title: A novel quantitative kinase assay using bacterial surface display and flow cytometry.
abstract: The inhibition of tyrosine kinases is a successful approach for the treatment of cancers and the discovery of kinase inhibitor drugs is the focus of numerous academic and pharmaceutical laboratories. With this goal in mind, several strategies have been developed to measure kinase activity and to screen novel tyrosine kinase inhibitors. Nevertheless, a general non-radioactive and inexpensive approach, easy to implement and adapt to a range of applications, is still missing. Herein, using Bcr-Abl tyrosine kinase, an oncogenic target and a model protein for cancer studies, we describe a novel cost-effective high-throughput screening kinase assay. In this approach, named the BacKin assay, substrates displayed on a Bacterial cell surface are incubated with Kinase and their phosphorylation is examined and quantified by flow cytometry. This approach has several advantages over existing approaches, as using bacteria (i.e. Escherichia coli) to display peptide substrates provides a self renewing solid support that does not require laborious chemical strategies. Here we show that the BacKin approach can be used for kinetic and mechanistic studies, as well as a platform to characterize and identify small-molecule or peptide-based kinase inhibitors with potential applications in drug development.
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia.
authors.forename: Sónia Troeira
authors.initials: ST
authors.lastname: Henriques
authors.forename: Louise
authors.initials: L
authors.lastname: Thorstholm
authors.forename: Yen-Hua
authors.initials: YH
authors.lastname: Huang
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Getz
authors.forename: Patrick S
authors.initials: PS
authors.lastname: Daugherty
authors.forename: David J
authors.initials: DJ
authors.lastname: Craik
mesh_terms: D001419:Bacteria / Q000378:metabolism*; D061505:Cell Surface Display Techniques; D004789:Enzyme Activation; D057075:Enzyme Assays / Q000379:methods*; D005434:Flow Cytometry; D016044:Fusion Proteins, bcr-abl / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D013379:Substrate Specificity
chemical_list: D010446:Peptide Fragments; D047428:Protein Kinase Inhibitors; D010770:Phosphotransferases; D011505:Protein-Tyrosine Kinases; D016044:Fusion Proteins, bcr-abl