High throughput flow cytometry based yeast two-hybrid array approach for large-scale analysis of protein-protein interactions

Authors

  • Jun Chen,

    Corresponding author
    1. University of New Mexico Center for Molecular Discovery, Albuquerque, New Mexico 87131
    2. University of New Mexico Cancer Center, Albuquerque, New Mexico 87131
    • UNM Cancer Center, 2325 Camino de Salud NE, MSC11 6020 CRF221, Albuquerque, NM 87131, USA
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  • Mark B. Carter,

    1. University of New Mexico Center for Molecular Discovery, Albuquerque, New Mexico 87131
    2. University of New Mexico Cancer Center, Albuquerque, New Mexico 87131
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  • Bruce S. Edwards,

    1. University of New Mexico Center for Molecular Discovery, Albuquerque, New Mexico 87131
    2. University of New Mexico Cancer Center, Albuquerque, New Mexico 87131
    3. Department of Pathology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131
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  • Hong Cai,

    1. Mesa Tech International LLC, Santa Fe, New Mexico 87507
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  • Larry A. Sklar

    Corresponding author
    1. University of New Mexico Center for Molecular Discovery, Albuquerque, New Mexico 87131
    2. University of New Mexico Cancer Center, Albuquerque, New Mexico 87131
    3. Department of Pathology, University of New Mexico Health Sciences Center, Albuquerque, New Mexico 87131
    • UNM Cancer Center, 2325 Camino de Salud NE, MSC11 6020 CRF221, Albuquerque, NM 87131, USA
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Abstract

The analysis of protein-protein interactions is a key focus of proteomics efforts. The yeast two-hybrid system (Y2H) has been the most commonly used method in genome-wide searches for protein interaction partners. However, the throughput of the current yeast two-hybrid array approach is hampered by the involvement of the time-consuming LacZ assay and/or the incompatibility of liquid handling automation due to the requirement for selection of colonies/diploids on agar plates. To facilitate large-scale Y2H assays, we report a novel array approach by coupling a GFP reporter based Y2H system with high throughput flow cytometry that enables the processing of a 96-well plate in as little as 3 min. In this approach, the yEGFP reporter has been established in both AH109 (MATa) and Y187 (MATα) reporter cells. It not only allows the generation of two copies of GFP reporter genes in diploid cells, but also allows the convenient determination of self-activators generated from both bait and prey constructs by flow cytometry. We demonstrate a Y2H array assay procedure that is carried out completely in liquid media in 96-well plates by mating bait and prey cells in liquid YPD media, selecting the diploids containing positive interaction pairs in selective media and analyzing the GFP reporter directly by flow cytometry. We have evaluated this flow cytometry based array procedure by showing that the interaction of the positive control pair P53/T is able to be reproducibly detected at 72 hr postmating compared with the negative control pairs. We conclude that our flow cytometry based yeast two-hybrid approach is robust, convenient, quantitative, and is amenable to large-scale analysis using liquid-handling automation. © 2011 International Society for Advancement of Cytometry

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