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.
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Fritz G, Mascher T (2014) A balancing act times two: sensing and regulating cell envelope homeostasis in Bacillus subtilis. Mol Micro 94:1201–1207CrossRefGoogle Scholar
Matsushita M, Janda KD (2002) Histidine kinases as targets for new antimicrobial agents. Bioorg Med Chem 10(4):855–867CrossRefPubMedGoogle Scholar
Gotoh Y, Eguchi Y, Watanabe T, Okamoto S, Doi A, Utsumi R (2010) Two-component signal transduction as potential drug targets in pathogenic bacteria. Curr Opin Microbiol 13(2):232–239CrossRefPubMedGoogle Scholar
Boyle-Vavra S, Yin S, Daum RS (2006) The VraS/VraR two-component regulatory system required for oxacillin resistance in community-acquired methicillin-resistant Staphylococcus aureus. FEMS Microbiol Lett 262(2):163–171CrossRefPubMedGoogle Scholar
Francis S, Wilke KE, Brown DE, Carlson EE (2013) Mechanistic insight into inhibition of two-component system signaling. Med Chem Commun 4(1):269–277CrossRefGoogle Scholar
Stephenson K, Yamaguchi Y, Hoch JA (2000) The mechanism of action of inhibitors of bacterial two-component signal transduction systems. J Biol Chem 275(49):38900–38904CrossRefPubMedGoogle Scholar
Kinoshita E, Kinoshita-Kikuta E, Takiyama K, Koike T (2006) Phosphate-binding tag, a new tool to visualize phosphorylated proteins. Mol Cell Proteomics 5(4):749–757CrossRefPubMedGoogle Scholar
Miroux B, Walker JE (1996) Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J Mol Bio 260(3):289–298CrossRefGoogle Scholar