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Methods Mol Biol. 2019;2025:439-476. doi: 10.1007/978-1-4939-9624-7_21.

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.

Author information

1
Architecture et Fonction des Macromolécules Biologiques (AFMB), Unité Mixte de Recherche (UMR) 7257, Centre National de la Recherche Scientifique (CNRS) Aix-Marseille Université, Marseille cedex 9, France.
2
Unité Récepteurs-Canaux, Département of Neuroscience, CNRS UMR 3571, Institut Pasteur, Paris Cedex 15, France.
3
Université Aix-Marseille, Inserm, CNRS, Institut Paoli-Calmettes, CRCM, Marseille Protéomique, Marseille, France.
4
Équipe Labellisée Ligue 2015, Department of Integrated Structural Biology, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), INSERM U1258/CNRS UMR 7104/Université de Strasbourg, Illkirch, France.
5
NZYTech Genes and Enzymes, Lisbon, Portugal.
6
Architecture et Fonction des Macromolécules Biologiques (AFMB), Unité Mixte de Recherche (UMR) 7257, Centre National de la Recherche Scientifique (CNRS) Aix-Marseille Université, Marseille cedex 9, France. Renaud.vincentelli@afmb.univ-mrs.fr.

Abstract

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.

KEYWORDS:

Domain-ligand interactions; High-throughput; PDZ Binding Motif; PDZ domains; Protein-protein interactions

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