In vitro renaturation of recombinant human pro-urokinase expressed in Escherichia coli

Objective Recombinant human pro-urokinase forms insoluble inclusion body when overexpress ed in Escherichia coli.It must be denatured and renatured in vitro so that it can acquire activity.This study aimed at increasing the renaturation yield of denaturant pro-urokinase.Methods We evaluated the basic renaturation conditions of pro-urokinase through qualita tive and quantitative analysis of pH, temperature, denatured concentration, prot ein concentration, and the ratio of reduced and oxidized thiol reagents.We als o compared the effects of nonspecific additives, step-wise dilution and urea gr adient dialysis.Results We defined the optimal conditions of pro-urokinase renaturation with a yield of about 20%-30%. Conclusion Different recombinant denatured proteins have different renaturation conditions due to their different molecular sizes, molecular constructions, disulfide bond numbers, and hydrophobicity.The renaturation yield can be increased by optimiz ing the renaturation conditions of a specific protein.