New Cell-Based Kinase Assays for TrkA and Other Receptor Tyrosine Kinases using PathHunter™ Protein-Protein Interaction Technology

New Cell-Based Kinase Assays for TrkA and Other Receptor Tyrosine Kinases using PathHunter™ Protein-Protein Interaction Technology
Version:
REV1_1008

Year:
2008

The PathHunter™ technology from DiscoveRx is an adaptation of Enzyme Fragment Complementation (EFC) that provides a novel, generic cell-based assay format for detecting protein-protein interactions. This approach has enabled development of a number of new screening and profiling assays for targets that have been difficult to address in a cell-based assay. One such class of targets, the receptor tyrosine kinases, are an example of a class of proteins for which we have now demonstrated productive, functional cell-based assays using protein-protein interaction as the detection mechanism. In this PathHunter assay approach, a small peptide epitope (ProLink) is expressed recombinantly on the target protein of interest, in this case the intracellular C-terminus of the RTK. This is co-expressed with a larger sequence, termed enzyme acceptor (EA) that is attached to a protein thought to interact with your target. A productive protein-protein interaction will generate an active beta-galactosidase enzyme, which is detected using a chemiluminescent substrate. We will present data illustrating the application of this approach to the TrkA kinase, an important target in neurological research. Our results show that this assay can be used to identify inhibitors of kinase activity in a simple, homogeneous screening assay that is cell-based and therefore more biologically relevant than traditional biochemical approaches.