HETEROLOGOUS EXPRESSION OF CYTOCHROME P-450

哺乳动物细胞色素P-450的数量不断增加 与已知的同时表达的Very 单个组织中紧密相关的形式(具有90%的序列同一性)， 有复杂的努力来严格净化和刻画个人 酶的各种形式。意识到密切相关的形式可能 Copurify可以具有非常不同的底物特性和催化作用 活动需要新的方法来提纯和 这些酶的特性。这些考虑，也许还有 最重要的是，进行结构-功能研究的愿望 这些细胞色素通过定点突变，促使我的 在异源宿主中表达这些酶的努力。为此， 拟研究的目标是合成和提纯 异源表达的单个细胞色素P-450亚型 系统，以及随后的结构-功能检查 通过定点突变研究的关系。最近的成功 本实验室利用细胞色素P-450IIE1在细胞中的表达 大肠杆菌已经为拟议的研究奠定了基础。 本研究的具体目的是：1)开发一种净化器 在大肠杆菌中表达细胞色素P-450IIE1的程序，部分基于 建立了从兔肝中分离P-450lIE1的方法 将提供足够清洁的酶来严格催化和 机制研究；2)确定负责的一个或多个部门 为了将细胞色素结合到内质网状膜上， 缺失分析和定点突变；3)鉴定 可能参与电子传递的细胞色素P-450残基 细胞色素P-450还原酶与P-450血红素组的比较 用催化和光谱分析进行定点突变， 最初专注于Trp-1 22和Phe430。

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.