Development of as table expression system for human drug metabolizing enzymes

酿酒酵母和糖酵母菌都含有与哺乳动物细胞相似的基因组和器官系统。后者富含内质网，可应用于哺乳动物细胞表达载体的基因修饰。因此，我们寻找S.pombe表达人P450的可行性。人类肝脏含有至少4种不同的P450 CYP2C亚家族成员（CYP2C8、CYP2C9、CYP2C18和CYP2C19）。这些形式参与各种化学物质的生物转化，包括临床上重要的药物。因此，我们从人肝脏cDNA文库中分离出了CYP2C9、CYP2C18和CYP2C19的cDNA克隆。分离的克隆最初通过ptl2载体导入S.pombe。转化体在内质网中产生了各自的人类CYP2C形式，但由于电子传递成分NADPH-cyt的水平较低，其催化活性仅为微量。P450还原酶。为了克服这种情况，我们引入了NADPH-cyt的cDNA。P450还原酶在S.pombe基因组中获得稳定的活性表达。获得的克隆显示出相当高的P450还原酶活性。使用表达还原酶的细胞，我们再次引入CYP2C形式，结果共表达的细胞显示出高水平的细胞色素P450和P450还原酶的产生。使用奥美拉唑和地西泮等药物的实验清楚地表明这些细胞系对药物代谢研究的有用性。

Both Saccharomyces cerevisiae and Shizosaccharomyces pombe contain genome and organe systems similar to mammalian cells. The latter is rich in endoplasmic reticulum and shown to be applicable for gene modification using expression vector for mammalian cells. Therefore, we looked for the feasibly of S.pombe for expression of human P450. Human livers contain at least 4 different members of CYP2C subfamilies of P450 (CYP2C8, CYP2C9, CYP2C18 and CYP2C19). These forms are involved in the biotrans formation of varies chemicals including clinically important drugs. We, thus, isolated cDNA clones of CYP2C9, CYP2C18, and CYP2C19 from human liver cDNA libraties. The isolated clones were initiallry introduced into S.pombe using pTL2-vectors. The transformants produced respective human CYP2C forms in endoplasmic reticulum but has only trace amounts of their catalytic activities, because of low levels of an electron transport component, NADPH-cyt. P450 reductase. To overcome this situation, we introduced cDNA of NADPH-cyt. P450 reductase in S.pombe genome to obtain stable expression of the activity. The clone obtained showed fairly high level of P450 reducatase activity. Using the reductase expressing cells, we introduced again CYP2C forms and the resultant co-expressed cells showed high levels of production of cytochrome P450 and P450 reductase. Experiments using drugs including omeprazole and dizepam clearly indicate the usefulness of these cell lines for drug metabolism studies.