Buters J T, Korzekwa K R, Kunze K L, Omata Y, Hardwick J P, Gonzalez F J
National Institutes of Health, National Cancer Institute, Bethesda, MD 20892.
Drug Metab Dispos. 1994 Sep-Oct;22(5):688-92.
A recombinant baculovirus containing the human CYP3A4 cDNA was constructed and used to express CYP3A4 in SF9 insect cells (0.46 +/- 0.13 nmol/mg protein, 103 +/- 29 nmol/liter, N = 15). The enzyme represented approximately 2-3% of total cellular protein and could be purified by a two-column procedure to a specific content of 12.7 nmol/mg protein. Catalytic activity of the purified enzyme after reconstitution was optimum using molar ratios of CYP3A4 to cytochrome b5 to NADPH-P450 oxidoreductase of 1:3:20, respectively. The enzyme metabolized cortisol, erythromycin, testosterone, and (R)-warfarin. Recombinant baculovirus expresses the highest amounts of all expression systems published to date of catalytically intact CYP3A4. This system is an excellent alternative for the isolation and characterization of P450 forms from human liver.
构建了一种含有人类CYP3A4 cDNA的重组杆状病毒，并用于在SF9昆虫细胞中表达CYP3A4（0.46±0.13 nmol/mg蛋白质，103±29 nmol/升，N = 15）。该酶约占细胞总蛋白的2 - 3%，可通过双柱法纯化至12.7 nmol/mg蛋白质的比活性。重组后纯化酶的催化活性在CYP3A4与细胞色素b5与NADPH - P450氧化还原酶的摩尔比分别为1:3:20时最佳。该酶可代谢皮质醇、红霉素、睾酮和（R）-华法林。重组杆状病毒表达的催化完整的CYP3A4是迄今为止所有已发表表达系统中量最高的。该系统是从人肝脏中分离和鉴定P450形式的极佳替代方法。
