Construction of Recombinant Prokaryotic Expression Plasmids of CYP2 B6 Gene and Expression in Escherichia coli

Objective: To construct the expression plasmids of CYP2 B6 gene,and to express CYP2 B6 protein in Escherichia coli[Rosetta2(DE3)pLysS].Methods: The CYP2 B6 gene was amplified by PCR technique from the template of the plasmid containing CYP2 B6 gene,then was inserted into plasmids of pET-22b(+),pET-28b(+),and pET-32a(+)respectively,and identified with PCR and sequencing.The recombinant expression plasmids containing CYP2 B6 gene were transformed into Rosetta2(DE3)pLysS and the target protein expression was induced by isopropyl-β-D-thiogalactopyranoside(IPTG).Results: The recombinant plasmids of pET-22b(+)-CYP2 B6,pET-28b(+)-CYP2 B6,and pET-32a(+)-CYP2 B6 were obtained and identified by PCR and sequencing.Commassie Blue staining method was used to detect the target protein expression. The pET-22b(+)-CYP2 B6 could not express target protein,the pET-28b(+)-CYP2 B6 could express CYP2 B6 protein accounting for about 5% of total protein;The target protein was greatly expressed by pET-32a(+)-CYP2 B6 and the expression level of CYP2 B6 protein was to be approximately 30% after 3 hours induction in the concentration of 50 μmol/L IPTG.The expressed protein was CYP2 B6 protein by the mass spectrometry results.Conclusion: CYP2 B6 is successfully constructed,which can highly express objective protein in Rosetta2(DE3)pLysS.