Recombinant expression and tryptophan-assisted analysis of human sweet taste receptor T1R3's extracellular domain in sweetener interaction studies.
Prep Biochem Biotechnol
; : 1-8, 2024 Apr 05.
Article
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| MEDLINE
| ID: mdl-38578840
ABSTRACT
The human palate can discern multiple tastes; however, it predominantly perceives five fundamental flavors sweetness, saltiness, sourness, bitterness, and umami. Sweetness is primarily mediated through the sweet taste receptor, a membrane-bound heterodimeric structure comprising T1R2-T1R3. However, unraveling the structural and mechanistic intricacies of the sweet taste receptor has proven challenging. This study aimed to address this knowledge gap by expressing an extracellular N-terminal domain encompassing the cysteine-rich domain of human hT1R3 (hT1R3-TMD) in Escherichia coli. The expressed protein was obtained as inclusion bodies, purified by metal affinity chromatography, and refolded using the dilution-refolding method. Through rigorous analysis, we confirmed the successful refolding of hT1R3-TMD and elucidated its structural characteristics using circular dichroism spectroscopy. Notably, the refolded protein was found to exist as either a monomer or a dimer, depending on its concentration. A tryptophan fluorescence quenching assay revealed that the dissociation constants for sucrose, sucralose, and brazzein were >9500 µM, 2380 µM and 14.3 µM, respectively. Our findings highlight the utility of this E. coli expression system for producing functional hT1R3-TMD for investigations and demonstrate the efficacy of the tryptophan fluorescence quenching assay in revealing complex interactions between sweet taste receptors and various sweeteners.
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MEDLINE
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En
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Prep Biochem Biotechnol
Asunto de la revista:
BIOQUIMICA
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BIOTECNOLOGIA
Año:
2024
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Article