Thermotolerance and molecular chaperone 107 Fig. 3 S.so-HSP20 prevents heat-induced denaturation of citrate synthase. Purified S.so-HSP20 was incubated at 43°C (a) with 150 nM citrate synthase, and turbidity was measured at 360 nm. S. so-HSP20 concentrations were 0.0 nM (black square), 150 nM (black triangle), 300 nM (open triangle). S.so-HSP20 was also incubated at 45°C (b) with 150 nM citrate synthase, and turbidity was measured at 320 nm. The S.so-HSP20 concentration was 0.0 nM (black triangle) or 150 nM (open triangle). BSA at 150 nM (black square) was used as a control. Data are representative of three trials and were expressed as the mean±SEM 70.4%, respectively (Fig. 3a). Similarly, heat-induced turbidity resulting from a 60-min incubation at 45°C was reduced by S.so-HSP20 by 38.3% (Fig. 3b). Heat-induced turbidity of the insulin B chain was reduced by S.so-HSP20 by 47% after 24 min at 40°C (Fig. 4). These results demonstrate that S.so- HSP20 exhibits chaperone activity in vitro. Discussion In the present study, we examined the thermotolerance and molecular chaperone properties of S. solfataricus P2 sHSP, HSP20. The coding region of the HSP20 gene of the thermoacidophilic archaeon, S. solfataricus P2, was cloned into a plasmid vector and overexpressed in bacteria. The Fig. 4 S.so-HSP20 has chaperone-like activity. Chaperone-like activity of S.so-HSP20 was determined by measuring turbidity during DTT-induced aggregation of the insulin B chain (0.2 mg/ml) for 24 min at 40°C; insulin B chain alone (black triangle), insulin B chain in the presence of 0.2 mg/ml BSA (black square), or the insulin B chain in the presence of 0.1 mg/ml S.so-HSP20 (open triangle). Data are representative of three trials and were expressed as the mean±SEM recombinant protein was used for structural and functional analysis. We analyzed mRNA expression of HSP20 in response to different culture temperatures. Many sHSPs are not constitutively expressed, and their expression is induced specifically in response to stress conditions, such as elevated temperatures. We found that the expression of S. so-HSP20 mRNA is markedly increased following culture of S. solfataricus P2 at temperatures far from the optimal growth temperature. This result suggests that S.so-HSP20 mRNA expression is stress-induced, and may protect S. solfataricus P2 from stress. Expression of S.so-HSP20 in E. coli also protected these cells from temperature stress including cold shock. E.coli HSPs have been suggested to play a major role in protecting cells from thermal killing. The function of S.so-HSP20 in E. coli may be to promote thermotolerance by enhancing protein synthesis in E. coli (including HSPs) or by protecting E. coli proteins from heat denaturation through its chaperone activity. We demonstrat- ed chaperone activity for S.so-HSP20 of S. solfataricus P2 at physiological and heat shock temperatures. The ability of S.so-HSP20 to protect against heat-induced aggregation of citrate synthase (Keisuke et al. 2001; Ding et al. 2008; Martin et al. 2008; Angelo et al. 2004) and insulin B chain (Azzoni et al. 2004; Satoru 2006) indicates that S.so-HSP20 possesses chaperone activity and the ability to recognize and bind unfolded proteins to prevent their aggregation in vitro. The relationship between cellular thermotolerance and S.so-HSP20 expression in an E. coli expression system has not previously been reported. The results of this study provide evidence that S.so-HSP20 can protect E. coli from thermostress and behave as a chaperone in vitro. Future studies will develop an S.so-HSP20 expression system in Sacharomyses cerevisiae, and will assess the ability of S.so- HSP20 to regulate apoptosis.