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<p>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 <i>Schizosaccharomyces pombe</i>. 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.</p>

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Development of a global screening system for detecting protein–protein interactions by luminescence complementation in fission yeast

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年1月12日

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  • Fereshteh Azadeh,
  • Atsushi Hashimoto,
  • Shinichi Nishimura,
  • Manabu Arioka,
  • Minoru Yoshida,
  • Akihisa Matsuyama

摘要

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.