title: Production of Recombinant Cholera Toxin B Subunit in Nicotiana benthamiana Using GENEWARE® Tobacco Mosaic Virus Vector.
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.
authors.affiliation: Owensboro Cancer Research Program, James Graham Brown Cancer Center, University of Louisville, Owensboro, KY, 42303, USA.
authors.forename: Lauren
authors.initials: L
authors.lastname: Moore
authors.affiliation: Owensboro Cancer Research Program, James Graham Brown Cancer Center, University of Louisville, Owensboro, KY, 42303, USA. krystal.hamorsky@louisville.edu.|Department of Medicine, University of Louisville School of Medicine, Louisville, KY, USA. krystal.hamorsky@louisville.edu.
authors.forename: Krystal
authors.initials: K
authors.lastname: Hamorsky
authors.affiliation: Owensboro Cancer Research Program, James Graham Brown Cancer Center, University of Louisville, Owensboro, KY, 42303, USA. n.matoba@louisville.edu.|Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, KY, USA. n.matoba@louisville.edu.
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Matoba
mesh_terms: D002772:Cholera Toxin / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D002845:Chromatography; D005822:Genetic Vectors; D030821:Plants, Genetically Modified; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification*; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism; D014027:Tobacco Mosaic Virus / Q000235:genetics*
chemical_list: D011994:Recombinant Proteins; D002772:Cholera Toxin
keywords: Ceramic hydroxyapatite chromatography; Cholera toxin B subunit; Nicotiana benthamiana; Plant virus vector; Plant-made pharmaceutical