# 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

## TL;DR

Researchers in India successfully produced a non-toxic cholera toxin subunit (CTB) using E. coli and a pET-24a(+) plasmid, addressing a global shortage caused by the pandemic.

## Contribution

This is the first reported production of recombinant CTB in a pET-based system in India.

## Key findings

- The CTB gene was cloned into pET-24a(+) and expressed in E. coli with optimal conditions of 1mM IPTG and 16°C for 16 hours.
- Purified rCTB was confirmed via LC-MS and matched the expected cholera toxin subunit B with a score of 82.
- The system yielded approximately 1 ± 0.1 mg of rCTB per liter of culture.

## Abstract

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 Escherichia coli. To the best of our knowledge, production of rCTB in a pET-based expression system in E. coli is reported for the first time in India.

The ctxB gene was amplified from toxigenic Vibrio cholerae and cloned into the pET-24a(+) plasmid and inserted into E. coli 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 E. coli 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 V. cholerae 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 E. coli pET-24a(+) plasmid-based expression system is a practical and efficient method for producing rCTB.