Chapter title |
Facile Method for the Production of Recombinant Cholera Toxin B Subunit in E. coli.
|
---|---|
Chapter number | 33 |
Book title |
Vaccine Design
|
Published in |
Methods in molecular biology, January 2016
|
DOI | 10.1007/978-1-4939-3389-1_33 |
Pubmed ID | |
Book ISBNs |
978-1-4939-3388-4, 978-1-4939-3389-1
|
Authors |
Krystal Hamorsky, Nobuyuki Matoba |
Editors |
Sunil Thomas |
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. |
Mendeley readers
Geographical breakdown
Country | Count | As % |
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Unknown | 2 | 100% |
Demographic breakdown
Readers by professional status | Count | As % |
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Student > Ph. D. Student | 1 | 50% |
Unknown | 1 | 50% |
Readers by discipline | Count | As % |
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Biochemistry, Genetics and Molecular Biology | 1 | 50% |
Unknown | 1 | 50% |