Expression And Characterization Of Acidithiobacillus Ferrooxidans ATCC23270 Heat Shock Protein Hsp90 (AtHtpG)

Heat shock protein (Hsp) 90 is an abundant molecular chaperone that is highly conserved. Hsp90 is mianly involved in the folding and conformational regulation of numerous client proteins and also mediates the refolding of stress-denatured proteins. Acidithiobacillus ferrooxidans is one of the most intensively studied bioleaching bacterias. During the bioleaching process, the microorganisms are always exposed to a series of adverse environmental changes, such as a sudden increase of temperature, which may restrain the activation of Acidithiobacillus ferrooxidans, and as a result, affect the bioleaching rates. In response to these environmental stresses, bacteria increase the genetic expression of a set of proteins so-called heat shock proteins, which may be related to Acidithiobacillus ferrooxidans' response or even the resistance to stressful condition. So, to study the characteration of Hsp90 from Acidithiobacillus ferrooxidans is very important.In this study, the gene encoding AtHtpG(AtHtpG) was amplified and the recombinant AtHtpG was expressed in E. coli BL21 (DE3), then purified with one-step Ni-NTA affinity column chromatography. The molecular mass was 71 kDa measured by SDS-PAGE analysis. The ATPase and the chaperon function of AtHtpG were tested. Found out that AtHtpG has ATPase activity at varied temperatures, and the ATPase activity of the AtHtpG gradually increased as the temperature ranged from 20℃to 50℃, but then decreased sharply at higher temperatures (above 50℃). AtHtpG can assist the refolding of denatured protein upon addition of GroE system.Microcalorimetric method was applied to study the interaction between the denatured protein and AtHtpG, and the influence factors to ATPase activities of AtHtpG were also elucidated. The results indicated that the appropriate concentration of Mg2+ and ATP could promote the hydrolysis of ATP by AtHtpG. In the present of GroE system and AtHtpG, the refolding reaction was an exothermic reaction, mainly due to hydrophobic interaction. And the result also illustrated that non-native proteins can refolding at a larger extent with the present of AtHtpG and GroE system, compared to with the present of ony GroE system.Also the functions of AtHtpG were elucidated in vivo. The investigation showed that AtHtpG-overexpression caused growth inhibition of E. coli BL21 (DE3) and proteins from E. coli cells producing Hsp90 exhibited lower thermostability. These results indicate that AtHtpG may assert a negative regulation to some impotant enzymes which were involved in thermal stress management through interacting with these enzymes, and as a result, leading growth inhibition.