EP0089626A2
Expired / Lapsed
EP
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Process for the preparation of polypeptides utilizing a charged amino acid polymer and exopeptidase
Assignee: GD Searle LLC
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Filing: 1983-03-17
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Granted: —
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Expires: 2003-03-17
Abstract
Abstract method for the production of polypeptides utilizing a polymer of charged amino acids and an exopeptidase. A DNA sequence coding for the charged amino acid polymer is inserted adjacent to one end of the polypeptide gene. The polypeptide-charged amino acid polymer fusion product is removed from most contaminents by selecting for the charged amino acid polymer during a first isolation step. Next the charged amino acid polymer is hydrolyzed using an exopeptidase which leaves the polypeptide active. A second isolation step removes the polypeptide from any remaining contaminents. An example using polyarginine and carboxypeptidase B to produce urogastrone is included.
Inventors
- 👤 Stephen James Brewer
- 👤 Helmut Max Sassenfeld
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-29 02:34
CPC Classifications
C; C12; C12P; C12P21/00; C12P21/02; C07; C07K; C07K14/00; C07K14/435; C07K14/475; C07K14/485; C12Q; C12Q1/00; C12Q1/34; C12Q1/37; C07K2319/00; C07K2319/01; C07K2319/02; C07K2319/50; C07K2319/70; C07K2319/74; C07K2319/75; Y; Y10; Y10S; Y10S435/00; Y10S435/803; Y10S930/00; Y10S930/01; Y10S930/12
Claims Analysis
Total Claims: 23
Claim 1
Independent Claim
114 words
In a method of producing a specific polypeptide involving a structural gene contained in a cloning vehicle such as a plasmid expressed in a prokaryotic or eukaryotic cell and the isolation of the said structural gene product the improvement comprising: (a) inserting adjacent to the said structural gene at either end or at both ends a DNA sequence coding for a polymer of charged amino acids; (b) expressing the said structural gene product and charged amino acid polymer as a fusion product; (c) separating the said fusion product from contaminents based upon the properties of the said polymer of changed amino acids; (d) removing the said polymer of charged amino acids using an exopeptidase.
Claim 2
Dependent Claim
114 words
In a method of producing a specific polypeptide involving a structural gene contained in a cloning vehicle such as a plasmid expressed in a prokaryotic or eukaryotic cell and the isolation of the said structural gene product the improvement comprising: (a) inserting adjacent to the said structural gene at either end or at both ends a DNA sequence coding for a polymer of charged amino acids; (b) expressing the said structural gene product and charged amino acid polymer as a fusion product; (c) separating the said fusion product from contaminents based upon the properties of the said polymer of changed amino acids; (d) removing the said polymer of charged amino acids using an exopeptidase.
Claim 3
Dependent Claim
30 words
The method of claim 1 wherein following step (d) an addition step is performed comprising isolating the said structural gene product free of the said polymer of charged amino acids.
Claim 4
Dependent Claim
15 words
The method of claim 1 or 2 wherein the charged amino acid polymer is polyarginine.
Claim 5
Dependent Claim
20 words
The method of claim 1 or 2 where in the said polymer of charged amino acids is 2 to 30.
Claim 6
Dependent Claim
18 words
The method of claim 1 or 2wherein the said polymer of charged amino acids is 3 to 12.
Claim 7
Dependent Claim
16 words
The method of claim 3 wherein the said polymer of charged amino acids is 5 arginines.
Claim 8
Dependent Claim
27 words
The method of claim 1 or 2 wherein the said DNA sequence coding for a polymer of charged amino acids comprises 2 to 30 consecutive triplet codons.
Claim 9
Dependent Claim
25 words
The method of claim 7 wherein the said DNA sequence coding for a polymer of charged amino acids comprises 3 to 12 consecutive triplet codons.
Claim 10
Dependent Claim
23 words
The method of claim 8 wherein the DNA sequence coding for a polymer of charged amino acids comprises five consecutive arginine triplet codons.
Claim 11
Dependent Claim
18 words
The method of claim 1 or 2 wherein the said structural gene is a gene coding for Urogastrone.
Claim 12
Dependent Claim
14 words
The method of claim 1 or 2 wherein the said exopeptidase is carboxypeptidase B.
Claim 13
Dependent Claim
16 words
The method of claim 1 or 2 wherein the said cloning vehicle is plasmid pWT 221.
Claim 14
Dependent Claim
15 words
The method of claim 1 or 2 wherein the said prokaryotic cell is Escherichia coli.
Claim 15
Dependent Claim
23 words
The method of claim 1 or 2 wherein separating the fusion product or isolating the structural gene product is accomplished using ion-exchange chromotography.
Claim 16
Dependent Claim
23 words
The method of claim 1 or 2 wherein separating the fusion product or isolating the structural gene product is accomplished using electrophoretic mobility.
Claim 17
Dependent Claim
29 words
The method of claim 1 or 2 wherein separating the fusion product or isolating the structural gene product is accomplished using antibody specific for the charged amino acid polymer.
Claim 18
Dependent Claim
23 words
The method of claim 1 or 2 wherein separating the fusion product or isolating the structural gene product is accomplished using gel filtration.
Claim 19
Independent Claim
52 words
A method of quantitating the presence of a polypeptide with a covalently attached polymer of charged amino acids comprising: a) hydrolyzing the covelently attached polymer of charged amino acids with an exopeptidase; b) quantitating the number of amino acids released by the exopeptidase hydrolysis as a measure of the said polypeptide present.
Claim 20
Independent Claim
52 words
A method of quantitating the presence of a polypeptide with a covalently attached polymer of charged amino acids comprising: a) hydrolyzing the covelently attached polymer of charged amino acids with an exopeptidase; b) quantitating the number of amino acids released by the exopeptidase hydrolysis as a measure of the said polypeptide present.
Claim 21
Dependent Claim
22 words
The method of claim 1 or 2 wherein separating the fusion product or isolating the structural product is accomplished using affinity chromatography.
Claim 22
Independent Claim
45 words
A method of quantitating the presence of a polypeptide with a covalent polymer of charged amino acids comprising: a) binding the covalently attached polymer of charged amino acids with a detectable ligand, b) quantitating the detectable ligand as a measure of the said polypeptide present.
Claim 23
Independent Claim
45 words
A method of quantitating the presence of a polypeptide with a covalent polymer of charged amino acids comprising: a) binding the covalently attached polymer of charged amino acids with a detectable ligand, b) quantitating the detectable ligand as a measure of the said polypeptide present.
References Cited (Backward Citations)
No citation records indexed.
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