-Asn-Pro-Cys-Cys-Asp-Ala-Ala-Thr-Cys-Lys-Leu-Thr-Pro-Gly-Ser-Gln-Cys-Ala-Glu-Gly-Leu-Cys-Cys-Asp-Gln-Cys-Lys-Phe-Ile-Lys-Ala-Gly-R-Ile-Cys-R-Arg-Ala-Arg-Gly-Asp-Asn-Y wherein Y and R have the same meaning as in Claim 1.
Claim 6
Dependent Claim
15 words
A substantially pure polypeptide of Claim 1 called "Bitistatin 1" having the following sequence: Ser-Pro-Pro-Val-Cys-Gly-Asn-Glu-Leu-Leu-Glu-Glu-Gly-Glu-Glu-Cys-Asp-Cys-Gly-Ser-Pro-Ala-Asn-Cys-Gln-Asp-Arg-Cys-Cys-Asn-Ala-Ala-Thr-Cys-Lys-Leu-Thr-Pro-Gly-Ser-Gln-Cys-Asn-His-Gly-Glu-Cys-Cys-Asp-Gln-Cys-Lys-Phe-Lys-Lys-Ala-Arg-Thr-Val-Cys-Arg-Ile-Ala-Arg-Gly-Asp-Trp-Asn-Asp-Asp-Tyr-Cys-Thr-Gly-Lys-Ser-Ser-Asp-Cys-Pro-Trp-Asn-His
Claim 7
Dependent Claim
15 words
A substantially pure polypeptide of Claim 1 called "Bitistatin 2" having the following sequence: Ser-Pro-Pro-Val-Cys-Gly-Asn-Lys-Ile-Leu-Glu-Gln-Gly-Glu-Asp-Cys-Asp-Cys-Gly-Ser-Pro-Ala-Asn-Cys-Gln-Asp-Gln-Cys-Cys-Asn-Ala-Ala-Thr-Cys-Lys-Leu-Thr-Pro-Gly-Ser-Gln-Cys-Asn-His-Gly-Glu-Cys-Cys-Asp-Gln-Asp-Lys-Phe-Lys-Lys-Ala-Arg-Thr-Val-Cys-Arg-Ile-Ala-Arg-Gly-Asp-Trp-Asn-Asp-Asp-Tyr-Cys-Thr-Gly-Lys-Ser-Ser-Asp-Cys-Pro-Trp-Asn-His
Claim 8
Dependent Claim
15 words
A substantially pure polypeptide of Claim 1 called "Bitistatin 3" having the following sequence: Val-Ser-Pro-Pro-Val-Cys-Gly-Asn-Lys-Ile-Leu-Glu-Gln-Gly-Glu-Asp-Cys-Asp-Cys-Gly-Ser-Pro-Ala-Asn-Cys-Gln-Asp-Gln-Cys-Cys-Asn-Ala-Ala-Thr-Cys-Lys-Leu-Thr-Pro-Gly-Ser-Gln-Cys-Asn-His-Gly-Glu-Cys-Cys-Asp-Gln-Cys-Lys-Phe-Lys-Lys-Ala-Arg-Thr-Val-Cys-Arg-Ile-Ala-Arg-Gly-Asp-Trp-Asn-Asp-Asp-Tyr-Cys-Thr-Gly-Lys-Ser-Ser-Asp-Cys-Pro-Trp-Asn-His
Claim 9
Dependent Claim
16 words
A substantially pure polypeptide of Claim 1 called "Eristostatin" having the following amino acid sequence: Gln-Glu-Glu-Pro-Cys-Ala-Thr-Gly-Pro-Cys-Cys-Arg-Arg-Cys-Lys-Phe-Lys-Arg-Ala-Gly-Lys-Val-Cys-Arg-Val-Ala-Arg-Gly-Asp-Trp-Asn-Asp-Asp-Tyr-Cys-Thr-Gly-Lys-Ser-Cys-Asp-Cys-Pro-Arg-Asn-Pro-Trp-Asn-His
Claim 10
Dependent Claim
13 words
A polypeptide according to Claim 1, having the following amino acid sequence: Glu-Cys-Glu-Ser-Gly-Pro-Cys-Cys-Arg-Asn-Cys-Lys-Phe-Leu-Lys-Glu-Gly-Thr-Ile-Cys-Lys-Arg-Ala-Arg-Gly-Asp-Phe-Met-Asp-Asp-Tyr-Cys-Asn-Gly-Lys-Thr-Cys-Asp-Cys-Pro-Arg-Asn-Pro-His-Lys-Gly-Pro-Ala-Thr
