Patent US4518526A successfully refreshed from Google Patents.
US4518526A
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Purification and activity assurance of precipitated heterologous proteins
Assignee: Genentech Inc
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Filing: 1984-06-01
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Granted: 1985-05-21
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Expires: 2004-06-01
Abstract
Abstract A process for isolating and purifying a precipitated heterologous protein from a host cell culture, including treating the host cell culture with a buffered solution of ionic strength suitable to solubilize most of the host protein but in which refractile heterologous protein is substantially insoluble, and disrupting the cells to form a supernatant and an insoluble fraction; treating the insoluble fraction with a strongly denaturing solution to solubilize the refractile heterologous protein; and recovering renatured heterologous protein.
Inventors
- 👤 Kenneth C. Olson
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-30 20:51
CPC Classifications
C; C12; C12N; C12N9/00; C12N9/14; C12N9/48; C12N9/50; C12N9/64; C12N9/6421; C12N9/6478; C12N9/6481; C07; C07K; C07K1/00; C07K1/107; C07K1/113; C07K1/1133; C12N9/6424; C12N9/6456; C12N9/6462; C12Y; C12Y304/00; C12Y304/21; C12Y304/21073; Y; Y10; Y10S; Y10S435/00; Y10S435/8215; Y10S435/948; Y10S530/00; Y10S530/806; Y10S530/808; Y10S530/82; Y10S530/825; Y10S530/826
Claims Analysis
Total Claims: 12
Claim 1
Independent Claim
90 words
A process for isolating and purifying a precipitated heterologous protein from host cell culture, which process comprises: (a) treating the host cell culture with a buffered solution of ionic strength suitable to solubilize most of the host protein, but in which refractile heterologous protein is substantially insoluble and disrupting the cells, to form a supernatant and an insoluble fraction; (b) treating the insoluble fraction from step (a) with a strongly denaturing solution to solubilize the refractile heterologous protein, and (c) recovering renatured heterologous protein from the solution of Step (b).
Claim 2
Dependent Claim
90 words
A process for isolating and purifying a precipitated heterologous protein from host cell culture, which process comprises: (a) treating the host cell culture with a buffered solution of ionic strength suitable to solubilize most of the host protein, but in which refractile heterologous protein is substantially insoluble and disrupting the cells, to form a supernatant and an insoluble fraction; (b) treating the insoluble fraction from step (a) with a strongly denaturing solution to solubilize the refractile heterologous protein, and (c) recovering renatured heterologous protein from the solution of Step (b).
Claim 3
Dependent Claim
22 words
The process of claim 1 wherein there is employed in step (a) a cell paste or a frozen and thawed cell paste.
Claim 4
Dependent Claim
16 words
The process of claim 1 wherein the host cell culture is a culture of E. coli.
Claim 5
Dependent Claim
27 words
The process of claim 1 wherein the heterologous protein is selected from the group consisting of hGH, pGH, bGH, bovine interferon, tPA, and FMD viral coat proteins.
Claim 6
Dependent Claim
20 words
The process of claim 1 which includes the prior step of killing the host culture with non-polar solvent or detergent.
Claim 7
Dependent Claim
24 words
The process of claim 1 wherein the buffered solution in (a) is about 0.01-2.0 M in ionic strength and buffered at about pH 4-10.
Claim 8
Dependent Claim
24 words
The process of claim 6 wherein the buffered solution in (a) is about 0.4-0.6 M in ionic strength and buffered at about pH 6-9.
Claim 9
Dependent Claim
22 words
The process of claim 1 wherein the strongly denaturing solution is guanidine HCl or sodium thiocyanate in a concentration of about 1-9M.
Claim 10
Dependent Claim
18 words
The process of claim 8 wherein the concentration of guanidine HCl or sodium thiocyanate is about 6-8 M.
Claim 11
Dependent Claim
11 words
The process of claim 1 wherein step (c) comprises buffer exchange.
Claim 12
Dependent Claim
8 words
Protein prepared by the process of claim 1.
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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) | 1988-05-20 | 1988-11-19 | 1989-05-21 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1992-05-20 | 1992-11-19 | 1993-05-21 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1996-05-20 | 1996-11-19 | 1997-05-21 | $7700.00 | Window Closed / Lapsed |