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Differential Radial Capillary Action of Ligand Assay (DRaCALA) for High-Throughput Detection of Protein–Metabolite Interactions in Bacteria

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Bacterial Pathogenesis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1535))

Abstract

Bacteria rely on numerous nucleotide second messengers for signal transduction such as cyclic AMP, cyclic-di-GMP, and cyclic-di-AMP. Although a number of receptors responsible for known regulated phenotypes have been established, the completeness of protein receptors in any given organism remains elusive. We have developed a method called differential radial capillary action of ligand assay (DRaCALA) that allows for an unbiased, systematic high-throughput screen for the detection of ligand binding proteins encoded by a genome. DRaCALA permits interrogation of ligand binding directly to an overexpressed protein in a cell lysate and bypasses the need of protein purification. Gateway-cloning-compatible open reading frame libraries are available for a diverse range of bacterial species and permits generation of the lysates overexpressing each open reading frame. These lysates can be assessed by DRaCALA in a 96-well format to allow rapid identification of protein–ligand interactions, including previously unknown proteins. Here, we present the protocols for generating the expression library, conducting the DRaCALA screen, data analysis, and hit validation.

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Author information

Authors and Affiliations

  1. Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, 20742, USA

    Mona W. Orr & Vincent T. Lee

  2. Maryland Pathogen Research Institute, University of Maryland, College Park, MD, 20742, USA

    Vincent T. Lee

  3. Biological Sciences Graduate Program, University of Maryland, College Park, MD, 20742, USA

    Mona W. Orr

Authors
  1. Mona W. Orr
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  2. Vincent T. Lee
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Corresponding author

Correspondence to Vincent T. Lee .

Editor information

Editors and Affiliations

  1. Department of Clinical Sciences, Lund University, Division of Infection Medicine, Lund, Sweden

    Pontus Nordenfelt

  2. Department of Clinical Sciences, Lund University, Division of Infection Medicine, Lund, Sweden

    Mattias Collin

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Orr, M.W., Lee, V.T. (2017). Differential Radial Capillary Action of Ligand Assay (DRaCALA) for High-Throughput Detection of Protein–Metabolite Interactions in Bacteria. In: Nordenfelt, P., Collin, M. (eds) Bacterial Pathogenesis. Methods in Molecular Biology, vol 1535. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6673-8_3

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  • DOI: https://doi.org/10.1007/978-1-4939-6673-8_3

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6671-4

  • Online ISBN: 978-1-4939-6673-8

  • eBook Packages: Springer Protocols

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