Studies On The Comprehensive Optimization Of Expression And Fermentation Of Recombinant Exendin-4

Exendin-4is a39amino acid peptide isolated from the salivary secretions ofHeloderma suspectum, a venomous lizard commonly known as the Gila monster. It shows53%sequence identity to mammalian glucagon-like peptide1(GLP-1) and it is the agonistof GLP-1’s receptor. Like GLP-1, Exendin-4could low plasma glucose. Exendin-4has arelatively long half-life (9.57h), GLP-1’s only2min, so Exendin-4has been widelyexplored as a potential therapy in metabolic disease.In this paper, we have carried on a systematic study on the express conditions andfermentation process of recombinant Escherichia coli (BL21(DE3)-pET-32a(+)-Exendin-4).2x YT is the best medium. Use the single-factor test and orthogonal test,study the affect of growth and Express on temperature, bacterial load, opportunity of induce,timing of induce, concentration of inducer, concentration of MgSO4,and so on. Theoptimal conditions of Fermentation are:32℃,3%bacterialload,OD600=1.5,0.1g/L lactose,induce9h.Use the optimal conditions, ferment13h,OD600achieve2.5, about10g/L wetbacteria, The fusion protein was30percent of total protein. We get the phage-resistantmutant with natural selection, the growth and expression of primary are the same to theoriginal strains, the expression of the mutant repeatedly passaged become lower. Ferment themutant in10L fermenter with batch fermentation and batch feed fermentation, study thefermentation process. Finally, batch ferment19h, OD600achieve5.28, about11.91g/L wetbacteria, the fusion protein was30percent of total protein. Batch feed ferment31h, OD600achieve32.25, about110.55g/L wet bacteria, the fusion protein was10percent of totalprotein.