Expression and purification of lipoprotein-associated phospholipase A2,a key enzyme involved in atherosclerosis
Expression and purification of lipoprotein-associated phospholipase A2,a key enzyme involved in atherosclerosis作者机构：State Key Laboratory of Drug Research Chinese Academy of Sciences Shanghai China Graduate School of the Chinese Academy of Sciences Chinese Academy of Sciences Shanghai China Department of Medicinal Chemistry Shanghai Institute of Materia Medica Shanghai Institutes for Biological Sciences Chinese Academy of Sciences Shanghai China
出 版 物：《Acta Pharmacologica Sinica》 (中国药理学报(英文版))
年 卷 期：2006年第27卷第6期
页 面：679-684页
核心收录：
学科分类：1007[医学-药学(可授医学、理学学位)] 1006[医学-中西医结合] 100706[医学-药理学] 100602[医学-中西医结合临床] 10[医学]
基 金：Project supported by the Research Foundation of the Chinese Academy of Sciences(№ KSCX1-SW-11-6)
主 题：lipoprotein-associated phospholipase A2 atherosclerosis coronary heart diseases cloning baculovirus purification
摘 要：Aim:To express and purify lipoprotein-associated phospholipase A2（Lp-PLA2）,and to establish a screening model for Lp-PLA2inhibitors using the expressed Lp-PLA2,Methods:We cloned the full-length cDNA of Lp-PLA2 from differentiatedTHP-I cells,and subcloned the cDNA into the baculovirus transfer *** addition,we introduced an N-terminal Kozak sequence for high-level translation initiation and a C-terminal sequence of 6 histidine residues forpurification,The fusion enzyme was expressed in Sf9 insect cells and purified byNi2+affinity *** Lp-PLA2activity was measured us-ing[3H]PAF as a substrate,and we examined the enzyme activity of recombinantLp-PLA2pretreated with the known Lp-PLA2inhibitor ***:Wesuccessfully cloned the full-length Lp-PLA2gene from differentiated THP-1 *** fusion enzyme was expressed in Sf9 insect cells at a high level and purifiedefficiently through a 2-step *** recombinant Lp-PLA2activity wasmeasured using[3H]PAF as a substrate,and proved to be enzymatically ***-PLA2inhibitor SB435495 produced a good inhibition curve for inhibition ofrecombinant Lp-PLA2with an IC50of 57±1μmol/***:We expressed andpurified Lp-PLA2at a high level in insect cell-baculovirus expression *** ratio was much greater than that obtained from human plasma and we estab-lished a screening model for Lp-PLA2inhibitors using the expressed Lp-PLA2.
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