Cloning, Expression and Purification of the Beta Subunit of Cholera Toxin Using Escherichia coli as an Expression Host and pET-24a(+) as a Cloning Vector

Cholera toxin has a biologically active A subunit and a binding B subunit. Being non-toxigenic, the Cholera toxin B (CTB) subunit has multiple applications in immunology and rapid diagnostics. A recombinant form of CTB (rCTB) is available for commercial use. However, the COVID-19 pandemic led to a severe global shortage in the production and distribution of commercial CTB, which posed a significant problem for researchers worldwide. Due to the non-availability of commercial rCTB post the COVID-19 pandemic, we attempted and successfully produced rCTB in a pET-based expression system in <i>Escherichia coli</i>. To the best of our knowledge, production of rCTB in a pET-based expression system in <i>E. coli</i> is reported for the first time in India. The <i>ctx</i>B gene was amplified from toxigenic <i>Vibrio cholerae</i> and cloned into the pET-24a(+) plasmid and inserted into <i>E. coli</i> DH5α. The plasmid was extracted, and the cloned gene from the pET-24a(+) vector was amplified and Sanger sequenced. The recombinant plasmid was transformed into <i>E. coli</i> BL21(DE3) and induced at different temperatures and concentrations of IPTG. The optimum time, temperature, and IPTG concentration were identified, and the rCTB protein was expressed in large culture and purified using Ni-NTA chromatography and dialysed. The protein was sequenced and confirmed as rCTB using LC-MS. Specific primers designed for the CTB gene amplified a product with the expected size of 341 bp. DNA sequencing confirmed the identity and correct orientation of CTB in the construct and translated using the Expasy translate tool. Optimum conditions for induction were 1mM IPTG, 16℃ for 16 hours. Confirmation of protein was done by liquid chromatography-mass spectrometry. The protein matched with cholera enterotoxin subunit B of <i>V. cholerae</i> serotype O1 (strain ATCC 39315) with a score of 82, and the induced protein was confirmed as rCTB. Multiple independent inductions yielded a total protein of ~1+/- 0.1mg per liter of induced culture. The <i>E. coli</i> pET-24a(+) plasmid-based expression system is a practical and efficient method for producing rCTB.