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Heterologous High-Level Gene Expression in the Photosynthetic Bacterium Rhodobacter capsulatus

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 824))

Abstract

The functional expression of heterologous genes in standard hosts such as Escherichia coli is often hampered by various limitations including improper folding, incomplete targeting, and missassembly of the corresponding enzymes. This observation led to the development of numerous expression systems that are based on alternative, metabolic versatile hosts. One such organism is the Gram-negative phototrophic nonsulfur purple bacterium Rhodobacter capsulatus. During photosynthetic growth, R. capsulatus exhibits several unique properties including the formation of an intracytoplasmic membrane system as well as the synthesis of various metal-containing cofactors. These properties make R. capsulatus a promising expression host particularly suited for difficult-to-express proteins such as membrane proteins. In this chapter, we describe a novel R. capsulatus expression system and its application.

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References

  1. Baneyx F (1999) Recombinant protein expression in Escherichia coli. Curr. Opin. Biotechnol. 10, 411–421.

    Article  PubMed  CAS  Google Scholar 

  2. Terpe K (2006) Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems. Appl. Microbiol. Biotechnol. 72, 211–222.

    Article  PubMed  CAS  Google Scholar 

  3. Wong SL (1995) Advances in the use of Bacillus subtilis for the expression and secretion of heterologous proteins. Curr. Opin. Biotechnol. 6, 517–522.

    Article  PubMed  CAS  Google Scholar 

  4. Weickert MJ, Doherty DH, Best EA, et al., (1996) Optimization of heterologous protein production in Escherichia coli. Curr. Opin. Biotechnol. 7, 494–499.

    Article  PubMed  CAS  Google Scholar 

  5. Li W, Zhou X, Lu P (2004) Bottlenecks in the expression and secretion of heterologous proteins in Bacillus subtilis. Res. Microbiol. 155, 605–610.

    Article  PubMed  CAS  Google Scholar 

  6. Cereghino JL, Cregg JM (2000) Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol. Rev. 24, 45–66.

    Article  PubMed  CAS  Google Scholar 

  7. Cregg JM, Cereghino JL, Shi J, et al., (2000) Recombinant protein expression in Pichia pastoris. Mol. Biotechnol. 16, 23–52.

    Article  PubMed  CAS  Google Scholar 

  8. León-Banares R, González-Ballester D, Galván A, et al., (2004) Transgenic microalgae as green cell-factories. Trends. Biotechnol. 22, 45–52.

    Article  PubMed  Google Scholar 

  9. De Schutter K, Lin Y-C, Tiels P, et al., (2009) Genome sequence of the recombinant protein production host Pichia pastoris. Nat. Biotechnol. 27, 561–566.

    Article  PubMed  Google Scholar 

  10. Nevalainen KM, Te’o VS, Bergquist PL (2005) Heterologous protein expression in filamentous fungi. Trends Biotechnol. 23, 468–474.

    Article  PubMed  CAS  Google Scholar 

  11. Punt PJ, van Biezen N, Conesa A, et al., (2002) Filamentous fungi as cell factories for heterologous protein production. Trends Biotechnol. 20, 200–206.

    Article  PubMed  CAS  Google Scholar 

  12. Katzke N, Arvani S, Bergmann R, et al., (2010) A novel T7 RNA polymerase dependent expression system for high-level protein production in the phototrophic bacterium Rhodobacter capsulatus. Protein. Expr. Purif. 69(2), 137.

    Article  PubMed  CAS  Google Scholar 

  13. Strnad H, Lapidus A, Paces J, et al., (2010) Complete genome sequence of the photosynthetic purple nonsulfur bacterium Rhodobacter capsulatus SB 1003. J. Bacteriol. doi:10.1128/JB.00366-10.

  14. De Smet L, Kostanjevecki V, Guisez Y, et al., (2001) A novel system for heterologous expression of flavocytochrome c in phototrophic bacteria using the Allochromatium vinosum rbcA promoter. Arch. Microbiol. 176, 19–28.

    Article  PubMed  Google Scholar 

  15. Drepper T, Arvani S, Rosenau F, et al., (2005) High-level transcription of large gene regions: a novel T(7) RNA-polymerase-based system for expression of functional hydrogenases in the phototrophic bacterium Rhodobacter capsulatus. Biochem. Soc. Trans. 33, 56–58.

    Article  PubMed  CAS  Google Scholar 

  16. Kappler U, McEwan AG (2002) A system for the heterologous expression of complex redox proteins in Rhodobacter capsulatus: Characterization of recombinant sulphite: cytochrome c oxidoreductase from Starkeya novella. FEBS Lett. 529, 208–214.

    Article  PubMed  CAS  Google Scholar 

  17. Laible PD, Scott HN, Henry L, et al., (2004) Towards higher-throughput membrane protein production for structural genomics initiatives. J. Struct. Funct. Genomics. 5, 167–172.

    Article  PubMed  CAS  Google Scholar 

  18. Klipp W, Masepohl B, Pühler A (1988) Identification and mapping of nitrogen fixation genes of Rhodobacter capsulatus: Duplication of a nifA-nifB region. J. Bacteriol. 170, 693–699.

    PubMed  CAS  Google Scholar 

  19. Simon R, Priefer U, Pühler A (1983) A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria. Biotechnology 1, 784–791.

    Article  CAS  Google Scholar 

  20. Sambrook J, Russell DW (ed.) (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

    Google Scholar 

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Correspondence to Thomas Drepper .

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Katzke, N., Bergmann, R., Jaeger, KE., Drepper, T. (2012). Heterologous High-Level Gene Expression in the Photosynthetic Bacterium Rhodobacter capsulatus . In: Lorence, A. (eds) Recombinant Gene Expression. Methods in Molecular Biology, vol 824. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-433-9_13

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  • DOI: https://doi.org/10.1007/978-1-61779-433-9_13

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-432-2

  • Online ISBN: 978-1-61779-433-9

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