HETEROLOGOUS EXPRESSION OF CYTOCHROME P-450

细胞色素 P-450 的异源表达

• 批准号: 3307102
• 负责人: TODD D PORTER
• 金额: $ 8.78万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-09-01 至 1994-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3307102
• 关键词: Escherichia coli; affinity chromatography; bacterial proteins; binding proteins; cytochrome P450; electron transport; enzyme mechanism; enzyme structure; gene expression; hemoprotein structure; isozymes; membrane proteins; molecular cloning; oxidoreductase; protein purification; protein structure function; site directed mutagenesis

The ever-increasing number of mammalian cytochromes P-450 that have been identified, coupled with the known concurrent expression of very closely related forms (having >90% sequence identity) in a single tissue, has complicated efforts to rigorously purify and characterize individual forms of the enzyme. The realization that closely related forms that may copurify can have very different substrate specificities and catalytic activities has necessitated new approaches for the purification and characterization of these enzymes. These considerations, and, perhaps most importantly, the desire to undertake structure-function studies on these cytochromes through site-directed mutagenesis, has prompted my efforts to express these enzymes in heterologous hosts. To this end, the objective of the proposed research is the synthesis and purification of an individual cytochrome P-450 isoform from a heterologous expression system, with the subsequent examination of structure-function relationships through site-directed mutagenesis studies. Recent success in this laboratory with the expression of cytochrome P-450IIE1 in Escherichia coli has laid the groundwork for the proposed studies. The specific aims of this research are: 1) to develop a purification procedure for cytochrome P-450IIE1 expressed in E. coli, based in part on the established procedure for isolating P-450lIE1 from rabbit liver, that will provide enzyme sufficiently clean for rigorous catalytic and mechanistic studies; 2) to identify the segment or segments responsible for binding the cytochrome to the endoplasmic reticular membrane by using deletion analysis and site-directed mutagenesis; 3) to identify those cytochrome P-450 residues likely to be involved in electron transfer between cytochrome P-450 reductase and the P-450 heme group by using site-directed mutagenesis with catalytic and spectroscopic assays, focusing initially on Trp-1 22 and Phe430.

哺乳动物细胞色素P-450的数量不断增加