Assessment of human drug metabolism by expression of cytochrome P-450 in yeasts.

本项目的目的是开发一种利用酵母细胞表达的细胞色素P-450的混合物来评估人类药物代谢的新方法。我们首次克隆了人类细胞色素P-450的主要基因--细胞色素P3A4，并在酵母细胞中进行了表达。另一方面，我们在酵母细胞中表达了人NADPH-细胞色素P-450还原酶和细胞色素b5。然后，我们对表达载体进行了改进，使不同的载体在同一细胞中共表达。我们成功地在同一细胞中共表达了CYP3A4、NADPH-细胞色素P-450还原酶和细胞色素b5，并观察到几种底物具有较高的酶活性。这些结果进一步表明了用这种方法表达几种不同的细胞色素P-450的可能性。然后，我们试图通过改变培养条件来提高酵母细胞中细胞色素P-450的产量。因此，我们得出结论，该方法是利用酵母细胞中表达的人细胞色素P-450的混合物来评估人类药物代谢的有用方法。

The purpose of this project is development of a new assessment method for human drug metabolism by using mixture of cytochrome P-450 expressed in yeast cells. We first cloned CYP3A4, a major human cytochrome P-450, and expressed it in yeast cells. On the other hand, we expressed human NADPH-cytochrome P-450 reductase and cytochrome b_5 in yeast cells. Then we improved the expression vectors to co-express different vectors in the same cells . We succeeded in co-expressing CYP3A4, NADPH-cytochrome P-450 reductase and cytochrome b_5 in the same cells and we observed high enzymic activities for several substrates. These results indicates further possible way to express sever al different cytochrome P-450 by this method.We, then, try to increase the yield of cytochrome P-450 in yeast cells changing by conditions of the incubation. Thus, we concluded that this approach is useful method for the assessment of human drug metabolism by using a mixture of human cytochrome P-450 expressed in yeast cells.