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Applied and Environmental Microbiology, January 2006, p. 102-111, Vol. 72, No. 1
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.1.102-111.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Production of Recombinant and Tagged Proteins in the Hyperthermophilic Archaeon Sulfolobus solfataricus

S.-V. Albers,¹ M. Jonuscheit,² S. Dinkelaker,³ T. Urich,⁴ A. Kletzin,⁴ R. Tampé,³ A. J. M. Driessen,¹ and C. Schleper^2*

Department of Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9751 NN Haren, The Netherlands,¹ Department of Biology, University of Bergen, Jahnebakken 5, Box 7800, N-5020 Bergen, Norway,² Institute for Microbiology and Genetics, Darmstadt University of Technology, Schnittspahnstr. 10, 64287 Darmstadt, Germany,⁴ Institute of Biochemistry, Biozentrum Frankfurt, Johann Wolfgang Goethe University, Marie-Curie-Strasse 9, 60439 Frankfurt am Main, Germany³

Received 10 July 2005/ Accepted 22 September 2005

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°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. solfataricus 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.

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* Corresponding author. Mailing address: Department of Biology, University of Bergen, Jahnebakken 5, Box 7800, N-5020 Bergen, Norway. Phone: 47-555 82665. Fax: 47-555 89671. E-mail: christa.schleper{at}bio.uib.no

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Applied and Environmental Microbiology, January 2006, p. 102-111, Vol. 72, No. 1
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.1.102-111.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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