Claim 11
Dependent Claim
13 words
A polypeptide according to Claim 1, having the following amino acid sequence: Glu-Cys-Glu-Ser-Gly-Pro-Cys-Cys-Arg-Asn-Cys-Lys-Phe-Leu-Lys-Glu-Gly-Thr-Ile-Cys-Lys-Arg-Ala-Arg-Gly-Asp-Phe-Leu-Asp-Asp-Tyr-Cys-Asn-Gly-Lys-Thr-Cys-Asp-Cys-Pro-Arg-Asn-Pro-His-Lys-Gly-Pro-Ala-Thr
Claim 12
Dependent Claim
13 words
A polypeptide according to Claim 1, having the following amino acid sequence: Glu-Cys-Glu-Ser-Gly-Pro-Cys-Cys-Arg-Asn-Cys-Lys-Phe-Leu-Lys-Glu-Gly-Thr-Ile-Cys-Lys-Arg-Ala-Arg-Gly-Asp-Trp-Met-Asp-Asp-Tyr-Cys-Asn-Gly-Lys-Thr-Cys-Asp-Cys-Pro-Arg-Asn-Pro-His-Lys-Gly-Pro-Ala-Thr
Claim 13
Dependent Claim
13 words
A polypeptide according to Claim 1, having the following amino acid sequence: Glu-Cys-Glu-Ser-Gly-Pro-Cys-Cys-Arg-Asn-Cys-Lys-Phe-Leu-Lys-Glu-Gly-Thr-Ile-Cys-Lys-Arg-Pro-Arg-Gly-Asp-Phe-Leu-Asp-Asp-Tyr-Cys-Asn-Gly-Lys-Thr-Cys-Asp-Cys-Pro-Arg-Asn-Pro-His-Lys-Gly-Pro-Ala-Thr
Claim 14
Dependent Claim
13 words
A polypeptide according to Claim 1, having the following amino acid sequence: Glu-Cys-Glu-Ser-Gly-Pro-Cys-Cys-Arg-Asn-Cys-Lys-Phe-Leu-Lys-Glu-Gly-Thr-Ile-Cys-Lys-Arg-Ala-Arg-Gly-Asp-Asp-Leu-Asp-Asp-Tyr-Cys-Asn-Gly-Lys-Thr-Cys-Asp-Cys-Pro-Arg-Asn-Pro-His-Lys-Gly-Pro-Ala-Thr
Claim 15
Dependent Claim
13 words
A polypeptide according to Claim 1, having the following amino acid sequence: Glu-Cys-Glu-Ser-Gly-Pro-Cys-Cys-Arg-Asn-Cys-Lys-Phe-Leu-Lys-Glu-Gly-Thr-Ile-Cys-Lys-Arg-Ala-Arg-Gly-Asp-Asp-Phe-Asp-Asp-Tyr-Cys-Asn-Gly-Lys-Thr-Cys-Asp-Cys-Pro-Arg-Asn-Pro-His-Lys-Gly-Pro-Ala-Thr
Claim 16
Dependent Claim
13 words
A polypeptide according to Claim 1, having the following amino acid sequence: Glu-Cys-Glu-Ser-Gly-Pro-Cys-Cys-Arg-Asn-Cys-Lys-Phe-Leu-Lys-Glu-Gly-Thr-Ile-Cys-Lys-Asn-Ala-Arg-Gly-Asp-Phe-Leu-Asp-Asp-Tyr-Cys-Asn-Gly-Lys-Thr-Cys-Asp-Cys-Pro-Arg-Asn-Pro-His-Lys-Gly-Pro-Ala-Thr
Claim 17
Dependent Claim
26 words
A method of inhibiting fibrinogen binding to mammalian platelets or fibrinogen-induced aggregation of mammalian platelets comprising incubating mammalian platelets with a polypeptide according to Claim 1.
