Construction of Recombinant Prokaryotic Expression Plasmid of CYP3A4 Gene and High Expression Induced by IPTG

The CYP3A4 gene was amplified by method of PCR technique from the template of pCWNF14 plasmid,then was cloned into pET-22b(+),pET-28b(+),and pET-32a(+)vector and identified with PCR and sequencing.The recombinant expression plasmid containing CYP3A4 gene was transformed into Rosetta(DE3)2pLysS and target protein expression was induced by IPTG.Only recombinant plasmid of pET-32a(+)-CYP3A4 could be induced by IPTG and express CYP3A4 protein.After 6 h induction in the concentration of 50 μmol/L,the recombinant CYP3A4 protein accounted for 40%of total Rosetta(DE3)2pLysS proteins detected by coomassie brilliant blue staining.The expressed target protein could be dissolved in 10 mmol/L 3-(3-Cholamidopropyl)dimethylammonio propanesulfonic acid(CHAPS)and 0.3%lauroyl sarcosine(SKL),but not in 8 mol/L urea.The recombinant CYP3A4 plasmid was successfully cloned and effectively expressed in Rosetta(DE3)2pLysS.