NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells
Open accessAbstract
Protein-fragment complementation assays (PCAs) are widely used for investigating protein interactions. However, the fragments used are structurally compromised and have not been optimized nor thoroughly characterized for accurately assessing these interactions. We took advantage of the small size and bright luminescence of NanoLuc to engineer a new complementation reporter (NanoBiT). By design, the NanoBiT subunits (i.e., 1.3 kDa peptide, 18 kDa polypeptide) weakly associate so that their assembly into a luminescent complex is dictated by the interaction characteristics of the target proteins onto which they are appended. To ascertain their general suitability for measuring interaction affinities and kinetics, we determined that their intrinsic affinity (KD = 190 μM) and association constants (kon = 500 M–1 s–1, koff = 0.2 s–1) are outside of the ranges typical for protein interactions. The accuracy of NanoBiT was verified under defined biochemical conditions using the previously characterized interaction between SME-1 β-lactamase and a set of inhibitor binding proteins. In cells, NanoBiT fusions to FRB/FKBP produced luminescence consistent with the linear characteristics of NanoLuc. Response dynamics, evaluated using both protein kinase A and β-arrestin-2, were rapid, reversible, and robust to temperature (21–37 °C). Finally, NanoBiT provided a means to measure pharmacology of kinase inhibitors known to induce the interaction between BRAF and CRAF. Our results demonstrate that the intrinsic properties of NanoBiT allow accurate representation of protein interactions and that the reporter responds reliably and dynamically in cells.
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NanoBiT splits NanoLuc luciferase into LgBiT and SmBiT subunits that associate with low intrinsic affinity (Kd ∼ 190 μM), producing luminescence only when the tagged proteins interact.
“The NanoLuc® Binary Technology (NanoBiT; Promega) splits NanoLuc luciferase into two subunits, Large BiT (LgBiT, 18 kDa) and Small BiT (SmBiT, 1.3 kDa), that associate with low intrinsic affinity (Kd ∼ 190 μM), producing luminescence only when the tagged proteins interact (26).”
From APOE Isoform-Dependent Self-Association Measured by a Split-Luciferase Complementation Assay: Differential Effects of Disease-Risk and Protective Variants (2026) · cites this paper as [26]
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- Authors
- Dixon, A. S., Schwinn, M. K., Hall, M. P., Zimmerman, K., Otto, P., Lubben, T. H., Butler, B. L., Binkowski, B. F., Machleidt, T., Kirkland, T. A., Wood, M. G., Eggers, C. T., Encell, L. P., & Wood, K. V.
- Affiliations
- Promega (United States) (US)
- Corresponding author
- Encell, Lance P.
- Venue
- ACS Chemical Biology
- Publisher
- American Chemical Society (ACS)
- Published
- 2015-11-16
- Volume
- 11
- Issue
- 2
- Pages
- 400-408
- ISSN
- 1554-8929 (ISSN-L)
- 1554-8937
- OpenAlex
- W2226712470
- Cited by
- 1,405
- References
- 22
- Citations by year
- 2026: 181 · 2025: 211 · 2024: 214 · 2023: 179 · 2022: 182 · 2021: 143 · 2020: 126 · 2019: 77 · 2018: 54 · 2017: 23 · 2016: 11 · 2013: 1
- FWCI
- 12.74
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Locations
- ACS Chemical Biology (publishedVersion, other-oa)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › bioluminescence and chemiluminescence research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Biotin and Related Studies
- Keywords
- NanoBiT, protein-fragment complementation assay, protein-protein interaction, luminescent reporter, interaction kinetics, binding affinity, kinase inhibitors, β-arrestin2, protein kinase A, response dynamics
- MeSH terms
- beta-Arrestins, beta-Arrestin 2, Amino Acid Sequence, beta-Lactamases, HeLa Cells, Humans, Kinetics, Luminescent Measurements, Models, Molecular, Molecular Sequence Data, Peptides, Cyclic AMP-Dependent Protein Kinases, Arrestins, Protein Interaction Mapping, Luminescent Agents, HEK293 Cells, Protein Interaction Maps
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