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Monitoring Intracellular Protein Interactions Using NanoLuc® Binary Technology (NanoBiT)


Protein:protein interactions (PPIs) are essential to the cellular signal transduction pathways that contribute to cancer. Although numerous approaches exist to monitor PPIs in vitro, methods for intracellular detection have been more limited. We developed NanoLuc® Binary Technology (NanoBiT), a two-subunit system based on NanoLuc® luciferase that can be applied to the intracellular detection of PPIs. Large BiT (LgBiT; 18 kDa) and Small BiT (SmBiT; 11 amino acid peptide) subunits are expressed as fusions to proteins of interest, where PPI facilitates subunit complementation to give a bright, luminescent enzyme. Unlike related approaches where an enzyme or protein is simply split, LgBiT was independently optimised for structural stability and SmBiT was selected from a peptide library specifically for the PPI
application. The result is a subunit pair that weakly associates (KD = 190 μM) yet shows only 3 fold lower activity at saturation vs. NanoLuc in vitro. In contrast to many split systems, the LgBiT:SmBiT interaction is reversible, allowing the detection of rapidly dissociating proteins. PPI dynamics can be followed in realtime in living cells using the Nano-Glo® Live Cell Reagent, a nonlytic detection reagent containing the cell-permeable furimazine substrate. Advantages over split systems include better sensitivity, allowing fusion protein expression at or near physiological levels, reversibility, fusion to a peptide or a small, structurally stable protein domain, real-time measurements using a non-lytic assay format, and subunits with reduced affinity for self-association. We have applied this system to several PPIs associated with cellular transformation.
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