OVEREXPRESSION OF XENOBIOTIC METABOLIZING ENZYMES

细胞色素P450是许多新陈代谢的主要酶 药物、原毒素和环境污染物。我们建议进一步 开发和优化一种高水平生产功能性、 利用杆状病毒表达催化活性人细胞色素P450 在昆虫细胞中。该方法包括细胞色素的增强。 细胞色素P450：NADPH共表达对P450的催化活性 氧化还原酶和细胞色素b5。人细胞色素CYP2C9、CYP3A4、 该方法将以细胞色素P450-2E1和细胞色素P450-2D6作为酶的原型 发展。催化常数、表观Km和成交量将为 对优化的杆状病毒表达酶进行测量，并与 从人的肝脏中分离出来的或用其他物质产生的相同的酶 异源表达系统。我们的最终目标是开发一种 杆状病毒表达的人细胞色素P450亚型的完整组合。 该系统的商业应用将是为了研究人类 异种生物代谢。 建议的商业应用： 大量生产具有催化活性的酶以允许 代谢物的结构测定，研究底物的代谢 周转率低时，确定P450的特定代谢和体能 特异性细胞色素P450与配体/底物相互作用的研究 酵素。

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.