Heat shock proteins (HSPs) are induced in response to various physiological or environmental stressors. However, the transcriptional activation of HSPs is regulated by a family of heat shock factors (HSFs). Fish models provide an ideal system for examining the biochemical and molecular mechanisms of adaptation to various temperatures and water environments. In this study, we examined the pattern differentials of heat shock factor 1 (HSF1) and expression of heat shock protein 70 (HSP70) in response to thermal stress in goldfish and mouse hepatocyte cultures by immune-blot analysis. Goldfish HSF1 (gfHSF1) changed from a monomer to a trimer at 33°C and showed slightly at 37°C, whereas mouse HSF1 (mHSF1) did so at 42°C. This experiment showed similar results to a previous study, indicating that gfHSF1 and mHSF1 play different temperature in the stress response. We also examined the activation conditions of the purified recombinant proteins in human HSF1 (hmHSF1) and gfHSF1 using CD spectroscopy and immune-blot analysis. The purified recombinant HSF1s were treated from 25°C to 42°C. Structural changes were observed in hmHSF1 and gfHSF1 according to the heat-treatment conditions. These results revealed that both mammal HSF1 (human and mouse HSF1) and fish HSF1 exhibited temperature-dependent changes; however, their optimal activation temperatures differed.

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Eukaryotic expression systems are used widely and have the advantages of protein processing, proteolytic cleavage, disulfide bond formation, and posttranslational modification in contrast to the prokaryotic expression system. In the present study, peptide gene (olive flounder beta‐defensin or hepcidin) was inserted into the vector of pPIC9K, which involved the secretion signal and promoter AOX1. The colonies with high copy numbers of the target gene for high‐level expression were selected by G418. Approximately 30 mg/L for beta‐defensin and 25 mg/L for hepcidin was obtained from the culture medium supernatant. An ammonium sulfate salting‐out method was used for purification; this one‐step purification simplified the procedures, and the purification effect was good in terms of the purity and yield. The proteins from yeast itself could be isolated easily using the ammonium sulfate salting‐out method.

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Peptides containing multiple disulfide bonds are usually problematic when expressed in Escherichia coli. We conducted the expression of β-defensin with three disulfide bonds, hepcidin with four disulfide bonds, and DkTx with six disulfide bonds using a small leader protein mosaic expression in the periplasm of E. coli, and purified them by affinity chromatography and characterized them by mass spectroscopy. The result showed that the expression level was high. A large amount of the pure recombinant peptide was also recovered after purification with a Ni 2+ affinity column. The mass results by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) indicated that the recombinant peptides had a folding structure, with the native status of all of the cysteines participating in disulfide bond formation. Moreover, after removing the tags, the result was identical to its natural form as well. We thus provide a method for producing large amounts of soluble peptides containing multiple disulfide bonds in E. coli.