6533b830fe1ef96bd1297837

RESEARCH PRODUCT

Expression and characterization of the human sweet taste receptor expressed in a mammalian inducible cell line

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description

International audience; Sweet taste perception is mediated by a heterodimeric receptor composed of the two distinct protein subunits, TAS1R2 and TAS1R3. TAS1R2 and TAS1R3 subunits are members of the small family of class C GPCRs. Class C GPCRs share a large N-terminal domain (NTD) linked to the heptahelical transmembrane domain by a cysteine-rich region. TAS1R2/TAS1R3 is the primary receptor for a diverse range of sweet compounds including natural sugars, sweet amino acids, artificial sweeteners and plant sweet-tasting proteins. In order to understand the molecular mechanisms that govern receptor – ligand interactions and the relative contribution of the two subunits to the detection of sweet compounds, we overexpressed TAS1R2/TAS1R3 using a stable tetracycline-inducible HEK293S cell line. Each TAS1R2 and TAS1R3 subunit were engineered by inserting two different N-terminal tags to allow the purification and detection of heterodimeric TAS1R2/TAS1R3 receptor. The functional activity of TAS1R2/TAS1R3 in heterologous HEK293S cells was analysed using calcium assay. A three-step affinity purification method was employed to purify the solubilized TAS1R2/TAS1R3 heterodimer. SDS-PAGE analysis showed that the receptor was pure. Circular dichroism demonstrated that the purified heterodimeric receptor was properly refolded and has expected secondary structures. Ligand binding was quantified using an intrinsic tryptophan fluorescence assay and revealed that solubilized TAS1R2/TAS2R3 was able to bind sucralose with an affinity in the high micromolar range. These results pave the way for future biophysical studies including NMR spectroscopy and electron microscopy.