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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Acknowledgements
We thank Lauren J. Moore for technical assistance. This work was supported by DoD/USAMRAA/TATRC/W81XWH-10-2-0082-CLIN2 and the Helmsley Charitable Trust Fund.
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Hamorsky, K., Matoba, N. (2016). Facile Method for the Production of Recombinant Cholera Toxin B Subunit in E. coli. In: Thomas, S. (eds) Vaccine Design. Methods in Molecular Biology, vol 1404. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-3389-1_33
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DOI: https://doi.org/10.1007/978-1-4939-3389-1_33
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Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-3388-4
Online ISBN: 978-1-4939-3389-1
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