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

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。

Abstract:

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.