High-Throughput Production of a New Library of Human Single and Tandem PDZ Domains Allows Quantitative PDZ-Peptide Interaction Screening Through High-Throughput Holdup Assay
2019
Duhoo, Yoan | Girault, Virginie | Turchetto, Jeremy | Ramond, Laurie | Durbesson, Fabien | Fourquet, Patrick | Nominé, Yves | Cardoso, Vânia | Sequeira, Ana Filipa | Brás, Joana | Fontes, Carlos | Travé, Gilles | Wolff, Nicolas | Vincentelli, Renaud | Architecture et fonction des macromolécules biologiques (AFMB) ; Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS) | Récepteurs Canaux - Channel Receptors ; Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS) | Centre de Recherche en Cancérologie de Marseille (CRCM) ; Aix Marseille Université (AMU)-Institut Paoli-Calmettes (IPC) ; Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS) | Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC) ; Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS) | NZYTech Genes & Enzymes | This work received institutional support from Centre National de la Recherche Scientifique (CNRS), Université de Strasbourg, Institut National de la Santé et de la Recherche Médicale (INSERM) and Région Alsace. The work was supported in part by a grants from Ligue contre le Cancer (équipe labellisée 2015). This work was supported by the Programme Transversal de Recherche from Institut Pasteur (PTR grant no. 483 to N.W.) and by the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INSB-05-01 for the AFMB. | Renaud Vincentelli | ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010)
International audience
显示更多 [+] 显示较少 [-]英语. PDZ domains recognize PDZ Binding Motifs (PBMs) at the extreme C-terminus of their partner proteins. The human proteome contains 266 identified PDZ domains, the PDZome, spread over 152 proteins. We previously developed the "holdup" chromatographic assay for high-throughput determination of PDZ-PBM affinities. In that work, we had used an expression library of 241 PDZ constructs (the "PDZome V.1"). Here, we cloned, produced, and characterized a new bacterial expression library ("PDZome V.2"), which comprises all the 266 known human PDZ domains as well as 37 PDZ tandem constructs. To ensure the best expression level, folding, and solubility, all construct boundaries were redesigned using available structural data and all DNA sequences were optimized for Escherichia coli expression. Consequently, all the PDZ constructs are produced in a soluble form. Precise quantification and quality control were carried out. The binding profiles previously published using "PDZome V.1" were reproduced and completed using the novel "PDZome V.2" library. We provide here the detailed description of the high-throughput protocols followed through the PDZ gene synthesis and cloning, PDZ production, holdup assay and data treatment.
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