Preparation, Modification And Bioactivity Of A Derivative Of Exendin-4

Exendin-4 is a 39-amino-acid peptide which was originally isolated from the salivary secretions of the lizard. Heloderma suspectum. Exendin-4 specifically interacts with glucagon-like peptide-1 (GLP-1) receptor in mammals and acts as a agonist of GLP-1 receptor. After binding to GLP-1 receptor it can stimulate insulin secretion in a glucose-dependent way, improve insulin sensitivity, protectβ-cell against apoptosis in response to different insults and promoteβ-cell proliferation. Because of its favorable spectrum of antidiabetic actions and high stability against dipeptidyl peptidase-IV (DPP-IV) it has received much attention as a possible therapeutic agent in the treatment of typeⅡdiabetes. Based on the sequence of Exendin-4 or more specifically of Exendin-4(1-30), we designed a novel peptide called E306 and expressed it in E. coli BL21(DE3). The purified E306 peptide was administrated to mice to analyze its biological activity. Using Biotin-NHS to modify the Lysines in E306, we got an orally available biotinylated E306.The study included three parts as follows:1,Cloning, expression and purification of E306Based on the findings on the relationship between structure and function of Exendin-4, we designed a novel peptide which added six Lysines at the N-teminal of Exendin-4 (1-30). The cDNA of E306 was obtained by PCR using proper primers and pET-32a(+)-E30 as template and then cloned into pET-32a(+) vector. The recombinant expression vector was then transformed into E.coli BL21 (DE3) to obtain genetically engineered strain pET-32a(+)-E306/BL21(DE3). The target fusion protein expressed after induction was soluble and exhibited a molecular weight of 21 kDa by SDS-PAGE analysis. Then the fusion protein was purified by Ni ion affinity chromatography. Purified fusion protein was then digested by enterokinase and again purified by Ni ion affinity chromatography to obtain E306. The production of E306 was calculated to be 0.64 mg/g bacterial cells.2,Characterization of E306E306 demonstrated significant improvement of glucose tolerance with a dose of 1 nmol/kg in intraperitoneal glucose tolerance tests both in healthy Kunming mice and diabetic db/db mice. The glucose area under the blood-glucose concentration curve during 120 min (AUC0-120min) is 41% (P<0.001),48% (P<0.001) lower than control group respectively. When administered intraperitoneally to Kunming mice, the hypoglycemic effect of E306 can lasts for 6 h with a dose of 1 nmol/kg. E306 dose-dependently prolonged the time of glucose lowering effect from 1 h (0.1 nmol/kg) to 7 h (10 nmol/kg) in this study.Further more, the secondary structure of E306 was determined by circular dichroism. The result showed that the amount ofα-helix,β-turn and random-coil in E306 was 68.8%,1.9% and 29.3% respectively. Under the same condition E30 exhibited 39.8% forα-helix,7.4% forβ-turn and 52.8% for random-coil. There was 29% moreα-helix in E306 than E30 which as a result of the extra six Lysines in E306.3,Biotinylation and biological activity of E306E30 was reacted with Biotin-NHS at a molar ration of 1:8 with gently mixing at room temperature for 1 h. The reaction mixture was then analyzed by Tricin-SDS-PAGE and high performance liquid chromatography (HPLC). The results indicated that Biotin-NHS attached to E30. The intraperitoneal glucose tolerance test showed that when administered orally biotinylated E306 significantly improve glucose tolerance with a dose of 300 nmol/kg. The glucose AUC0-120min values of these two tests are 38%(P<0.01),21%(P>0.05) lower than control group respectively.Taken together, in this study we constructed an expression vector pET-32a(+)-E306 and obtained genetically engineered strain pET-32a(+)-E306/ BL21(DE3). Purified E306 demonstrated significant improvement on glucose tolerance both in healthy Kunming mice and diabetic db/db mice. With a dose of 1 nmol/kg the hypoglycemic effect of E306 in Kunming mice can last for 6 h when administered intraperitoneally. Using Biotin-NHS to modify the Lysines in E306 then got an orally available biotinylated E306, which can significantly improve glucose tolerance in Kunming mice. The results of the study can offer certain reference for the development of diabetes drugs.