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+ 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 43 000 was purified and its purity was up to 98%.