Open AccessA Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions
Author(s) -
Tabitha A. Peterson,
Mark Stamnes,
Robert C. Piper
Publication year - 2018
Publication title -
journal of visualized experiments
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.596
H-Index - 91
ISSN - 1940-087X
DOI - 10.3791/57801
Subject(s) - yeast , computational biology , plasmid , encode , protein–protein interaction , two hybrid screening , saccharomyces cerevisiae , population , computer science , genomic library , biology , synthetic biology , tandem affinity purification , genetics , dna , gene , base sequence , biochemistry , demography , enzyme , sociology , affinity chromatography
Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the discovery of high-affinity interactors that are highly enriched in the library of interacting candidates. In a traditional format, the yeast 2-hybrid assay can yield too many colonies to analyze when conducted at low stringency where low affinity interactors might be found. Moreover, without a comprehensive and complete interrogation of the same library against different bait plasmids, a comparative analysis cannot be achieved. Although some of these problems can be addressed using arrayed prey libraries, the cost and infrastructure required to operate such screens can be prohibitive. As an alternative, we have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing a strategy termed DEEPN (Dynamic Enrichment for Evaluation of Protein Networks), which incorporates high-throughput DNA sequencing and computation to follow the evolution of a population of plasmids that encode interacting partners. Here, we describe customized reagents and protocols that allow a DEEPN screen to be executed easily and cost-effectively.