Facile Method for the Production of Recombinant Cholera Toxin B Subunit in E. coli
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
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Introduction CTB is the nontoxic subunit of cholera holotoxin. The protein forms stand-alone, stable homopentamers with a molecular mass of about 55 kDa [1]. Bacterially produced recombinant CTB is currently used as an active component of internationally licensed, World Health Organization-prequalified oral cholera vaccine (Dukoral®, Crucell) to induce holotoxin-neutralizing antibodies in the gut [2]. CTB is a strong vaccine adjuvant and has been used as a scaffold for vaccine development against bacterial and viral pathogens (reviewed in ref. 3). In addition, studies have revealed that CTB induces antiinflammatory responses and suppresses immunopathological reactions in allergy and autoimmune diseases (reviewed in ref. 3). Commercially available nonrecombinant CTB contains trace amounts of cholera toxin and cholera toxin A subunit [4], which can influence the biological activity of CTB [3]. Therefore, high-quality recombinant CTB is necessary for immunological research. We have developed a simple E. coli-based expression and twostep purification scheme to produce high-purity recombinant CTB. The pET expression system was used for CTB expression. pET-22b(+) expression vector contains the T7 promoter which is known to drive high expression levels of recombinant proteins. Moreover, pET-22b(+) contains the N-terminal pelB leader sequence to target recombinant proteins to the periplasm. In this case, CTB was secreted into the bacterial culture medium, allowing for facile isolation and purification of the protein. Immobilized metal affinity chromatography (IMAC) and ceramic hydroxyapatite (CHT) chromatography were used to purify CTB. CTB is known to bind to immobilized Ni2+ ions through internal histidine resides
Sunil Thomas (ed.), Vaccine Design: Methods and Protocols, Volume 2: Vaccines for Veterinary Diseases, Methods in Molecular Biology, vol. 1404, DOI 10.1007/978-1-4939-3389-1_33, © Springer Science+Business Media New York 2016
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Krystal Hamorsky and Nobuyuki. Matoba
[5]; therefore, CTB can be purified to high purity using IMAC. CHT is a multimodal resin that utilizes cation exchange and metal affinity and is known to offer unique selectivity and often separates biomolecules that appear homogenous using other chromatographic methods. Furthermore, CHT aids in removing nonproteinous contaminants such as DNA and endotoxins. The expression and purification scheme described herein allows for an easy and efficient way to manufacture recombinant CTB, which may facilitate immunological research and vaccine development.
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Materials Prepare all solutions using ultrapure Milli-Q water (Milli-Q Synthesis, Millipore, 18.2 MΩ cm at 25 °C) and analytical grade reagents. Prepare and store all reagents at room temperature (unless indicated otherwise). CTB purified product is stored at 4 °C until use.
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CTB Expression
1. pET22b-CTB: pET-22b(+) (Novagen) vector containing the coding sequence for Vibrio cholerae CTB gene (GenBank accession no. AAC34728) (obtained via standard molecular biology/subcloning proced
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