A surface display yeast two-hybrid screening system for high-throughput protein interactome mapping.
Despite the wide acceptance of yeast two-hybrid (Y2H) system for protein-protein interaction analysis and discovery, conventional Y2H assays are not well suited for high-throughput screening of the protein interaction network ("interactome") on a genomic scale due to several limitations, including labor-intensive agar plating and colony selection methods associated with the use of nutrient selection markers, complicated reporter analysis methods associated with the use of LacZ enzyme reporters, and incompatibility of the liquid handling robots. We recently reported a robust liquid culture Y2H system based on quantitative analysis of yeast-enhanced green fluorescent protein (yEGFP) reporters that greatly increased the analysis throughput and compatibility with liquid handling robots. To further advance its utility in high-throughput complementary DNA (cDNA) library screening, we report the development of a novel surface display Y2H (sdY2H) library screening system with uniquely integrated surface display hemagglutination (sdHA) antigen and yEGFP reporters. By introduction of a surface reporter sdHA into the yEGFP-based Y2H system, positive Y2H targets are quickly isolated from library cells by a simple magnetic separation without a large plating effort. Moreover, the simultaneous scoring of multiple reporters, including sdHA, yEGFP, and conventional nutrient markers, greatly increased the specificity of the Y2H assay. The feasibility of the sdY2H assay on large cDNA library screening was demonstrated by the successful recovery of positive P53/T interaction pairs at a target-to-background ratio of 1:1,000,000. Together with the massive parallel DNA sequencing technology, it may provide a powerful proteomic tool for high-throughput interactome mapping on a genomic scale.
Chen
Jun
J
Biosciences Division, National Flow Cytometry Resource, Los Alamos National Laboratory, Los Alamos, NM 87545, USA.
Zhou
Jianhong
J
Sanders
Claire K
CK
Nolan
John P
JP
Cai
Hong
H
D045744:Cell Line, Tumor; D015723:Gene Library; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006367:HeLa Cells; D006384:Hemagglutination; D006801:Humans; D008280:Magnetics; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods; D017422:Sequence Analysis, DNA / Q000379:methods; D013499:Surface Properties; D020798:Two-Hybrid System Techniques
D049452:Green Fluorescent Proteins