Expression and characterization of chaperonin fromSulfolobus solfataricus P2

Abstract:

[Objective] To elucidate the structure and functional mechanism of b subunit of chaperonin from the thermoacidophilic archaeon Sulfolobus solfataricus P2. [Methods] Molecular cloning of the b subunit gene of chaperonin from the thermoacidophilic archaeon Sulfolobus solfataricus P2 was performed by using PCR technique. The gene was expressed in BL21(DE3) of Escherichia coli. After purified and assembled in vitro, the structure of the b subunit homo-oligomer was observed by transmission electron microscope (TEM). The function of this homo-oligomer as a chaperonin was evaluated. [Results] The gene encoding b subunit of chaperonin was amplified by PCR from the genomic DNA of Sulfolobus solfataricus P2 and expressed in BL21(DE3) of E. coli. In vitro, the purified β monomer could assemble to a homo-oligomer in the presence of ATP and Mg2+. As observed by transmission electron microscope(TEM), the b subunit homo-oligomer (b16mer) showed a double-ring structure, which is typical in groupⅡchaperonins. The optimum temperature for ATPase activity of the b16mer was 80℃. The β16mer was able to promote the refolding of denatured GFP and improve the thermostability of xylanase. [Conclusion] According to the prediction and analysis of the chaperonin sequence from thermoacidophilic archaeon Sulfolobus solfataricus P2 genome, we cloned and expressed the b subunit of chaperonin from P2. This subunit formed a homo-oligomer in vitro and showed a typical structure of groupⅡchaperonins. We found that the b16mer was able to function correctly when promoting the refolding and improving the thermostability of some other proteins. Our research has laid a foundation for the further study on the molecular mechanism of ther-moacidophilic archaeon.