Thermotolerance and molecular chaperone Table 2 Strains and plasmids used in this work *HSP20, NCBI reference sequence: NP_343781.1 (She et al. 2001) Strain or plasmid Strains Sulfolobus solfataricus p2 E.coli BL21(DE3) E.coli P E.coli H E.coli HR E.coli NusG Plasmids pET-28a-HSP20 pUCm-T-HSP-RT pUCm-T-NusG Relevant genotype or features Sulfolobus solfataricus DSMZ1617 E.coli B (DE3)[F−dcm ompT hsdSB (rB−mB−)] E.coli BL21(DE3) with pET-28a E.coli BL21(DE3) with pET-28a-HSP20 E.coli DH5α with pUCm-T-HSP-RT E.coli DH5α with pUCm-T-NusG-RT pET28a(+) with HSP20* pUCm-T with HSP-RT DNA pUCm-T with NusG-RT DNA 105 Reference DSMZ Studier et al. 1990 This work This work This work This work This work This work This work Preps Kit and EZ-10 Spin Column Gel Extraction Kit (BIO BASIC INC). All samples were prepared according to the manufacturer's instructions and DNA concentration was measured using a Genova UV/visible spectrophotometer at 260 nm. Cloning, expression, and purification of recombinant S.so-HSP20 The open reading frame of S.so-HSP20 was amplified from the chromosomal DNA of S. solfataricus P2. The forward primer (HSP20F) contained a BamH I site (indicated lowercase, Table 1) and the reverse primer (HSP20R) contained a unique XhoI site (indicated lowercase, Table 1) with stop codon. The PCR product was digested with BamHI and XhoI, and ligated into the pET-28a vector (Novogen, USA) digested with BamHI and XhoI, generat- ing pET-28a-S.so-HSP20, which encodes S.so-HSP20 with an N-terminal His6 extension. pET-28a-S.so-HSP20 was transformed into E. coli BL21 (DE3), and His6-S.so-HSP20 was induced by addition of isopropyl-β-D-Thiogalactoside (IPTG; 1-mM final concentration) to exponentially growing cultures of E. coli (pET -28a-S.so-HSP20) in Lauria-Bertani (LB) medium containing 50 μg/ml of kanamycin. His6- tagged protein was purified by Ni-NTA (Qiagen, USA) according to the manufacturer's instructions. S.so-HSP20 was purified to homogeneity, based on Coomassie blue staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Thermotolerance experiments with transformed E. coli For thermotolerance experiments, E. coli (pET-28a-S.so- HSP20) was cultured as described above. IPTG (1 mM) was added to mid-log phase cultures (OD600 = 0.8), and the cells were incubated at 30°C for 2 h. The cultures were incubated at 50°C for 0, 15, 30, 45, and 60 min and at 4°C for 1, 2, 3, and 4 d. The cultures were then diluted to 5.0 × 106 cells/ml and 50 μl samples were plated in triplicate on LB plates containing 50 μg/ml of kanamycin. The plates were incubated overnight at 37°C before scoring colony formation to determine the percentage of survivors. For each treatment, the proportion of surviving E. coli colonies was calculated by dividing the number of viable cells per plate in the treated samples versus untreated samples (no exposure to heat or cold shock). All samples were analyzed in triplicate. Statistical significance was performed using the two-tailed paired Student's t test. Assay of molecular chaperone activity Citrate synthase (Sigma, USA) was diluted with 40 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) KOH buffer at pH 7.5 to a final concentration of 150 nM in 1.0-ml cuvettes and heated at 43°C with 150 nM or 300 nM of purified S.so-HSP20 or at 45°C with 150 nM of purified S.so-HSP20. Aggregation of citrate synthase was monitored by measuring turbidity at 360 and 320 nm with a SPECTRA Max PLUS spectrophotometer every 5 and 10 min for 1 h. Bovine serum albumin (BSA) was used at 150 nM to evaluate non-specific protection of citrate synthase (Chen et al. 2007). Chaperone-like activity of S.so-HSP20 was deter- mined by measuring its ability to prevent dithiothreitol (DTT)-induced aggregation of insulin B chain in 50 mM Tris–HCl (pH 7.4) buffer with 0.15 M NaCl for 25 min at 40°C. The DTT was used at a final concentration of 20 mM. The turbidity of insulin B chain was measured using a SPECTRA max PLUS spectrophotometer at 360 nm. The ratio of S.so-HSP20 to insulin concentration for the assay was 1:2.