OVEREXPRESSION OF XENOBIOTIC METABOLIZING ENZYMES

细胞色素P450是许多代谢的主要酶。 药物、原毒素和环境污染物。我们建议进一步 开发和优化高水平生产功能性， 使用杆状病毒表达的催化活性人细胞色素P450 在昆虫细胞中。该方法包括增强细胞色素 通过细胞色素P450：NADPH的共表达的P450催化活性 氧化还原酶和细胞色素b5。 人细胞色素CYP 2C 9、CYP 3A 4、 CYP 2 E1和CYP 2D 6将用作方法的原型酶 发展催化常数，表观Km和转换数，将是 测量优化的杆状病毒表达的酶，并与 从人类肝脏中分离或使用其他方法产生的相同酶 异源表达系统。 我们的最终目标是开发一个 杆状病毒表达的人细胞色素P450亚型的完整组。 该系统的商业应用将是对人类的研究。 异生物代谢 拟定商业应用： 生产大量的催化活性酶， 代谢物结构测定，研究底物代谢 低周转，确定P450形成特定的代谢和物理 特异性细胞色素P450的配体/底物相互作用研究 内切酶

Cytochromes P450 are the principal enzymes for the metabolism of many drugs, protoxins and environmental pollutants. We propose to further develop and optimize a method for high level production of functional, catalytically active human cytochromes P450 using baculovirus-expression in insect cells. This proposed method includes enhancement of cytochrome P450 catalytic activity through the co-expression of cytochrome P450:NADPH oxidoreductase and cytochrome b5. Human cytochromes CYP2C9, CYP3A4, CYP2E1 and CYP2D6 will be used as prototype enzymes for method development. Catalytic constants, apparent Km and turnover number, will be measured for the optimized baculovirus-expressed enzymes and compared to the same enzyme isolated from human liver or produced using other heterologous expression systems. Our eventual goal is to develop a complete panel of baculovirus-expressed human cytochrome P450 isoforms. The commercial applications of this system would be for the study of human xenobiotic metabolism. PROPOSED COMMERCIAL APPLICATION: Production of large quantities of catalytically active enzyme to allow structural determination of metabolites, study metabolism of substrates with low turnover, determine P450 form specific metabolism and physical studies of ligand/substrate interaction with specific cytochrome P450 enzymes.