In Vitro Expression And Catalytic Activity Identification Of Human Hepatic CYP2B6

Cytochromes P450 is a superfamily of hemethiolate-containing proteins involved in the oxidative metabolism of a large variety of xenobiotics and endobiotics. Many P450 enzymes participate in the conversion of carcinogens, environmental pollutants, and drugs to more polar metabolites, thereby facilitating their excretion and preventing the accumulation of these potentially harmful compounds. Predominantly expressed in liver, members of the CYP1-3 families exhibit broad substrate specificity and metabolize the majority of administered drugs.Human CYP2B6 is one of the less well characterized forms, probably because it was initially thought to be expressed at low levels and in only a fraction of human liver samples both at the mRNA and protein level, also because of the lack of suitable probes. And the levels of CYP2B6 have been underestimated because of poor selectivity and sensitivity of previously used antibodies to detect CYP2B6 protein. Moreover, recent studies using more selective and specific immunochemical detection methods demonstrate that the average relative abundance of CYP2B6 in human liver ranges from 2 to 10% of the total P450 content compared with an earlier report of 0.2%. Further characterization of CYP2B6 revealed higher mRNA and protein levels than previously observed. Recently, CYP2B6 has gained more attention since it was shown to be significantly involved in the metabolic activation and inactivation of a number of clinically important drugs. Furthermore, the antidepressant and antismoking agent bupropion is one of the most specific substrates of CYP2B6 and, therefore, is a preferred in vitro probe drug for the enzyme. The in vitroexpression of CYP2B6 is of great importance to investigate the role of CYP2B6 in drug metabolism. In this study, CYP2B6 recombinant enzyme was obtained using baculovirus expression system and its catalytic activity was measured using bupropion as probe. 1. Cloning of human CYP2B6 cDNATotal RNA was isolated from a single human liver sample and a full length cDNA encoding human CYP2B6 was transcripted from mRNA in a Chinese human liver by reverse transcriptase polymerase chain reaction (RT-PCR). The specific primers were designed based on the published human CYP2B6 cDNA sequence (GenBank accession NO. NM₀00767). The PCR products were isolated and ligated with pGEM-T vector by T4 DNA ligase. The recombinant pGEM-CYP2B6 was sequenced and the sequence of the clone was differed from the published human CYP2B6 cDNA sequence.The variations were changed by gene mutation using TaKaRa BKL Kit. The harvested recombinants were cleaved with endonucleases BamHIIXhoI, and the segments CYP2B6 were subcloned into the vector pFastBac. The recombinants pFastBac-CYP2B6 were then transformed into E.coli DHlOBac in which recombinant Bacmid would generate by transposition. After Bacmid-CYP2B6 transfected the Sf9 cells, the recombinant virus would generate.2. Co-infection of Sf9 cells with recombinant viruses of human CYP2B6 and CYPORSf9 cells were co-infected with the homologous recombinant baculoviruses containing the human CYP2B6 cDNAs and CYPOR cDNAs, respectively. The ratio of CYP2B6 to CYPOR was adjusted during the course of infection. Hemin stock solution was added to a final concentration of 3 μg/ml. The infected Sf9 cells were pelleted approximately 3 days postinfection, resuspended and washed three times by PBS, repelleted and socinated to obtain the cell homogenate containing the recombinant CYP2B6, stored at -80 "C until further use.3. Validation of the recombinant CYP2B6 expressionProteins were separated on a 10% SDS-polyacrylamide gel and transferred to 0.45^m pore size PVDF membrane. Membranes were incubated for 16 h in blocking buffer. Mouse anti-His antibody was added at a dilution 1:750 with blocking buffer, incubated for 1 h, and followed by additional washes with PBS containing 0.05% Tween 20. The membrane was then soaked in blocking buffer with a 1:2500 peroxidase-conjugated goat anti-mouse IgG, incubated for 1 h. Peroxidase activities were detected by incubating the membrane in510 ml PBS buffer at pH 7.5 containing 0.15 mg/mL DAB deaminobenzidine and 60 μL of 10% H2O2. The His-tagged recombinant CYP2B6 was detected by Western Blot analysis. The P450 content was assayed by reduced CO difference spectrum.Dithionite was added, the samples were gently bubbled with CO for 30s, and the reduced carbonyl spectrum was recorded between 400 and 500 nm on a Unicam UV/VIS spectrophotometer. There was a 420-nm peak observed in the reduced CO difference spectrum, perhaps it was a denatured formofCYP2B6. 4. Enzyme activities of cell lysate with dual expression of C YP2B6 and CYPORThe enzyme activity was identified using bupropion as a probe substrate which will be measured by HPLC. The reaction was carried out in an incubation mixture of 200μL total volume containing buffer, NADPH-generating system, the different concentrations of bupropion, and the desired amounts of cell lysates. Kinetic parameters for bupropion hydroxylation were calculated from a Lineweaver-Burk plot. The apparent Km and Vmax for bupropion hydroxylation were 138 ± 42umol/L and 0.676 ± 0.21nmol/min/mg pro., respectively.In a word, high-level expression of human P450 2B6 (CYP2B6) was achieved using a baculovirus expression system. Active recombinant human CYP2B6 obtained will facilitate further examination of the role of this enzyme in drug metabolism.