Skip to main content
SpringerLink
Log in

Measuring Protein–Protein Interactions in Cells using Nanoluciferase Bioluminescence Resonance Energy Transfer (NanoBRET) Assay

  • Protocol
  • First Online:
Chemogenomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2706))

Abstract

Protein–protein interactions (PPIs) are increasingly recognized for their roles in functional cellular networks and their importance in disease-targeting contexts. Assessing PPI in the native cellular environment is challenging and requires specific and quantitative methods. Bioluminescence resonance energy transfer (BRET) is a biophysical process that can be used to quantify PPI. With Nanoluciferase bioluminescent protein as a donor and a fluorescent chloroalkane ligand covalently bound to HaloTag protein as an acceptor, NanoBRET provides a versatile and robust system to quantitatively measure PPI in living cells. BRET efficiency is proportional to the distance between the donor and acceptor, allowing for the measurement of PPI in real time. In this paper, we describe the use of NanoBRET to study specific interactions between proteins of interest in living cells that can be perturbed by using small-molecule antagonists and genetic mutations. Here, we provide a detailed protocol for expressing NanoLuc and HaloTag fusion proteins in cell culture and the necessary optimization of NanoBRET assay conditions. Our example results demonstrate the reliability and sensitivity of NanoBRET for measuring interactions between proteins, protein domains, and short peptides and quantitating the PPI antagonist compound activity in living cells.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 169.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 219.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Nooren IMA, Thornton JM (2003) Diversity of protein-protein interactions. EMBO J 22(14):3486–3492. https://doi.org/10.1093/emboj/cdg359

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Arkin M (2005) Protein-protein interactions and cancer: small molecules going in for the kill. Curr Opin Chem Biol 9(3):317–324. https://doi.org/10.1016/j.cbpa.2005.03.001

    Article  CAS  PubMed  Google Scholar 

  3. Jin L, Wang W, Fang G (2014) Targeting protein-protein interaction by small molecules. Ann Rev Pharmacol Toxicol 54:435–456. https://doi.org/10.1146/annurev-pharmtox-011613-140028

    Article  CAS  Google Scholar 

  4. Li B, Rong D, Wang Y (2019) Targeting protein-protein interaction with covalent small-molecule inhibitors. Curr Top Med Chem 19(21):1872–1876. https://doi.org/10.2174/1568026619666191011163410

    Article  CAS  PubMed  Google Scholar 

  5. Pfleger KDG, Eidne KA (2006) Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET). Nat Methods 3(3):165–174. https://doi.org/10.1038/nmeth841

    Article  CAS  PubMed  Google Scholar 

  6. Coriano C, Powell E, Xu W (2016) Monitoring ligand-activated protein-protein interactions using bioluminescent resonance energy transfer (BRET) assay. Method Mol Biol (Clifton, NJ) 1473:3–15. https://doi.org/10.1007/978-1-4939-6346-1_1

    Article  Google Scholar 

  7. Wu P, Brand L (1994) Resonance energy transfer: methods and applications. Anal Biochem 218(1):1–13. https://doi.org/10.1006/abio.1994.1134

    Article  CAS  PubMed  Google Scholar 

  8. Boute N, Jockers R, Issad T (2002) The use of resonance energy transfer in high-throughput screening: BRET versus FRET. Trends Pharmacol Sci 23(8):351–354. https://doi.org/10.1016/s0165-6147(02)02062-x

    Article  CAS  PubMed  Google Scholar 

  9. Machleidt T, Woodroofe CC, Schwinn MK et al (2015) NanoBRET – a novel BRET platform for the analysis of protein-protein interactions. ACS Chem Biol 10(8):1797–1804. https://doi.org/10.1021/acschembio.5b00143

    Article  CAS  PubMed  Google Scholar 

  10. Dale NC, Johnstone EKM, White CW, Pfleger KDG (2019) NanoBRET: the bright future of proximity-based assays. Front Bioeng Biotechnol 7:56. https://doi.org/10.3389/fbioe.2019.00056

    Article  PubMed  PubMed Central  Google Scholar 

  11. Groß VE, Gershkovich MM, Schöneberg T et al (2022) NanoBRET in C. elegans illuminates functional receptor interactions in real time. BMC Mol Cell Biol 23(1):8. https://doi.org/10.1186/s12860-022-00405-w

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Phillipou AN, Lay CS, Carver CE et al (2020) Cellular target engagement approaches to monitor epigenetic reader domain interactions. SLAS Discov Adv Life Sci R & D 25(2):163–175. https://doi.org/10.1177/2472555219896278

    Article  CAS  Google Scholar 

  13. Hall MP, Unch J, Binkowski BF et al (2012) Engineered luciferase reporter from a deep-sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chem Biol 7(11):1848–1857. https://doi.org/10.1021/cb3002478

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Los GV, Encell LP, McDougall M et al (2008) HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem Biol 3(6):373–382. https://doi.org/10.1021/cb800025k

    Article  CAS  PubMed  Google Scholar 

  15. Weihs F, Wang J, Pfleger KDG, Dacres H (2020) Experimental determination of the bioluminescence resonance energy transfer (BRET) Förster distances of NanoBRET and red-shifted BRET pairs. Analytica Chimica Acta: X 6:100059. https://doi.org/10.1016/j.acax.2020.100059

