DiscoverRx recently updated a platform developed years ago by its CEO for cell-based assays. “We’ve put some new improvements and twists on it and those are what have made it amenable to measuring protein-protein interaction events in live cells,” explains Keith Olson, Ph.D., vp of R&D.
The PathHunter™ assays incorporate a target protein fused to a b-galactosidase (b-gal) peptide expressed in the cytoplasm. Another portion of b-gal (enzyme activator) is expressed only in the nucleus. When these two interact, active enzyme is formed, generating signal. This can detect protein translocation to the nucleus or nuclear envelope degradation during mitosis.
All the assays run on any chemiluminescence-capable plate reader with a single reagent addition step, do not require special instrumentation or antibody labeling. An advantage of using an enzyme approach is that it amplifies the signal—up to a twenty or thirty signal to background ratio. “One of the challenges of FRET and HRET, the two most common ways to address protein-protein interactions, is that they don’t generate a lot of signal. After you finish building an assay, you may only have a two- to threefold signal to background ratio,” Dr. Olson states.
In addition, the system is tunable. “We’ve been able to modulate the b-gal reporter system so we have essentially no background association of the two components. The only way to generate signal is to have productive protein-protein interactions.”
The company also offers the PathHunter b-Arrestin Assays for over 100 G-protein coupled receptors (GPCRs). These are high-throughput screening assays for monitoring GPCR activation following ligand stimulation, without imaging instrumentation, fluorescent protein tags, or radioactivity. It provides direct measure of b-Arrestin binding to the GPCR of interest during activation, making it useful for de-orphanizing novel GPCRs. Dr. Olson says the company has recently started a program to look at transcription factors and cytoplasmic kinases involved in general cell signaling events.