title: Fluorescence-based optimization of human bitter taste receptor expression in Saccharomyces cerevisiae.
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 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.
authors.affiliation: Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.
authors.forename: Taishi
authors.initials: T
authors.lastname: Sugawara
authors.forename: Keisuke
authors.initials: K
authors.lastname: Ito
authors.forename: Mitsunori
authors.initials: M
authors.lastname: Shiroishi
authors.forename: Natsuko
authors.initials: N
authors.lastname: Tokuda
authors.forename: Hidetsugu
authors.initials: H
authors.lastname: Asada
authors.forename: Takami
authors.initials: T
authors.lastname: Yurugi-Kobayashi
authors.forename: Tatsuro
authors.initials: T
authors.lastname: Shimamura
authors.forename: Takumi
authors.initials: T
authors.lastname: Misaka
authors.forename: Norimichi
authors.initials: N
authors.lastname: Nomura
authors.forename: Takeshi
authors.initials: T
authors.lastname: Murata
authors.forename: Keiko
authors.initials: K
authors.lastname: Abe
authors.forename: So
authors.initials: S
authors.lastname: Iwata
authors.forename: Takuya
authors.initials: T
authors.lastname: Kobayashi
mesh_terms: 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
chemical_list: D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D000098706:Taste Receptors, Type 2; D049452:Green Fluorescent Proteins