假单胞菌株M18 gacA基因的克隆、表达及蛋白纯化  

Cloning and Expression of Pseudomonas sp. M18 gacA Gene and Its Purification

作　　者：任慧娟[1] 许煜泉[1] 董德贤[1] 李荣秀[1] 

机构地区：[1]上海交通大学生命科学技术学院生物制造实验室,上海200240

出　　处：《微生物学通报》2010年第3期401-406,共6页Microbiology China

基　　金：国家863计划项目(No.2006AA02Z228)

摘　　要：GacA是GacS/A双元调控系统的一个组分,克隆假单胞菌株M18中的gacA基因。测序结果同Pseudomonas sp.PAO1比较,该基因2个碱基的改变未引起氨基酸的改变。将该基因克隆到表达质粒pET28b(+)中,将重组质粒经热激转化至大肠杆菌BL21(DE3)中,IPTG诱导表达。表达产物经金属螯合层析柱纯化,蛋白纯度约为98%,质谱鉴定证明纯化的蛋白为GacA蛋白。CD谱分析GacA包含了4%α-helix,48%β-sheet和48%无规则卷曲。GacA蛋白的获得为进一步研究其晶体结构、生物学性能以及GacS/A双元调控系统奠定了基础。GacA is a component of GacS/GacA two-component system.In this study,Pseudomonas sp.M18 gacA gene had been successfully cloned and expressed in vector pET28b （＋）.Plasmid sequencing showed that two bases were different from Pseudomonas sp.PAO1 but did not change the amino acid sequence.The constructed recombinant plasmid was transformed to E.coli BL21 （DE3） by heat shock method and expressed under induction of IPTG.Ni-NTA was used to purify this his-tag GacA.Its purity reached to 98%.By Peptide Mass Fingerprinting,the obtained protein was confirmed as GacA.Circular dichroism data showed that GacA contained 4% α-helix,48% β-sheet and 48% random coil.Purified GacA protein could be used for further analysis such as crystal structure,biological characteristics and GacS/A two-component system.

关 键 词：GACA 克隆 表达 纯化 质谱鉴定 圆二色谱 

分 类 号：Q78[生物学—分子生物学]