Cloning And Expression Of Pseudomonas Sp. M18GacS, GacA Gene And Its Purification

Sensor kinase GacS (global activator sensor kinase) and responseregulator GacA (global antibiotic and cyanide control) aretwo-component regulatory system. GacS and GacA, as members of awidely occurring two-component system, control exoprotein productionin E. carotovora subsp. Carotovora. In many gram-negative bacteria, theGacS/GacA two-component system regulates the expression ofextracellular products, and these can be virulence determinants inpathogenic species. Whether gacS/gacA mutants could competitivelydisplace the wild-type populations on roots and thus pose a threat to theefficacy of biological control. Surprisingly, despite extensive studies ofthe gacS, gacA mutation, GacS and GacA proteins have not be expessedand purified. Thus, gacS, gacA cloning, expression and purification andin-depth research are significant area of research.In this study, Pseudomonas sp. M18gacA gene had beensuccessfully cloned and expressed in vector pET28b(+). The constructedrecombinant plasmid was transformed to E. coli BL21(DE3) by heatshock method and expressed under induction of IPTG. Ni-NTA was used to purify this his-tag GacA. Its purity reached to95%. By Peptide MassFingerprinting, the obtained protein was confirmed as Pseudomonas M18GacA.Thermal stability analysis of GacA is also done. GacA protein willbe denaturated under40℃for10min, proving it to have low heatresistance. By applying bacterial proteins of M18to SepharoseNTA-Ni-GacA, Chitinase,50S ribosomal protein L25/general stressprotein Ctc, heat shock protein90, bifunctional aconitate hydratase,dihydrolipoamide dehydrogenase, alkyl hydroperoxide reductase subunitC and elongation factor G were found that may interact with GacA Theexperiment is only a preliminary exploration, and lots of experimentsneeded to do to confirm.UCLA-DOE predicted that gacS gene contains rare codons. Thus theconstructed recombinant plasmid pET/gacS can not be expressed in E.coli BL21(DE3). The recombinant plasmid was transformed to E. coliBL21-CodonPlus(DE3)-RP by heat shock method and expressed underinduction of IPTG. Ni-NTA was used to purify this his-tag GacS. Itspurity reached to90%. TMHMM2.0was used to predict transmembranestructure of GacS protein, and it shows that amino acids11-33and168-187are the GacS transmembrane structure.