Gene expression and molecular characterization of a chaperone protein HtpG from Bacillus licheniformis

• The putative HtpG gene of B. licheniformis is cloned and expressed in E. coli.
• The recombinant HtpG exists as a mixture of monomer, dimer and other oligomers in solution.
• The protein conformation is stable at higher temperatures under neutral pH, but is thermally sensitive at alkaline conditions.
• It also experiences a significant change in the molecular architecture upon the addition of ATP and certain concentrations of organic solvents.

Heat shock protein 90 (Hsp90/HtpG) is a highly abundant and ubiquitous ATP-dependent molecular chaperone consisting of three flexibly linked regions, an N-terminal nucleotide-binding domain, middle domain, and a C-terminal domain. Here the putative htpG gene of Bacillus licheniformis was cloned and heterologously expressed in Escherichia coli M15 cells. Native-gel electrophoresis, size exclusion chromatography, and cross-linking analysis revealed that the recombinant protein probably exists as a mixture of monomer, dimer and other oligomers in solution. The optimal conditions for the ATPase activity of B. licheniformis HtpG (BlHtpG) were 45 °C and pH 7.0 in the presence of 0.5 mM Mg2+ ions. The molecular architecture of this protein was stable at higher temperatures with a transition point (Tm) of 45 °C at neutral pH, whereas the Tm value was reduced to 40.8 °C at pH 10.5. Acrylamide quenching experiment further indicated that the dynamic quenching constant (Ksv) of BlHtpG became larger at higher pH values. BlHtpG also experienced a significant change in the protein conformation upon the addition of ATP and organic solvents. Collectively, our experiment data may provide insights into the molecular properties of BlHtpG and identify the alteration of protein structure to forfeit the ATPase activity at alkaline conditions.