Prokaryotic expression of recombinant GIP protein and optimization of fermentation process

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

Abstract：Glucose-dependent insulin stimulating polypeptide, also known as gastric inhibitory peptide, is a gastrointestinal regulatory peptide composed of 42 amino acids, which can inhibit gastric acid secretion, stimulate insulin release, promote nerve cell proliferation, and has clinical application value. Synthetic or biochemical separation technology to obtain GIP is costly and not suitable for large-scale production, so it is important to use genetic engineering technology to express GIP polypeptides. In this study, the recombinant GIP fusion protein was obtained through the synthesis of DDDDK restriction site and GIP polypeptide gene fragment, and the prokaryotic expression was carried out by pET35b(+) system. In this paper, recombinant Escherichia coli BL21(DE3) /pET35b-GIP expressing GIP polypeptide was fermented and optimized to increase its expression level by using engineering strains constructed in laboratory. Based on TB medium, this paper optimizes each component of the medium at the shaking bottle level through single factor experiment and response surface experiment. Then, the fermentation process conditions were optimized on the basis of the optimal medium. Finally, a preliminary scale-up test was carried out in a 15 L fermenter. The results showed that the expression of fusion protein could reach (1.37± 0.02) g/L after optimized medium, which was 50.55% higher than the initial medium. After the fermentation process optimization, the expression of fusion protein could reach (1.68 ± 0.06) g/L, which was increased to 1.73 times of that before the optimization. In the preliminary amplification test of 15 L fermenter, the highest expression level reached (3.44 ± 0.02) g/L, which was 2.05 times that of the shaker. By optimizing the fermentation medium and process at shaker level and scaling up in a 15 L fermenter, the expression capacity of GIP polypeptides was greatly improved, laying a foundation for scaling up production.

Keywords： GIP polypeptide E. coli Fermentation. optimization Response surface