人细胞周期蛋白D1在大肠杆菌BL21中的表达及纯化

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Ni²⁺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， Western-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.

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

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(Ni~(2+)) 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%.