Eukaryotic Expression And Activity Detection Of GLP-1-IgG2σ Fc Fusion Protein

Glucagon-like peptide-1 (GLP-1) is an incretin hormone encoded by the proglucagon gene. This 30-amino acid protein exerts its biological effects by activating a G-protein-coupled receptor. GLP-1 improves glycemic control by reducing their postprandial and fasting glucose levels. Its major pharmacological activities include the promotion of first- and second-phase insulin secretion, the suppression of glucagon activity under hyperglycemic conditions, and delaying the gastric emptying rate. Since GLP-1 has the effect of lowering blood glucose and reducing weight, so when the drug as a treatment for Type Ⅱ diabetes can effectively prevent the occurrence of hypoglycemia and insulin resistance. GLP-1 is rapidly degraded in vivo and has a plasma elimination half-life (t1/2) of 2 min because of its rapid enzymatic degradation by dipeptidyl peptidase Ⅳ (DPP-Ⅳ) and rapid kidney clearance. Cleavage site replacement and the addition of a macromolecular protein or an aliphatic chain were used in the structural modification in order to extend the half-life of GLP-1. Some novel GLP-1 agonists have been approved for use in the treatment of type 2 diabetes, such as Exenatide、Liraglutide and Dulaglutide. In this study, GLP-1-IgG2σ-Fc was constructed by fusing the human IgG2σ constant heavy-chain with a GLP-1 variant (A8G/G26E/R36G). GLP-1-IgG2σ-Fc fusion protein was expressed using CHO-K1 mammalian cell expression platform. GLP-1-IgG2σ-Fc was purified by chromatography and the relative molecular mass, purity, and antigen specificity of the expressed fusion protein were studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting.The bioactivities of fusion protein was tested in vivo and vitro.Part I:Construction of GLP-1 Fc fusion protein expression plasmids and stably expressing cell lineA sequence encoding GLP-1-IgG2σ-Fc was designed using the nucleotide sequences of GLP-1 and IgG2σ and synthesized. This sequence was then digested by the restriction enzymes and inserted into the mammalian SGLs expression vector. The recombinant SGLs-GLP-1-IgG2σ-Fc plasmid was verified by DNA sequencing (forward primer:5’-CAGGACCACGTCGTGCCAGT-3’). Stably expressing cell lines were obtained by electroporation with the linearized plasmid. Highly expressing clones were selected based on SDS-PAGE and ELISA analyses. After screening the expression level of protein had reached 1.2g/L. Analysis of the continues culture medium by SDS-PAGE indicated that these cells secreted the purest GLP-1-IgG2σ-Fc on day 12. Finally, in this part we Constructed the GLP-1 Fc fusion protein expression plasmids and stably expressing cell line. It provides the basis for subsequent experiments.Part II:Purification and identification of GLP-1 Fc fusion proteinThe object of this part is to obtain pure protein after purified by chromatography, then to identify whether the protein was the target product. The expression medium was harvested and filtered at Dayl2. The GLP-1-IgG2σ-Fc fusion protein was then purified from the medium by protein A affinity chromatography and Hydrophobic interaction chromatography.Western blotting analysis confirmed that the expressed protein showed good antigenicity to both GLP-1 and human IgG antibodies and had an apparent molecular weight of 68 kDa. Finally, in this part we obtained the purified GLP-1 fusion protein which has a good antigenicity to both GLP-1 and human IgG antibodies.Part Ⅲ:The biological activity of GLP-1 Fc fusion protein in vivo andvitroTests of glucose-induced insulin secretion (GSIS) were performed using INS-1 cells and isolated rat islet exposed to GLP-1-IgG2σ-Fc and glucose. As a result, GLP-1-IgG2σ-Fc stimulated insulin secretion in a glucose-dependent manner.To investigate this effect on insulin secretion further, mRNA was isolated from the INS-1 cells. Insulin mRNA expressionevaluated by RT-PCR indicated that the fusion protein promotes the synthesis of insulin. Further investigation of OGTT tests was performed in C57/BL6J mice. Compared with the vehicle group, mice treated with either GLP-1-IgG2σ-Fc showed reduced glucose levels. Then KKAy mice were treated with intraperitoneal GLP-1-IgG2σ-Fc once every three days for 4 weeks. During this period, GLP-1-IgG2σ-Fc significantly reduced the plasma glucose levels. Furthermore, GLP-1-IgG2σ-Fc produced a more sustained hypoglycemic effect in KKAy mice than did GLP-1-IgG4-Fc.Our findings suggested that GLP-1-IgG2σ-Fc has a lower immunogenicity and a longer half-life.The pharmacokinetics of GLP-1-IgG2σ-Fc were studied in cynomolgus monkeys after a single dose of 0.1 mg/kg. The t1/2 of GLP-1-IgG2σ-Fc was approximately 57.1h.These superior features indicated that GLP-1-IgG2σ-Fc could provide a potential long-acting GLP-1 receptor agonist for the treatment of type 2 diabetes.In summary, this study successfully constructed the stably expressing cell lines. We have obtained GLP-1 biological activity in vitro and vivo after purification.The hypoglycemic effects of GLP-1-IgG2σ-Fc were sustained for longer than those of GLP-1-IgG4-Fc in KKAy mice. Pharmacokinetic and pharmacodynamic analyses of GLP-1-IgG2σ-Fc confirmed that is had a prolonged t1/2 and could reduce body weight. These superior features indicated that the study of GLP-1-IgG2σ-Fc worth further exploration.