Chapter title |
Differential Radial Capillary Action of Ligand Assay (DRaCALA) for High-Throughput Detection of Protein-Metabolite Interactions in Bacteria.
|
---|---|
Chapter number | 3 |
Book title |
Bacterial Pathogenesis
|
Published in |
Methods in molecular biology, January 2017
|
DOI | 10.1007/978-1-4939-6673-8_3 |
Pubmed ID | |
Book ISBNs |
978-1-4939-6671-4, 978-1-4939-6673-8
|
Authors |
Mona W. Orr, Vincent T. Lee |
Editors |
Pontus Nordenfelt, Mattias Collin |
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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