Development of a global screening system for detecting protein-protein interactions by luminescence complementation in fission yeast.
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.
Azadeh F, Hashimoto A, Nishimura S, Arioka M, Yoshida M, Matsuyama A