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Functional screening system for GPCR‐agonists using peptide‐secreting yeasts (654.5)
Author(s) -
Shigemori Tomohiro,
Kuroda Kouichi,
Ueda Mitsuyoshi
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.654.5
Subject(s) - g protein coupled receptor , yeast , saccharomyces cerevisiae , peptide , receptor , signal peptide , biochemistry , phage display , transmembrane protein , agonist , biology , chemistry , gene , peptide sequence
G‐protein coupled receptors (GPCRs) which feature a seven‐transmembrane structure are involved in various diseases as well as in regulating physiological functions. Phage display has been used for discovering bioactive peptides, but the methodology is never effective for screening GPCR‐agonists because phage‐displayed peptides are screened based only on binding ability and are different from their final soluble form. In this study, we established a functional screening system for GPCR‐activating peptides by combining peptide‐secreting yeasts with GPCRs‐producing mammalian cells. Glucagon‐like peptide‐1 receptor (GLP1R) and its peptide agonist exendin‐4 (Ex4) were used as model molecules. Ex4‐secreting yeast (Ex4‐yeast) was constructed by introducing the Ex4‐encoding gene linked to secretion signal genes of glucoamylase or yeast α‐factor into Saccharomyces cerevisiae. Detection of yeast‐secreted Ex4 was performed by evaluating cAMP production in GLP1R‐producing CHO cells exposed to yeast culture medium incubated in glass tube and microplate. Model screening of Ex4‐yeast from mixture containing Ex4‐ and control‐yeasts (ratio of each cell number, 1:9) was carried out on microplate. The results indicated that the functional screening system with peptide‐secreting yeasts would be promising for efficient discovery of novel peptide‐based GPCR agonists [1]. [1] Shigemori et al. submitted.