Fermentation Optimization of Recombinant Semaglutide Precursor Protein

• 1、School of bioengineering, Jiangnan University, Wuxi 214122, China

Abstract：Semaglutide is a long-acting GLP-1 receptor agonist that promotes insulin release in a glucose-dependent manner, suppresses glucagon secretion, and delays gastric emptying. With its excellent safety profile, broad indications, and long half-life, it has emerged as a standout among similar hypoglycemic drugs and has become a focus of clinical research. The traditional solid-phase peptide synthesis method requires large amounts of organic solvents, causing significant environmental pollution, making the semi-recombinant approach a preferable alternative.In this study, artificially synthesized enzyme cleavage sites such as DDDDK enterokinase, Kex2, and CPB, along with the GLP-1 (9-37) peptide gene fragment, were used for prokaryotic expression via the pET22b(+) system, resulting in a trivalent recombinant fusion protein. This paper focuses on constructing a successful engineered strain and optimizing the fermentation of recombinant E. coli BL21(DE3)/pET22b-GLP to enhance the expression of the GLP-1 (9-37) peptide.The research began with TB medium as the base, optimizing its components through single-factor and response surface experiments at the shake flask level. Subsequently, fermentation process conditions were optimized based on the optimal medium. Finally, preliminary scale-up verification was conducted in a 15 L fermenter.The results showed that after medium optimization, the fusion protein expression reached (5.43 ± 0.05) g/L, representing a 65.04% increase compared to the initial medium. Following fermentation process optimization, the expression level remained at (5.43 ± 0.13) g/L. In the 15 L fermenter scale-up experiment, the maximum expression level achieved was (8.50 ± 0.16) g/L, 1.56 times higher than the shake flask level.By optimizing the fermentation medium and process at the shake flask level and validating the scale-up in a 15 L fermenter, the expression capacity of the GLP-1 (9-37) peptide was significantly enhanced, laying a solid foundation for large-scale production.

Keywords： Semaglutide GLP-1 Peptide Escherichia coli Fermentation Optimization