Claim 18
Dependent Claim
24 words
The use of a polypeptide according to Claim 1 for the manufacture of a medicament useful for inhibiting aggregation of platelets in a mammal.
Claim 19
Independent Claim
48 words
A method for purifying a polypeptide of the following sequence: Glu-Cys-Glu-Ser-Gly-Pro-Cys-Cys-Arg-Asn-Cys-Lys-Phe-Leu-Lys-Glu-Gly-Thr-Ile-Cys-Lys-Arg-Ala-Arg-Gly-Asp-Asp-Met-Asp-Asp-Tyr-Cys-Asn-Gly-Lys-Thr-Cys-Asp-Cys-Pro-Arg-Asn-Pro-His-Lys-Gly-Pro-Ala-Thr comprising (a) dissolving lyophilized Echis carinatus venom in slightly basic solution; (b) centrifuging the solution to obtain supernatant; (c) loading supernatant onto a column; and (d) determining fractions containing platelet aggregation inhibitory activity and concentrating under vacuum.
Claim 20
Independent Claim
48 words
A method for purifying a polypeptide of the following sequence: Glu-Cys-Glu-Ser-Gly-Pro-Cys-Cys-Arg-Asn-Cys-Lys-Phe-Leu-Lys-Glu-Gly-Thr-Ile-Cys-Lys-Arg-Ala-Arg-Gly-Asp-Asp-Met-Asp-Asp-Tyr-Cys-Asn-Gly-Lys-Thr-Cys-Asp-Cys-Pro-Arg-Asn-Pro-His-Lys-Gly-Pro-Ala-Thr comprising (a) dissolving lyophilized Echis carinatus venom in slightly basic solution; (b) centrifuging the solution to obtain supernatant; (c) loading supernatant onto a column; and (d) determining fractions containing platelet aggregation inhibitory activity and concentrating under vacuum.
Claim 21
Dependent Claim
50 words
A gene comprising a recombinant DNA molecule encoding a polypeptide having the following amino acid sequence: H₂-(Ch)-Cys-R-R-R-Arg-Gly-Asp-R-R-R-R-R-Cys-(Cx)-H wherein Ch represents at least one amino acid; Cx represents at least one amino acid; and each R, either the same or different, represents an amino acid, wherein said polypeptide inhibits platelet aggregation.
Claim 22
Dependent Claim
50 words
A gene comprising a recombinant DNA molecule encoding a polypeptide having the following amino acid sequence: H₂-(Ch)-Cys-R-R-R-Arg-Gly-Asp-R-R-R-R-R-Cys-(Cx)-H wherein Ch represents at least one amino acid; Cx represents at least one amino acid; and each R, either the same or different, represents an amino acid, wherein said polypeptide inhibits platelet aggregation.
Claim 23
Dependent Claim
15 words
A gene according to Claim 19, wherein the amino acid sequence is as follows: Glu-Cys-Glu-Ser-Gly-Pro-Cys-Cys-Arg-Asn-Cys-Lys-Phe-Leu-Lys-Glu-Gly-Thr-Ile-Cys-Lys-Arg-Ala-Arg-Gly-Asp-Phe-Leu-Asp-Asp-Tyr-Cys-Asn-Gly-Lys-Thr-Cys-Asp-Cys-Pro-Arg-Asn-Pro-His-Lys-Gly-Pro-Ala-Thr
Claim 24
Dependent Claim
73 words
A replicable microbial expression vector comprising a promoter-operator sequence capable of expressing heterologous proteins in a microorganism followed by DNA encoding a platelet aggregation inhibitor having an amino acid sequence H₂N-(Ch)-Cys-R-R-R-Arg-Gly-Asp-R-R-R-R-R-Cys-(Cx)-H wherein Ch represents at least one amino acid; Cx represents at least one amino acid; and each R, either the same or different, represents an amino acid, wherein transcription of said DNA in a transformant microorganism is under control of said promoter-operator.
