[Expression and characterization of chaperonin from Sulfolobus solfataricus P2].

OBJECTIVE:To elucidate the structure and functional mechanism of beta subunit of chaperonin from the thermoacidophilic archaeon Sulfolobus solfataricus P2. METHODS:Molecular cloning of the beta 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 beta 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 beta 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 beta monomer could assemble to a homo-oligomer in the presence of ATP and Mg2+. As observed by transmission electron microscope(TEM), the beta subunit homo-oligomer (beta16mer) showed a double-ring structure, which is typical in group II chaperonins. The optimum temperature for ATPase activity of the beta16mer was 80 degrees C. The beta16mer 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 beta subunit of chaperonin from P2. This subunit formed a homo-oligomer in vitro and showed a typical structure of group II chaperonins. We found that the beta16mer 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 thermoacidophilic archaeon.

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