Journal
BIOPHYSICAL CHEMISTRY
Volume 96, Issue 2-3, Pages 305-318Publisher
ELSEVIER
DOI: 10.1016/S0301-4622(02)00023-6
Keywords
G-protein coupled receptor; inclusion bodies; renaturation; ligand binding; disulfide connectivity
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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. (C) 2002 Elsevier Science B.V. All rights reserved.
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