초록 ▼

The present invention discloses improved methods of disaggregating protein aggregates, and refolding denatured proteins, using high pressure. In particular, the present invention provides for the use of agitation, high temperature, "stepped" depressurization, dialysis and dilution under pressure to

The present invention discloses improved methods of disaggregating protein aggregates, and refolding denatured proteins, using high pressure. In particular, the present invention provides for the use of agitation, high temperature, "stepped" depressurization, dialysis and dilution under pressure to increase the speed and extent of aggregate dissolution and protein refolding.

대표청구항 ▼

The invention claimed is: 1. A method of producing biologically active disaggregated protein from protein aggregates comprising: (i) providing a protein aggregate; (ii) mixing said protein aggregate with a reducing agent in an amount sufficient to reduce disulfide bonds therein; (iii) subjecting th

The invention claimed is: 1. A method of producing biologically active disaggregated protein from protein aggregates comprising: (i) providing a protein aggregate; (ii) mixing said protein aggregate with a reducing agent in an amount sufficient to reduce disulfide bonds therein; (iii) subjecting the mixture of step (ii) to increased pressure of about 0.25 kbar to about 12 kbar, whereby said protein aggregate dissolves and/or disaggregates; (iv) dialyzing the mixture under pressure, whereby said reducing agent is removed and disulfide bonds reform; and (v) removing the dissolved and/or disaggregated protein from increased pressure, whereby said protein refolds such that biological activity is retained. 2. The method of claim 1, further comprising the step of agitating said protein aggregate and/or dissolved protein and/or disaggregated protein to enhance dissolution and/or disaggregation and/or refolding. 3. The method of claim 1, further comprising subjecting said protein aggregate, prior to refolding, to a temperature of about 30° C. to about 125° C. 4. The method of claim 3, wherein said method does not utilize a chaotropic agent. 5. The method of claim 1, wherein said reducing agent is dithiothreitol, glutathione, dithioerythritol, or β-mercaptoethanol. 6. The method of claim 1, wherein said increased pressure comprises about 500 atmospheres to about 10,000 atmospheres. 7. The method of claim 1, wherein steps (ii)-(v) are performed in about 3 hours to about 12 hours. 8. The method of claim 7, wherein steps (ii)-(v) are performed in about 6 hours. 9. The method of claim 1, wherein steps (ii) and (iii) are performed in the presence of a chaotropic agent, and said method further comprises the step of removing said chaotropic agent. 10. The method of claim 8, wherein said chaotropic agent is guanidine, guanidine sulfate, guanidine hydrochloride, urea, thiocyanate, sarcosyl, sodium dodecyl sulfate, or sodium octyl sulfate. 11. The method of claim 1, wherein said protein aggregate comprises inclusion bodies, soluble and insoluble precipitates, soluble non-native oligomers, gel, fibrils, films, filaments, protofibrils, amyloid deposits, plaques, or dispersed non-native intracellular oligomers. 12. A method of producing biologically active disaggregated protein from protein aggregates comprising: (i) providing a protein aggregate; (ii) subjecting the said protein aggregate to increased pressure of about 0.25 kbar to about 12 kbar, and high temperature of about 30° C. to about 125° C., whereby said protein aggregate dissolves and/or disaggregates; and (iii) removing the dissolved and/or disaggregated protein from increased pressure and high temperature, whereby said protein refolds such that biological activity is retained. 13. The method of claim 12, wherein said method does not include use of a chaotropic agent. 14. The method of claim 12, further comprising the step of agitating said protein aggregate and/or dissolved protein and/or disaggregated protein to enhance dissolution and/or disaggregation and/or refolding. 15. The method of claim 12, further comprising mixing said protein aggregate with a reducing agent in an amount sufficient to reduce disulfide bonds therein. 16. The method of claim 15, further comprising dialyzing said mixture under pressure, whereby said reducing agent is removed and disulfide bonds reform. 17. The method of claim 15, further comprising removing said reducing agent by diafiltration or ultrafiltration. 18. The method of claim 15, further comprising negating the effect of said reducing agent by addition of an oxidizing agent. 19. The method of claim 15, wherein said reducing agent is dithiothreitol, glutathione, dithioerythritol, or β-mercaptoethanol. 20. The method of claim 12, wherein said increased pressure comprises about 0.5 kbar to about 10 kbar. 21. The method of claim 12, wherein steps (ii)-(iii) are performed in about 3 hours to about 12 hours. 22. The method of claim 21, wherein steps (ii)-(iii) are performed in about 6 hours. 23. The method of claim 12, wherein said protein aggregate comprises inclusion bodies, soluble and insoluble precipitates, soluble non-native oligomers, gel, fibrils, films, filaments, protofibrils, amyloid deposits, plaques, or dispersed non-native intracellular oligomers. 