人CDK4基因的原核表达及重组蛋白的纯化和复性

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.

Prokaryotic Expression,Purification and Renaturation of Recombinant Human CDK4

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 Ni2...