Fluorescence-based optimization of human bitter taste receptor expression in Saccharomyces cerevisiae.
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 1mg 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-beta-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.
Sugawara
Taishi
T
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
Ito
Keisuke
K
Shiroishi
Mitsunori
M
Tokuda
Natsuko
N
Asada
Hidetsugu
H
Yurugi-Kobayashi
Takami
T
Shimamura
Tatsuro
T
Misaka
Takumi
T
Nomura
Norimichi
N
Murata
Takeshi
T
Abe
Keiko
K
Iwata
So
S
Kobayashi
Takuya
T
D002850:Chromatography, Gel; D003001:Cloning, Molecular; D005453:Fluorescence; D005818:Genetic Engineering / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D006801:Humans; D043562:Receptors, G-Protein-Coupled / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012995:Solubility; D055697:Taste Perception; D000098706:Taste Receptors, Type 2
D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D000098706:Taste Receptors, Type 2; D049452:Green Fluorescent Proteins