Category: Cellular Technologies
Here we present mechanistic cellular studies on several proteins targeted for degradation via the ubiquitin proteasome pathway in mammalian cells. Using bioluminescence resonance energy transfer with NanoLuc and HaloTag fusions, termed NanoBRET, we can monitor changes in interactions of proteins targeted for degradation, including dynamic recruitment to E3 ligases and real-time trafficking to the 26S proteasome after treatment with inhibitors or PROTAC compounds. We show also the ability to quantitate over a high dynamic range total protein levels by monitoring NanoLuc luminescent levels (RLUs) of several proteins, either expressed exogenously or as endogenous knock-in fusion proteins using CRISPR/Cas9. For the endogenous knock-in experiments, we utilize our NanoLuc binary complementation (NanoBiT) technology consisting of a 11 amino acid high affinity tag (HiBiT) that is very efficiently inserted using CRISPR/Cas9, and generate luminescence by complementation with the large NanoBiT subunit (LgBiT). Live cell monitoring of protein:protein interactions and degradation is shown for β-catenin, cMyc, BRD4, and HIF1α. These combined approaches deconvolute the complicated processes involved in proteosomal recruitment and are a powerful strategy for understanding and following protein degradation.
Steven Edenson– Strategic Collaborations Manager, Promega Corporation, Madison, Wisconsin