人S100A2-GST融合蛋白原核表达和纯化  

Expression and purification of human S100A2-GST fusion protein in prokaryotic cells

作　　者：卫佳[1] 王胜[1] 苗静琨[1] 陈英华[1] 李星星[1] 吴丽美[1] 左国伟[1] 何通川[2] 周兰[1] 

机构地区：[1]重庆医科大学生物医学工程系,重庆400016 [2]美国芝加哥大学医学中心分子肿瘤研究室,美国Chicago IL 60637

出　　处：《重庆医科大学学报》2008年第12期1427-1430,共4页Journal of Chongqing Medical University

基　　金：国家自然科学基金(30340039);教育部“春晖计划”科研启动经费资助项目(2003年);2004年重庆市留学回国人员启动基金

摘　　要：目的:制备人S100A2-GST融合蛋白(Human S100A2-GST fusion protein,hS100A2-GST),为进一步研究人hS100A2蛋白的功能及其与其它因子或系统的相互作用奠定基础。方法:从pHAHA-hS100A2中双酶切获取hS100A2基因,亚克隆至原核表达载体pGST-moluc,构建pGST-moluc-hS100A2,转化BL21后酶切鉴定,IPTG诱导重组菌表达融合蛋白hS100A2-GST,再经过Glutathion-Sepharose4B球珠分离纯化、SDS-PAGE及Western Blot鉴定,Bradford法蛋白定量。结果:XhoⅠ和EcoRⅠ双酶切该重组质粒后获得2个片段,分别约300bp和5kb,提示pGST-moluc-hS100A2构建正确;重组菌株经过IPTG诱导后,表达分子量约为36kD的蛋白,产率达5mg/L菌液;Glutathion-Sepharose4B球珠纯化后,该融和蛋白纯度达92%;Western Blot显示该蛋白可以被S100A2的抗体特异识别,证实其为hS100A2-GST融合蛋白。结论:成功构建了hS100A2-GST原核表达质粒pGST-moluc-hS100A2;该质粒可在大肠杆菌中诱导表达hS100A2-GST融合蛋白,产率高;应用Glutathion-Sepharose4B球珠可获得纯度达92%的该蛋白。为后续有关hS100A2的研究打下了基础。Objective： To obtain human S100A2-GST fusion protein for further research on human S100A2（hS100A2）protein function and its interactions with other proteins and systems. Methods： hS100A2 gene from pHAHA-hS100A2 was subcloned into an expression vector pGST-moluc to construct pGST-moluc-hS10OA2.The new plasmid was identified by digestion with Xho Ⅰ and EcoR Ⅰ .The recombinant BL21 was induced with IPTG to express recombinant fusion protein hS100A2-GST,which was purified by Glutathion-Sepharose 4B ball beads,identified by SDS-PAGE and Western Blot,and quantified by Bradford method. Results： After the digestion,the recombinant plasmid was cutted into two fragments,300 hp and 5kb.After treated with IPTG,the recombinant BL21 strain expressed a protein,which was about 36 kD and was recognized specifically by anti-hS100A2. Its yield was 5 mg/L bacterial culture. After isolated by Glutathion-Sepharose 4B ball beads,its purity was 92%. Conclusion： hS100A2-GST expression plasmid pGST-moluc-hSlOOA2 was constructed successfully, hS100A2-GST fusion protein could be expressed in Escherichia coli with higher yield and isolated with higher purity,which lays the foundation for the follow-up of the hSIOOA2 research.

关 键 词：人S100A2-GST融合蛋白 诱导表达 蛋白纯化 蛋白鉴定 

分 类 号：Q786[生物学—分子生物学]