人CDK4基因的原核表达及重组蛋白的纯化和复性  

Prokaryotic Expression,Purification and Renaturation of Recombinant Human CDK4

作　　者：曹玉华[1] 许晶晶[1] 陈勇[1] 王琪[1] 冯晶[1] 郝东云[1] 李桂英[1] 

机构地区：[1]吉林大学分子酶学工程教育部重点实验室,长春130021

出　　处：《吉林大学学报（理学版）》2008年第5期992-996,共5页Journal of Jilin University:Science Edition

基　　金：国家自然科学基金(批准号:30671934);吉林省杰出青年基金(批准号:200600107)

摘　　要：将人CDK4基因克隆入原核表达载体pET28a(+)中,经酶切和测序鉴定正确的重组质粒pET28a-CDK4,转化E.coliBL21(DE3)后获得表达菌株.该表达菌株经IPTG诱导后,高效表达出带有组氨酸标签的以包涵体形式存在的融合蛋白,表达量占菌体总蛋白的52.6%,包涵体经过洗涤、尿素变性溶解、His Trap HP Kit柱纯化、稀释复性,获得纯度达98%以上的蛋白.SDS-PAGE及Western blot分析表明,在分子量34 000处有一特异性蛋白条带.结果表明,已成功的表达和纯化纯度达98%的重组人CDK4蛋白.The expression vector pET28a-CDK4 was constructed by inserting human CDK4 cDNA into pET28a（＋） and was identified by digestion with restriction enzymes and sequence analysis. Then an expression strain was selected after transformation of the recombined plasmid into E. coli BL21（DE3）, fusion protein with His-tag was efficiently expressed in the form of inclusion body after IPTG induction and its content was approximately 52.6% of total bacteria proteins. The inclusion body was washed, dissolved and purified by Ni^2＋ chelate chromatography under denatured condition. The purified inclusion body protein was renatured by the gradual removal of urea via dialysis in solubilization buffer. SDS-PAGE analysis and Western blotting with an anti-CDK4 antibody showed that the fusion protein with a molecular weight of about 43000 was purified and its purity was up to 98%.

关 键 词：人CDK4 原核表达 包涵体 融合蛋白 亲和纯化 

分 类 号：Q786[生物学—分子生物学]