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%.