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A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions
Published on: June 6, 2018
A surface display yeast two-hybrid screening system for high-throughput protein interactome mapping
Jun Chen1, Jianhong Zhou, Claire K Sanders
1Biosciences Division, National Flow Cytometry Resource, Los Alamos National Laboratory, Los Alamos, NM 87545, USA.
Analytical Biochemistry
|March 21, 2009
Summary
A novel surface display yeast two-hybrid (sdY2H) system enhances high-throughput screening of protein interactions. This method uses magnetic separation and multiple reporters for faster, more specific interactome analysis on a genomic scale.
Area of Science:
- Proteomics
- Molecular Biology
- Biotechnology
Background:
- Conventional yeast two-hybrid (Y2H) systems have limitations for high-throughput interactome screening.
- These limitations include labor-intensive methods, complex reporter analysis, and poor robot compatibility.
Purpose of the Study:
- To develop a novel surface display Y2H (sdY2H) system for high-throughput screening of cDNA libraries.
- To improve the efficiency, specificity, and throughput of Y2H assays for interactome mapping.
Main Methods:
- Development of a sdY2H system integrating surface display hemagglutination (sdHA) and yeast-enhanced green fluorescent protein (yEGFP) reporters.
- Utilized magnetic separation for rapid isolation of positive Y2H targets from library cells.
- Employed simultaneous scoring of multiple reporters (sdHA, yEGFP, nutrient markers) to enhance assay specificity.
Main Results:
- Demonstrated feasibility of sdY2H for large-scale cDNA library screening.
- Successfully recovered P53/T interaction pairs with a high target-to-background ratio (1:1,000,000).
- The sdY2H system significantly increased analysis throughput and compatibility with liquid handling robots.
Conclusions:
- The sdY2H system offers a powerful tool for high-throughput interactome mapping on a genomic scale.
- Integration of surface display and multiple reporters enhances Y2H assay specificity and efficiency.
- This technology, combined with DNA sequencing, advances proteomic discovery.