Claim 25
Dependent Claim
73 words
A replicable microbial expression vector comprising a promoter-operator sequence capable of expressing heterologous proteins in a microorganism followed by DNA encoding a platelet aggregation inhibitor having an amino acid sequence H₂N-(Ch)-Cys-R-R-R-Arg-Gly-Asp-R-R-R-R-R-Cys-(Cx)-H wherein Ch represents at least one amino acid; Cx represents at least one amino acid; and each R, either the same or different, represents an amino acid, wherein transcription of said DNA in a transformant microorganism is under control of said promoter-operator.
Claim 26
Dependent Claim
18 words
An expression vector of Claim 21, wherein the amino acid sequence has the following amino acid sequence: Glu-Cys-Glu-Ser-Gly-Pro-Cys-Cys-Arg-Asn-Cys-Lys-Phe-Leu-Lys-Glu-Gly-Thr-Ile-Cys-Lys-Arg-Ala-Arg-Gly-Asp-Asp-Leu-Asp-Asp-Tyr-Cys-Asn-Gly-Lys-Thr-Cys-Asp-Cys-Pro-Arg-Asn-Pro-His-Lys-Gly-Pro-Ala-Thr
Claim 27
Dependent Claim
14 words
A viable cell culture comprising cells transformed with the expression vector of Claim 22.
Claim 28
Dependent Claim
170 words
A process for preparing a cysteine-rich polypeptide containing the following sequence: H₂N-(Ch)-Cys-R-R-R-Arg-Gly-Asp-R-R-R-R-R-Cys-(Cx)-H wherein Ch represents at least one amino acid; Cx represents at least one amino acid; and each R, either the same or different, represents an amino acid, wherein: (a) the C-terminal first amino acid of the sequence is attached to a solid cross-linked polystyrene resin to form an amino acyl polymer, the C-terminal first amino acid blocking group is removed from the amino acyl polymer, the next sequential second amino acid is coupled to the C-terminal amino acid residue, subsequent amino acids are coupled in step-wise fashion to form the polypeptide sequence, and the N-terminal Boc group protecting the N-terminal amino acid is removed; (b) the polypeptide sequence is treated with HF and one or more thio-containing scavengers; (c) the treated sequence is washed and transferred from the column to a large volume of dilute acetic acid to minimize undesirable crosslinking; and (d) the solution resulting from step (c) is adjusted to a pH of about 8.0 stirred.
Claim 29
Dependent Claim
170 words
A process for preparing a cysteine-rich polypeptide containing the following sequence: H₂N-(Ch)-Cys-R-R-R-Arg-Gly-Asp-R-R-R-R-R-Cys-(Cx)-H wherein Ch represents at least one amino acid; Cx represents at least one amino acid; and each R, either the same or different, represents an amino acid, wherein: (a) the C-terminal first amino acid of the sequence is attached to a solid cross-linked polystyrene resin to form an amino acyl polymer, the C-terminal first amino acid blocking group is removed from the amino acyl polymer, the next sequential second amino acid is coupled to the C-terminal amino acid residue, subsequent amino acids are coupled in step-wise fashion to form the polypeptide sequence, and the N-terminal Boc group protecting the N-terminal amino acid is removed; (b) the polypeptide sequence is treated with HF and one or more thio-containing scavengers; (c) the treated sequence is washed and transferred from the column to a large volume of dilute acetic acid to minimize undesirable crosslinking; and (d) the solution resulting from step (c) is adjusted to a pH of about 8.0 stirred.
References Cited (Backward Citations)
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