噬夏孢欧文氏菌类crtB基因的克隆及在大肠杆菌中的表达

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。

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

The gene crtB of Erwinia uredovora,encoding the phytoene synthase,was amplified by PCR and cloned into pET-15b expression vector.The recombinant 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 40% 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 Ni~(2 ) 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.