TY - JOUR
T1 - Fluorescence-based optimization of human bitter taste receptor expression in Saccharomyces cerevisiae
AU - Sugawara, Taishi
AU - Ito, Keisuke
AU - Shiroishi, Mitsunori
AU - Tokuda, Natsuko
AU - Asada, Hidetsugu
AU - Yurugi-Kobayashi, Takami
AU - Shimamura, Tatsuro
AU - Misaka, Takumi
AU - Nomura, Norimichi
AU - Murata, Takeshi
AU - Abe, Keiko
AU - Iwata, So
AU - Kobayashi, Takuya
N1 - Funding Information:
We thank Dr. Per Ljungdahl of the Department of Cell Biology at the Wenner-Gren Institute, Stockholm University, for kindly providing the FGY217 yeast strain. This study was supported in part by the ERATO Iwata Human Receptor Crystallography Project (JST) (to S.I.), the Targeted Proteins Research Program (to S.I. and T.M.), Grant-in-Aid for Scientific Research (B) (20370035) (to T.K.), a grant from the Research and Development Program for New Bioindustry Initiatives (to K.A.), and Grants-in-Aid for Scientific Research (S) (to K.A.) in Japan.
PY - 2009/5/15
Y1 - 2009/5/15
N2 - 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.
AB - 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.
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U2 - 10.1016/j.bbrc.2009.03.089
DO - 10.1016/j.bbrc.2009.03.089
M3 - Article
C2 - 19306845
AN - SCOPUS:64749091175
SN - 0006-291X
VL - 382
SP - 704
EP - 710
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 4
ER -