线虫烯脂酰CoA水合酶的克隆、表达、纯化及初步晶体学分析  

Cloning,Expression,Purification and Preliminary Crystallographic Analysis of Caenorhabditis elegans Enoyl-CoA Hydratase

作　　者：李志杰[1,2] 张凯[1] 翟宇佳[1] 周强军[1] 耿运琪[2] 孙飞[1] 

机构地区：[1]中国科学院生物物理研究所,生物大分子国家重点实验室,北京100101 [2]南开大学生命科学学院,教育部分子微生物学与技术重点实验室,天津300071

出　　处：《生物物理学报》2010年第1期37-48,共12页Acta Biophysica Sinica

基　　金：国家重点基础研究发展计划("973"计划)(2006BC806506;2006CB911001);国家自然科学基金创新群体(30721003)~~

摘　　要：生物体β-氧化循环是脂肪酸氧化分解的主要途径,许多代谢疾病都与其密切相关。线虫β-氧化循环与人类相似,但与其相关的研究报道却很少。线虫基因C32E8.9编码的蛋白被WormBase命名为烯脂酰CoA水合酶(WormBase ID:CE29693),被推测具有催化β-氧化循环第二步反应的功能。作者将C32E8.9基因克隆到原核表达载体上,在大肠杆菌中获得高效表达,并分别纯化了母体蛋白以及硒代蛋白衍生物。多角度静态光散射实验表明该蛋白的聚合状态为三聚体。该蛋白在沉淀剂2-甲基-2,4-戊二醇的作用下形成可供衍射分析的六棱柱形状晶体,空间群为P21,晶胞参数为a=79.0,b=82.4,c=79.2,α=γ=90.0°,β=120°,数据分析表明该晶体非单晶,是一种罕见的蛋白质"三晶"——包含三套晶格。The β-oxidation spiral is the main pathway of fatty acid degradation in mammalian cells and many metabolic diseases are related to the disorders of the β-oxidation spiral. The β-oxidation spiral of Caenorhabditis elegans （C.elegans） is similar to human but is rarely reported. The protein encoded by C.elegans C32E8.9 gene is named as enoyl-CoA hydratase （cECH） in WormBase （ID： CE29693）, which is considered to catalyze the second step of the β-oxidation spiral in C.elegans. C32E8.9 gene was cloned into a prokaryotic expression vector pEXS-DH. Recombinant cECH and its selenium derivative （Se-cECH） were expressed and purified intensively with high purity, cECH was found to form hexamer according to size exclusion chromatography with multi-angle light scattering detection. Both cECH and Se-cECH were successfully crystallized by using the precipitant, 2-Methyl-2, 4-pentanediol. The crystal belongs to space group P21 with unit cell a=79.0A^°, b=82.4A^°, c=79.2A^°, α=γ=90.0°,β=120°.Data analysis indicated that the crystal was not a monocrystal but an unusual tricrystal that contains three sets of lattices.

关 键 词：脂肪酸β-氧化循环 烯脂酰CoA水合酶 线虫 多角度静态光散射 三晶 

分 类 号：Q71[生物学—分子生物学]