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Fluorescence-based optimization of human bitter taste receptor expression in Saccharomyces cerevisiae.

DOI: 10.1016/j.bbrc.2009.03.089 DOI Help

Authors: T. Sugawara (University of Tokyo) , K. Ito (RIKEN SPring-8 Center) , M. Shiroishi (Japan Science and Technology Agency) , N. Tokuda (Kyoto University) , H. Asada (Japan Science and Technology Agency) , T. Yurugi-kobayashi (Japan Science and Technology Agency) , T. Shimamura (Japan Science and Technology Agency) , T. Misaka (University of Tokyo) , N. Nomura (Japan Science and Technology Agency) , T. Murata (Japan Science and Technology Agency) , K. Abe (University of Tokyo) , T. Kobayashi (Iwata Human Receptor Crystallography Project) , S. Iwata (Diamond Light Source; Iwata Human Receptor Crystallography Project; Kyoto University; Imperial College London)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Biochemical And Biophysical Research Communications , VOL 382 (4) , PAGES 704-710

State: Published (Approved)
Published: May 2009

Abstract: Human TAS2 receptors (hTAS2Rs) perceive bitter tastants, but few studies have explored the structure–function relationships of these receptors. In this paper, we report our trials on the large-scale preparations of hTAS2Rs for structural analysis. Twenty-five hTAS2Rs were expressed using a GFP-fusion yeast system in which the constructs and the culture conditions (e.g., the signal sequence, incubation time and temperature after induction) were optimized by measuring GFP fluorescence. After optimization, five hTAS2Rs (hTAS2R7, hTAS2R8, hTAS2R16, hTAS2R41, and hTAS2R48) were expressed at levels greater than 1 mg protein/L of culture, which is a preferable level for purification and crystallization. Among these five bitter taste receptors, hTAS2R41 exhibited the highest detergent solubilization efficiency of 87.1% in n-dodecyl-?-d-maltopyranoside (DDM)/cholesteryl hemisuccinate (CHS). Fluorescence size-exclusion chromatography showed that hTAS2R41 exhibited monodispersity in DDM/CHS without aggregates, suggesting that hTAS2R41 is a good target for future crystallization trials.

Journal Keywords: Bitter Taste Receptor; Pre-Crystallization Screening; Saccharomyces Cerevisiae; G Protein-Coupled Receptor; Fluorescence Size-Exclusion Chromatography; Monodispersity

Subject Areas: Biology and Bio-materials


Instruments: NONE-No attached Diamond beamline

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