104 Small heat shock proteins (sHSPs) are a family of stress- inducible molecular chaperones that range in size from 12 to 43 kDa (Arrigo 1998; Ehrnsperger et al. 1998; MacRae 2000; Van Montfort et al. 2001) and that form oligomers consisting 9 to 50 subunits (Boston et al. 1996; Waters et al. 1996; She et al. 2001). The ability of sHSPs to form oligomers contributes to their thermal stability and ability to avoid denaturation in response to high temperatures. sHSPs have chaperone-like activity in vitro and protect organisms from various stresses. At denaturing temperatures, sHSPs can prevent the aggregation of proteins by binding to, and forming a stable complex with, folding intermediates of their substrate proteins. In some cases, sHSPs can also promote renaturation of unfolded polypeptides (Brock et al. 1972; Esposito et al. 1998). In this study, we subcloned the coding sequence of the small heat shock protein, HSP20 (SSO2427) from the thermoacidophilic archaeon S. solfataricus P2 into a bacterial expression vector and produced recombinant protein for structural and functional analysis. To investigate the level of HSP20 mRNA in S. solfataricus P2, we measured its mRNA expression profile in response to variations in culture temperature, including cold shock. Escherichia coli transformed with HSP20 was protected from a thermal stress of 50°C and cold stress of 4°C. Purified S.so-HSP20 behaved as a molecular chaperone by inhibiting the thermal aggregation of citrate synthase and insulin B chain. These results indicate that HSP20 plays an important role in the response to thermal stress. Materials and methods Culturing S. solfataricus P2 at different temperature and cold-shock treatment For the cultivation of S. solfataricus P2, a standard culture medium (yeast extract, 2.0 g; KH2PO4, 3.10 g; (NH4)2SO4, 2.50 g; MgSO4 ×7H2O, 0.20 g; CaCl2 × 2H2O, 0.25 g; Distilled water, 1,000.00 ml) was used. The pH of the medium was adjusted to 3.5 at room temperature with 10 N H2SO4 prior to autoclaving. S. solfataricus P2 was cultured at different temperatures (60°C, 75°C, 80°C). The cells were harvested during the log growth phase at an OD600= 0.6. For cold-shock treatment, S. solfataricus P2 was exposed to 4°C for 2 h after culturing at 75°C. RNA extraction All samples were homogenized in Trizol Reagent (Invi- trogen, CA, USA), and total RNA was prepared according to the manufacturer's instructions. Total RNA was quantified on a Genova UV/visible spectrophotometer at 260 nm. D.-C. Li et al. Measurement of S.so-HSP20 expression by quantitative real-time PCR Complementary DNA was synthesized from 2 μg of total RNA from each sample using random decamer primers and Murine Molony Leukemia Virus reverse transcriptase (Promega, Madison, WI, USA) in a 25-μl reaction. The expression pattern of S.so-HSP20 was analyzed in various samples using gene-specific primers HSP20RT-F, HSP20RT- R (Table 1) and the MiniOpticon TM System (Bio-Rad, USA). Each polymerase chain reaction (PCR) reaction mix contained 12.5 μl of Supermix, 10 μM primer HSP20RT-F, 10 M primer HSP20RT-R, and 1 μl RT-products. PCR products were detected using the iQ SYBR Green Supermix kit (Bio-Rad). A standard curve was constructed using tenfold serial dilutions (1:104, 1: 105, 1: 106, 1: 107, and 1: 108) of purified plasmids subcloned with S.so-HSP20 together with a non-template control. A standard curve for NusG (Samson et al. 2008, 2011) was constructed using a purified plasmid subcloned with the NusG gene fragment that had been amplified with primers NusGF and NusGR (Table 1). This plasmid was then diluted in a tenfold series (1:105, 1:106, 1:107, 1:108, and 1:109). All samples were analyzed in triplicate. Statistical significance was performed using the two-tailed paired Student's t test. S.so-HSP20 gene expression profiles obtained from real-time PCR were normalized with the NusG transcript to correct for differ- ences in the starting amount of RNA and in the efficiency of the reverse transcription reactions. Melting curve analysis of the PCR reaction was performed to assess specificity. Genomic DNA extraction and plasmid purification S. solfataricus P2 was cultured at 75°C for 2 days using a standard culture medium. Cells were collected from 30 ml of culture by centrifugation at 4,200×g for 10 min. S. solfataricus P2 genomic DNA and Plasmids (Table 2) were extracted and purified using EZ-10 Spin Column Genome DNA isolation Kit, EZ-10 Spin Column Plasmid Mini- Table 1 Primers used in this work Primer HSP20F HSP20R NusGF NusG HSP20RT-F HSP20RT-R Sequence(5′-3′) CGggatccATGCCCAAGAGGGAAGAAAAGGATA CCGctcgagTTACTCAACTTTTATGTCAACACCA GTGGTTGGAGGGCAAGAAATAA GGGACCAGAAATTACTTCTACTACATC CAGACGGTGTGCCGAAAATAG TAGCCTTAGCCGCCTTCTCG Lowercase letters indicate restriction endonuclease recognition sequences (BamHI, XhoI)