title: High-throughput de novo screening of receptor agonists with an automated single-cell analysis and isolation system.
abstract: Reconstitution of signaling pathways involving single mammalian transmembrane receptors has not been accomplished in yeast cells. In this study, intact EGF receptor (EGFR) and a cell wall-anchored form of EGF were co-expressed on the yeast cell surface, which led to autophosphorylation of the EGFR in an EGF-dependent autocrine manner. After changing from EGF to a conformationally constrained peptide library, cells were fluorescently labeled with an anti-phospho-EGFR antibody. Each cell was subjected to an automated single-cell analysis and isolation system that analyzed the fluorescent intensity of each cell and automatically retrieved each cell with the highest fluorescence. In ~3.2 × 10(6) peptide library, we isolated six novel peptides with agonistic activity of the EGFR in human squamous carcinoma A431 cells. The combination of yeast cells expressing mammalian receptors, a cell wall-anchored peptide library, and an automated single-cell analysis and isolation system might facilitate a rational approach for de novo drug screening.
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan.
authors.forename: Nobuo
authors.initials: N
authors.lastname: Yoshimoto
authors.affiliation: The Institute of Scientific and Industrial Research, Osaka University, Mihogaoka, Ibaraki, Osaka 567-0047, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Tatematsu
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan.
authors.forename: Masumi
authors.initials: M
authors.lastname: Iijima
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan.
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Niimi
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan.
authors.forename: Andrés D
authors.initials: AD
authors.lastname: Maturana
authors.affiliation: Graduate School of Science, Osaka Prefecture University, Gakuen-cho, Naka-ku, Sakai, Osaka 599-8570, Japan.
authors.forename: Ikuo
authors.initials: I
authors.lastname: Fujii
authors.affiliation: Graduate School of Engineering, Kobe University, Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501, Japan.
authors.forename: Akihiko
authors.initials: A
authors.lastname: Kondo
authors.affiliation: The Institute of Scientific and Industrial Research, Osaka University, Mihogaoka, Ibaraki, Osaka 567-0047, Japan.
authors.forename: Katsuyuki
authors.initials: K
authors.lastname: Tanizawa
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8601, Japan.
authors.forename: Shun'ichi
authors.initials: S
authors.lastname: Kuroda
mesh_terms: D001681:Biological Assay / Q000295:instrumentation* / Q000379:methods; D002469:Cell Separation / Q000295:instrumentation* / Q000379:methods; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation* / Q000379:methods; D004867:Equipment Design; D019544:Equipment Failure Analysis; D005434:Flow Cytometry / Q000295:instrumentation* / Q000379:methods; D057166:High-Throughput Screening Assays / Q000295:instrumentation* / Q000379:methods; D018160:Receptors, Cytoplasmic and Nuclear / Q000037:antagonists & inhibitors*; D012371:Robotics / Q000295:instrumentation* / Q000379:methods
chemical_list: D018160:Receptors, Cytoplasmic and Nuclear