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An eYFP Reporter Gene for the Yeast Two-hybrid System

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Abstract

The yeast two-hybrid system is a powerful tool for detecting binary protein interactions, widely used in large-scale interactome mapping. We modified two yeast strains commonly used in yeast two-hybrid experiments by integrating into their genomes a new reporter gene encoding the enhanced yellow fluorescent protein eYFP. The suitability of this reporter gene for interaction screening was evaluated by fluorescence microscopy and fluorescence-activated cell sorting analysis. The gene shows good potential as a two-hybrid reporter gene for detecting strong interactions. Gal4 transcriptional activation gives rise to sufficient fluorescence for detection with a flow cytometer, but the eYFP reporter is not sensitive enough for detecting weak or moderate interactions. This study highlights the advantages of a fluorescent reporter gene in yeast two-hybrid screening.

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Abbreviations

AD:

Activation domain

DB:

DNA-binding domain

eYFP :

Enhanced yellow fluorescent protein

FACS:

Fluorescence-activated cell sorting

G418r :

Resistance

Y2H:

Yeast two-hybrid

YPD:

Yeast peptone dextrose

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Acknowledgments

We wish to thank Luc Willems (Centre de Biologie cellulaire et moléculaire, Gembloux Agro-Bio Tech, Université de Liège, Gembloux, Belgium) for providing access to the fluorescence microscope and FACS instrument. This work was funded by Gembloux Agro-Bio Tech (Project PLANTINTERACT). Jean-Claude Twizere is senior research associate at the FNRS (Fonds National de la Recherche Scientifique in Belgium).

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Authors and Affiliations

  1. Unité de Biologie Animale et Microbienne, Gembloux Agro-Bio Tech, Université de Liège, 6 avenue du Maréchal Juin, 5030, Gembloux, Belgium

    Coralie Damon, Daniel Portetelle & Micheline Vandenbol

  2. Centre de Biologie cellulaire et moléculaire, Gembloux Agro-Bio Tech, Université de Liège, Gembloux, Belgium

    Mathieu Boxus & Jean-Claude Twizere

Authors
  1. Coralie Damon
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  2. Mathieu Boxus
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  3. Jean-Claude Twizere
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  4. Daniel Portetelle
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  5. Micheline Vandenbol
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Corresponding author

Correspondence to Coralie Damon.

Electronic Supplementary Material

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Supplementary material 1 (DOC 32 kb) Methodology for eYFP reporter gene construction

10930_2013_9467_MOESM2_ESM.doc

Supplementary material 2 (DOC 27 kb) Plasmid pairs of Y2H controls 1–4, used in this study to test the new reporter gene (adapted from [4])

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Damon, C., Boxus, M., Twizere, JC. et al. An eYFP Reporter Gene for the Yeast Two-hybrid System. Protein J 32, 126–130 (2013). https://doi.org/10.1007/s10930-013-9467-4

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