Refolding transforming growth factor beta family proteins
View Patent ↗Compositions and methods for folding proteins belonging to the transforming growth factor beta superfamily are disclosed. The compositions and methods allow for the folding of such proteins when produced in an expression system that does not yield a properly folded, biologically active product.
1. A method of inducing folding of a denatured polypeptide, the method comprising:
providing a denatured polypeptide comprising a transforming growth factor beta (TGF beta) superfamily member; and
contacting the polypeptide with an amount of a refolding buffer effective to induce folding of the polypeptide, wherein the refolding buffer comprises (i) potassium phosphate or sodium phosphate at a concentration of 25 mM to 150 mM with a pH of 5.8 to 8.0, (ii) guanidine-HCl at a concentration of 0.3 M to 2 M, (iii) L-Arginine at a concentration of 0.25 M to 1 M, (iv) Tween-80 at a concentration of 0.05% to 1%, and (v) oxidized glutathione at a concentration of 1 mM to 4 mM and reduced glutathione at a concentration of 0.05 mM to 0.8 mM, wherein the ratio of oxidized to reduced glutathione is from 5:1 to 20:1.
2. The method of claim 1 , wherein the refolding buffer comprises L-Arginine at a concentration of 0.30 M to 0.5M.
3. The method of claim 1 , wherein the refolding buffer comprises L-Arginine at a concentration of at least 0.30 M.
4. The method of claim 1 , wherein the refolding buffer comprises L-Arginine at a concentration of at least 0.35 M.
5. The method of claim 1 , wherein the refolding buffer comprises L-Arginine at a concentration of 0.35 M.
6. The method of claim 1 , wherein the refolding buffer comprises Tween-80 at a concentration of 0.1% to 1%.
7. The method of claim 1 , wherein the refolding buffer comprises Tween-80 at a concentration of 0.1% to 0.5%.
8. The method of claim 1 , wherein the refolding buffer comprises Tween-80 at a concentration of at least 0.1%.
9. The method of claim 1 , wherein the refolding buffer comprises Tween-80 at a concentration of 0.1%.
10. The method of claim 1 , wherein the refolding buffer comprises oxidized and reduced glutathione at a ratio of from 5:1 to 10:1.
11. The method of claim 1 , wherein the refolding buffer comprises oxidized and reduced glutathione at a ratio of 5:1.
12. The method of claim 1 , wherein the refolding buffer comprises oxidized glutathione at a concentration of 1 mM to 2 mM.
13. The method of claim 1 , wherein the refolding buffer comprises oxidized glutathione at a concentration of 1 mM.
14. The method of claim 1 , wherein the refolding buffer comprises guanidine-HCl at a concentration of 0.5 M to 1.0 M.
15. The method of claim 1 , wherein the refolding buffer comprises guanidine-HCl at a concentration of at least 0.5 M.
16. The method of claim 1 , wherein the refolding buffer comprises guanidine-HCl at a concentration of 0.5 M.
17. The method of claim 1 , wherein the refolding buffer comprises potassium phosphate at a concentration of 25 mM to 100 mM.
18. The method of claim 1 , wherein the refolding buffer comprises potassium phosphate at a concentration of 25 mM to 75 mM.
19. The method of claim 1 , wherein the refolding buffer comprises potassium phosphate at a concentration of at least 50 mM.
20. The method of claim 1 , wherein the refolding buffer comprises potassium phosphate at a concentration of 50 mM.
21. The method of claim 1 , wherein the refolding buffer comprises potassium phosphate at a pH of 7.0 to 8.0.
22. The method of claim 1 , wherein the refolding buffer comprises potassium phosphate at a pH of 7.5 to 8.0.
23. The method of claim 1 , wherein the refolding buffer comprises potassium phosphate at a pH of about 7.8.
24. The method of claim 1 , wherein the refolding buffer does not consist of (i) potassium phosphate pH 7.8 at a concentration of 50 mM, (ii) guanidine-HCl at a concentration of 0.5 M, (iii) L-Arginine at a concentration of 0.35 M, (iv) Tween-80 at a concentration of 0.1%, (v) oxidized glutathione at a concentration of 1 mM, and (vi) reduced glutathione at a concentration of 0.2 mM.
25. The method of claim 1 , wherein the refolding buffer comprises (i) potassium phosphate pH 7.8 at a concentration of 50 mM, (ii) guanidine-HCl at a concentration of 0.5 M, (iii) L-Arginine at a concentration of 0.35 M, (iv) Tween-80 at a concentration of 0.1%, (v) oxidized glutathione at a concentration of 1 mM, and (vi) reduced glutathione at a concentration of 0.2 mM.
26. The method of claim 1 , wherein the refolding buffer consists of (i) potassium phosphate pH 7.8 at a concentration of 50 mM, (ii) guanidine-HCl at a concentration of 0.5 M, (iii) L-Arginine at a concentration of 0.35 M, (iv) Tween-80 at a concentration of 0.1%, (v) oxidized glutathione at a concentration of 1 mM, and (vi) reduced glutathione at a concentration of 0.2 mM.
27. The method of claim 1 , wherein the refolding buffer lacks urea.
28. The method of claim 1 , wherein the refolding buffer lacks glycine.
29. The claim 1 , wherein the TGF beta superfamily member is a glial cell line-derived neurotrophic factor (GDNF) family member.
30. The method of claim 29 , wherein the GDNF family member is a neublastin protein.
31. The method of claim 30 , wherein the neublastin protein comprises amino acid residues 122-220 of SEQ ID NO:1.
32. The method of claim 30 , wherein the neublastin protein comprises amino acid residues 117-220 of SEQ ID NO:1.
33. The method of claim 30 , wherein the neublastin protein comprises amino acid residues 108-220 of SEQ ID NO:1.
34. The method of claim 1 , further comprising expressing the polypeptide in bacteria prior to inducing folding with the refolding buffer.
35. The method of claim 34 , wherein the bacteria is E. coli.
36. The method of claim 34 , wherein the polypeptide is expressed in bacteria in an insoluble form and, prior to inducing folding with the refolding buffer, the insoluble polypeptide is contacted with an amount of a solubilization buffer effective to denature the polypeptide.