Expression Human Cytochrome P450 2C9 In Baculovirus-Insect Cell Expression System

Cytochrome 450 proteins (CYP, P450) are major enzymes taking part in drug metabolism in humans. The monooxygenase systems are responsible for the hydroxylation of a great variety of endogenous and xenobiotic substrates. Cytochrome P450 2C9 (CYP2C9), a member of the CYP2C enzyme subfamily, ranks amongst the most important drug metabolizing enzymes in human. It makes up about 20% of the total Cytochrome P450 protein in liver microsomes, and hydroxylates about 16% of drugs in current clinical use. Human CYP2C9 has been shown to exhibit geneticly polymorphic. In addition to the wild-type protein CYP2C9*1, at least thirty single nucleotide polymorphisms (SNPs) have been reported within the coding region of the CYP2C9 gene producing the variant allozymes, CYP2C9*2-CYP2C9*30. Most of the variant alleles are associated with altered enzyme activity. CYP2C9*13 was discovery and reported by our laborary for the first time. Primary investigation have demonstrated significantly lower intrinsic clearance of all investigated CYP2C9 substrates in cells expressing the allele CYP2C9*13 compared with wild-type. The preliminary study with enzymes expressed in COS cells suggested that much higher levels of expression would be needed to conduct kinetic studies, to further characterize the catalytic activity of CYP2C9*13, explain the mechanism CYP2C9*13-mediated reduction of drug metabolism and the relationship between structure and function. Therefore, we adapted the baculovirus-insect cell system for the high-level expression of CYP2C9, in order to isolate sufficient enzyme to permit analysis of drugs metabolism at physiolog relevant substrate concentrations. P450s are not self-sufficent enzyme, they all need NADPH-cytochrome P450 oxidoreductase (CPR) as an electron donor. P450 reductase is a unique universal donor of electrons to practically all known microsomal cytochrome P450s. Due to high-level expression of gene of interest in the baculovirus-insect system, the CPR in the cell is impossible to support the P450 enzyme reaction. So we need to add CPR or construct a co-expression system.In this paper, with reverse transcription polymerase chain reaction (RT-PCR) and DNA recombinant technique, a full-length cDNA encoding the CPR from Hep2 cell was cloned into pGEMT Easy plasmid as a primary vector. The plasmid was analyzed by restriction enzyme to confirm the presence and correct orientation of the insert. Then the target cDNA segment was analyzed by DNA sequencing. There happened to be a mutation site where two extra bases A and G were inserted about 200bp downstream the initiating ATG. So we introduced a site-directed mutation in order to obtain wide type sequence. The correctness of the site-directed deletion and the absence of undesired modifications were confirmed by automatic DNA sequencing. Then the CPR cDNA was subcloned into a mammalian expression vector pFastBac Dual, located downstream of the PH promoter. The pFASTBAC DUAL vector has two promoters and cloning sites, allowing expression of two genes, one from the polyhedron promoter and one from the p10 promoter. The cDNA of CYP2C9*1/*3/*13 that were already constructed on pcDNA3.1+ vector were subcloned into pFastBac Dual vector respectively, located downstream of another promotor p10. The co-expression plasmid pFastBacDual–CPR-CYP2C9*1/*3/*13 were constructed. The co-expressing CYP 2C9*1/*3/*13 and CPR couldn't quantify by CO-difference spectroscopy. The pFastBacDual-CPR-CYP2C9*1/*3/*13 plasmids were reconstructed by cutting the PH promoter and CPR cDNA to form pFastBac-CYP2C9*1/*3/*13 plasmids. The recombinant pFASTBAC? plasmid was transformed into DH10BAC? competent cells which contain the bacmid with a mini-attTn7 target site and the helper plasmid. The mini-Tn7 element on the pFASTBAC plasmid can transpose to the mini-attTn7 target site on the bacmid in the presence of transposition proteins provided by the helper plasmid. Colonies containing recombinant bacmids were identified by antibiotic selection and blue/white screening, since the transposition results in disruption of the lacZαgene. High molecular weight mini-prep DNA was prepared from selected E. coli clones containing the recombinant bacmid, this DNA were then used to transfect insect cells with polyethylenimine (25 kDa, branched). Harvest P1 virus after 3 days transfection. The infectious potency of a stock of baculovirus is determined by examining and counting plaque formations in an immobilized monolayer culture. The P1 virus stock had a confirmed titer of 1×106 to 1×107 pfu/mL. Enlarge virus at MOI 0.1. Infect cells at a high virus: cell ratio (multiplicity of infection 10-20). Freshly prepared hematin solution was added 1 day before cell harvested. Cells were harvested at 3 days postinfection, and Sonics fragmentation, Ultra-filtrate. P450 was quantified by CO-difference spectroscopy.In this lab Guo had the COS cells expression levels of CYP2C9*1 protein was 7.51 pmol/mg,CYP2C9*3 and CYP2C9*13 proteins were CYP2C9*1's 69.9% and 35.5%, which were quantified by immunoblotting. In this work, the Sf9 cells expression levels were 41pmol/mg to 120pmol/mg, which were quantified by CO-difference spectroscopy. The expression level had ten times up.In this work, we used PEI as the transfection reagent, and demonstrated PEI can transfect Sf9 insect cells. The cost of PEI reagent of transfected cells is very close to zero. The CPR and P450s were treated with CHAPS for reconstitution and optimization of catalytic activity. The result of diclfenac catalytic activity showed CYP2C9*13 had lower intrinsic clearance than CYP2C*3 due to more increase in Km and more decrease in Vmax. The CYP2C9 came from the COS cells expression system had the similar result. This demonstrated the P450-CPR system was correct.In this work, we established the baculovirus-insect cell system for the high-level expression of CYP2C9 and CPR successfully, which can conduct kinetic studies, to further characterize the catalytic activity of CYP2C9*13, explain the mechanism CYP2C9*13-mediated reduction of drug metabolism and the relationship between structure and function.