Mammalian G protein-coupled receptor expression in Escherichia coli: II. Refolding and biophysical characterization of mouse cannabinoid receptor 1 and human parathyroid hormone receptor 1
| العنوان: | Mammalian G protein-coupled receptor expression in Escherichia coli: II. Refolding and biophysical characterization of mouse cannabinoid receptor 1 and human parathyroid hormone receptor 1 |
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| المؤلفون: | Marie-Eve Gravière, Rainer Rudolph, Giuliano Sciara, Julie Lichière, Marina Isabella Siponen, Céline Huyghe, Renaud Wagner, Kerstin Michalke, Christian Cambillau, Christine Magg, Aline Desmyter, Isabelle Lepaul |
| المساهمون: | Architecture et fonction des Macromolécules Biologiques - UMR 6098 (AFMB), Université de Provence - Aix-Marseille 1-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS), Aix Marseille Université (AMU), Centre National de la Recherche Scientifique (CNRS)-Université de Provence - Aix-Marseille 1 |
| المصدر: | Analytical Biochemistry Analytical Biochemistry, 2010, 401 (1), pp.74-80. ⟨10.1016/j.ab.2010.02.017⟩ Analytical Biochemistry, Elsevier Masson, 2010, 401 (1), pp.74-80. ⟨10.1016/j.ab.2010.02.017⟩ |
| بيانات النشر: | Elsevier BV, 2010. |
| سنة النشر: | 2010 |
| مصطلحات موضوعية: | Protein Folding, [SDV]Life Sciences [q-bio], Biophysics, 010402 general chemistry, 01 natural sciences, Biochemistry, Inclusion bodies, Mice, 03 medical and health sciences, Receptor, Cannabinoid, CB1, Cell surface receptor, Protein purification, Escherichia coli, Animals, Humans, Refolding, 5-HT5A receptor, G protein-coupled receptor, Receptor, Molecular Biology, Receptor, Parathyroid Hormone, Type 1, 030304 developmental biology, Inclusion Bodies, Cyclodextrins, 0303 health sciences, biology, Sciences du Vivant [q-bio]/Biotechnologies, Cell Biology, Recombinant Proteins, 0104 chemical sciences, Hormone receptor, Rhodopsin, biology.protein, Protein expression, Protein Binding |
| الوصف: | G protein-coupled receptors (GPCRs) represent approximately 3% of the human proteome. They are involved in a large number of diverse processes and, therefore, are the most prominent class of pharmacological targets. Besides rhodopsin, X-ray structures of classical GPCRs have only recently been resolved, including the beta1 and beta2 adrenergic receptors and the A2A adenosine receptor. This lag in obtaining GPCR structures is due to several tedious steps that are required before beginning the first crystallization experiments: protein expression, detergent solubilization, purification, and stabilization. With the aim to obtain active membrane receptors for functional and crystallization studies, we recently reported a screen of expression conditions for approximately 100 GPCRs in Escherichia coli, providing large amounts of inclusion bodies, a prerequisite for the subsequent refolding step. Here, we report a novel artificial chaperone-assisted refolding procedure adapted for the GPCR inclusion body refolding, followed by protein purification and characterization. The refolding of two selected targets, the mouse cannabinoid receptor 1 (muCB1R) and the human parathyroid hormone receptor 1 (huPTH1R), was achieved from solubilized receptors using detergent and cyclodextrin as protein folding assistants. We could demonstrate excellent affinity of both refolded and purified receptors for their respective ligands. In conclusion, this study suggests that the procedure described here can be widely used to refold GPCRs expressed as inclusion bodies in E. coli. |
| تدمد: | 0003-2697 1096-0309 |
| الوصول الحر: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::522c2d66690a2c7d7111b990e4322ab5Test https://doi.org/10.1016/j.ab.2010.02.017Test |
| حقوق: | OPEN |
| رقم الانضمام: | edsair.doi.dedup.....522c2d66690a2c7d7111b990e4322ab5 |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 00032697 10960309 |
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