Expression And Characterization Of Recombinant Human CYP2E1*1, CYP2E1*2, CYP2E1*3 And CYP2E1*4

This project utilized the baculovirus-insect cells system to express CYP2E1 wild type recombinant protein,as well as CYP2E1*2,CYP2E1*3 and CYP2E1*4 variants to study the metabolism kinetics of those CYP2E1s and,to help the research of phase I drug metabolism.Aims To express CYP2E1 wild type and CYP2E1*2,CYP2E1*3 and CYP2E1*4 variants,then compare the metabolism kinetics of those enzymes,and to acquire recombinant enzymes to help the study of phase I drug metabolism.Methods Using human liver RNA as templates to obtain CYP2E1 cDNA by RT-PCR.And 3 CYP2E1 variants were generated using site mutant PCR.Then the correct CYP2E1s cDNA verified by sequencing was connected with pFastBac vector to generate recombinant pFastBac-CYP2E1s,which were then transformed into E.coli DH 10Bac cells.Recombinant Bacmid-CYP2E1s were generated by transposition.Then Spodoptera frugiperda(Sf9) insect cells were infected with Bacmid-CYP2E1s to generate recombinant baculoviruses carrying human CYP2E1s cDNA,respectively.Finally,Sf9 insect cells were tri-infected with recombinant baculoviruses carrying human CYP2E1s,CYPOR and CYPb5 to obtain active recombinant CYP2E1 enzyme mix.The expression conditions were optimized to obtain the highest activity,including the multiplicity of infection(MOI) of total viruses,MOI ratio among the 3 viruses and the infection time.The activity of the recombinant enzymes was determined using chlorzoxazone(czx) and 7-methoxy-4-trifluoromethylcoumarin(7-MFC) as substrates,respectively.6-OH hydroxylation activity of czx was measured by HPLC analysis. 7-hydroxy-4-trifluoromethyl coumarin(7-HFC),the metabolite of 7-MFC,was measured by fluorescent analysis.Results Successful recombinant viruses containing genes of interest were verified by each construction step.After Sf9 cells were infected with each recombinant CYP2E1s,the RT-PCR products suggested that the genes of interest were expressed on the mRNA level,and the western blot results suggested that the protein of interest were expressed on the level of translation.Active recombinant CYP2E1 wild type and CYP2E1*2,CYP2E1*3 and CYP2E1*4 variants were confirmed by using chlorzoxazone and 7-methoxy-4-trifluoromethylcoumarin as substrates.Their K_m for chlorzoxazone were 72.4,40.83,37.16 and 32.66μmol·L^-1, respectively,and V_max were 0.402,0.374,0.356 and 0.354μmol·min^-1·g^-1 protein, respectively;their K_m for 7-methoxy-4-trifluoromethylcoumarin were 5.3,10.6,19.2 and 14.22μmol·L^-1,respectively,and V_max were 0.023,0.034,0.036 and 0.033μmol·min^-1·g^-1 protein,respectively.Conclusions As the results showed,there were no significant differences among recombinant CYP2E1 wild type and CYP2E1*2,CYP2E1*3 and CYP2E1*4 variants,however,the K_m of the 3 variants were lower than the wild type.The recombinant proteins could be used as enzyme sources for phase I drug metabolism.