谷氨酰t-RNA还原酶基因(hemA)的高效表达  

High-level Expression of Bacillus subtilis hemA gene in Escherichia coli and the Effect on the 5-ALA Biosynthes is Pathway

作　　者：李爽[1] 李恒鑫[1] 刘辉[1] 袁新宇[2] 杨明明[1] 龚月生[1] 

机构地区：[1]西北农林科技大学动物科技学院,杨凌712100 [2]湖北工业大学生物工程学院

出　　处：《中国生物工程杂志》2007年第6期82-86,共5页China Biotechnology

摘　　要：枯草芽孢杆菌(Bacillus subtilis)中的hemA基因编码谷氨酰tRNA还原酶,该酶是B.subtilis代谢途径中由谷氨酸到5-氨基乙酰丙酸(5-ALA)反应的限速酶。将B.subtilis的hemA基因克隆到pET28a载体上,并在大肠杆菌(Escherichia coli)BL21(DE3)中诱导表达,SDS-PAGE电泳分析,表达的目的蛋白占总蛋白的20%。通过分离纯化得到谷氨酰tRNA还原酶。重组菌发酵液上清中5-ALA含量达40.2mg/L,菌液呈红色,过筛试验和紫外分光光度检测验证显色物质为卟啉类,实验表明,表达的重组蛋白促进了5-ALA的合成和代谢。Bacillus subtilis glutamyl-tRNA reductase encoded by hemA gene, which is the rate-limitation enzyme in biosynthesis of 5-ALA. 5-ALA is the precursor of the biosynthesis of terapyrroles as well as an important functional molecular in medical and agricultural application. The hemA gene was polymerase chain reaction amplified from B. subtilis genome DNA and cloned into the expression vector pET28a. The hemA gene was High-level expressed in Escherichia coli BL21 （DE3） when induced by IPTG, in which the recombinant protein makes up 20% of total soluble protein through SDS-PAGE analysis. We also obtained the purified protein of HemA through the Ni^2＋ -NAT affinity chromatography resin. Biosynthesis level of 5-ALA was enhanced responding to expression of the recombinant protein. The production of 5-ALA reached 40.2mg/L. The catabolite metabolism of 5-ALA porphyrins was also accelerated.

关 键 词：枯草芽孢杆菌 谷氨酰t-RNA还原酶基因 高效表达 5-ALA 

分 类 号：Q78[生物学—分子生物学]