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

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.