Expression, Purification Of Recombinant Cytochrome P450nor2 And Preparation Of Polyclonal Antibodies

Cytochrome P450 (P450) is a heme enzyme, whose CO complex gives Soret absorption at 450 nm in its optical absorption spectrum. Cytochrome P450nor ( P450nor) is a unique cytochrome P450 ( P450), which plays a key role in fungal denitrification. P450nor is a nitric oxide (NO) reductase (Nor) found in eukaryotic microorganisms, which reduces NO to nitrous oxide (Na O) by directly accepting electrons from NADH or NADPH. Unlike other P450s, P450nor has no monooxygenase activity although it belongs to the P450 super family (CYP55A). Several P450nor genes have been cloned from fungi and yeast, indicating a wide distribution of P450nor.Cytochrome P450nor gene was cloned into the expression vector pET-28 to yield the recombinant expression plasmid pET-P450nor, which could direct the synthesis of a eukaryotic derived protein in Escherichia coli BL21.The vector allows overproduction and single-step purification of Hisg-tagged cytochrome P450nor by the facilitation of metal (Ni24) chelate affinity chromatography. The expression level of cytochrome P450nor was high at 30 癈 after IPTG induction. SDS-PAGE and Western blot analysis showed a specific band (about 43 KD). The overproduced cytochrome P450nor was purified to electrophoretic homogeneity within 2.5 h and about 20.8 mg purified protein was obtained from 2L cell culture. The proliferation of SSMC-7721 cell line could be inhibited by cytochrome P450nor. Rabbit polyclonal antibodies (titer over 64,000) were produced against recombinant cytochrome P450nor and proved to be very useful for immunoblotting study. This expression system and specific antibodies will be beneficial to the understanding of the structure of cytochrome P450nor.The Bac-to-Bac baculovirus expression system is a novel gene expression system that allows the rapid and efficient generation of recombinant baculovirus DNA by site-specific transposition in E.coli, rather than homologous recombinationin insect cells. The recombinant vector, pFast-P450nor, was constructed by inserting the cytochrome P450nor gene from Cylindrocarpon tonkinense into the BamH I/Xba I sites of the transposing vector pFastBacl in the correct orientation with respect to the polyhedrin promoter. After transformation, pFast-P450nor was introduced into the competent cells (E.coli DHlOBac) containing a shuttle vector, Bacmid. Recombinant bacmid pAc-P450nor was constructed by transposing a mini-Tn7 element from the donor plasmid, pFast-P450nor, to the mini-attTn7 attachment site on the bacmid, where the Tn7 transposition functions were provided in trans by a helper plasmid. The recombinant bacmid DNA was isolated and transfected into the insect cells (Spodoptera frugiperda, Sf9) to produce the recombinant virus, rAc-P450nor. Fresh insect Sf9 cells were infected with the recombinant virus containing cytochrome P450nor to express the target protein. Result showed that the recombinant bacmid expression vector, pAc-P450nor, was constructed successfully and the cytochrome P450nor gene was highly expressed in Sf9 insect cells. A specific band (about 43 kD) was detected by SDS-PAGE and confirmed by Western-blot analysis. Availability of this expression system should facilitate characterization of the role of cytochrome P450nor in the metabolism of NO.