GST-Dot4融合蛋白的表达·纯化及鉴定  

Expression,Purification and Identification of GST-Dot4 Fusion Protein

作　　者：张艳梅[1] 丛文娟[1] 郭建军[1] 刘中来[1] 

机构地区：[1]华中师范大学生命科学学院,湖北武汉430079

出　　处：《安徽农业科学》2009年第26期12414-12417,共4页Journal of Anhui Agricultural Sciences

摘　　要：[目的]为了提高Dot4蛋白在大肠杆菌中的可溶性表达并获得较高纯度的蛋白。[方法]以pGEX-KG-Dot4重组质粒转化E.coliBL21后,用IPTG诱导重组菌表达GST-Dot4融合蛋白,采用SDS-PAGE及W estern B lot法鉴定表达产物,用G lutathione Sepharose4B亲和层析柱分离纯化。[结果]pGEX-KG-Dot4重组表达载体在大肠杆菌中获得高效表达;经G lutathione Sepharose4B亲和层析柱纯化获得了高纯度的Dot4融合蛋白;W estern B lot表明,该蛋白可与GST标签抗体反应,表明获得了目的蛋白。[结论]在大肠杆菌中表达纯化后得到较高纯度的GST-Dot4融合蛋白,为Dot4蛋白的结构、功能及作用机制研究奠定了基础。[ Objective] The paper aimed to improve the soluble expression of Dot4p in E. coli and to obtain higher purity protein. [ Method ] The pGEX-KG-Dot4 recombinant plasmid was transformed to E. coli BI21 and the expression of GST-Dot4 fusion protein was induced with IPTG. The expression product was identified by SDS- PAGE and Western Blot, and separated and purified by Glutathione Sepharose4B affinity chromatography column. [ Result ] The pGEX-KG-Dot4 recombinant expression vector was successfully expressed in E. coli; and obtained high purified Dot4 fusion protein through purifying Glutathione Scpharose4B affinity chromatography column; Western blot analysis showed that the fusion protein interacted with GST-tagged antibody. [ Conclusion ] GST-Dot4 fusion protein with high purity could be obtained after it puri- fied in E. coil ,which laid a foundation of its structure,function and mechanism of Dot4p.

关 键 词：融合蛋白 可溶性表达 蛋白纯化 

分 类 号：S188[农业科学—农业基础科学]