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

Objective Recombinant human pro-urokinase forms insoluble inclusion body wh en overexpressed 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 thr ough qualitative and quantitative analysis of pH, temperature, denatured co ncentration, protein concentration, and the ratio of reduced and oxidized t hiol reagents. We also compared the effects of nonspecific additives, step- wise dilution and urea gradient dialysis. Results We defined the optimal conditions of pro-urokinase renaturation wit h a yield of about 20% - 30%. Conclusion Different recombinant denatured proteins have different renatura tion conditions due to their different molecular sizes, molecular construct ions, disulfide bond numbers, and hydrophobicity. The renaturation yield ca n be increased by optimizing the renaturation conditions of a specific prot ein.