采用TEV酶法进行整合膜蛋白拓扑学分析

为了研究膜蛋白的跨膜结构，进行拓扑学分析是十分重要的.有许多分析膜蛋白拓扑结构的方法，本文采用烟草蚀斑病毒（TEV）酶特异性切割测试蛋白中跨膜片段的前段或后端所插入的tev识别序列EXXYXQ(S/G)，如果TEV酶能够切割，表明该序列位于目标蛋白的细胞 质外.将Tev识别序列ENLYFQG 分别插入到拟南芥整合膜蛋白的的跨膜区域，然后转化进入酿酒酵母中. 消解酶(zymolyase)酶破除酵母的细胞壁后，TEV酶消化球状体，最后通过Western免疫印迹法来分析结果.有关该方法的注意事项在结果中进行了讨论.

Topology Analysis of Integral Membrane Proteins by Using TEV Protease

Determination of membrane topology is important for understanding the structure of a transmembrane protein of interest. There are several methods to determine membrane topology. In this method, the tobacco etch virus (TEV) protease are utilized to specifically cleave the consensus sequence EXXYXQ(S/G) (tev). When TEV protease eventually cleaves the test protein at the tev sequence, it can be suggested that the tev-inserted region of the protein is located at the outside of the plasma membrane. The tev sequence ENLYFQG were inserted individually before and after a putative transmembrane segment of an integral plasma membrane protein of Arabidopsis thaliana. Each of the tev-inserted proteins was expressed in cells of the yeast Saccharomyces cerevisiae, and intact spheroplasts prepared from the cells by digesting the cell wall with zymolyase were subjected to TEV protease, followed by Western blot analysis. Technical care points of this method were discussed based on the results.