Urban P, Cullin C, Pompon D
Centre de Génétique Moléculaire du CNRS, Laboratoire Propre associé à l'Université Pierre-et-Marie-Curie, Gif-sur-Yvette, France.
Biochimie. 1990 Jun-Jul;72(6-7):463-72. doi: 10.1016/0300-9084(90)90070-w.
Cytochrome P-450s constitute a superfamily of mono-oxygenases which require the association with specific redox enzymes bound to the endoplasmic reticulum membrane for their activity. Conditions for the functional expression of these mammalian enzymes in yeast cells and the respective merits and limitations of currently used P-450 expression systems, are considered. The dependence of the mouse P-450 IA1 specific activity on the cytochrome expression level in yeast microsomes is studied and results demonstrate that the low amounts of endogenous NADPH-cytochrome P-450 reductase and cytochrome b5 which are naturally present, are limiting for the heterologous monooxygenase activities. The sequences encoding human liver cytochrome b5, the native and a modified form of the yeast NADPH-cytochrome P-450 reductase were cloned by making use of PCR techniques, over-expressed in yeast as functional forms, and characterized. New vectors allowing a high level of mammalian P-450 expression upon induction were also constructed and tested. A strategy for the construction of a co-expression system allowing maximal activity of mammalian cytochrome P-450s is discussed.
细胞色素P - 450构成了一个单加氧酶超家族，其活性需要与结合在内质网膜上的特定氧化还原酶相关联。本文考虑了这些哺乳动物酶在酵母细胞中功能表达的条件以及当前使用的P - 450表达系统各自的优缺点。研究了小鼠P - 450 IA1比活性对酵母微粒体中细胞色素表达水平的依赖性，结果表明，天然存在的少量内源性NADPH - 细胞色素P - 450还原酶和细胞色素b5限制了异源单加氧酶的活性。利用PCR技术克隆了编码人肝细胞色素b5、酵母NADPH - 细胞色素P - 450还原酶的天然形式和修饰形式的序列，在酵母中以功能形式进行了过量表达并进行了表征。还构建并测试了新的载体，这些载体在诱导后可实现高水平的哺乳动物P - 450表达。本文讨论了构建一个能使哺乳动物细胞色素P - 450发挥最大活性的共表达系统的策略。
