Development of Heterologous Expression System and Optimization of the Method of Cholera Toxin β-Subunit Production in E. coli

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Hamesd H. Jamgochian¹, Mikhail V. Zamakhaev¹, Nikolai N. Sluchanko¹, Anna V. Goncharenko^1,a*, and Mikhail S. Shumkov¹

¹Bach Institute of Biochemistry, Federal Research Center for Biotechnology, Russian Academy of Sciences, 119071 Moscow, Russia

^* To whom correspondence should be addressed.

Received May 18, 2023; Revised August 14, 2023; Accepted August 15, 2023

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Cholera is a deadly infection disease, which is usually associated with low hygiene levels and limited access to high-quality drinking water. An effective way to prevent cholera is the use of vaccines. Among active vaccine components there is the CtxB protein (cholera toxin β-subunit). In the current work, we have developed a genetic system for production of the recombinant CtxB in E. coli cells and studied conditions for synthesis and purification of the target product at the laboratory scale. It has been found that the optimal algorithm for isolation of the recombinant protein is to grow E. coli culture in the synthetic M9 medium with glycerol, followed by CtxB purification out of the spent culture medium using Ni²⁺-chelate affinity chromatography techniques. Forty-eight hours after induction of CtxB expression, concentration of the target product could be up to 50 mg/liter in the culture medium. The CtxB protein retains its pentameric structure during expression and through purification. The latter makes it possible to consider the developed system as a promising tool for the industrial-level production of recombinant CtxB for medical and research purposes.

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KEY WORDS: cholera toxin β-subunit, CtxB, expression, recombinant protein

DOI: 10.1134/S0006297923090109

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