Blake J A, Pritchard M, Ding S, Smith G C, Burchell B, Wolf C R, Friedberg T
Biomedical Research Centre, Ninewells Hospital and Medical School, Dundee, UK.
FEBS Lett. 1996 Nov 18;397(2-3):210-4. doi: 10.1016/s0014-5793(96)01196-9.
The catalytic activities of recombinant cytochrome P450s expressed in E. coli have been impeded by the absence of endogenous P450 reductase. To solve this problem, we coexpressed P450 reductase with CYP3A4. Membranes from this strain contained 215 pmol P450/mg protein and a reductase activity of 1315 nmol cytochrome c reduced/min per mg. We detected 6beta-hydroxylation of testosterone and oxidation of nifedipine in vivo with turnover numbers of 15.2 and 17.3 min(-1), respectively. These values compare favourably with those obtained using an optimally reconstituted system. Our data demonstrate that a catalytically efficient human P450 system can be generated in E. coli.
在大肠杆菌中表达的重组细胞色素P450的催化活性因缺乏内源性P450还原酶而受到阻碍。为了解决这个问题，我们将P450还原酶与CYP3A4共表达。该菌株的膜含有215 pmol P450/mg蛋白质，还原酶活性为1315 nmol细胞色素c还原/分钟·mg。我们在体内检测到睾酮的6β-羟基化和硝苯地平的氧化，周转数分别为15.2和17.3 min⁻¹。这些值与使用最佳重组系统获得的值相比具有优势。我们的数据表明，可以在大肠杆菌中产生催化效率高的人P450系统。
