噬夏孢欧文氏菌crtY基因在大肠杆菌中的表达及其产物的纯化  

Expression of crtY from Erwinia uredovora in Escherichia coli and Purification of Its Gene Product

作　　者：李丽[1] 吴柘[1] 郭道森[1] 汪靖超[1] 李荣贵[1] 

机构地区：[1]青岛大学生物系,山东青岛266071

出　　处：《食品科学》2007年第5期203-207,共5页Food Science

基　　金：山东省博士基金项目(2003);青岛市自然科学基金项目(05-1-JC-91)

摘　　要：噬夏孢欧文氏菌(Erwinia uredovora)可以累积类胡萝卜素,合成过程涉及的一系列反应都是在相关酶的催化下完成的,其中番茄红素环化酶由基因crtY编码,催化由番茄红素转化为β-胡萝卜素的酶促反应。本研究以噬夏孢欧文氏菌基因组DNA为模板,通过聚合酶链式反应扩增获得crtY基因,测序正确后克隆进表达载体pET-15b,构建表达质粒pET-15b-crtY,重组质粒转化E．coli BL21(DE3),构建工程菌,经IPTG诱导后,重组番茄红素环化酶在大肠杆菌中实现了高效表达,通过镍离子树脂亲和层析和Superdex 75凝胶过滤层析,得到了电泳纯的重组噬夏孢欧文氏菌番茄红素环化酶,该蛋白体外具有催化番茄红素转化为β-胡萝卜素的活性。Carotenoids can be accumulated in Erwinia uredovora. Several genes are related to the synthesis of carotenoids. One of the genes, crtY, encodes lycopene cyclase which catalyzes the conversion from lycopene to β-carotene. CrtY is amplified by PCR and cloned into pET-15b to construct the expressing plasmid pET-15b-crtY, which is introduced into E.coli BL21（DE3） to construct the engineering bacteria. Overexpression of crtY in bacteria is achieved by ~ induction. Recombinant protein can be purified by chromatography on nickel column and Superdex 75 column. The protein is homogeneous as judged by SDS-PAGE, and can catalyze the conversion from lycopene to 13-carotene.

关 键 词：ERWINIA uredovora crty 番茄红素环化酶 纯化 活性 

分 类 号：Q562[生物学—生物化学]