Recombinant expression and tryptophan-assisted analysis of human sweet taste receptor T1R3's extracellular domain in sweetener interaction studies
- Authors
- Jin, Soo-Bin; Kim, Hyun-A; Shin, Ji-Ae; Jung, Na-Hee; Park, Seo-Young; Hong, Sungguan; Kong, Kwang-Hoon
- Issue Date
- Apr-2024
- Publisher
- TAYLOR & FRANCIS INC
- Keywords
- Expression in Escherichia coli; N-terminal domain of hT1R3; human sweet taste receptor; refolding process; tryptophan assay
- Citation
- Preparative biochemistry & biotechnology
- Journal Title
- Preparative biochemistry & biotechnology
- URI
- https://scholarworks.bwise.kr/cau/handle/2019.sw.cau/73303
- DOI
- 10.1080/10826068.2024.2336985
- ISSN
- 1082-6068
1532-2297
- 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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Collections - College of Natural Sciences > Department of Chemistry > 1. Journal Articles
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