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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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      Abstract

      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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      Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications.

      A set of yeast strains based on Saccharomyces cerevisiae S288C in which commonly used selectable marker genes are deleted by design based on the yeast genome sequence has been constructed and analysed. These strains minimize or eliminate the homology to the corresponding marker genes in commonly used vectors without significantly affecting adjacent gene expression. Because the homology between commonly used auxotrophic marker gene segments and genomic sequences has been largely or completely abolished, these strains will also reduce plasmid integration events which can interfere with a wide variety of molecular genetic applications. We also report the construction of new members of the pRS400 series of vectors, containing the kanMX, ADE2 and MET15 genes.
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        Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis.

        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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           A Jean,  John Woods,  R Gietz (1992)
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            Author and article information

            Affiliations
            [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.

            Contributors
            Role: Editor
            Journal
            PLoS One
            PLoS ONE
            plos
            plosone
            PLoS ONE
            Public Library of Science (San Francisco, USA)
            1932-6203
            2012
            18 May 2012
            : 7
            : 5
            3356411
            22623985
            PONE-D-11-25420
            10.1371/journal.pone.0037136
            (Editor)
            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.
            Counts
            Pages: 10
            Categories
            Research Article
            Biology
            Biochemistry
            Cytochemistry
            Cell Membrane
            Membrane Proteins
            Proteins
            Transmembrane Proteins
            Biomacromolecule-Ligand Interactions
            Drug Discovery
            Biophysics
            Biomacromolecule-Ligand Interactions
            Biotechnology
            Bioengineering
            Biological Systems Engineering
            Biomimetics
            Genetic Engineering
            Genetically Modified Organisms
            Applied Microbiology
            Drug Discovery
            Chemistry
            Analytical Chemistry
            Chemical Analysis
            Colorimetric Analysis
            Chemical Biology
            Engineering
            Bioengineering
            Biological Systems Engineering
            Biomimetics

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