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Mol Syst Biol. 2016 Apr 22;12(4):863. doi: 10.15252/msb.20156660.

Pooled-matrix protein interaction screens using Barcode Fusion Genetics.

Author information

1
Donnelly Centre, University of Toronto, Toronto, ON, Canada Lunenfeld-Tanenbaum Research Institute Mt. Sinai Hospital, Toronto, ON, Canada Synthetic Biology Division, Research Center for Advanced Science and Technology The University of Tokyo, Tokyo, Japan Institute for Advanced Bioscience, Keio University, Tsuruoka, Yamagata, Japan PRESTO, Japan Science and Technology Agency (JST), Tokyo, Japan yachie@synbiol.rcast.u-tokyo.ac.jp fritz.roth@utoronto.ca.
2
Donnelly Centre, University of Toronto, Toronto, ON, Canada Lunenfeld-Tanenbaum Research Institute Mt. Sinai Hospital, Toronto, ON, Canada.
3
Donnelly Centre, University of Toronto, Toronto, ON, Canada Lunenfeld-Tanenbaum Research Institute Mt. Sinai Hospital, Toronto, ON, Canada Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
4
Département de Virologie, Unité de Génétique Moléculaire des Virus à ARN Institut Pasteur, Paris, France.
5
Joint IRB-BSC Program in Computational Biology, Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain.
6
Center for Cancer Systems Biology (CCSB) and Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA Department of Genetics, Harvard Medical School, Boston, MA, USA.
7
Donnelly Centre, University of Toronto, Toronto, ON, Canada.
8
Weill Institute for Cell and Molecular Biology, Cornell University, Ithaca, NY, USA.
9
Center for Cancer Systems Biology (CCSB) and Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA Department of Genetics, Harvard Medical School, Boston, MA, USA Department of Plant Systems Biology, Technische Universität München Wissenschaftszentrum Weihenstephan, Freising, Germany.
10
Donnelly Centre, University of Toronto, Toronto, ON, Canada Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
11
Lunenfeld-Tanenbaum Research Institute Mt. Sinai Hospital, Toronto, ON, Canada Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
12
Joint IRB-BSC Program in Computational Biology, Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain.
13
Donnelly Centre, University of Toronto, Toronto, ON, Canada Lunenfeld-Tanenbaum Research Institute Mt. Sinai Hospital, Toronto, ON, Canada Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada Center for Cancer Systems Biology (CCSB) and Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA Canadian Institute for Advanced Research, Toronto, ON, Canada Department of Computer Science, University of Toronto, Toronto, Ontario, Canada yachie@synbiol.rcast.u-tokyo.ac.jp fritz.roth@utoronto.ca.

Abstract

High-throughput binary protein interaction mapping is continuing to extend our understanding of cellular function and disease mechanisms. However, we remain one or two orders of magnitude away from a complete interaction map for humans and other major model organisms. Completion will require screening at substantially larger scales with many complementary assays, requiring further efficiency gains in proteome-scale interaction mapping. Here, we report Barcode Fusion Genetics-Yeast Two-Hybrid (BFG-Y2H), by which a full matrix of protein pairs can be screened in a single multiplexed strain pool. BFG-Y2H uses Cre recombination to fuse DNA barcodes from distinct plasmids, generating chimeric protein-pair barcodes that can be quantified via next-generation sequencing. We applied BFG-Y2H to four different matrices ranging in scale from ~25 K to 2.5 M protein pairs. The results show that BFG-Y2H increases the efficiency of protein matrix screening, with quality that is on par with state-of-the-art Y2H methods.

KEYWORDS:

DNA barcode; interactome; next‐generation sequencing; protein interaction; yeast two‐hybrid

PMID:
27107012
PMCID:
PMC4848762
DOI:
10.15252/msb.20156660
[Indexed for MEDLINE]
Free PMC Article

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