Study On The Biosynthesis Of Exendin-4 And Its Bioactivity

Exendin-4 is a 39 amino acid peptide isolated from the salivary secretions of Heloderma suspectum, a venomous lizard commonly known as the Gila monster. It shows 53% sequence identity to mammalian glucagon-like peptide 1 (GLP-1) and it is the agonist of GLP-1's receptor. Like GLP-1, Exendin-4 could low plasma glucose. Exendin-4 has a relatively long half-life (9.57h), GLP-1's only 2 min, so Exendin-4 has been widely explored as a potential therapy in metabolic disease.For the sake of a high expression of Exendin-4 in Escherichia coli, the original Exendin-4 gene was modified in light of its amino acid sequence and the code bias of E. coli. Not only 70% of the codes were changed, but also the sites of restriction enzyme and enterokinase were added to the two terminals of Exendin-4 respectively. The recombinant expression vector of pRSET B-Exendin-4 and pET-32a(+)- Exendin-4 was constructed and transformed into E. coli, only pET-32(a+)- Exendin-4 expressed. The fusion protein was 32 percent of total protein in E. coli after induction with 1 mmol/L IPTG for 6 hours at 30℃. With Ni-NTA chromatography, we obtained 6.44 mg exendin-4 fusion protein from one gram wet bacteria. After the fusion protein was digested by enterokinase, affinity chromatography was used to bind the N terminals of 6×His tag and purify Exendin-4.The yield of Exendin-4 was up to 1.59 mg per gram wet bacteria with the purity of 99%. The experiment in BKS.Cg-m +/+ Lepr db/J mice revealed that intraperitoneal injection of Exendin-4 had prominent glucose-lowering effects. The optimal dose was 7μg/kg and the maximal decrease in plasma glucose was up to 51.93% at 4 h after intraperitoneal injection and the effect of low plasma glucose is up to 8h after intraperitoneal injection. When intraperitoneal injection of Exendin-4 was 10μg/kg, plasma glucose could keep in low concentration and HbA1c decrease 13.51%.