葡激酶突变体Y_1-Sak的体外变复性研究

Refolding of Recombinant Staphylokinase Variant Y_1-Sak from Escherichia coli

具有低免疫原性的双功能葡激酶突变体Y1Sak(△15,S16K,K74A,E75A,R77A,K109R,F111D)在大肠杆菌中的表达产物为包涵体,必须进行体外变复性才能得到活性.详细研究了复性方式、pH、温度、复性的初始蛋白浓度和添加L精氨酸对Y1Sak的体外变复性的影响,发现复性温度和添加的L精氨酸对Y1Sak的复性有重要的影响.经过工艺优化后,Y1Sak的纯化活性回收率达到40%左右,蛋白比活性由20 kU/mg提高到53 kU/mg.用动态光散射分析发现,工艺优化后复性的高比活性Y1Sak的水合动力学半径与野生型葡激酶相近,分子基本处于单体状态.

Recombinant staphylokinase variant Y1-Sak （△ 15, S16K, K74A, E75A, R77A, K109R, F111D） forms insoluble inclusion body when overexpressed in Escherichia coll. In order to acquire protein activity,in vitro denaturation and renaturation of Y1-Sak from inclusion bodies is required. The effects of various factors,including refolding methods, pH, temperature, the initial Y1-Sak concentration and L-arginine used as additive,on the refolding of Y1-Sak are studied. The results show that L-arginine plays a critical role in increasing refolding protein activity and the specific activity of Y1-Sak is greatly affected by refolding temperature. After process optimization, the specific activity of renatured Y1-Sak increased from 20 kU/mg to 53 kU/mg. Analysis of the products by dynamic light scattering shows the majority of Y1-Sak molecules with high specific activity exist as monomer and fewer Y1-Sak molecules aggregate to form dimmer and polymer.