24. The method of claim 12, wherein steps (ii) and (iii) are performed in the presence of a chaotropic agent, and said method further comprises the step of removing said chaotropic agent. 25. The method of claim 24, wherein said chaotropic agent is guanidine, guanidine sulfate, guanidine hydrochloride, urea, thiocyanate, sarcosyl, sodium dodecyl sulfate, or sodium octyl sulfate. 26. A method of producing biologically active disaggregated protein from protein aggregates comprising: (i) providing a protein aggregate; (ii) mixing said protein aggregate with a reducing agent in an amount sufficient to reduce disulfide bonds therein; (iii) subjecting the mixture of step (ii) to increased pressure of about 0.25 kbar to about 12 kbar, high temperature of about 30° C. to about 125° C., and agitation, whereby said protein aggregate dissolves and/or disaggregates; (iv) removing or neutralizing said reducing agent, whereby disulfide bonds reform; and (v) removing the dissolved and/or disaggregated protein from increased pressure and high temperature, whereby said protein refolds such that biological activity is retained. 27. The method of claim 26, wherein said method does not include use of a chaotropic agent. 28. The method of claim 26, further comprising dialyzing said mixture under pressure, whereby said reducing agent is removed and disulfide bonds reform. 29. The method of claim 26, further comprising removing said reducing agent by diafiltration or ultrafiltration. 30. The method of claim 26, further comprising negating the effect of said reducing agent by addition of an oxidizing agent. 31. The method of claim 26, wherein said reducing agent is dithiothreitol, glutathione, dithioerythritol, or β-mercaptoethanol. 32. The method of claim 26, wherein said increased pressure comprises about 0.5 kbar to about 10 kbar. 33. The method of claim 26, wherein steps (ii)-(iii) are performed in about 1 hour to about 12 hours. 34. The method of claim 33, wherein steps (ii)-(iii) are performed in about 6 hours. 35. The method of claim 26, wherein said protein aggregate comprises inclusion bodies, soluble and insoluble precipitates, soluble non-native oligomers, gel, fibrils, films, filaments, protofibrils, amyloid deposits, plaques, or dispersed non-native intracellular oligomers. 36. The method of claim 26, wherein steps (ii) and (iii) are performed in the presence of a chaotropic agent, and said method further comprises the step of removing said chaotropic agent. 37. The method of claim 36, wherein said chaotropic agent is guanidine, guanidine sulfate, guanidine hydrochloride, urea, thiocyanate, sarcosyl, sodium dodecyl sulfate, or sodium octyl sulfate. 38. A method of refolding denatured protein comprising: (i) providing a denatured protein composition; (ii) mixing said protein composition with a reducing agent in an amount sufficient to reduce disulfide bonds therein; (iii) subjecting the mixture of step (ii) to increased pressure of about 0.25 kbar to about 12 kbar; (iv) dialyzing the mixture under pressure, whereby said reducing agent is removed and disulfide bonds reform; and (v) removing the protein composition from increased pressure, whereby said protein refolds such that biological activity is retained. 39. A method of refolding denatured protein comprising: (i) providing a protein composition; (ii) subjecting the said protein composition to increased pressure of about 0.25 kbar to about 12 kbar, and high temperature of about 30° C. to about 125° C.; and (iii) removing the protein composition from increased pressure and high temperature, whereby said protein refolds such that biological activity is retained. 40. A method of refolding denatured protein comprising: (i) providing a protein composition; (ii) mixing said protein composition with a reducing agent in amount sufficient to reduce disulfide bonds therein; (iii) subjecting the mixture of step (ii) to increased pressure of about 0.25 kbar to about 12 kbar, high temperature of about 30° C. to about 125° C., and agitation; (iv) removing or neutralizing said reducing agent, whereby disulfide bonds reform; and (v) removing the protein composition from increased pressure and high temperature, whereby said protein refolds such that biological activity is retained. 41. A method of rendering a protein aggregate susceptible to dissolution and/or disaggregation by detergents and/or increased temperature comprising subjecting said protein aggregate to high pressure of about 0.25 kbar to about 12 kbar in combination with one or more of detergents and/or increased temperature. 42. A method of producing biologically active disaggregated protein from protein aggregates comprising: (i) providing a protein aggregate; (ii) subjecting the mixture of step (i) to increased pressure of about 0.25 kbar to about 12 kbar, high temperature of about 30° C. to about 125° C. and agitation, whereby said protein aggregate dissolves and/or disaggregates; (iii) altering the pH of the mixture of step (ii) by dialysis; and (iv) removing the dissolved and/or disaggregated protein from increased pressure and high temperature, whereby said protein refolds such that biological activity is retained.