OVEREXPRESSION, PURIFICATION AND CHARACTERIZATION OF HUMAN RECOMBINANT 15-LIPOXYGENASE
OVEREXPRESSION, PURIFICATION AND CHARACTERIZATION OF HUMAN RECOMBINANT 15-LIPOXYGENASE
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DOI：
10.1016/0005-2760(93)90085-n
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发表时间：
1993-07-21
期刊：
影响因子：
--
通讯作者：
SIGAL, E
中科院分区：
文献类型：
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作者：
KUHN, H;BARNETT, J;SIGAL, E
人15-脂氧合酶高水平表达(约。细胞蛋白的20%)在杆状病毒/昆虫细胞表达系统。在Mono Q柱上，用阴离子交换层析法，用约为0的色谱柱，很容易地从细胞质中纯化出催化活性酶（纯度为90-95%）。酶活性回收率为95%。通常情况下，每L培养物的纯酶产量为25-50 mg，比活性为7.1-21 μ mol 13-羟基十八烯二烯酸(13-HODE)/mg。周转率最小（8.4-25 s-1）。在生产过程中，在纯化酶或昆虫细胞培养基中添加亚铁离子均能显著提高酶的比活性和酶的铁含量。等电点为5.85，n端氨基酸序列与cDNA预测结果一致。纯化后的重组酶对花生四烯酸具有双重位置特异性（以12:1的比例生成15S-和12s -羟二糖四烯酸（12S-HETE））。双氧产物14R，15S-和各种8,15-二hete异构体也被鉴定。以亚油酸为底物，确定最适ph为7.0，K(M)为3mum。该酶在脂肪酸氧合过程中经历自杀性失活，对标准脂氧合酶抑制剂敏感，并对磷脂、胆固醇酯、生物膜和人低密度脂蛋白进行氧合。与先前对兔酶的研究相反，未检测到糖基化。
Human 15-lipoxygenase was expressed to high levels (approx. 20% of cellular protein) in a baculovirus/insect cell expression system. Catalytically active enzyme was readily purified (90-95% pure) from cytosolic fractions by anion-exchange chromatography on a Mono Q column with approx. 95% recovery of enzymatic activity. Routinely, a yield of 25-50 mg of pure enzyme per L of culture and a specific activity of 7.1-21 mumol 13-hydroxyoctadecadienoic acid (13-HODE)/mg.min (turnover rate of 8.4-25 s-1) were obtained. Both the specific activity and the enzyme's iron content was significantly increased by the addition of ferrous ions to either the purified enzyme or to the insect cell culture medium during production. An isoelectric point of 5.85 was determined and the N-terminal amino acid sequence was found to be identical to that predicted from the cDNA. The purified recombinant enzyme exhibits a dual positional specificity with arachidonic acid (formation of 15S- and 12S-hydroxyeicosatetraenoic acid (12S-HETE) in a ratio of 12: 1). Double oxygenation products 14R,15S- and various 8,15-DiHETE isomers were also identified. With linoleic acid as substrate, a pH-optimum of 7.0 and a K(M) of 3 muM were determined. The enzyme undergoes suicidal inactivation during fatty acid oxygenation, is sensitive to standard lipoxygenase inhibitors, and oxygenates phospholipids, cholesterol esters, biomembranes and human low-density lipoprotein. Contrary to prior studies on the rabbit enzyme, no glycosylation was detected.