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Article dans une revue
Biophysical and functional characterization of the human olfactory receptor OR1A1 expressed in a mammalian inducible cell line
Abstract : Olfactory receptors (ORs) play a crucial role in detecting the odorant molecules present in the surrounding environment. These receptors, which belong to class A G-protein-coupled receptors, constitute the largest transmembrane protein family in the human genome. Functional studies showed that the OR family includes members that are able to respond to a large set of odorants and members that are activated by a relatively small number of related odorants. To understand the molecular mechanisms that govern the receptor-ligand interactions, we overexpressed the human OR hOR1A1 in a stable tetracycline-inducible HEK293S cell line. This receptor was engineered by inserting a C-terminal rholD4 epitope tag and an N-terminal FLAG epitope tag to allow its purification and detection. The functional activity of the FLAG-rholD4-tagged hOR1A1 in heterologous HEK293S cells was analysed using a real-time cAMP assay. A two-step purification using monoclonal anti-FLAG immunoaffinity purification and gel filtration was then employed to purify the detergent-solubilized receptor. A size exclusion chromatography-multi-angle light scattering analysis showed the presence of monomeric and dimeric forms of FLAG-rholD4-tagged hOR1A1. The amounts of the monomeric and dimeric forms purified from sixty T175 flasks were approximately 1.6 and 1.1 mg, respectively. The circular dichroism analysis showed that the purified receptor was properly folded. Ligand binding was quantified using an intrinsic tryptophan fluorescence assay and revealed that the detergent-solubilized FLAG-rholD4-tagged hOR1A1 bound its cognate odorant, dihydrojasmone, with an affinity in the micromolar range. These results pave the way for future crystallographic and NMR studies.
https://hal-univ-bourgogne.archives-ouvertes.fr/hal-01412444
Contributeur : Csga - Université de Bourgogne Connectez-vous pour contacter le contributeur
Soumis le : jeudi 8 décembre 2016 - 14:22:11
Dernière modification le : mardi 9 mars 2021 - 03:30:02
Contributeur : Csga - Université de Bourgogne Connectez-vous pour contacter le contributeur
Soumis le : jeudi 8 décembre 2016 - 14:22:11
Dernière modification le : mardi 9 mars 2021 - 03:30:02