title: Bacterial Histidine Kinases: Overexpression, Purification, and Inhibitor Screen.
abstract: Bacterial histidine kinases are promising targets for new antimicrobial agents. In antibacterial therapy such agents could inhibit bacterial growth by targeting essential two-component regulatory systems or resensitize bacteria to known antibiotics by blocking stress responses like the cell wall stress response. However, (1) activity assays using the truncated phosphorylation domains have been shown to produce artifacts and (2) the purification of the full-length histidine kinases is complicated. Here, we describe a standard protocol for the recombinant expression and purification of functional full-length histidine kinases and other membrane proteins from gram-positive bacteria that do not harbor more than two trans-membrane domains using an Escherichia coli host. This guide also presents in vitro phosphorylation assays to screen for new antimicrobial compounds that target bacterial histidine kinases using radioactively labeled ATP and, as a novel approach, Phos-tag acrylamide gel electrophoresis to detect phosphorylated proteins by mobility shift in the polyacrylamide gel.
authors.affiliation: Institute of Medical Microbiology, Immunology and Parasitology, University of Bonn, Sigmund-Freud-Str. 25, Bonn, 53127, Germany.
authors.forename: Mike
authors.initials: M
authors.lastname: Gajdiss
authors.affiliation: Institute of Medical Microbiology, Immunology and Parasitology, University of Bonn, Sigmund-Freud-Str. 25, Bonn, 53127, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Türck
authors.affiliation: Institute of Medical Microbiology, Immunology and Parasitology, University of Bonn, Sigmund-Freud-Str. 25, Bonn, 53127, Germany. g.bierbaum@uni-bonn.de.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Bierbaum
mesh_terms: D020106:Acrylamide; D000255:Adenosine Triphosphate / Q000378:metabolism; D001681:Biological Assay / Q000379:methods*; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology*; D000071677:Histidine Kinase / Q000037:antagonists & inhibitors* / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D011868:Radioisotopes; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D011868:Radioisotopes; D011994:Recombinant Proteins; D020106:Acrylamide; D000255:Adenosine Triphosphate; D000071677:Histidine Kinase
keywords: Antimicrobial compound; Full-length; Histidine kinase; Kinase inhibitor; Phos-tag; Phosphorylation; Purification; Two-component regulatory system