Methods for protein refolding

• US 8,067,201 B2
• Filed: 04/19/2010
• Issued: 11/29/2011
• Est. Priority Date: 04/17/2009
• Status: Active Grant

• Current Assignee: Bristol-Myers Squibb Company
• Original Assignee: Bristol-Myers Squibb Company
• Inventors: Lin, Zheng, Morin, Paul
• Primary Examiner(s): NASHED, NASHAAT T
• Application Number:
  US12/763,168
• Publication Number:
  US 20100273216A1
• Time in Patent Office: 589 Days
• Field of Search: None
• US Class Current: 435/69.1
• CPC Class Codes: C07K 1/1136 by reversible modification of the secondary, tertiary or quarternary structure, e.g. using denaturating or stabilising agents
• CPC Class Codes: C12P 21/02 having a known sequence of two or more amino acids, e.g. glutathione

Abstract

Provided herein are methods for refolding proteins. The methods involve covalently modifying a denatured protein with a nonproteinaceous polymer and then renaturing the modified protein.

Claims

1. A method for refolding a protein comprising covalently modifying a denatured protein with a nonproteinaceous polymer and renaturing the covalently modified protein, wherein the method comprises removing the nonproteinaceous polymer after renaturing the covalently modified protein.

2. The method of claim 1, wherein the nonproteinaceous polymer is polyethylene glycol (PEG) polymer.

3. The method of claim 2, wherein the PEG has a molecular weight of 20 kDa to 80 kDa.

4. The method of claim 2, wherein the PEG is linear.

5. The method of claim 2, wherein the PEG is branched.

6. The method of claim 1, wherein the nonproteinaceous polymer is attached to the protein through a cleavable linker.

7. The method of claim 1, wherein the protein is covalently modified with the nonproteinaceous polymer at a site specific location.

8. The method of claim 2, wherein the PEG is covalently attached to the protein at a cysteine residue.

9. The method of claim 1, wherein the denatured protein was expressed in bacterial cells.

10. The method of claim 9, wherein the bacterial cells are E. coli.

11. The method of claim 9, wherein the denatured protein was isolated from inclusion bodies.

12. The method of claim 1, wherein the protein was denatured by addition of detergents, or chaotropic agents.

13. The method of claim 12, wherein the disulfide bonds of the protein were chemically cleaved.

14. The method of claim 12, wherein the protein was denatured using a chaotropic agent.

15. The method of claim 14, wherein the protein is renatured by removing the chaotropic agent.

16. The method of claim 15, wherein the chaotropic agent is removed using dialysis.

17. The method of claim 1, wherein the protein comprises a tenth fibronectin Type III (¹⁰Fn3) domain.

18. The method of claim 17, wherein the protein comprises two ¹⁰Fn3 domains.

19. A method for producing a protein, comprising:

a) expressing a recombinant protein in bacterial cells,

b) isolating the recombinant protein from inclusion bodies,

c) covalently modifying the recombinant protein with PEG,

d) renaturing the pegylated recombinant protein, and

e) removing the PEG from the recombinant protein.

20. The method of claim 19, wherein the recombinant protein comprises a tenth fibronectin Type III (¹⁰Fn3) domain.

21. A method for refolding a protein comprising covalently modifying a denatured protein with a nonproteinaceous polymer and renaturing the covalently modified protein, wherein the nonproteinaceous polymer is attached to the protein through a protease cleavable linker.

22. The method of claim 21, wherein the nonproteinaceous polymer is polyethylene glycol (PEG) polymer.

23. The method of claim 21, wherein the protein comprises a tenth fibronectin Type III (¹⁰Fn3) domain.

24. A method for producing a pegylated protein, comprising:

a) expressing a recombinant protein in bacterial cells,

b) isolating the recombinant protein from inclusion bodies,

c) covalently modifying the recombinant protein with PEG, and

d) renaturing the pegylated recombinant protein,

wherein the PEG is attached to the recombinant protein through a protease cleavable linker.

25. The method of claim 24, wherein the recombinant protein comprises a tenth fibronectin Type III (¹⁰Fn3) domain.

26. A method for producing a pegylated protein, comprising:

a) expressing a recombinant protein in bacterial cells,

b) isolating the recombinant protein from inclusion bodies,

c) exposing the recombinant protein to a chaotropic agent,

d) covalently modifying the recombinant protein with PEG,

e) renaturing the pegylated recombinant protein by removing the chaotropic agent,

f) purifying the refolded, pegylated, recombinant protein, and

g) removing the PEG from the recombinant protein.

27. The method of claim 26, wherein the recombinant protein comprises a tenth fibronectin Type III (¹⁰Fn3) domain.