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

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。

Cloning and Expression of crtE from Erwinia uredovora in Escherichia coli

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 Ni2 chelating resin column. The purified protein with a N-terminal His-tag shows a molecular weight of 34 kDa and pI of 6.3.