title: Differential Radial Capillary Action of Ligand Assay (DRaCALA) for High-Throughput Detection of Protein-Metabolite Interactions in Bacteria.
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.
authors.affiliation: Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, 20742, USA.|Biological Sciences Graduate Program, University of Maryland, College Park, MD, 20742, USA.
authors.forename: Mona W
authors.initials: MW
authors.lastname: Orr
authors.affiliation: Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD, 20742, USA. vtlee@umd.edu.|Maryland Pathogen Research Institute, University of Maryland, College Park, MD, 20742, USA. vtlee@umd.edu.
authors.forename: Vincent T
authors.initials: VT
authors.lastname: Lee
mesh_terms: D001419:Bacteria / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D057166:High-Throughput Screening Assays / Q000379:methods*; D008024:Ligands; D016366:Open Reading Frames; D011485:Protein Binding; D015203:Reproducibility of Results; D015398:Signal Transduction; D014169:Transformation, Bacterial
chemical_list: D001426:Bacterial Proteins; D008024:Ligands
keywords: DRaCALA; High-throughput screen; Nucleotide signals; ORFeome; Protein–ligand interaction; Receptors