噬夏孢欧文氏菌(Erwinia uredovora)类胡萝卜素合成相关基因crtE的克隆及其在大肠杆菌中的表达  

Cloning and Expression of crtE from Erwinia uredovora in Escherichia coli

作　　者：汪靖超[1] 孙东平[1] 杜桂彩[2] 郭道森[1] 李荣贵[1] 

机构地区：[1]青岛大学生物系,山东青岛266071 [2]青岛大学天然色素山东省重点实验室,山东青岛266071

出　　处：《食品科学》2007年第7期276-279,共4页Food Science

基　　金：山东省优秀中青年科学家奖励基金项目(2003);青岛市自然科学基金项目(05-1-JC-91)

摘　　要：噬夏孢欧文氏菌基因crtE编码GGPP合成酶。通过PCR扩增获得crtE基因,克隆进表达载体,构建表达质粒pET-15bcrtE。重组质粒转化E.coli BL21(DE3),构建工程菌;重组GGPP合成酶在大肠杆菌中实现了高效表达,表达量占菌体总蛋白的42%。重组蛋白以包含体形式存在,包含体经洗涤、尿素溶解、复性并经镍离子亲和层析树脂纯化,得到了电泳纯的重组噬夏孢欧文氏菌GGPP合成酶,带有His-tag的该蛋白分子量为34kDa,pI值为6.3。Gene crtE of Erwinia uredovora, encoding the GGPP synthase, was amplified by PCR, and cloned into pET-15b expression vector. The recombinant plasmid was transformed into E.coli to construct engineering bacterium. Overexpression of recombinant GGPP synthase was achieved in engineering bacterium by IPTG induction, The expression level was up to 42% of the total cellular proteins. The recombinant protein was found mainly in inclusion bodies, after being solved in 8 mol/L urea and refolded. The recombinant GGPP synthase in inclusion bodies was purified on a Ni^2＋ chelating resin column. The purified protein with a N-terminal His-tag shows a molecular weight of 34 kDa and pI of 6.3.

关 键 词：噬夏孢欧文氏菌 crtE GGPP合成酶 表达 纯化 

分 类 号：Q785[生物学—分子生物学] Q786