Fluorescence-based optimization of human bitter taste receptor expression in Saccharomyces cerevisiae
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- 作者：Taishi Sugawara ; Keisuke Ito ; Mitsunori Shiroishi ; Natsuko Tokuda ; Hidetsugu Asada ; Takami Yurugi-Kobayashi ; Tatsuro Shimamura ; Takumi Misaka ; Norimichi Nomura ; Takeshi Murata ; Keiko Abe ; So Iwata ; Ta
- 关键词：Bitter taste receptor ; Pre-crystallization screening ; Saccharomyces cerevisiae ; G protein-coupled receptor ; Fluorescence size-exclusion chromatography ; Monodispersity
- 刊名：Biochemical and Biophysical Research Communications
- 出版年：2009
- 出版时间：15 May 2009
- 年：2009
- 卷：382
- 期：4
- 页码：704-710
- 全文大小：403 K
文摘
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.