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dc.contributor.authorShivhare, Devendraen_US
dc.contributor.authorMusialak-Lange, Magdalenaen_US
dc.contributor.authorJulca, Ireneen_US
dc.contributor.authorGluza, Pawelen_US
dc.contributor.authorMutwil, Mareken_US
dc.identifier.citationShivhare, D., Musialak-Lange, M., Julca, I., Gluza, P. & Mutwil, M. (2021). Removing auto-activators from yeast-two-hybrid assays by conditional negative selection. Scientific Reports, 11(1), 5477-.
dc.description.abstractYeast-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.en_US
dc.description.sponsorshipMinistry of Education (MOE)en_US
dc.description.sponsorshipNanyang Technological Universityen_US
dc.relation.ispartofScientific Reportsen_US
dc.rights© 2021 The Author(s). This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit
dc.subjectScience::Biological sciencesen_US
dc.titleRemoving auto-activators from yeast-two-hybrid assays by conditional negative selectionen_US
dc.typeJournal Articleen
dc.contributor.schoolSchool of Biological Sciencesen_US
dc.description.versionPublished versionen_US
dc.subject.keywordsHigh-Throughput Screeningen_US
dc.subject.keywordsProtein–Protein Interaction Networksen_US
dc.description.acknowledgementThis article was supported by Max Planck Gesellschaft and is funded by NTU Start-Up Grant and Singaporean Ministry of Education grant MOE2018-T2-2-053.en_US
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