Cited by 1,405bioluminescence and chemiluminescence researchAdvanced Fluorescence Microscopy TechniquesBiotin and Related Studies

NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells

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Dixon, Andrew S., Schwinn, Marie K., Hall, Mary P., Zimmerman, Kris, Otto, Paul, Lubben, Thomas H., Butler, Braeden L., Binkowski, Brock F. +6 more·2016·ACS Chemical Biology

Abstract

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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Crossref · 10.1021/acschembio.5b00753

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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
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
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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