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Monitoring Intracellular Protein Interactions Using NanoLuc® Binary Technology (NanoBiT™)
Poster Title: Monitoring Intracellular Protein Interactions Using NanoLuc® Binary Technology (NanoBiT™)
Submitted on 04 Feb 2016
Author(s): Brock Binkowski(1) , Andrew Dixon(2) , Marie Schwinn(1) , Mary Hall (1) , Kris Zimmerman(1 ), Paul Otto(1) , Thomas Lubben(1) , Braeden L. Butler(1) , Thomas Machleidt(1) , Monika G. Wood(1) , Christopher Eggers(1) , Lance P. Encell(1) , Frank Fan(1) & Keith Wood(1)
Affiliations: (1)Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711-5399; (2)Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, UT 84112
This poster was presented at AACR 2015
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Poster Information
Abstract: 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; 17.6 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 optimized 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 real-time in living cells using the NanoGlo® Live Cell Reagent, a non-lytic detection reagent containing the cell-permeable furimazine substrate. Advantages over split systems include better sensitivity, 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.Summary: NanoBiT is extremely bright, small & stable, and flexible
- >1,000 fold brighter than split firefly luciferase at 37 °C
- Express fusion partners at low levels, minimizing potential artifacts
- LgBiT, 17.6kDa, evolved for increased structural stability; SmBiT, 11 amino acids
- NanoBiT is reversible
- Monitor protein interaction dynamics at a single time point or continuously for 1-2 hours
- Room temperature or 37 °C measurements
- Validated in 96-, 384- & 1536-well fo
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