HETEROLOGOUS EXPRESSION OF CYTOCHROME P-450

越来越多的哺乳动物细胞色素P-450， 已经确定，加上已知的并发表达非常 在单一组织中密切相关的形式（具有>90%的序列同一性）， 严格净化和描述个体特征的努力是复杂的 酶的形式。 认识到密切相关的形式， 共纯化可以具有非常不同的底物特异性和催化活性。 活动需要新的方法来净化和 这些酶的特性。 这些考虑，也许， 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 在异源宿主中表达这些酶的努力。 为此中央 本研究的目的是合成和纯化 来自异源表达的单个细胞色素P-450同种型 系统，随后检查结构-功能 通过定点突变研究的关系。 最近的成功 在这个实验室中，细胞色素P-450 IIE 1在 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）开发一种纯化方法， 细胞色素P-450 IIE 1在E.大肠杆菌，部分基于 建立了从兔肝中分离P-450 lIE 1的方法， 将为严格的催化提供足够清洁的酶， 机制研究; 2）确定负责的部分或部分 用于通过使用将细胞色素结合到内质网膜， 缺失分析和定点突变; 3）鉴定那些 可能参与电子转移的细胞色素P-450残基 细胞色素P-450还原酶和P-450血红素组之间的关系， 用催化和光谱分析定点诱变， 最初集中于Trp-122和Phe 430。

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.