Expression And Pharmacogenetics Of Recombinant Human CYP2C8*1, CYP2C8*2, CYP2C8*3 And CYP2C8*4

The HEK293FT cell lines stably expressing human CYP2C8 were established by lentivector-mediated gene transfer system. Human recombinant protein of CYP2C8 wild type, CYP2C8*2, CYP2C8*3 and CYP2C8*4 variants were expressed using the baculovirus-insect cells system to establish the in vitro models for studying drug metabolism and pharmacogenetics of CYP2C8s.Aims To express CYP2C8 wild type and CYP2C8*2, CYP2C8*3 and CYP2C8*4 variants, compare the metabolism kinetics of those enzymes, and to provide recombinant enzymes for study on phase I drug metabolism and pharmacogenetics in vitro.Methods Human liver total RNA was utilized as template to obtain CYP2C8 cDNA by RT-PCR. CYP2C8 variants were then generated by site-directed mutant PCR. The correct CYP2C8s cDNA verified by sequencing was connected with either pCDH-EF1-MCS-T2A-copGFP vector or pFastBac vector. The recombinant pCDH-EF1-CYP2C8s-T2A-copGFP was cotransfected HEK293FT cells with package vectors to generate recombinant lentivirus, which was then used to transduce HEK293FT cells. The stable transfected cell clones were estabolished by limiting dilution assay. The recombinant pFastBac-CYP2C8s were then transformed into competent E. coli DH 10Bac cells. Recombinant Bacmid-CYP2C8s were generated by transposition. Then Spodoptera frugiperda (Sf9) insect cells were transfected with Bacmid-CYP2C8s to generate recombinant baculoviruses carrying human CYP2C8s cDNA, respectively. CYP2C8 need to coexist with POR and b5 in order to fulfill its metabolic activity. Sf9 insect cells were tri-infected with recombinant baculoviruses carrying human v-CYP2C8, v-POR, v-b5 and heme to obtain active recombinant CYP2C8 enzyme as the basic expression level of POR, b5 and heme in Sf9 insect cells is low. The expression conditions were optimized to obtain the highest activity, including the multiplicity of infection (MOI) of total viruses, MOI ratio among the three viruses and the collection time. The activity of the recombinant enzymes was determined using paclitaxel, repaglinide and ibuprofen as substrates, respectively.6-a hydroxylation activity of paclitaxel was measured by HPLC analysis, while the metabolites of repaglinide and ibuprofen were measured by LC-MS analysis.Results Two HEK293FT-CYP2C8 cell lines with high fluorescence were estabolished and real-time PCR was employed to prove the expression of mRNA. Recombinant viruses containing genes of interest were verified at each construction step. After Sf9 cells were infected with each recombinant v-CYP2C8s, Western blot was employed to verify that the protein of interest were expressed. Active recombinant CYP2C8 wild type and CYP2C8*2, CYP2C8*3 and CYP2C8*4 variants were confirmed by using paclitaxel, repaglinide and ibuprofen as substrates. Their Km values for paclitaxel 6a-hydroxylation were 8.65,9.65,16.25 and 11.81μmol·L-1, Vmax were 69.83,36.95,84.15 and 28.90 pmol·min-1·pmol-1 protein, respectively. The Km values for repaglinide were 12.01,11.34,7.43 and 11.38μmol·L-1, Vmax were 15.69,11.56,12.74 and 11.91*10"6 U·min-1·pmol-1 protein, respectively. The Km values for R-ibuprofen were 341.3,479.2,230.1 and 295.3μmol·L-1, Vmax were 4.921,7.632,2.401 and 1.814*10-3 U·min-1·pmol-1 protein, respectively. The Km values for S-ibuprofen were 388.8,544.9,269.5 and 351.2μmol·L-1, Vmax were 3.017,5.269,1.744 and 1.278*10*3 U·min-1·pmol-1 protein, respectively.Conclusions CYP2C8*4 showed significantly lower activity comparing to the wild type for all the substrates. But CYP2C8*2 and CYP2C8*3 showed different activities toward different substrates comparing to the wild type. Furthermore, no significantly difference for the stereo selective formation of (R)- and (S)-2-OH ibuprofen were found among CYP2C8 allozymes.