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

Biophys Chem. 2002 May 2;96(2-3):305-18. doi: 10.1016/s0301-4622(02)00023-6.

Abstract

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.

MeSH terms

  • Amino Acid Sequence
  • Calorimetry
  • Circular Dichroism
  • Disulfides / chemistry*
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Glucagon / metabolism
  • Glucagon-Like Peptide 1
  • Glucagon-Like Peptide-1 Receptor
  • Humans
  • 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
  • Titrimetry

Substances

  • Disulfides
  • GLP1R protein, human
  • Glucagon-Like Peptide-1 Receptor
  • Peptide Fragments
  • Protein Precursors
  • Receptors, Glucagon
  • Glucagon-Like Peptide 1
  • Glucagon