EXPRESSION OF MODIFIED HUMAN CYTOCHROME-P450 3A4 IN ESCHERICHIA-COLI AND PURIFICATION AND RECONSTITUTION OF THE ENZYME
EXPRESSION OF MODIFIED HUMAN CYTOCHROME-P450 3A4 IN ESCHERICHIA-COLI AND PURIFICATION AND RECONSTITUTION OF THE ENZYME
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DOI：
10.1006/abbi.1993.1401
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发表时间：
1993-08-15
影响因子：
3.9
通讯作者：
GUENGERICH, FP
中科院分区：
文献类型：
--
作者：
GILLAM, EMJ;BABA, T;GUENGERICH, FP
将人细胞色素P450（P450）3A 4全长cDNA克隆及N端修饰的4个衍生物插入pCW载体，转化大肠杆菌DH 5 α细胞。用天然序列观察到很少的表达;用缺失残基3-12的构建体（NF 14）实现了最高水平的表达（范围为40-110 nmol P450升-1）。在所有构建体中，P450主要在膜中发现。修饰的P450 3A 4（构建体NF 14）显示典型的P450血红素蛋白谱。在五步程序中将蛋白质纯化至电泳均一性[标称23 nmol P450（mg蛋白质）-1]。对于大多数目的，发现在简单的两步过程中将修饰的P450 3A 4纯化至≥ 70%的均一性[标称15 nmol P450（mg蛋白质）-1]更实用。修饰的P450 3A 4（NF 14）或从人肝中纯化的P450 3A 4可与兔肝NADPH-P450还原酶混合，以获得几乎与人肝微粒体中发现的那些一样高的催化活性（以nmol P450为基础），但最佳重构条件不仅包括磷脂酰丝氨酸、L-α-二月桂酰-和L-α-二油酰-sn-甘油-3-磷酸胆碱的混合物，胆酸盐和细胞色素B5，但也谷胱甘肽在预孵育。发现其他几种硫醇在这一作用中不能取代。该酶对硝苯地平氧化、睾酮6β-羟基化、黄曲霉毒素B1的8，9-环氧化和3α-羟基化反应具有良好的催化活性。这些方法提供了生产、纯化和重构P450 3A 4的催化活性和有用的衍生物的相对方便和可靠的手段，P450 3A 4是一种在药物和其它异生素化学品的氧化中具有许多作用的人P450酶。
A full-length human cytochrome P450 (P450) 3A4 cDNA clone and four derivatives in which the N-terminus was modified were inserted into a pCW vector and used to transformEscherichia coliDH5α cells. Little expression was seen with the native sequence; the highest level of expression (range of 40-110 membrane-bound nmol P450 liter−1) was achieved with a construct (NF14) in which residues 3-12 were deleted. In all of the constructs P450 was found primarily in the membranes. The modified P450 3A4 (construct NF14) showed typical P450 hemoprotein spectra. The protein was purified to electrophoretic homogeneity in a five-step procedure [nominally 23 nmol P450 (mg protein)−1]. For most purposes it was found to be more practical to purify the modified P450 3A4 to ∼70% homogeneity [nominally 15 nmol P450 (mg protein)−1] in a simple two-step process. The modified P450 3A4 (NF14) or P450 3A4 purified from human liver could be mixed with rabbit liver NADPH-P450 reductase to achieve catalytic activities nearly as high as those found in human liver microsomes (on a nmol P450 basis), but the optimal reconstitution conditions included not only a mixture of phosphatidylserine, L-α-dilauroyl- and L-α-dioleoyl-sn-glycero-3-phosphocholines, cholate, and cytochromeb5suggested by others but also glutathione during the preincubation. Several other thiols were found not to substitute in this role. Good catalytic activity was seen for nifedipine oxidation, testosterone 6β-hydroxylation, and the 8,9-epoxidation and 3α-hydroxylation of aflatoxin B1, reactions previously ascribed to the enzyme. These procedures provide a relatively convenient and reliable means of producing, purifying, and reconstituting a catalytically active and useful derivative of P450 3A4, a human P450 enzyme that has many roles in the oxidation of drugs and other xenobiotic chemicals.