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Updated: Mar 22, 2026

Overexpressing and Purifying a Toxic Nuclease from Escherichia coli
Published on: August 29, 2025
Facile Method for the Production of Recombinant Cholera Toxin B Subunit in E. coli
Krystal Hamorsky1,2, Nobuyuki Matoba3,4
1Owensboro Cancer Research Program, University of Louisville James Graham Brown Cancer Center, 1020 Breckenridge Street Suite 201, Owensboro, KY, 42303, USA. krystal.hamorsky@louisville.edu.
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.
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Cholera
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