硫矿硫化叶菌P2分子伴侣基因的克隆表达及其性质

Expression and characterization of chaperonin fromSulfolobus solfataricus P2

摘要:

【目的】研究重组表达的硫矿硫化叶菌P2分子伴侣b亚基体外同源聚合体的结构和生化功能。【方法】利用PCR技术从硫矿硫化叶菌P2的基因组DNA中克隆得到分子伴侣b亚基的基因，将该基因克隆到表达载体pET-21a(+)上并在大肠杆菌BL21(DE3)中实现了表达。对纯化后的b亚基单体进行体外聚合，利用透射电镜观察b分子伴侣的结构，并对其促蛋白折叠性质进行了研究。【结果】硫矿硫化叶菌P2分子伴侣b亚基基因在大肠杆菌BL21中实现了高效表达，纯化后的分子伴侣b亚基单体在ATP和Mg2+存在的条件下可自组装形成分子伴侣聚合体。透射电镜观察表明：该b分子伴侣具有Ⅱ型分子伴侣典型的双层面包圈结构，每个环由8个亚基构成。该b分子伴侣具有ATPase活性，最适反应温度为80℃；它不仅能够促进变性的绿色荧光蛋白（GFP）重新折叠，而且还能有效的提高木聚糖酶的热稳定性。【结论】本文根据P2基因组序列分析预测的分子伴侣基因设计引物，克隆表达了硫矿硫化叶菌P2分子伴侣的b亚基, 纯化后对其进行体外聚合，透射电镜观察表明该聚合体具有Ⅱ型分子伴侣的经典结构，功能分析表明该b分子伴侣能够在体外促进异源蛋白质的折叠、提高其它酶分子的热稳定性。这为进一步深入研究嗜热古菌耐热抗逆的分子机制，奠定了良好的基础。

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.