DEVELOPMENT OF A FLOW CYTOMETRIC YEAST TWO HYBRID SCREENING SYSTEM

Los Alamos Nat Secty-Los Alamos Nat Lab, Los Alamos NM

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. To discover protein interactome maps at genomic scales, yeast two hybrid-based screening system has been used extensively in the past decade. Despite the tremendous effort in adapting liquid handling robot and array instrumentation, the current Y2H mapping approach is still labor intensive and low throughput attributed to the agar plating-based nutrient marker selection and LacZ reporter analysis. Our recent development of a yEGFP-based yeast two hybrid system enabled the quantitative analysis of GFP fluorescence of the Y2H assay cultures by flow cytometry without plating. With the recent instrumentation advancement of the HyperCyt[unreadable] high-throughput flow cytometry sampling platform, a plate of 384 Y2H liquid culture samples can be quantitatively analyzed in less than 20 minutes. While such system is well-suited for analyzing binary protein-protein interaction pairs, it does not have adequate throughput to perform large cDNA library screening, e.g. sorting a large human cDNA library cells (typically ~109 cells) could take days. To address this limitation, we are establishing a novel sdY2H system that incorporates the surface display reporter as an additional selection marker allowing rapid enrichment of positive cell populations via a simple magnetic separation step. This new surface reporter together with the previously established yEGFP reporter system enable rapid cDNA library screening in liquid cultures, providing a high throughput alternative to conventional plating-based screening and analysis.