Recombinant Expression, in Vitro Refolding, and Biophysical Characterization of the Human Glucagon-like Peptide-1 Receptor
| العنوان: | Recombinant Expression, in Vitro Refolding, and Biophysical Characterization of the Human Glucagon-like Peptide-1 Receptor |
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| المؤلفون: | Christian Ihling, Rainer Rudolph, Kai Tittmann, Andrea Sinz, Kathrin Schröder-Tittmann, Eva Bosse-Doenecke, Steffen Reedtz-Runge |
| المصدر: | Biochemistry. 49:7956-7965 |
| بيانات النشر: | American Chemical Society (ACS), 2010. |
| سنة النشر: | 2010 |
| مصطلحات موضوعية: | Agonist, Circular dichroism, medicine.drug_class, Recombinant Fusion Proteins, Ligands, medicine.disease_cause, Biochemistry, Glucagon-Like Peptide-1 Receptor, Inclusion bodies, 03 medical and health sciences, 0302 clinical medicine, Escherichia coli, Receptors, Glucagon, medicine, Humans, Receptor, 030304 developmental biology, 0303 health sciences, biology, Chemistry, Ligand (biochemistry), Cytosol, Chaperone (protein), biology.protein, 030217 neurology & neurosurgery |
| الوصف: | Activation of the glucagon-like peptide-1 receptor (GLP-1R) upon ligand binding leads to the release of insulin from pancreatic cells. This strictly glucose-dependent process renders the receptor and its ligands useful in the treatment of type II diabetes mellitus. To enable a biophysical characterization in vitro, we expressed the human full-length GLP-1R in the cytosol of Escherichia coli as inclusion bodies. After purification, refolding of the SDS-solubilized receptor was achieved by the exchange of SDS against the detergent Brij78 using an artificial chaperone system. Far-UV circular dichroism spectroscopic studies revealed that the receptor adopts a characteristic alpha-helical structure in Brij78 micelles. Ligand binding of the renatured protein was quantified by fluorescence quenching and surface plasmon resonance spectroscopy. In the presence of Brij micelles, the refolded receptor binds the agonist exendin-4 with an apparent dissociation constant of approximately 100 nM in a reversible one-step mechanism. To demonstrate that the detected ligand binding activity is not only due to an autonomously functional N-terminal domain (nGLP-1R) but also due to additional contacts with the juxtamembrane part, we separately expressed and refolded the extracellular domain relying on identical protocols established for the full-length GLP-1R. In support of the suggested multidomain binding mode, the nGLP-1R binds exendin-4 with a lower affinity (K(app) in the micromolar range) and a different kinetic mechanism. The lower ligand affinity of the nGLP-1R results entirely from a decreased kinetic stability of the receptor-ligand complex, dissociation of which is approximately 40-fold faster in the case of the nGLP-1R compared to the full-length GLP-1R. In summary, a framework was developed to produce functional human full-length GLP-1R by recombinant expression in E. coli as a prerequisite for eventual structure determination and a rigorous biophysical characterization including protein variants. |
| تدمد: | 1520-4995 0006-2960 |
| الوصول الحر: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::f33765c37e0da8f87a28c9ebaaca6c82Test https://doi.org/10.1021/bi101159sTest |
| رقم الانضمام: | edsair.doi.dedup.....f33765c37e0da8f87a28c9ebaaca6c82 |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 15204995 00062960 |
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