RGD-葡激酶的凝胶过滤层析法复性及其纯化  

The Renaturation and Purification of RGD-Staphylokinase by Gel Filtration

作　　者：程鏖[1,2] 宋刚[1,2] 苏华波 于敏[1,2] 李育阳 宋后燕 

机构地区：[1]复旦大学教育部分子医学重点实验室,上海200032 [2]复旦大学生命科学院,上海200433

出　　处：《生物工程学报》2002年第6期693-697,共5页Chinese Journal of Biotechnology

基　　金：国家 8 6 3生物高技术研究发展计划项目 (No .86 3 2 0 0 1AA2 15 2 91)资助~~

摘　　要：构建的溶栓和抗栓双重功能的RGD 葡激酶突变体 (RGD Sak)在大肠杆菌中高表达 ,目的蛋白质以包涵体形式存在。为获得有活性的蛋白质 ,需要对包涵体进行变复性。利用凝胶层析方法对包涵体中RGD Sak进行复性 ,并与稀释复性法进行比较 ,发现凝胶柱复性方法具有操作周期短、简便、成本低而高效等优点。复性后蛋白质用Q SepharoseFF离子交换进一步纯化 ,纯度达 95 % ,酪蛋白凝胶板活性测定表明两种复性法得到的蛋白质比活性相当。圆二色谱测定显示两种复性法得到的蛋白质的二级结构成份和谱形一致 ,说明在两种复性过程中完成了RGDA recombinant RGD-Staphylokinase(RGD-Sak) with thrombolytic and anti-thrombolytic bifunction was expressed in E.coli. The expression product accumulates as inclusion bodies. In order to obtain active molecule, the RGD-Sak in the inclusion body should be denatured and then renatured. The renaturation of RGD-Sak was performed by gel filtration. Comparing with the traditional way of dilution renaturation, gel filtration way is better than the traditional one, since there are some advantages, such as simple processing, high recovery, low cost and higher purity after renaturation. After renaturation, RGD-Sak was purified by Q-Sepharose FF, and the purity was more than 95%. Analysis of CD spectra showed that the final product from the two renaturation ways have similar CD spectra. It was demostrated that RGD-Sak molecules proceeded correct refolding through gel filtration or dilution renaturation process.

关 键 词：RGD-葡激酶 凝胶过滤层析法 复性 纯化 抗栓 溶栓 

分 类 号：Q55[生物学—生物化学]