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 composition comprising an amount of a refolding buffer effective to, when diluted by a factor of 1 to 10, induce folding of a neublastin polypeptide, wherein the refolding buffer comprises the following components at 1 to 10 times the stated concentrations: (i) potassium phosphate or sodium phosphate at a concentration of 25 mM to 150 mM with a pH ranging from 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 composition of claim 1 , wherein the refolding buffer comprises L-Arginine at 1 to 10 times a concentration of 0.30 M to 0.5M.
3. The composition of claim 1 , wherein the refolding buffer comprises L-Arginine at 1 to 10 times a concentration of at least 0.30 M.
4. The composition of claim 1 , wherein the refolding buffer comprises L-Arginine at 1 to 10 times a concentration of at least 0.35 M.
5. The composition of claim 1 , wherein the refolding buffer comprises L-Arginine at 1 to 10 times a concentration of 0.35 M.
6. The composition of claim 1 , wherein the refolding buffer comprises Tween-80 at 1 to 10 times a concentration of 0.1% to 1%.
7. The composition of claim 1 , wherein the refolding buffer comprises Tween-80 at 1 to 10 times a concentration of 0.1% to 0.5%.
8. The composition of claim 1 , wherein the refolding buffer comprises Tween-80 at 1 to 10 times a concentration of at least 0.1%.
9. The composition of claim 1 , wherein the refolding buffer comprises Tween-80 at 1 to 10 times a concentration of 0.1%.
10. The composition of claim 1 , wherein the refolding buffer comprises oxidized and reduced glutathione at a ratio of from 5:1 to 10:1.
11. The composition of claim 1 , wherein the refolding buffer comprises oxidized and reduced glutathione at a ratio of 5:1.
12. The composition of claim 1 , wherein the refolding buffer comprises oxidized glutathione at 1 to 10 times a concentration of 1 mM to 2 mM.
13. The composition of claim 1 , wherein the refolding buffer comprises oxidized glutathione at 1 to 10 times a concentration of 1 mM.
14. The composition of claim 1 , wherein the refolding buffer comprises guanidine-HCl at 1 to 10 times a concentration of 0.5 M to 1.0 M.
15. The composition of claim 1 , wherein the refolding buffer comprises guanidine-HCl at 1 to 10 times a concentration of at least 0.5 M.
16. The composition of claim 1 , wherein the refolding buffer comprises guanidine-HCl at 1 to 10 times a concentration of 0.5 M.
17. The composition of claim 1 , wherein the refolding buffer comprises potassium phosphate at 1 to 10 times a concentration of 25 mM to 100 mM.
18. The composition of claim 1 , wherein the refolding buffer comprises potassium phosphate at 1 to 10 times a concentration of 25 mM to 75 mM.
19. The composition of claim 1 , wherein the refolding buffer comprises potassium phosphate at 1 to 10 times a concentration of at least 50 mM.
20. The composition of claim 1 , wherein the refolding buffer comprises potassium phosphate at 1 to 10 times a concentration of 50 mM.
21. The composition of claim 1 , wherein the refolding buffer comprises potassium phosphate at a pH of 7.0 to 8.0.
22. The composition of claim 1 , wherein the refolding buffer comprises potassium phosphate at a pH of 7.5 to 8.0.
23. The composition of claim 1 , wherein the refolding buffer comprises potassium phosphate at a pH of about 7.8.
24. The composition 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 composition of claim 1 , wherein the refolding buffer comprises the following components at 1 to 10 times the stated concentrations: (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 composition of claim 1 , wherein the refolding buffer consists of the following components at 1 to 10 times the stated concentrations: (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 composition of claim 1 , wherein the refolding buffer lacks urea.
28. The composition of claim 1 , wherein the refolding buffer lacks glycine.