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Development of as table expression system for human drug metabolizing enzymes

人药物代谢酶as表表达系统的开发

基本信息

批准号：
07557149
负责人：
YAMAZOE Yasushi
金额：
$ 7.62万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1997
项目状态：
已结题

项目摘要

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.

酿酒酵母和粟酒裂殖酵母都含有类似于哺乳动物细胞的基因组和器官系统。后者富含内质网，并显示出适用于使用哺乳动物细胞的表达载体进行基因修饰。因此，我们寻找粟酒裂殖酵母表达人P450的可行性。人类肝脏含有至少4种不同的P450 CYP 2C亚家族成员（CYP 2C 8、CYP 2C 9、CYP 2C 18和CYP 2C 19）。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cDNA文库中分离出CYP 2C 9、CYP 2C 18和CYP 2C 19的cDNA克隆。使用pTL 2载体将分离的克隆最初引入粟酒裂殖酵母中。转化体在内质网中产生相应的人CYP 2C形式，但由于电子传递组分NADPH-cyt水平低，因此仅具有痕量的催化活性。P450还原酶。为了克服这种情况，我们引入了NADPH-cyt的cDNA。P450还原酶在粟酒裂殖酵母基因组中获得稳定表达的活性。获得的克隆显示出相当高水平的P450还原酶活性。使用还原酶表达细胞，我们再次引入CYP 2C形式，并且所得共表达细胞显示高水平的细胞色素P450和P450还原酶的产生。使用包括奥美拉唑和地西泮在内的药物的实验清楚地表明这些细胞系用于药物代谢研究的有用性。