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首页> 外文期刊>Protein Expression and Purification >Human CYP1A1 allelic variants: baculovirus expression and purification, hydrodynamic, spectral, and catalytical properties and their potency in the formation of all-trans-retinoic acid
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Human CYP1A1 allelic variants: baculovirus expression and purification, hydrodynamic, spectral, and catalytical properties and their potency in the formation of all-trans-retinoic acid

机译：人CYP1A1等位基因变体：杆状病毒的表达和纯化，流体动力学，光谱和催化特性及其在形成全反式维甲酸中的功效

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Three human cytochrome P450 1A1 (CYP1A1) allelic variants, namely wild-type (CYP1A1.1), CYP1A1.2 (I462V), and CY-P1A1.4 (T461N), we re expressed as C-terminal His-tagged fusions including a thrombin cleavage site in Spodoptera frugiperda insect cells by baculovirus infection. The variants were expressed with 30-90 nmol (1.8-5.4 mg) spectrally active cytochrome MO per one liter of culture and purified to electrophoretic homogeneity by Ni-agarose chromatography. The recombinant variants were structurally characterized by UV/Vis, ultracentrifugation, and EPR. Optical and EPR spectra showed all three variants predominantly in high spin state; moreover, EPR indicated changes in the electronic structure of the heme iron of the two mutant variants. Sedimentation equilibrium experiments demonstrated the purified variants in dimeric state in the presence of 0.2% emulgen+0.05% cholate. Higher detergent concentration, the presence of imidazole, and cleavage of the His-tag led to monomerization. Catalytic activity of all purified variants was reconstituted with purified human NADPH-P450 reductase and dilaurylphosphatidylcholine. Enzyme kinetics of ethoxyresorufin O-deethylation revealed similar K-m (approximate to 0.4 muM) for all variants but slightly different V-max values (CYP1A1.1: 4.2, CYP1A1.2: 7.0, and CYP1A1.4: 3.0 nmol/minmol CYP1A1). The extended C-terminus influenced the enzymatic activity only slightly. All three variants are able to produce significant amounts of all-trans-retinoic acid from all-trans-retinal with V-max of 4.0, 3.3, and 5.6 nmol/minmol CYP1A1 and K-m values of 111, 83, and 250 muM for CYP1A1.1, CYP1A1.2, and CYP1A1.4, respectively. Availability of the three purified human CYP1A1 variants should facilitate further characterization of their role in metabolism of endogenous and exogenous compounds as well as structural studies. (C) 2002 Elsevier Science (USA). All rights reserved. [References: 30]
机译：三种人类细胞色素P450 1A1（CYP1A1）等位基因变体，即野生型（CYP1A1.1），CYP1A1.2（I462V）和CY-P1A1.4（T461N），我们以C末端带有His标记的融合蛋白表示，包括通过杆状病毒感染在草地贪夜蛾昆虫细胞中的凝血酶裂解位点。每升培养物用30-90 nmol（1.8-5.4 mg）具有光谱活性的细胞色素MO表达变体，并通过Ni-琼脂糖层析纯化至电泳均一。通过UV / Vis，超速离心和EPR对重组变体进行结构表征。光学和EPR光谱显示这三个变体主要处于高自旋状态;此外，EPR表明两个突变体变体血红素铁的电子结构发生了变化。沉降平衡实验表明，在0.2％乳原+ 0.05％胆酸盐的存在下，纯化的二聚体变异体处于二聚状态。较高的洗涤剂浓度，咪唑的存在以及His标签的裂解导致单体化。用纯化的人NADPH-P450还原酶和二月桂基磷脂酰胆碱重建所有纯化的变体的催化活性。乙氧基间苯二酚O-脱乙基化的酶动力学显示所有变体的Km相似（约0.4μM），但V-max值略有不同（CYP1A1.1：4.2，CYP1A1.2：7.0和CYP1A1.4：3.0nmol / min / nmol CYP1A1）。延伸的C末端仅轻微影响酶活性。这三个变体均能从全反式视网膜中产生大量全反式维甲酸，V-max为4.0、3.3和5.6 nmol / min / nmol CYP1A1，Km值为111、83和250μM分别适用于CYP1A1.1，CYP1A1.2和CYP1A1.4。三个纯化的人CYP1A1变体的可用性应有助于进一步表征其在内源性和外源性化合物的代谢以及结构研究中的作用。 （C）2002 Elsevier Science（美国）。版权所有。 [参考：30]

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