Chapter title |
Production of Recombinant Cholera Toxin B Subunit in Nicotiana benthamiana Using GENEWARE ® Tobacco Mosaic Virus Vector
|
---|---|
Chapter number | 9 |
Book title |
Recombinant Proteins from Plants
|
Published in |
Methods in molecular biology, January 2016
|
DOI | 10.1007/978-1-4939-3289-4_9 |
Pubmed ID | |
Book ISBNs |
978-1-4939-3288-7, 978-1-4939-3289-4
|
Authors |
Lauren Moore, Krystal Hamorsky, Nobuyuki Matoba |
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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