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Production of Recombinant Cholera Toxin B Subunit in Nicotiana benthamiana Using GENEWARE® Tobacco Mosaic Virus Vector

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Recombinant Proteins from Plants

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1385))

Abstract

Here, we describe a method to produce a recombinant cholera toxin B subunit in Nicotiana benthamiana plants (CTBp) using the GENEWARE® tobacco mosaic virus vector system. Infectious transcripts of the vector RNA are generated in vitro and inoculated on N. benthamiana seedlings. After 11 days, CTBp is extracted in a simple tris buffer at room temperature. No protease inhibitor is required. The leaf homogenate is treated with mild heat and a pH shift to selectively precipitate host-derived proteins. CTBp is purified to >95 % homogeneity by two-step chromatography using immobilized metal affinity and ceramic hydroxyapatite resins. This procedure yields on average 400 mg of low-endotoxin CTBp from 1 kg of fresh leaf material.

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References

  1. Jelinek T, Kollaritsch H (2008) Vaccination with Dukoral against travelers’ diarrhea (ETEC) and cholera. Expert Rev Vaccines 7(5):561–567

    Article  CAS  PubMed  Google Scholar 

  2. Sanchez J, Holmgren J (2011) Cholera toxin—a foe & a friend. Indian J Med Res 133:153–163

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Sun JB, Czerkinsky C, Holmgren J (2010) Mucosally induced immunological tolerance, regulatory T cells and the adjuvant effect by cholera toxin B subunit. Scand J Immunol 71(1):1–11

    Article  CAS  PubMed  Google Scholar 

  4. Matoba N, Davis KR, Palmer KE (2011) Recombinant protein expression in Nicotiana. Methods Mol Biol 701:199–219

    Article  CAS  PubMed  Google Scholar 

  5. Hamorsky KT, Kouokam JC, Bennett LJ et al (2013) Rapid and scalable plant-based production of a cholera toxin B subunit variant to aid in mass vaccination against cholera outbreaks. PLoS Negl Trop Dis 7(3):e2046

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Shivprasad S, Pogue GP, Lewandowski DJ et al (1999) Heterologous sequences greatly affect foreign gene expression in tobacco mosaic virus-based vectors. Virology 255(2):312–323

    Article  CAS  PubMed  Google Scholar 

  7. Pogue GP, Vojdani F, Palmer KE et al (2010) Production of pharmaceutical-grade recombinant aprotinin and a monoclonal antibody product using plant-based transient expression systems. Plant Biotechnol J 8(5):638–654

    Article  CAS  PubMed  Google Scholar 

  8. Dertzbaugh MT, Cox LM (1998) The affinity of cholera toxin for Ni2+ ion. Protein Eng 11(7):577–581

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was supported by DoD/USAMRAA/TATRC/W81XWH-10-2-0082-CLIN2 and the Helmsley Charitable Trust Fund.

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Authors and Affiliations

  1. Owensboro Cancer Research Program, James Graham Brown Cancer Center, University of Louisville, Owensboro, KY, 42303, USA

    Lauren Moore, Krystal Hamorsky & Nobuyuki Matoba

  2. Department of Medicine, University of Louisville School of Medicine, Louisville, KY, USA

    Krystal Hamorsky

  3. Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY, USA

    Nobuyuki Matoba

Authors
  1. Lauren Moore
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  2. Krystal Hamorsky
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  3. Nobuyuki Matoba
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Corresponding authors

Correspondence to Krystal Hamorsky or Nobuyuki Matoba .

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Editors and Affiliations

  1. Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, Ontario, Canada

    Jacqueline MacDonald

  2. Department of Biology, Western University, London, Ontario, Canada

    Igor Kolotilin

  3. London, Ontario, Canada

    Rima Menassa

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Moore, L., Hamorsky, K., Matoba, N. (2016). Production of Recombinant Cholera Toxin B Subunit in Nicotiana benthamiana Using GENEWARE® Tobacco Mosaic Virus Vector. In: MacDonald, J., Kolotilin, I., Menassa, R. (eds) Recombinant Proteins from Plants. Methods in Molecular Biology, vol 1385. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3289-4_9

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  • DOI: https://doi.org/10.1007/978-1-4939-3289-4_9

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3288-7

  • Online ISBN: 978-1-4939-3289-4

  • eBook Packages: Springer Protocols

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