Isolation, Purification And Properties Of A Novel Small Heat Shock Protein From The Hyperthermophile Sulfolobus Solfataricus

The small heat shock proteins are found widely in the cytosol of eukaryotes and archaea. They have important physiological functions in promoting protein folding and preventing the aggregation of denatured protein and play important roles in vitro and in vivo.As a strain of extreme thermophilic archaea, Sulfolobus solfataricus which grows optimally at about 80°C belongs to Sulfolobus, Crenarcheaota. A sHsp had been identified through annotation of its genome. However, there is no report on the functions and properties of the protein. Here we report the characterization of a sHsp expressed from the putative sHsp gene. The sHsp is composed of 124 amino acids encoded by an open reading frame of 375 nucleotides. The putative molecular mass and the isoelectric point of it are 14.1kDa and 8.84, respectively.In order to study its characterization, the gene encoded this protein was cloned from the genome of Sulfolobus solfataricus and constructed the recombinant plasmid using the gene of SsHsp14.1 and pET-28a, which was cloned and expressed in E.coli cells. The target protein could be optimally overexpressed within the E.coli under 37°C for 8h with 1.0mM IPTG and the recombinant SsHsp14.1 expressed was mainly in soluble form. Because of the His Tags on the pET-28a, pure target protein could be able to get through the Ni2+ affinity chromatography and SephadexG50. Results showed that the fusion protein SsHsp14.1 was successfully purified with buffer containing 300mM imidazole using the affinity chromatography and we get the target protein to near homogeneity finally.In this study, the characteristics and functions of the sHsp were futher studied. The results suggested that the expression of SsHsp14.1 could prevent heat-induced proteins aggregation in vivo and it contribute to cellular thermotolerance of E.coli. Chaperone-like activity of SsHsp14.1 was proved by the result of measuring its activity to DTT-induced aggregation of insulin B-chain. We also showed that SsHsp14.1 could interact with purified CALB (Candida antarctica lipase B) in vitro and prevented aggregation of this lipase at 60°C. The result proves that SsHsp14.1 alone provides thermal protection to aggregation-prone proteins without the need for auxiliary proteins. This confirms that SsHsp14.1 has the same chaperone-like activity that has been shown for other sHsps from bacteria and eukaryotes. We also indicated that SsHsp14.1 could protect the catalytic activity of enzymes against thermal stress in vitro, including the bromelain and EcoR I. SsHsp14.1 exhibited broad substrate specificity. SsHsp14.1 itself was very stable in extreme conditions such as high temperature, the heated sHsp was associated with an increase in the thermostability of other protein. This study laid a sound foundation for the further study of the mechanism of sHsps and the relationship between sHsps and thermophilic archaea.