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

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, Takuya Kobayashi

Research output: Contribution to journalArticlepeer-review

13 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)704-710
Number of pages7
JournalBiochemical and Biophysical Research Communications
Volume382
Issue number4
DOIs
Publication statusPublished - May 15 2009

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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