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Antibiotics pp 247-259 | Cite as

Bacterial Histidine Kinases: Overexpression, Purification, and Inhibitor Screen

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1520)

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.

Key words

Two-component regulatory system Histidine kinase Purification Full-length Kinase inhibitor Antimicrobial compound Phosphorylation Phos-tag 

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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Mike Gajdiss
    • 1
  • Michael Türck
    • 1
  • Gabriele Bierbaum
    • 1
  1. 1.Institute of Medical Microbiology, Immunology and ParasitologyUniversity of BonnBonnGermany

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