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Cell Wall Trapping of Autocrine Peptides for Human G-Protein-Coupled Receptors on the Yeast Cell Surface

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      G-protein-coupled receptors (GPCRs) regulate a wide variety of physiological processes and are important pharmaceutical targets for drug discovery. Here, we describe a unique concept based on yeast cell-surface display technology to selectively track eligible peptides with agonistic activity for human GPCRs (Cell Wall Trapping of Autocrine Peptides (CWTrAP) strategy). In our strategy, individual recombinant yeast cells are able to report autocrine-positive activity for human GPCRs by expressing a candidate peptide fused to an anchoring motif. Following expression and activation, yeast cells trap autocrine peptides onto their cell walls. Because captured peptides are incapable of diffusion, they have no impact on surrounding yeast cells that express the target human GPCR and non-signaling peptides. Therefore, individual yeast cells can assemble the autonomous signaling complex and allow single-cell screening of a yeast population. Our strategy may be applied to identify eligible peptides with agonistic activity for target human GPCRs.

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      Most cited references 41

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        The functions of many open reading frames (ORFs) identified in genome-sequencing projects are unknown. New, whole-genome approaches are required to systematically determine their function. A total of 6925 Saccharomyces cerevisiae strains were constructed, by a high-throughput strategy, each with a precise deletion of one of 2026 ORFs (more than one-third of the ORFs in the genome). Of the deleted ORFs, 17 percent were essential for viability in rich medium. The phenotypes of more than 500 deletion strains were assayed in parallel. Of the deletion strains, 40 percent showed quantitative growth defects in either rich or minimal medium.
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            Author and article information

            [1]Organization of Advanced Science and Technology, Kobe University, 1-1 Rokkodai, Nada, Kobe, Japan
            [2]Department of Structural Molecular Biology, Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka, Japan
            [3]Graduate School of Bioagricultural Sciences, Nagoya University, Furo, Chikusa, Nagoya, Japan
            [4]Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe, Japan
            University of Connecticut, United States of America
            Author notes

            Conceived and designed the experiments: JI NY KT SK CO HF AK. Performed the experiments: JI. Analyzed the data: JI. Contributed reagents/materials/analysis tools: JI. Wrote the paper: JI AK.

            Role: Editor
            PLoS One
            PLoS ONE
            PLoS ONE
            Public Library of Science (San Francisco, USA)
            18 May 2012
            : 7
            : 5
            Ishii et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
            Pages: 10
            Research Article
            Cell Membrane
            Membrane Proteins
            Transmembrane Proteins
            Biomacromolecule-Ligand Interactions
            Drug Discovery
            Biomacromolecule-Ligand Interactions
            Biological Systems Engineering
            Genetic Engineering
            Genetically Modified Organisms
            Applied Microbiology
            Drug Discovery
            Analytical Chemistry
            Chemical Analysis
            Colorimetric Analysis
            Chemical Biology
            Biological Systems Engineering



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