HiBiT-RR and nLuc^K0 Tagging Systems for Monitoring Targeted Protein Degradation

Targeted protein degradation (TPD) has become a powerful modality in drug discovery. Central to developing TPD molecules like PROTACs is the ability to accurately and sensitively measure the degradation of a target protein. The NanoLuciferase (nLuc) and the complementary split-nLuc system are highly sensitive reporter technologies ideal for this purpose. However, the presence of lysine residues on these tags can lead to ubiquitination of the tag itself, creating potential artifacts where the tag, not the target protein, drives degradation. This chapter details protocols for the generation and use of lysine-free variants, HiBiT-RR and nLuc^K0, where all lysine residues are replaced with arginine. These alternative tools mitigate the risk of tag-driven degradation, thereby increasing the reliability of TPD screening assays. We describe methods for their use in cellular degradation assays with both transiently expressed and endogenously tagged proteins, and validation via immunoblotting.