H Zhu1, W Liu, W Shi, Y Xue, L Kuai, Z Ma. 1. State Key Laboratory of Pharmaceutical Biotechnology, Department of Biochemistry, Nanjing University, Nanjing 210093, China.
Abstract
OBJECTIVE: Recombinant human pro-urokinase forms insoluble inclusion body when 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 through qualitative and quantitative analysis of pH, temperature, denatured concentration, protein concentration, and the ratio of reduced and oxidized thiol 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 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 optimizing the renaturation conditions of a specific protein.
OBJECTIVE: Recombinant human pro-urokinase forms insoluble inclusion body when 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 through qualitative and quantitative analysis of pH, temperature, denatured concentration, protein concentration, and the ratio of reduced and oxidized thiol 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 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 optimizing the renaturation conditions of a specific protein.
Authors: Yaroslav Gursky; Robert Bibilashvili; Mikchail Minashkin; Alex Krasnov; Alex Deikin; Tatyana Ermolkevich; Andrey Popov; Lilia Verbovaya; Nicolai Rutkevich; Alexsander Shevelev; Sofia Georgieva; Sergey V Razin; Igor Goldman; Elena Sadchikova Journal: Transgenic Res Date: 2009-04-25 Impact factor: 2.788