RRC ID 30717
Author Yoshimoto N, Tatematsu K, Iijima M, Niimi T, Maturana AD, Fujii I, Kondo A, Tanizawa K, Kuroda S.
Title High-throughput de novo screening of receptor agonists with an automated single-cell analysis and isolation system.
Journal Sci Rep
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.
Volume 4
Pages 4242
Published 2014-2-28
DOI 10.1038/srep04242
PII srep04242
Description S.cerevisiaeでGal1プロモーター制御下での目的遺伝子の発現に使用できます。本研究ではアゴニスト探索のためにEGFRを酵母で発現するために使用されました。
PMID 24577528
PMC PMC3937795
MeSH Biological Assay / instrumentation* Biological Assay / methods Cell Separation / instrumentation* Cell Separation / methods Drug Evaluation, Preclinical / instrumentation* Drug Evaluation, Preclinical / methods Equipment Design Equipment Failure Analysis Flow Cytometry / instrumentation* Flow Cytometry / methods High-Throughput Screening Assays / instrumentation* High-Throughput Screening Assays / methods Receptors, Cytoplasmic and Nuclear / antagonists & inhibitors* Robotics / instrumentation* Robotics / methods
IF 4.011
Times Cited 6
WOS Category BIOCHEMISTRY & MOLECULAR BIOLOGY
Resource
DNA material pGMH20 (RDB01956).