Gillam E M, Baba T, Kim B R, Ohmori S, Guengerich F P
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
Arch Biochem Biophys. 1993 Aug 15;305(1):123-31. doi: 10.1006/abbi.1993.1401.
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 transform Escherichia coli DH5 alpha 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 approximately 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-alpha-dilauroyl- and L-alpha-dioleoyl-sn-glycero-3-phosphocholines, cholate, and cytochrome b5 suggested 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 6 beta-hydroxylation, and the 8,9-epoxidation and 3 alpha-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.
将一个全长人细胞色素P450（P450）3A4 cDNA克隆及4个N端经修饰的衍生物插入到pCW载体中，并用于转化大肠杆菌DH5α细胞。天然序列的表达量很低；通过缺失3至12位残基的构建体（NF14）实现了最高水平的表达（范围为40 - 110膜结合nmol P450升-1）。在所有构建体中，P450主要存在于细胞膜中。修饰后的P450 3A4（构建体NF14）呈现出典型的P450血红蛋白光谱。该蛋白通过五步程序纯化至电泳纯[名义上为23 nmol P450（mg蛋白）-1]。对于大多数用途而言，发现在一个简单的两步过程中将修饰后的P450 3A4纯化至约70%的纯度[名义上为15 nmol P450（mg蛋白）-1]更为实用。修饰后的P450 3A4（NF14）或从人肝脏纯化的P450 3A4可与兔肝脏NADPH - P450还原酶混合，以实现几乎与人肝脏微粒体中相当的催化活性（基于nmol P450），但最佳重组条件不仅包括其他人所建议的磷脂酰丝氨酸、L-α-二月桂酰-和L-α-二油酰-sn-甘油-３-磷酸胆碱、胆酸盐和细胞色素b5的混合物，还包括预孵育期间的谷胱甘肽。发现其他几种硫醇不能替代其在该作用中的地位。对于硝苯地平氧化、睾酮6β-羟基化以及黄曲霉毒素B1的8,9-环氧化和3α-羟基化反应，观察到了良好的催化活性，这些反应以前被归因于该酶。这些方法提供了一种相对方便且可靠的手段，用于生产、纯化和重组P450 3A4的具有催化活性且有用的衍生物，P450 3A4是一种在药物和其他外源性化学物质氧化中具有多种作用的人P450酶。
