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US4532207A
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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: 1983-03-03
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Granted: 1985-07-30
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Expires: 2003-03-03
Abstract
Abstract A 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 contaminants 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 J. Brewer
- 👤 Helmut M. Sassenfeld
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-25 20:01
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
113 words
In a method of producing a 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 polypeptide 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 polypeptide and charged amino acid polymer as a fusion product; (c) separating the said fusion product from contaminants based upon the properties of the said polymer of charged amino acids; (d) removing the said polymer of charged amino acids using an exopeptidase.
Claim 2
Dependent Claim
113 words
In a method of producing a 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 polypeptide 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 polypeptide and charged amino acid polymer as a fusion product; (c) separating the said fusion product from contaminants based upon the properties of the said polymer of charged 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 additional step is performed comprising isolating the said structural gene polypeptide 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
22 words
The method of claim 1 or 2 where in the acid polymer of charged amino acids is 2 to 30 amino acids.
Claim 6
Dependent Claim
21 words
The method of claim 1 or 2 wherein the said polymer of charged amino acids is 3 to 12 amino acids.
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
21 words
The method of claim 1 or 2 wherein the said structural gene is a gene coding for gamma or beta 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
15 words
The method of claim 1 or 2 wherein the said prokaryotic cell is Escherichia coli.
Claim 14
Dependent Claim
15 words
The method of claim 1 wherein separating the fusion product is accomplished using affinity chromatography.
Claim 15
Dependent Claim
15 words
The method of claim 1 wherein separating the fusion product is accomplished using ion-exchange chromatography.
Claim 16
Dependent Claim
15 words
The method of claim 1 wherein separating the fusion product is accomplished using electrophoretic mobility.
Claim 17
Dependent Claim
21 words
The method of claim 1 wherein separating the fusion product is accomplished using antibody specific for the charged amino acid polymer.
Claim 18
Dependent Claim
15 words
The method of claim 1 wherein separating the fusion product is accomplished using gel filtration.
Claim 19
Dependent Claim
16 words
The method of claim 2 wherein isolating the structural gene polypeptide is accomplished using ion-exchange chromatography.
Claim 20
Dependent Claim
16 words
The method of claim 2 wherein isolating the structural gene polypeptide is accomplished using electrophoretic mobility.
Claim 21
Dependent Claim
22 words
The method of claim 2 wherein isolating the structural gene polypeptide is accomplished using antibody specific for the charged amino acid polymer.
Claim 22
Dependent Claim
16 words
The method of claim 2 wherein isolating the structural gene polypeptide is accomplished using gel filtration.
Claim 23
Dependent Claim
16 words
The method of claim 2 wherein isolating the structural gene polypeptide is accomplished using affinity chromatography.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1988-07-29 | 1989-01-28 | 1989-07-30 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1992-07-29 | 1993-01-28 | 1993-07-30 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1996-07-29 | 1997-01-28 | 1997-07-30 | $7700.00 | Window Closed / Lapsed |