ArticleScientific reports2021
Removing auto-activators from yeast-two-hybrid assays by conditional negative selection.
Article in Scientific reports, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 10 papers.
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10 citing papers in PubMed, 13 citations in OpenAlex.
- Amplicon/Protein Bead Display enables quantitativebioRxiv : the preprint server for biology · 2026Article
- Saccharomyces cerevisiae as a model for mammalian diseases.FEMS yeast research · 2026Review
- MS-Based Proteomics Approach in Mapping Protein-Protein Interaction.Advances in experimental medicine and biology · 2026Review
- The Role of CRABS CLAW Transcription Factor in Floral Organ Development in Plants.International journal of molecular sciences · 2025Review
- Functional identification ofPhysiology and molecular biology of plants : an international journal of functional plant biology · 2025Article
- Review
- Decoding Plant-Pathogen Interactions: A Comprehensive Exploration of Effector-Plant Transcription Factor Dynamics.Molecular plant pathology · 2025Review
- Massively parallel measurement of protein-protein interactions by sequencing using MP3-seq.Nature chemical biology · 2024Article
- Next-Generation Yeast Two-Hybrid Screening to Discover Protein-Protein Interactions.Methods in molecular biology (Clifton, N.J.) · 2023Article
- AnBioTechniquesArticle
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Authors and funding
5 authors at 2 institutions in 3 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Yeast-two-hybrid (Y2H) is widely used as a strategy to detect protein-protein interactions (PPIs). Recent advancements have made it possible to generate and analyse genome-wide PPI networks en masse by coupling Y2H with next-generation sequencing technology. However, one of the major challenges of yeast two-hybrid assay is the large amount of false-positive hits caused by auto-activators (AAs), which are proteins that activate the reporter genes without the presence of an interacting protein partner. Here, we have developed a negative selection to minimize these auto-activators by integrating the pGAL2-URA3 fragment into the yeast genome. Upon activation of the pGAL2 promoter by an AA, yeast cells expressing URA3 cannot grow in media supplemented with 5-Fluoroorotic acid (5-FOA). Hence, we selectively inhibit the growth of yeast cells expressing auto-activators and thus minimizing the amount of false-positive hits. Here, we have demonstrated that auto-activators can be successfully removed from a Marchantia polymorpha cDNA library using pGAL2-URA3 and 5-FOA treatment, in liquid and solid-grown cultures. Furthermore, since URA3 can also serve as a marker for uracil autotrophy, we propose that our approach is a valuable addition to any large-scale Y2H screen.
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