Advertisement

A Practical Guide to Quantitative Interactor Screening with Next-Generation Sequencing (QIS-Seq)

  • Yunchen Gong
  • Darrell Desveaux
  • David S. Guttman
  • Jennifer D. LewisEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1613)

Abstract

Yeast two-hybrid screens are a powerful approach to identify protein-protein interactions; however, they are typically limited in the number of interactions identified, and lack quantitative values to ascribe confidence scores to the interactions that are obtained. We have developed a high-throughput, quantitative, yeast two-hybrid screening approach coupled with next-generation sequencing. This strategy allows the identification of interacting proteins that are preferentially associated with a bait of interest, and helps eliminate nonspecific interacting proteins. The method is high-throughput, allowing many more baits to be tested and many more candidate interacting proteins to be identified. Quantitative data allows the interactors to be ascribed confidence scores based on their enrichment with particular baits, and can identify both common and rare interacting proteins.

Key words

Yeast two-hybrid screen Next-generation sequencing High-throughput screening Quantitative 

Notes

Acknowledgments

We would like to thank Maël Baudin, Jana A. Hassan, and Vasanth Singan for critically reviewing the manuscript. This work was supported by Natural Sciences and Engineering Research Council of Canada awards to D.S.G. and D.D; a Canada Research Chair in Plant-Microbe Systems Biology (D.D.) or Comparative Genomics (D.S.G.); the Centre for the Analysis of Genome Evolution and Function (D.D. and D.S.G.); United States Department of Agriculture Agricultural Research Service 5335-21000-040-00D (J.D.L).

References

  1. 1.
    Fields S, Song OK (1989) A novel genetic system to detect protein-protein interactions. Nature 340:245–246CrossRefPubMedGoogle Scholar
  2. 2.
    Gyuris J, Golemis E, Chertkov H, Brent R (1993) CDI1, a human G1-phase and S-phase protein phosphatase that associates with CDK2. Cell 75:791–803CrossRefPubMedGoogle Scholar
  3. 3.
    Johnsson N, Varshavsky A (1994) Split ubiquitin as a sensor of protein interactions in vivo. Proc Natl Acad Sci U S A 91:10340–10344CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Harbers M (2008) The current status of cDNA cloning. Genomics 91:232–242CrossRefPubMedGoogle Scholar
  5. 5.
    Lewis JD, Wan JR, Ford R, Gong Y, Fung P, Wang P, Desveaux D, Guttman DS (2012) Quantitative interactor screening with next-generation sequencing (QIS-Seq) identifies Arabidopsis thaliana MLO2 as a target of the Pseudomonas syringae type III effector HopZ2. BMC Genomics 13:8CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Hancock JF, Paterson H, Marshall CJ (1990) A polybasic domain or palmitoylation is required in addition to the CAAX motif to localize p21ras to the plasma membrane. Cell 63:133–139CrossRefPubMedGoogle Scholar
  7. 7.
    Yeung T, Gilbert GE, Shi J, Silvius J, Kapus A, Grinstein S (2008) Membrane phosphatidylserine regulates surface charge and protein localization. Science 319:210–213CrossRefGoogle Scholar
  8. 8.
    Iyer K, Burkle L, Auerback D, Thaminy S, Dinkel M, Engels K, Stagljar I (2005) Utilizing the split-ubiquitin membrane yeast two-hybrid system to identify protein-protein interactions of integral membrane proteins. Sci STKE 2005:I3Google Scholar
  9. 9.
    Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJE (2015) The Phyre2 web portal for protein modeling, prediction and analysis. Nat Protoc 10:845–858CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Yunchen Gong
    • 1
    • 2
  • Darrell Desveaux
    • 1
    • 2
  • David S. Guttman
    • 1
    • 2
  • Jennifer D. Lewis
    • 3
    • 4
    Email author
  1. 1.Department of Cell and Systems BiologyUniversity of TorontoTorontoCanada
  2. 2.Centre for the Analysis of Genome Evolution and FunctionUniversity of TorontoTorontoCanada
  3. 3.Plant Gene Expression Center, United States Department of AgricultureAlbanyUSA
  4. 4.Department of Plant and Microbial BiologyUniversity of California BerkeleyBerkeleyUSA

Personalised recommendations