METHODS FOR PROTEIN REFOLDING

• US 20100273216A1
• Filed: 04/19/2010
• Published: 10/28/2010
• 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
• Granted Patent: US 8,067,201 B2
• Time in Patent Office: Days
• US Class Current: 435/70.100
• 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.

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 2, wherein the PEG is a comb-type PEG.

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

8. The method of claim 1, wherein the method further comprises removing the nonproteinaceous polymer after renaturing the covalently modified protein.

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

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

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

12. The method of claim 11, wherein the bacterial cells are E. coli.

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

14. The method of claim 1, wherein the protein was denatured mechanically, by high temperature, by low temperature, suboptimal pH, addition of detergents, high salt concentration, use of chaotropic agents, or chemically cleaving disulfide bonds.

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

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

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

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

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

20. The method of claim 18, wherein the Fn3 domain is a tenth Fn3 domain.

21. 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.

22. 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, and

f) purifying the refolding, pegylated, recombinant protein.

23. 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.