Lysineless HiBiT and NanoLuc Tagging Systems as Alternative Tools for Monitoring Targeted Protein Degradation

Target protein degradation (TPD) has emerged as a revolutionary approach in drug discovery, leveraging the cell’s intrinsic machinery to selectively degrade disease-associated proteins. Nanoluciferase (nLuc) fusion proteins and the NanoBiT technology offer two robust and sensitive screening platforms to monitor the subtle changes in protein abundance induced by TPD molecules. Despite these advantages, concerns have arisen regarding potential degradation artifacts introduced by tagging systems due to the presence of lysine residues on them, prompting the development of alternative tools. In this study, we introduce HiBiT-RR and nLuc^K0, variants devoid of lysine residues, to mitigate such artifacts. Our findings demonstrate that HiBiT-RR maintains a similar sensitivity and binding affinity with the original HiBiT. Moreover, the comparison between nLuc^WT and nLuc^K0 constructs reveals variations in degradation patterns induced by certain TPD molecules, emphasizing the importance of choosing appropriate tagging systems to ensure the reliability of experimental outcomes in studying protein degradation processes.