    Article  CAS  PubMed  Google Scholar 

  16. Azad T, Tashakor A, Hosseinkhani S (2014) Split-luciferase complementary assay: applications, recent developments, and future perspectives. Anal Bioanal Chem 406(23):5541–5560. https://doi.org/10.1007/s00216-014-7980-8

    Article  CAS  PubMed  Google Scholar 

  17. Mo X-L, Luo Y, Ivanov AA et al (2016) Enabling systematic interrogation of protein-protein interactions in live cells with a versatile ultra-high-throughput biosensor platform. J Mol Cell Biol 8(3):271–281. https://doi.org/10.1093/jmcb/mjv064

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Sampaio NG, Kocan M, Schofield L et al (2018) Investigation of interactions between TLR2, MyD88 and TIRAP by bioluminescence resonance energy transfer is hampered by artefacts of protein overexpression. PLoS One 13(8):e0202408. https://doi.org/10.1371/journal.pone.0202408

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Bradley WD, Arora S, Busby J et al (2014) EZH2 inhibitor efficacy in non-Hodgkin’s lymphoma does not require suppression of H3K27 monomethylation. Chem Biol 21(11):1463–1475. https://doi.org/10.1016/j.chembiol.2014.09.017

    Article  CAS  PubMed  Google Scholar 

  20. Szewczyk MM, Ishikawa Y, Organ S et al (2020) Pharmacological inhibition of PRMT7 links arginine monomethylation to the cellular stress response. Nat Commun 11(1):2396. https://doi.org/10.1038/s41467-020-16271-z

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Dilworth D, Hanley RP, Ferreira de Freitas R et al (2022) A chemical probe targeting the PWWP domain alters NSD2 nucleolar localization. Nat Chem Biol 18(1):56–63. https://doi.org/10.1038/s41589-021-00898-0

    Article  CAS  PubMed  Google Scholar 

  22. Wu Y, Zhou X, Barnes CO et al (2016) The DDB1-DCAF1-Vpr-UNG2 crystal structure reveals how HIV-1 Vpr steers human UNG2 toward destruction. Nat Struct Mol Biol 23(10):933–940. https://doi.org/10.1038/nsmb.3284

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Varshavsky A (2019) N-degron and C-degron pathways of protein degradation. Proc Natl Acad Sci 116(2):358–366. https://doi.org/10.1073/pnas.1816596116

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Liu H, Pfirrmann T (2019) The Gid-complex: an emerging player in the ubiquitin ligase league. Biol Chem 400(11):1429–1441. https://doi.org/10.1515/hsz-2019-0139

    Article  CAS  PubMed  Google Scholar 

  25. Maitland MER, Lajoie GA, Shaw GS, Schild-Poulter C (2022) Structural and functional insights into GID/CTLH E3 ligase complexes. Int J Mol Sci 23(11). https://doi.org/10.3390/ijms23115863

  26. Dong C, Zhang H, Li L et al (2018) Molecular basis of GID4-mediated recognition of degrons for the Pro/N-end rule pathway. Nat Chem Biol 14(5):466–473. https://doi.org/10.1038/s41589-018-0036-1

    Article  CAS  PubMed  Google Scholar 

  27. Hao B, Oehlmann S, Sowa ME et al (2007) Structure of a Fbw7-Skp1-cyclin E complex: multisite-phosphorylated substrate recognition by SCF ubiquitin ligases. Mol Cell 26(1):131–143. https://doi.org/10.1016/j.molcel.2007.02.022

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The Structural Genomics Consortium is a registered charity (no: 1097737) that receives funds from Bayer AG, Boehringer Ingelheim, Bristol Myers Squibb, Genentech, Genome Canada through Ontario Genomics Institute [OGI-196], EU/EFPIA/OICR/McGill/KTH/Diamond Innovative Medicines Initiative 2 Joint Undertaking [EUbOPEN grant 875510], Janssen, Merck KGaA (aka EMD in Canada and US), Pfizer, and Takeda.

Author information

Authors and Affiliations

  1. Structural Genomics Consortium, University of Toronto, Toronto, ON, Canada

    Magdalena M. Szewczyk, Dominic D. G. Owens & Dalia Barsyte-Lovejoy

  2. Department of Pharmacology and Toxicology, University of Toronto, Toronto, ON, Canada

    Dalia Barsyte-Lovejoy

Authors
  1. Magdalena M. Szewczyk
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Dominic D. G. Owens
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Dalia Barsyte-Lovejoy
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Dalia Barsyte-Lovejoy .

Editor information

Editors and Affiliations

  1. Department of Pharmacy, Ludwig-Maximilians-Universität München, Munich, Germany

    Daniel Merk

  2. Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Frankfurt am Main, Germany

    Apirat Chaikuad

Rights and permissions

Reprints and permissions

Copyright information

© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Szewczyk, M.M., Owens, D.D.G., Barsyte-Lovejoy, D. (2023). Measuring Protein–Protein Interactions in Cells using Nanoluciferase Bioluminescence Resonance Energy Transfer (NanoBRET) Assay. In: Merk, D., Chaikuad, A. (eds) Chemogenomics. Methods in Molecular Biology, vol 2706. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3397-7_10

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-3397-7_10

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3396-0

  • Online ISBN: 978-1-0716-3397-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation