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Fluorescence-based optimization of human bitter taste receptor expression in Saccharomyces cerevisiae |
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| Authors: | Taishi Sugawara Keisuke Ito Mitsunori Shiroishi Hidetsugu Asada Tatsuro Shimamura Norimichi Nomura Keiko Abe Takuya Kobayashi |
| Affiliation: | a Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan b Iwata Human Receptor Crystallography Project, ERATO, JST, Konoe-cho, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan c Department of Medical Chemistry, Kyoto University Faculty of Medicine, Konoe-cho, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan d Division of Molecular Biosciences, Membrane Protein Crystallography Group, Imperial College London, London SW7 2AZ, UK e Diamond Light Source, Harwell Science and Innovation Campus, Didcot, Chilton, Oxfordshire OX11 0DE, UK |
| 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-β-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. |
| Keywords: | Bitter taste receptor Pre-crystallization screening Saccharomyces cerevisiae G protein-coupled receptor Fluorescence size-exclusion chromatography Monodispersity |
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