John G H, Hasler J A, He Y A, Halpert J R
Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson 85721.
Arch Biochem Biophys. 1994 Nov 1;314(2):367-75. doi: 10.1006/abbi.1994.1455.
Dog CYP2B11, rat CYP2B1, and rabbit CYP2B5 have been expressed in Escherichia coli from cDNAs modified at the N-terminus (Barnes et al., 1991, Proc. Natl. Acad. Sci. USA 88, 5597-5601). Using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps), solubilized membranes representing > 100 nmol of P450 2B11, > 35 nmol of P450 2B1, and > 7 nmol of P450 2B5 were efficiently extracted (40-70% yield) from a 1-liter culture. Chaps-solubilized preparations produced a reduced CO/reduced difference spectrum devoid of P420 and were used directly in a reconstituted system. The E. coli-expressed 2B enzymes retained the same functional characteristics as the purified hepatic enzymes or enzymes expressed in COS cells in terms of androstenedione metabolite profiles. Hydroxylation rates were determined under a variety of conditions, including two concentrations of NADPH-cytochrome P450 reductase (2 and 16 nmol/nmol P450) and the absence or presence of cytochrome b5 (2 nmol/nmol P450). The androstenedione hydroxylase activities of expressed 2B1 and 2B5 were stimulated by cytochrome b5, whereas P450 2B11 was inhibited slightly by cytochrome b5. Purified expressed 2B11 (specific content, 8 nmol/mg protein) had similar activities as the Chaps-solubilized membrane preparation. The solubilized membranes containing 2B11 were also tested with 2,2',4,4',5,5'-hexachlorobiphenyl (245-HCB). Three major metabolites, 2-hydroxy-4,5,2',4',5'-pentachlorobiphenyl, 3-hydroxy-2,4,5,2',4',5'-hexachlorobiphenyl, and 2-hydroxy-3,4,5,2',4',5'-hexachlorobiphenyl were produced from 245-HCB. These metabolites are identical to those produced by 2B11 purified from liver microsomes. The 245-HCB hydroxylation rates were similar for E. coli-expressed 2B11, dog liver microsomes, and purified liver 2B11. When only the second codon in the 2B1 was changed to GCT, > 25 nmol of P450 was extracted from a 1-liter culture, suggesting that the full Barnes et al. modification scheme may not be necessary for high-level expression. An efficient method of expressing, extracting, and analyzing different P450 2B enzymes has thus been achieved. In addition, rabbit P450 2B5, which has never been purified from liver, as well as different P450 2B mutants can now be expressed at much higher levels than previously reported. The ability to express different 2B wild-type and mutant P450s in E. coli provides an excellent opportunity to study the molecular basis of species differences in substrate metabolism.
犬CYP2B11、大鼠CYP2B1和兔CYP2B5已通过在N端修饰的cDNA在大肠杆菌中表达（Barnes等人，1991年，《美国国家科学院院刊》88，5597 - 5601）。使用3 - [(3 - 胆酰胺丙基)二甲基铵] - 1 - 丙烷磺酸盐（Chaps），从1升培养物中高效提取（产率40 - 70%）了代表超过100 nmol的P450 2B11、超过35 nmol的P450 2B1和超过7 nmol的P450 2B5的溶解膜。Chaps溶解的制剂产生了没有P420的还原型CO/还原型差光谱，并直接用于重组系统。就雄烯二酮代谢物谱而言，大肠杆菌表达的2B酶保留了与纯化的肝酶或在COS细胞中表达的酶相同的功能特性。在多种条件下测定了羟化速率，包括两种浓度的NADPH - 细胞色素P450还原酶（2和16 nmol/nmol P450）以及细胞色素b5的存在与否（2 nmol/nmol P450）。细胞色素b5刺激了表达的2B1和2B5的雄烯二酮羟化酶活性，而细胞色素b5对P450 2B11有轻微抑制作用。纯化的表达2B11（比活性，8 nmol/mg蛋白）与Chaps溶解的膜制剂具有相似的活性。还用2,2',4,4',5,5'-六氯联苯（245 - HCB）测试了含有2B11的溶解膜。从245 - HCB产生了三种主要代谢物，2 - 羟基 - 4,5,2',4',5'-五氯联苯、3 - 羟基 - 2,4,5,2',4',5'-六氯联苯和2 - 羟基 - 3,4,5,2',4',5'-六氯联苯。这些代谢物与从肝微粒体纯化的2B11产生的代谢物相同。大肠杆菌表达的2B11、犬肝微粒体和纯化的肝2B11的245 - HCB羟化速率相似。当仅将2B1中的第二个密码子改为GCT时，从1升培养物中提取了超过25 nmol的P450，这表明完整的Barnes等人的修饰方案对于高水平表达可能不是必需的。因此实现了一种表达、提取和分析不同P450 2B酶的有效方法。此外，从未从肝脏中纯化过的兔P450 2B5以及不同的P450 2B突变体现在可以以比以前报道的更高水平表达。在大肠杆菌中表达不同的2B野生型和突变型P450的能力为研究底物代谢中物种差异的分子基础提供了极好的机会。
