噬夏孢欧文氏菌类crtB基因的克隆及在大肠杆菌中的表达

Cloning and Expression of Gene crtB from Erwinia Uredovora in Escherichia Coli

噬夏孢欧文氏菌类胡萝卜素合成相关基因crtB,编码八氢番茄红素合成酶。利用PCR技术扩增出crtB基因,克隆进表达载体,构建表达质粒pET-15bcrtB。重组质粒转化大肠杆菌,构建工程菌株。经IPTG诱导,工程菌高效表达了重组八氢番茄红素合成酶,表达量占菌体总蛋白的40%。重组蛋白以包含体形式存在,包含体经洗涤、尿素溶解、复性并经镍离子树脂亲和层析、Superdex 75凝胶层析柱纯化,得到了电泳纯的重组八氢番茄红素合成酶,带有His-tag的该蛋白分子量为35 kDa,pI值为7.3。

The gene crtB of Erwinia uredovora, encoding the phytoene synthase, was amplified by PCR and cloned into pET-15b expression vector. The recomhinant plasmid was transformed into E. coli BL21 （DE3） to construct engineering bacterium. By IPTG induction, overexpression of recombinant phytoene synthase was achieved, with the expression level up to 400% of the total cellular proteins. The recombinant protein was found mainly in inclusion bodies, after solving in urea and refolding, the recombinant phytoene synthase in inclusion bodies was purified to homogeneity on a Ni2＋ chelating resin column and superdex 75 column. The purified protein with an N-terminal His-tag showed a molecular weight of 35 kDa and pI of 7.3.