In vitro folding, functional characterization, and disulfide pattern of the extracellular domain of human GLP-1 receptor

• Published in: Biophysical chemistry 2002-05, Vol.96 (2), p.305-318
• Main Authors: Bazarsuren, Ariuna, Grauschopf, Ulla, Wozny, Manfred, Reusch, Dietmar, Hoffmann, Eike, Schaefer, Wolfgang, Panzner, Steffen, Rudolph, Rainer
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Calorimetry; Circular Dichroism; Disulfide connectivity; Disulfides - chemistry; Escherichia coli - genetics; Escherichia coli - metabolism; G-protein coupled receptor; Glucagon - metabolism; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Humans; Inclusion bodies; Ligand binding; Molecular Sequence Data; Peptide Fragments - metabolism; Protein Binding; Protein Folding; Protein Precursors - metabolism; Protein Structure, Tertiary; Receptors, Glucagon - chemistry; Receptors, Glucagon - isolation & purification; Receptors, Glucagon - metabolism; Renaturation; Titrimetry
• ISSN: 0301-4622; 1873-4200
• DOI: 10.1016/S0301-4622(02)00023-6

The N-terminal, extracellular domain of the receptor for glucagon-like peptide 1 (GLP-1 receptor) was expressed at a high level in E. coli and isolated as inclusion bodies. Renaturation with concomitant disulfide bond formation was achieved from guanidinium-solubilized material. A soluble and active fraction of the protein was isolated by ion exchange chromatography and gel filtration. Complex formation with GLP-1 was shown by cross-linking experiments, surface plasmon resonance measurements, and isothermal titration calorimetry. The existence of disulfide bridges in the N-terminal receptor fragment was proven after digestion of the protein with pepsin. Further analysis revealed a disulfide-binding pattern with links between cysteines 46 and 71, 62 and 104, and between 85 and 126.