Purification and activity assurance of precipitated heterologous proteins

• US 4,518,526 A
• Filed: 06/01/1984
• Issued: 05/21/1985
• Est. Priority Date: 12/22/1982
• Status: Expired due to Term

• Current Assignee: Genentech, Inc. (Roche Holding AG)
• Original Assignee: Genentech, Inc. (Roche Holding AG)
• Inventors: Olson, Kenneth C.
• Primary Examiner(s): SCHAIN, HOWARD
• Application Number:
  US06/615,682
• Time in Patent Office: 354 Days
• Field of Search: 260/112 R, 424/85, 435/68, 435/70, 435/172, 435/240, 435/241, 435/948, 436/548
• US Class Current: 530/351
• CPC Class Codes: C07K 1/1133 by redox-reactions involving cystein/cystin side chains
• CPC Class Codes: C12N 9/6462 u-Plasminogen activator (3.4.21.73), i.e. urokinase
• CPC Class Codes: C12N 9/6481 Pepsins (3.4.23.1; 3.4.23.2; 3.4.23.3)
• CPC Class Codes: C12Y 304/21073 u-Plasminogen activator (3.4.21.73), i.e. urokinase
• CPC Class Codes: Y10S 435/948 using viruses or cell lines
• CPC Class Codes: Y10S 530/806 Antigenic peptides or proteins
• CPC Class Codes: Y10S 530/808 Materials and products related to genetic engineering or hybrid or fused cell technology, e.g. hybridoma, monoclonal products
• CPC Class Codes: Y10S 530/825 Bacteria
• CPC Class Codes: Y10S 530/826 Viruses

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.

Claims

1. 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).

2. The process of claim 1 wherein there is employed in step (a) a cell paste or a frozen and thawed cell paste.

3. The process of claim 1 wherein the host cell culture is a culture of E. coli.

4. 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.

5. The process of claim 1 which includes the prior step of killing the host culture with non-polar solvent or detergent.

6. 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.

7. 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.

8. The process of claim 1 wherein the strongly denaturing solution is guanidine HCl or sodium thiocyanate in a concentration of about 1-9M.

9. The process of claim 8 wherein the concentration of guanidine HCl or sodium thiocyanate is about 6-8 M.

10. The process of claim 1 wherein step (c) comprises buffer exchange.

11. Protein prepared by the process of claim 1.