Construction And Activity Comparison Of Human CYP3A4 And Its Four Mutants Tri-expressed With POR And Cyt B5

We employed the bac-to-bac expression system to express CYP3A4 wild typerecombinant protein, and CYP3A4.3, CYP3A4.4, CYP3A4.5 and CYP3A4.18 variantproteins. The metabolic kinetics of those recombinant proteins was compared and theiratypical kinetics was studied.Aims To express CYP3A4 wild type and CYP3A4.3, CYP3A4.4, CYP3A4.5 andCYP3A4.18 variants, compare the metabolism kinetics of those enzymes, and providethe recombinant enzymes for study on phase I drug metabolism in vitro.Methods The human CYP3A4 gene was used as template to obtain CYP3A4alleles by site-directed mutation PCR. The correct CYP3A4 alleles were verified bysequencing, and connected with pFastBac vector to generate recombinantpFastBac-CYP3A4s, which were then transformed into E. coli DH10Bac. RecombinantBacmid-CYP3A4s were generated by transposition. Then Spodoptera frugiperda (Sf9)insect cells were infected with Bacmid-CYP3A4s to generate recombinant baculovirusescarrying human CYP3A4 gene, respectively. CYP3A4 need to coexist with the POR,and b₅ in order to fulfill its metabolic activity. The basic expression level of POR and b₅in Sf9 insect cells is low enough to need artificially addition, as well as heme. Thenfinally, Sf9 insect cells were tri-infected with recombinant baculoviruses carrying human CYP3A4, POR, b₅ and heme to obtain active recombinant CYP3A4 enzyme.The expression conditions were optimized to obtain the highest activity, including themultiplicity of infection (MOI) of total viruses. The activity of the recombinantenzymes was determined using testosterone (Tst) and 7-benzyloxy-4-trifluoromethylcoumarin (7-BFC) as substrates. The metabolite of Tst, 6-β-OH Tstwas measured by HPLC analysis to compare the metabolic kinetic parameters betweenwild type and the variants. Hill equation was employed to study the atypical kineticsamong the recombinant proteins.Results Recombinant viruses containing genes of interest were verified at eachconstruction step. After Sf9 cells were infected with each recombinant virus, RT-PCRwas employed to verify that genes of interest were expressed on the mRNA level, andon the level of translation the protein of interest expression is confirmed by westernblot. Active recombinant CYP3A4 wild type and CYP3A4.3, CYP3A4.4, CYP3A4.5and CYP3A4.18 variants were confirmed by using Tst as substrate. Their K_m against Tstwere 136, 147, 192, 430 and 110 umol·L^-1. V_max were 64, 120, 187, 73 and 212pmol·min^-1·g^-1protein, respectively. Results from the regression using Hill equation,their S₅₀ were 130, 150, 170,180 and 130 umol·L^-1, V_max were 63,122, 180,44 and 228pmol·min^-1·g^-1 protein, n value were 1.03, 0.98, 1.10, 1.38 and 0.93, respectively.Conclusion The results suggested that CYP3A4.5 showed lower activitycomparing to the wild type, but CYP3A4.3, CYP3A4.4 and CYP3A4.18 showed highactivity comparing to the wild type, especially CYP3A4.18. After the atypical kineticstudy, there are clues that CYP3A4.4 and CYP3A4.5 show positive cooperativity, andCYP3A4.18 shows negative cooperativity.