Production of recombinant and tagged proteins in the hyperthermophilic archaeon Sulfolobus solfataricus
Production of recombinant and tagged proteins in the hyperthermophilic archaeon Sulfolobus solfataricus
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DOI：
10.1128/aem.72.1.102-111.2006
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发表时间：
2006-01-01
影响因子：
4.4
通讯作者：
Schleper, C
中科院分区：
文献类型：
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作者：
Albers, SV;Jonuscheit, M;Schleper, C
许多系统可用于在细菌和真核模式生物中生产重组蛋白，这使我们能够研究其天然宿主中的蛋白质并鉴定蛋白质-蛋白质相互作用伙伴。相比之下，针对古细菌只开发了少数转化系统，并且针对超嗜热生物不存在高水平基因表达的系统。最近，为crenarchaeote Sulfolobus solfataricus开发了具有报告基因的基于病毒的穿梭载体，crenarchaeote Sulfolobus solfataricus是在80摄氏度下最佳生长的超嗜热古菌的模式生物（M. Jonuscheit，E. Martusewitsch，K. M. Stedman和C.施莱珀山Microbiol. 48：1241-1252，2003）。在这里，我们已经完善了这个系统的高水平基因表达在S。在两种不同的启动子的帮助下，主要伴侣蛋白，嗜热因子55的热诱导型启动子和阿拉伯糖结合蛋白AraS的阿拉伯糖诱导型启动子，将其转化到soffiataricus中。证明了异源和同源基因的功能性表达，包括从两价酸菌产生细胞质硫加氧酶还原酶，从S. solfataricus，和两个膜相关的ATP酶可能参与蛋白质的分泌。通过融合His或Strep标签获得蛋白质的单步纯化。据我们所知，这些是应用表达载体系统在超嗜热古菌中产生大量重组蛋白和标记蛋白的第一个例子。
Many systems are available for the production of recombinant proteins in bacterial and eukaryotic model organisms, which allow us to study proteins in their native hosts and to identify protein-protein interaction partners. In contrast, only a few transformation systems have been developed for archaea, and no system for high-level gene expression existed for hyperthermophilic organisms. Recently, a virus-based shuttle vector with a reporter gene was developed for the crenarchaeote Sulfolobus solfataricus, a model organism of hyperthermophilic archaea that grows optimally at 80 degrees C (M. Jonuscheit, E. Martusewitsch, K. M. Stedman, and C. Schleper, Mol. Microbiol. 48:1241-1252, 2003). Here we have refined this system for high-level gene expression in S. soffiataricus with the help of two different promoters, the heat-inducible promoter of the major chaperonin, thermophilic factor 55, and the arabinose-inducible promoter of the arabinose-binding protein AraS. Functional expression of heterologous and homologous genes was demonstrated, including production of the cytoplasmic sulfur oxygenase reductase from Acidianus ambivalens, an Fe-S protein of the ABC class from S. solfataricus, and two membrane-associated ATPases potentially involved in the secretion of proteins. Single-step purification of the proteins was obtained via fused His or Strep tags. To our knowledge, these are the first examples of the application of an expression vector system to produce large amounts of recombinant and also tagged proteins in a hyperthermophilic archaeon.