Expression of porcine leukocyte 12-lipoxygenase in a baculovirus/insect cell system and its characterization.
Expression of porcine leukocyte 12-lipoxygenase in a baculovirus/insect cell system and its characterization.
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猪白细胞 12-脂氧合酶在杆状病毒/昆虫细胞系统中的表达及其表征。
DOI：
10.1006/abbi.1994.1302
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发表时间：
1994
影响因子：
3.9
通讯作者：
Marnett,LJ
中科院分区：
文献类型：
--
作者：
Reddy,RG;Yoshimoto,T;Yamamoto,S;Funk,CD;Marnett,LJ
使用杆状病毒表达载体在昆虫细胞中表达来自猪白细胞的花生四烯酸 12-脂氧合酶 (12-LO)。重组 12-LO 表达为 N 端融合蛋白，具有 31 个氨基酸的多肽，带有 6 个组氨酸标签和一个肠激酶切割位点。重组病毒感染草地贪夜蛾细胞后48小时观察到最大胞内酶活性和蛋白水平。裂解细胞并通过 Ni2+-次氮基三乙酸酯柱层析一步纯化重组蛋白。纯化的酶在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上迁移为单条带。重组酶催化形成12-氢过氧-5,8,10,14-二十碳四烯酸和少量15-氢过氧-5,8,11,13-二十碳四烯酸。手性相 HPLC 分析表明 12-(S) 对映体是主要产物。纯化的重组12-脂加氧酶将亚油酸氧化至花生四烯酸氧化程度的约19％。去甲二氢愈创木酸和5,8,11,14-二十碳四烯酸抑制重组酶，IC50分别为2.2和0.06 μM。克隆猪白细胞12-LO inS 的表达。 frugiperdacells 和 Ni2+-次氮基三乙酸色谱纯化提供了一种分离毫克量的这种形式 12-LO 的简单方法。
Arachidonate 12-lipoxygenase (12-LO) from porcine leukocytes was expressed in insect cells using a baculovirus expression vector. The recombinant 12-LO was expressed as an N-terminal fusion protein with a 31-amino acid polypeptide carrying a six-histidine tag and an enterokinase cleavage site. Maximal intracellular enzyme activity and protein levels were observed 48 h after infection ofSpodoptera frugiperdacells with the recombinant virus. Cells were lysed and the recombinant protein was purified in a single step by Ni2+-nitrilotriacetate column chromatography. The purified enzyme migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Recombinant enzyme catalyzed the formation of 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid and a small amount of 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid. Chiral-phase HPLC analysis indicated that the 12-(S) enantiomer was the predominant product. The purified recombinant 12-lipoxygenase oxygenated linoleic acid to about 19% of the extent of oxygenation of arachidonic acid. Nordihydroguaiaretic acid and 5,8,11,14-eicosatetraynoic acid inhibited the recombinant enzyme with IC50′s of 2.2 and 0.06 μM, respectively. Expression of cloned porcine leukocyte 12-LO inS. frugiperdacells and purification by Ni2+-nitrilotriacetate chromatography provides a straightforward method for isolation of milligram quantities of this form of 12-LO.