Abstract
Deciphering protein–protein interactions (PPIs) is crucial for a comprehensive understanding of biological processes, yet current methodologies often provide incomplete interactome maps. Here, we present a sensitive bimolecular NanoBiT-based protein complementation assay platform for robust PPI detection in the fission yeast Schizosaccharomyces pombe. Our platform utilizes two NanoLuc moieties, SmBiT and LgBiT, fused to interacting protein partners, generating a quantifiable luminescent signal upon interaction. To maximize the chances of detection and mitigate potential issues arising from tag position-dependent inactivation of bait proteins, our system enables simultaneous expression of two distinct bait constructs within a single cell: one with the LgBiT-tag fused at its N-terminus and another at its C-terminus. For the prey, we constructed a comprehensive ORFeome library of fission yeast proteins, each fused with SmBiT at its C-terminus, leveraging homologous recombination tools. We established efficient high-throughput methods for cloning and selection of single-copy integrants, enabling the rapid construction of the prey library and reliable identification of true yeast transformants. Validating the platform, high-throughput screening using the general transcription elongation factor Tfs1 successfully identified previously undetectable interactors. This versatile platform not only significantly expands the scope of interactome discovery but also offers a powerful tool for future protein-compound interaction studies.
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Data availability
The authors confirm that all data required to support the conclusions of this article are included within the text, figures, and tables. Protein–protein interaction screening data generated using the NanoBiT system in this study have been submitted to the IMEx (http://www.imexconsortium.org) consortium through IntAct [X]44 and assigned the identifier IM-30540. Strains and plasmids are available upon request or from the Yeast Genetic Resource Center (YGRC/NBRP) of Japan.
Abbreviations
- NanoBiT:
-
Nanoluciferase binary technology
- ORF:
-
Open reading frame
- PCA:
-
Protein complementation assay
- PCR:
-
Polymerase chain reaction
- PPI:
-
Protein–protein interaction
- Y2H:
-
Yeast-two-hybrid
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Acknowledgements
This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Numbers 22K05361 and 23H05473 and by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) KAKENHI Grant Number 23H04882 for Transformative Research Areas (A). Grammar and linguistic refinement of this manuscript were kindly assisted by Gemini 1.5 Pro (Google Cloud AI Studio).
Funding
This work was supported by the Japan Society for the Promotion of Science (Grant Number: 22K05361 and 23H05473), and the Ministry of Education, Culture, Sports, Science and Technology (Grant Number: 23H04882). The authors declare that no other funding was received for this study.
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This work was conceptualized by A.M. and M.Y. All experiments were done by A.M., A.H., and F.A. A.M. and F.A. prepared the original draft. S.N., M.A. and M.Y. edited the manuscript. All authors reviewed the manuscript.
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Azadeh, F., Hashimoto, A., Nishimura, S. et al. Development of a global screening system for detecting protein–protein interactions by luminescence complementation in fission yeast. Sci Rep (2026). https://doi.org/10.1038/s41598-026-35430-8
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DOI: https://doi.org/10.1038/s41598-026-35430-8
