Study On Expression Of Optimized RolGLP-1in E.Coli And Its Application

Up to now, there are nearly300million diabetics over the world, most of which are type2diabetes mellitus. T2DM is a major risk factor for people’s health, a quick development that has followed rapid economic growth and changes in lifestyle. Although many therapies have become available for T2DM, such as diet control, appropriate exercise and useof pharmacolo-gical agents, a number of T2DM patients may still require exogenous insulin for absolute defect of endogenous insulin as a result of progressively degenerating β cells. Therefore, some new agents offering such protection and restoration for β cells may be a promising approach for T2DM.Incretin, also known as glucagon-like peptide-1(GLP-1), can significantly enhance the biosynthesis and secretion of insulin, promote the regeneration of β cells, inhibit the apoptosis of β cells and improve the function of islet by binding to the GLP-1receptor, a member of the G protein-coupled receptor family. It also can reduce glucagon secretion, delay gastric emptying, induce satiety, and improve peripheral insulin sensitivity. Especially, there is no hypoglycemia appeared in the treatment of T2DM with GLP-1because the insulinotropic activity of GLP-1depends on the concentration of blood glucose. Due to its antidiabetic characteristics, GLP-1is at the forefront focus of the pharmaceutical industry’s attention.Unfortunately, GLP-1is limited as a therapeutic agent for its short biological half-life due to DPP Ⅳ-mediated degradation. GLP-1-(7-37) or GLP-1-(7-36) is the main active forms of GLP-1in the body. The amide bond (-NHCO-) of Ala8at N-terminal in GLP-1(7-37) or GLP-1(7-36) is very susceptive to DPP Ⅳ cleavage, resulting in the truncated peptide, GLP-1(9-37) or GLP-1(9-36), a process that is so rapid that the estimated half-life of GLP-1in the circulation is only about1minute. Therefore, more attention needs to be paid on searching for more stable GLP-1analog.In light of this, our laboratory has developed the recombinant orally long-acting GLP-1(rolGLP-1) by point mutation, a new GLP-1analog, which is resistant to both DPP-IV and trypsin. Hou et al proved that GLP-M containing10tandem repeated rolGLP-1could significantly reduce the level of blood glucose and improve the abnormal signs caused by high concentration of blood glucose in the diabetic rats. However, there was a defect in the previous design, due to the redundant amino acids of GLP-M caused by the linker (Proline-Isoleucine-Arginine) between each copy of rolGLP-1and purification tag (6copies of Histidine). Although there were no obvious side-effects in the short-time use of these redundant amino acids, but the safety of long-term use is not guaranteed. Moreover, the redundant amino acids will affect the approval of this drug by Food and Drug Administration. In view of this, to ensure seamless connections between each copy of rolGLP-1, we synthesized the gene of ten tandem rolGLP-1(Tsingke Biological Technology Co., Ltd, Beijing) and constructed optimized rolGLP-1(Opt-rolGLP-1) expressed in E.coli BL(DE3), using the techniques of molecular cloning and site-directed mutagenesis.The transformed engineering strains E.coli BL21(DE3)/Opt-rolGLP-1was induced with isopropyl-β-D-thiogalactopyranoside (IPTG) and then analysised by SDS-PAGE electrophoresis and Western Blotting. The results showed that Opt-rolGLP-1expressed efficiently in E.coli BL21(DE3). The expression condition of Opt-rolGLP-1was optimized and the results showed that, the best expression condition of Opt-rolGLP-1was under induction by IPTG with the final concentration of1.0mM, and incubated at25℃for8h. The harvested cells were disrupted by ultrasonication after resuspension with PBS buffer. The supernatant fraction was purified by Anion Exchange Chromatography and Gel Filtration Chromatography. It’s pleased that the purity of was high to98%and this purity can meet the requirements of animal experiments.The digestion assay of Opt-rolGLP-1in vitro demonstrated tha Opt-rolGLP-1can be cut into single copy rolGLP-1to play a role, for that the last amino acid of C terminal in the Opt-rolGLP-1is arginine, the recognition and cleavage site for trypsin.High fat and sugar diet and STZ (100mg/kg, intraperitoneal injection) were employed to construct the mice model of type2diabetes. The mice model of type2diabetes was constructed successfully twice by this method, which proved that the method is mature and stable. Diabetic mice were treated by Opt-rolGLP-1and rolGLP-1oral administration or subcutaneous injection of rolGLP-1. The average food intake and water intake decreased after treatment. The diabetic group showed a trend of decreased body weight, while the Opt-rolGLP-1or rolGLP-1treated groups remained unaltered. It’s important that he levels of fasting plasma glucose, cholesterol, triglycerides and glycosylated hemoglobin were lowered significantly after treatment. Moreover, hyperinsulinemia and oral glucose tolerance were inproved obviously by Opt-rolGLP-1or rolGLP-1. Another important finding was that the levels of GLUT2, INSR, GCK mRNA in liver were raise by Opt-rolGLP-1or rolGLP-1, while the expression of PGC-1α mRNA in liver decreased, which might be the part of the mechanism for Opt-rolGLP-1or rolGLP-1to cure the type2diabetes.The effect of Opt-rolGLP-1on food intake, water intake and weight in normal mice was also studied. The results showed that Opt-rolGLP-1can reduce the food intake and water intake of normal Kunming mice, and delay weight gain in mice, indicated that Opt-rolGLP-1maintain the activity of suppressing appetite and delaying gastric emptying.This study provides the satisfactory evidences for the research of orally protein drugs and settles the foundation for further clinical application of Opt-rolGLP-1.