| Small heat shock proteins (sHSPs) are ubiquitous in the organisms. It has been reported that sHSPs could improve thermal stability of cells in vivo, prevent heterogenous proteins from thermal-aggregation and help denatured proteins to re-fold in vitro. So far, much more attention has been focused on the sHSPs from thermophiles because of their high thermal stability. Fewer literatures have been reported on the structure and mechanism of sHSPs, so that studies on sHSPs protein will be valuable for the basic research and applied biotechnology.In this study, the coding sequence of SsHSP14.1 from the hyper-thermophilic archeaon, Sulfolobus solfataricus (S. solfataricus), has been firstly cloned. The recombinant SsHSP14.1 was expressed and purified. The function and mechanism of SsHSP14.1 were further investigated.Cloning, expression and purification of SsHSP14.1: The coding sequence of SsHSP14.1 was amplified by PCR with genome DNA as template, and was ligated to the vector pET28a. E. coli BL21(DE3) harboring the expression vector was screened. The optimum conditions for the expression of SsHSP14.1 were:5% of inoculums amount, 1mM of final concentration of IPTG and 6h of induction time. The protein was purified by Nickle column and anion exchange chromatography, 27.62% of SsHSP14.1 was yielded with a purity of 92%. Study on the function and characteristic of SsHSP14.1: 1) The cell viability of E.coli with and without SsHSP14.1 expression at high temperature was compared. SsHSP14.1 has been proved to be able to improve the thermal stability of E. coli. After 1h of incubation at 50℃, cell viability was 19.9% when no SsHSP14.1 was expressed in the cells, while the viability increased to 31.2% when SsHSP14.1 was expressed.2) SsHSP14.1 could prevent proteins from aggregation. Experimental results showed that 90% of E. coli proteins were aggregated after 2h at 80℃, but 46.8% of them remained soluble under the same condition when SsHSP14.1 was added. With the addition of SsHSP14.1, the content of soluble CALB increased two times after 20min incubation at 50℃.3) SsHSP14.1 could improve the themal stability of enzymes. Bromelain was totally inactivated after being incubated at 60℃for 40min, while 40% of relative activity remained with the addition of SsHSP14.1, and 30% of which was still remained after 1h of incubation. The addition of SsHSP14.1 could reduce the inactivation rate, and the refolding of inactivated EcoRI was promoted by SsHSP14.1.4) The on ATP-dependence, the thermal stability and the oligomerization of SsHSP14.1 were investigated. It was indicated that SsHSP14.1 was ATP-independent, and existed as large oligomers (360kD), its oligomerization state was affected by the concentration of salt. The high thermal stability of SsHSP14.1 was proved, and the chaperone function of SsHSP14.1 could be remained after being heated in the boiling water for 15min.Study on the mechanism of SsHSP14.1: The structural model of SsHSP14.1 dimer was predicted by homology modeling. On the basis of the model, key amino acid residues (L109,E33,K75,K75-Q79) were mutated and cloned. The corresponding mutants (L109D,K75E,E33K/K75E,DEL75-79) were expressed and purified, and their structure and function were studied, and were compared to that of SsHSP14.1. The results showed that: 1) L109, K75 and E33 were crucial to the structure and function of SsHSP14.1. The mutant proteins, L109D and K75E, both existed as monomers without chaperone activities. While K75E/E33K could form large polymers with EcoRI and had similar function to SsHSP14.1. 2) The domain L5-7 was important to improve the thermal stability of enzymes. Deletion of this domain had little effect on the oligomerization of protein. The mutant DEL75-79 was as effective as SsHSP14.1 in preventing proteins from aggregating, but it could not protect EcoRI and bromelain from thermal inactivation. Results of ANS binding experiment showed that the hydrophobicity of DEL75-79 was lower than that of SsHSP14.1, indicating that the change of its function was related to its structure.
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