title: Expression, purification and preliminary characterization of glucagon receptor extracellular domain.
abstract: Glucagon is a pancreatic hormone that plays pivotal roles in regulating glucose homeostasis and metabolism. Glucagon exerts its action by binding to its receptor, glucagon receptor (GCGR), one of class B G-protein coupled receptors (GPCRs). Diabetes is a bihormonal disease in which excessive glucagon secretion is a major contributor in the pathogenesis of this disease; elucidation of how glucagon binds to GCGR will facilitate the rational design of the GCGR antagonist for treating diabetic hyperglycemia. Here we report the successful expression and purification of the GCGR extracellular domain (GCGR-ECD) and its fusion protein with the glucagon peptide at its C-terminus (GCGR-ECD-Gc). We utilized the maltose binding protein (MBP) fusion method and disulfide bond isomerase DsbC co-expression approach for the success of the soluble expression of both GCGR-ECD and GCGR-ECD-Gc in Escherichia coli. We also obtained a high yield production of secreted GCGR-ECD with the baculovirus expression system by optimizing its N-terminal secreting signal. We first utilized isothermal titration calorimetry approach to determine the in vitro binding affinities of glucagon to the GCGR-ECD. No significant differences were found between the prokaryotic expressed GCGR-ECD (7.6μM) and the eukaryotic glycosylated one (6.6μM). The observation of the intra ligand-receptor binding within the fusion protein GCGR-ECD-Gc suggests it as a good candidate for further structural study.
authors.affiliation: Department of Endocrinology and Metabolism, Huashan Hospital, Fudan University, Shanghai 200040, China.
authors.forename: Lili
authors.initials: L
authors.lastname: Wu
authors.forename: Yujia
authors.initials: Y
authors.lastname: Zhai
authors.forename: Jiuwei
authors.initials: J
authors.lastname: Lu
authors.forename: Qinghua
authors.initials: Q
authors.lastname: Wang
authors.forename: Fei
authors.initials: F
authors.lastname: Sun
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D005934:Glucagon / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007313:Insecta; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D018027:Receptors, Glucagon / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D010455:Peptides; D021382:Protein Sorting Signals; D018027:Receptors, Glucagon; D011993:Recombinant Fusion Proteins; D005934:Glucagon