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

目的　重组人尿激酶原在大肠杆菌中过量表达时形成不溶物包涵体 ,需经体外变复性后才能获得生物活性。本文旨在提高包涵体中变性尿激酶原的复性效率。方法　通过对pH、温度、变性剂种类及浓度、蛋白浓度、以及巯基氧化还原对比率等的定性定量分析 ,研究重组人尿激酶原体外变复性的基本条件 ,并比较了添加一些非特异有效成分、脉冲稀释、梯度透析等方法对提高重组人尿激酶原体外变复性效率的作用。结果　确定了重组人尿激酶原体外变复性的基本方法 ,其复性效率可达 30 %左右。结论　不同的包涵体蛋白的体外变复性效率因蛋白的分子大小、二巯键数目、疏水程度等而异 ,对特定蛋白复性条件的优化可提高其复性效率

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.