106 Fig. 1 S.so-HSP20 mRNA expression in S. solfataricus P2 analyzed by real-time PCR. The level of S.so-HSP20 mRNA was measured in cells cultured at the indicated growth temperature or cold shock at 4°C for 2 h, and normalized against NusG (Internal control) levels. The mRNA expression value at 75°C was set to 1 and the other values were normalized against this. Data are representative of three trials and were expressed as the mean±SEM. Statistical significance was performed using the two-tailed paired Student's t test. (**p<0.01) Results S.so-HSP20 mRNA expression in S. solfataricus P2 at different growth temperatures and under cold-shock conditions The expression of S.so-HSP20 mRNA in S. solfataricus P2 was measured at different culture temperatures (Fig. 1). S. so-HSP20 mRNA expression was strongly increased at culture temperatures far from the optimal growth tempera- ture of 75°C. The increases in S.so-HSP20 mRNA expression at temperatures that were lower (60°C) or higher D.-C. Li et al. (80°C) than the optimum culture temperature (75°C) were 2.83- and 2.5-fold, respectively. S.so-HSP20 mRNA expression also increased 3.23-fold in response to cold- shock treatment (4°C). Survival after thermal stress in E. coli expressing S.so-HSP20 To determine whether S.so-HSP20 could enhance the thermotolerance of E. coli, the effect of heat stress on the growth of E. coli cells transformed with S.so-HSP20 was examined. E. coli expressing S.so-HSP20 was more resistant to heat stress than cells containing only the pET- 28a vector. As shown in Fig. 2, after exposure to 50°C, the survival of cells transformed with pET-28a only dropped to about 1.5% at 1 h, whereas that of the cells with pET-28a-S. so-HSP20 was 37.9%. Transformation with S.so-HSP20 also protected E. coli from exposure to cold (4°C), with survival recorded as 18.8% after 3-days cold exposure, compared to just 1.2% in E. coli transformed with pET-28a only. These results show that expression of S.so-HSP20 in E. coli cells increases cellular thermotolerance. S.so-HSP20 exhibits chaperone activity in vitro A key characteristic of molecular chaperones is their ability to suppress the aggregation of non-native proteins. To test whether S.so-HSP20 exhibits chaperone activity, we per- formed thermal unfolding assays using citrate synthase and insulin B chain as model substrates (Figs. 3 and 4). Purified S.so-HSP20 effectively protected citrate synthase against heat-induced denaturation. At concentrations of 150 or 300 nM, S.so-HSP20 reduced heat-induced turbidity result- ing from incubation at 43°C for 60 min by 48.1% and Fig. 2 Thermotolerance assays were performed at 50°C (a) and 4°C (b). E. coli P (with pET-28a) and E. coli H (with pET-28a-S.so-HSP20) were cultured as described and subjected to 50°C treatment for up to 60 min and 4°C treatment for up to 4 days. After heat or cold treatment, samples were taken at the time indicated, diluted, and immediately plated on LB plates with kanamycin. Survival of E. coli H cells (black boxes) and E. coli P cells (open boxes) are shown, and are expressed as a percentage of the survival obtained in LB plates. Data are representative of three trials and were expressed as the mean±SEM. Statistical significance was performed using the two- tailed paired Student's t test. (*p<0.05; **p<0.01)