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