The present disclosure provides an effective method for the refolding of denatured proteins in solution so that properly folded, biologically active protein in solution is recovered in high yield. The refolding takes place at pressures between about 0.25 kbar to about 3.5 kbar, advantageously at about 1.5 kbar to about 3 kbar. Typically a chaotropic agent is present at a concentration which is not effective for denaturing protein at atmospheric pressure, and optionally, oxidation-reduction reagents can be incorporated in the refolding solution so that native intramolecular disulfide bonds can be formed where that is desired. The method is applicable to substantially all proteins, especially after solubilization and/or denaturation of insoluble protein aggregates, inclusion bodies, or abnormal oligomeric (soluble) aggregates.
CROSS REFERENCE TO RELATED APPLICATIONS
This is a divisional of application Ser. No. 09/350,327 filed Jul. 9, 1999, now U.S. Pat. No. 6,489,450, which claims priority to U.S. Provisional Application Ser. No. 60/350,327, filed Jul. 9, 1998.
This application claims benefit of U.S. Provisional Application No. 60/092,208, filed Jul. 9, 1998, which is incorporated by reference herein to the extent that it is consistent with the present disclosure.
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.
