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      Desired Alteration of Protein Affinities: Competitive Selection of Protein Variants Using Yeast Signal Transduction Machinery

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          Abstract

          Molecules that can control protein-protein interactions (PPIs) have recently drawn attention as new drug pipeline compounds. Here, we report a technique to screen desirable affinity-altered (affinity-enhanced and affinity-attenuated) protein variants. We previously constructed a screening system based on a target protein fused to a mutated G-protein γ subunit (Gγ cyto) lacking membrane localization ability. This ability, required for signal transmission, is restored by recruiting Gγ cyto into the membrane only when the target protein interacts with an artificially membrane-anchored candidate protein, thereby allowing interacting partners (Gγ recruitment system) to be searched and identified. In the present study, the Gγ recruitment system was altered by integrating the cytosolic expression of a third protein as a competitor to set a desirable affinity threshold. This enabled the reliable selection of both affinity-enhanced and affinity-attenuated protein variants. The presented approach may facilitate the development of therapeutic proteins that allow the control of PPIs.

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          Most cited references 48

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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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            A novel genetic system to detect protein-protein interactions.

            Protein-protein interactions between two proteins have generally been studied using biochemical techniques such as crosslinking, co-immunoprecipitation and co-fractionation by chromatography. We have generated a novel genetic system to study these interactions by taking advantage of the properties of the GAL4 protein of the yeast Saccharomyces cerevisiae. This protein is a transcriptional activator required for the expression of genes encoding enzymes of galactose utilization. It consists of two separable and functionally essential domains: an N-terminal domain which binds to specific DNA sequences (UASG); and a C-terminal domain containing acidic regions, which is necessary to activate transcription. We have generated a system of two hybrid proteins containing parts of GAL4: the GAL4 DNA-binding domain fused to a protein 'X' and a GAL4 activating region fused to a protein 'Y'. If X and Y can form a protein-protein complex and reconstitute proximity of the GAL4 domains, transcription of a gene regulated by UASG occurs. We have tested this system using two yeast proteins that are known to interact--SNF1 and SNF4. High transcriptional activity is obtained only when both hybrids are present in a cell. This system may be applicable as a general method to identify proteins that interact with a known protein by the use of a simple galactose selection.
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              Improved method for high efficiency transformation of intact yeast cells.

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

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2014
                22 September 2014
                : 9
                : 9
                Affiliations
                [1 ]Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Kobe, Japan
                [2 ]Organization of Advanced Science and Technology, Kobe University, Kobe, Japan
                University of South Florida College of Medicine, United States of America
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: MK NF JI AK. Performed the experiments: MK NF. Analyzed the data: MK JI. Wrote the paper: MK JI.

                [¤]

                Current address: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan

                Article
                PONE-D-14-12562
                10.1371/journal.pone.0108229
                4171513
                25244640

                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.

                Page count
                Pages: 15
                Funding
                This work was supported in part by a Research Fellowship for Young Scientists from the Japan Society for the Promotion of Science, the Naito Foundation, and Special Coordination Funds for Promoting Science and Technology, Creation of Innovation Centers for Advanced Interdisciplinary Research Areas (Innovative Bioproduction Kobe; iBioK) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, Commission for Development of Artificial Gene Synthesis Technology for Creating Innovative Biomaterial from the Ministry of Economy, Trade and Industry (METI) of Japan. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Biotechnology
                Bioengineering
                Synthetic Bioengineering
                Macromolecular Engineering
                Protein Engineering
                Directed Evolution
                Molecular Biology
                Molecular Biology Techniques
                Molecular Biology Assays and Analysis Techniques
                Library Screening
                Protein Interaction Assays
                Engineering and Technology
                Research and Analysis Methods
                Model Organisms
                Yeast and Fungal Models
                Custom metadata
                The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.

                Uncategorized

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