关键词: 人CDK4, 原核表达, 包涵体, 融合蛋白, 亲和纯化

Abstract: 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.coliBL21 (DE3), fusion protein with His tag was efficiently expressed in the form of inclusion body af ter IPTG induction and its content was approximately 52.6% of total bacteria pro teins. The inclusion body was washed, dissolved and purified by Ni²⁺ 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%.

Key words: human CDK4, prokaryotic expression, inclusion body, fusion protein, chelate purification