人细胞周期蛋白D1在大肠杆菌BL21中的表达及纯化  

Expression and Purification of Recombinant Human CyclinD1 in E.coli BL21

作　　者：李桂英[1] 邹德生[1] 周立宏[1] 曹玉华[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）》2006年第5期839-843,共5页Journal of Jilin University:Science Edition

基　　金：国家自然科学基金青年基金(批准号:30200256);吉林省科技发展计划项目基金(批准号:20050410-3)

摘　　要：将人细胞周期蛋白D1全长cDNA克隆入原核表达载体pET-28c(+)中,经酶切和测序鉴定正确的重组质粒转化E.coli BL21(DE3)后获得表达菌株.该菌株经IPTG诱导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23%.包涵体经洗涤和溶解,在变性条件下利用N i2+螯合柱纯化、尿素梯度复性后,得到纯度达98%以上的纯化蛋白.SDS-PAGE显示纯化蛋白的分子量约为43 000,W estern-blot分析表明,在相应分子量处有一特异性条带,说明成功表达和纯化重组人细胞周期蛋白D1.The expression vector pET-28c-cycD was constructed by inserting human cyclinD1 cDNA into pET- 28c（ ＋ ） and was identified by digestion with restriction enzymes and sequence analysis. Then an expression strain was selected after the 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 23% of total bactrerial proteins. The inclusion body was washed, dissolved and purified by Ni2^＋ chelate chromatography under denatured condition. The inclusion body protein was renatured by gradual removal of urea through dialysis to obtain the purified fusion protein. SDS-PAGE analysis and Western blotting with an anti-cyclinD1 antibody showed that fusion protein with a molecular weight of about 43 000 was purified and its purity was up to 98%.

关 键 词：人细胞周期蛋白D1 表达 包涵体 融合蛋白 蛋白质纯化 大肠杆菌 

分 类 号：Q786[生物学—分子生物学]