title: Facile Method for the Production of Recombinant Cholera Toxin B Subunit in E. coli.
abstract: Herein, we report an Escherichia coli-based expression and purification method of recombinant cholera toxin B subunit (CTB). The CTB gene (E. coli codon optimized) is cloned into commercial pET-22b(+) vector using standard molecular biology techniques and the resulting vector is transformed into BL21(DE3) electrocompetent cells. The bacterial cells are grown and induction with isopropyl β-D-1-thiogalactopyranoside (IPTG) results in accumulation of CTB in the culture medium. CTB is purified from the culture medium using a simple two-step chromatography process: immobilized metal affinity chromatography (IMAC) followed by ceramic hydroxyapatite (CHT). CTB is purified to >95 % homogeneity with a yield of over 10 mg per liter of culture. Depending on the application, endotoxin is removed using a commercially available endotoxin removal resin to <1 EU/mg.
authors.affiliation: Owensboro Cancer Research Program, University of Louisville James Graham Brown Cancer Center, 1020 Breckenridge Street Suite 201, 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, University of Louisville James Graham Brown Cancer Center, 1020 Breckenridge Street Suite 201, 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* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D002846:Chromatography, Affinity; D017886:Durapatite / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D011994:Recombinant Proteins; D002772:Cholera Toxin; D017886:Durapatite
keywords: Ceramic hydroxyapatite chromatography; Cholera toxin B subunit; Immobilized metal affinity chromatography; Mucosal vaccine; pET expression system; pelB signal sequence