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Journal of Biomolecular Screening
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High Throughput Autophosphorylation Assay for Bacterial Protein Histidine Kinases

Siddhartha Roychoudhury

Procter & Gamble Pharmaceuticals, Research Division, Cincinnati, OH 45040

Susan M. Collins

Procter & Gamble Pharmaceuticals, Research Division, Cincinnati, OH 45040

Barbara A. Hynd

Procter & Gamble Pharmaceuticals, Research Division, Cincinnati, OH 45040

Christian N. Parker

Procter & Gamble Pharmaceuticals, Research Division, Cincinnati, OH 45040

Protein histidine kinases play a major role in bacteria as sensor components in the so-called "two-component" systems involved in signal transduction. We describe a high throughput assay for these kinases using CheA, the bacterial chemotaxis-regulating kinase from Escherichia coil. The purpose of the assay was to monitor ATP-dependent autophosphorylation of the kinase resulting in the phosphorylation of the conserved histidine residue. Unlike most eukaryotic protein kinases, "two-component" kinases are not known to phosphorylate histones and small peptide substrates. This limits the level of phosphorylation and consequently the signal generated in these assays. We, therefore, established the desirable reaction conditions first using the conventional method involving the radio labeling of CheA by [y-32P]ATP followed by gel electrophoresis-based analysis. Next, we converted the assay to a high throughput format in which CheA, autophosphorylated and radiolabeled with [y-33P]ATP, was trapped in a filter via anionic or hydrophobic interaction using diethyl aminoethyl or nitrocellulose-based 96-well filter plates, respectively. Free [y-33P]ATP was removed by washing the wells with high salt buffers. The dried plates were then analyzed for radioactivity associated with the wells by scintillation counting. Finally, we performed and validated the assay in a partially automated format using nitrocellulose-based filter plates.

Journal of Biomolecular Screening, Vol. 2, No. 2, 85-90 (1997)
DOI: 10.1177/108705719700200206
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D. K. Saini and J. S. Tyagi
High-Throughput Microplate Phosphorylation Assays Based on DevR-DevS/Rv2027c 2-Component Signal Transduction Pathway to Screen for Novel Antitubercular Compounds
J Biomol Screen, April 1, 2005; 10(3): 215 - 224.
[Abstract] [PDF]