Skip to main content
SpringerLink
Log in

Matrix-Based Yeast Two-Hybrid Screen Strategies and Comparison of Systems

  • Protocol
  • First Online:
Two Hybrid Technologies

Part of the book series: Methods in Molecular Biology ((MIMB,volume 812))

Abstract

Today, matrix-based screens are used primarily for smaller and medium-size clone collections in combination with automation and cloning techniques that allow for reliable and fast interaction screening. Matrix-based yeast two-hybrid screens are an alternative to library-based screens. However, intermediary forms are possible too and we compare both strategies, including a detailed discussion of matrix-based screens. Recent improvement of matrix screens (also called array screens) uses various pooling strategies as well as novel vectors that increase their efficiency while decreasing false-negative rates and increasing reliability.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Abbreviations

3-AT:

3-Amino-1,2,4-triazole

AD:

Activation domain

DBD:

DNA-binding domain

GFP:

Green fluorescent protein

GO:

Gene ontology

ORF:

Open reading frame

STD:

Shifted transversal design

Y2H:

Yeast two hybrid

References

  1. Fields, S., and Song, O. (1989) A novel genetic system to detect protein-protein interactions. Nature 340, 245–6.

    Article  PubMed  CAS  Google Scholar 

  2. Drees, B. L. (1999) Progress and variations in two-hybrid and three-hybrid technologies. Curr Opin Chem Biol 3, 64–70.

    Article  PubMed  CAS  Google Scholar 

  3. Johnsson, N., and Varshavsky, A. (1994) Ubiquitin-assisted dissection of protein transport across membranes. Embo 13, 2686–98.

    CAS  Google Scholar 

  4. Bartel, P. L., Roecklein, J. A., SenGupta, D., and Fields, S. (1996) A protein linkage map of Escherichia coli bacteriophage T7. Nat Genet 12, 72–7.

    Article  PubMed  CAS  Google Scholar 

  5. Fossum, E., Friedel, C. C., Rajagopala, S. V., Titz, B., Baiker, A., Schmidt, T., Kraus, T., Stellberger, T., Rutenberg, C., Suthram, S., Bandyopadhyay, S., Rose, D., von Brunn, A., Uhlmann, M., Zeretzke, C., Dong, Y. A., Boulet, H., Koegl, M., Bailer, S. M., Koszinowski, U., Ideker, T., Uetz, P., Zimmer, R., and Haas, J. (2009) Evolutionarily conserved herpesviral protein interaction networks. PLoS Pathog 5, e1000570.

    Article  PubMed  Google Scholar 

  6. Uetz, P., Dong, Y. A., Zeretzke, C., Atzler, C., Baiker, A., Berger, B., Rajagopala, S. V., Roupelieva, M., Rose, D., Fossum, E., and Haas, J. (2006) Herpesviral protein networks and their interaction with the human proteome. Science 311, 239–42.

    Article  PubMed  CAS  Google Scholar 

  7. Uetz, P., Giot, L., Cagney, G., Mansfield, T. A., Judson, R. S., Knight, J. R., Lockshon, D., Narayan, V., Srinivasan, M., Pochart, P., Qureshi-Emili, A., Li, Y., Godwin, B., Conover, D., Kalbfleisch, T., Vijayadamodar, G., Yang, M., Johnston, M., Fields, S., and Rothberg, J. M. (2000) A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Nature 403, 623–627.

    Article  PubMed  CAS  Google Scholar 

  8. Koegl, M., and Uetz, P. (2007) Improving yeast two-hybrid screening systems. Brief Funct Genomic Proteomic 6, 302–12.

    Article  PubMed  CAS  Google Scholar 

  9. Yu, H., Braun, P., Yildirim, M. A., Lemmens, I., Venkatesan, K., Sahalie, J., Hirozane-Kishikawa, T., Gebreab, F., Li, N., Simonis, N., Hao, T., Rual, J. F., Dricot, A., Vazquez, A., Murray, R. R., Simon, C., Tardivo, L., Tam, S., Svrzikapa, N., Fan, C., de Smet, A. S., Motyl, A., Hudson, M. E., Park, J., Xin, X., Cusick, M. E., Moore, T., Boone, C., Snyder, M., Roth, F. P., Barabasi, A. L., Tavernier, J., Hill, D. E., and Vidal, M. (2008) High-quality binary protein interaction map of the yeast interactome network. Science 322, 104–10.

    Article  PubMed  CAS  Google Scholar 

  10. Boxem, M., Maliga, Z., Klitgord, N., Li, N., Lemmens, I., Mana, M., de Lichtervelde, L., Mul, J. D., van de Peut, D., Devos, M., Simonis, N., Yildirim, M. A., Cokol, M., Kao, H. L., de Smet, A. S., Wang, H. D., Schlaitz, A. L., Hao, T., Milstein, S., Fan, C. Y., Tipsword, M., Drew, K., Galli, M., Rhrissorrakrai, K., Drechsel, D., Koller, D., Roth, F. P., Iakoucheva, L. M., Dunker, A. K., Bonneau, R., Gunsalus, K. C., Hill, D. E., Piano, F., Tavernier, J., van den Heuvel, S., Hyman, A. A., and Vidal, M. (2008) A protein domain-based interactome network for C-elegans early embryogenesis. Cell 134, 534–545.

    Article  PubMed  CAS  Google Scholar 

  11. Vollert, C. S., and Uetz, P. (2004) The phox homology (PX) domain protein interaction network in yeast. Mol Cell Proteomics 3, 1053–64.

    Article  PubMed  CAS  Google Scholar 

  12. Flajolet, M., Rotondo, G., Daviet, L., Bergametti, F., Inchauspe, G., Tiollais, P., Transy, C., and Legrain, P. (2000) A genomic approach of the hepatitis C virus generates a protein interaction map. Gene 242, 369–79.

    Article  PubMed  CAS  Google Scholar 

  13. Bader, J. S., Chaudhuri, A., Rothberg, J. M., and Chant, J. (2004) Gaining confidence in high-throughput protein interaction networks. Nat Biotechnol 22, 78–85.

    Article  PubMed  CAS  Google Scholar 

  14. Jin, F. L., Avramova, L., Huang, J., and Hazbun, T. (2007) A yeast two-hybrid smart-pool-array system for protein-interaction mapping. Nature Methods 4, 405–407.

    PubMed  CAS  Google Scholar 

  15. Jin, F. L., Hazbun, T., Michaud, G. A., Salcius, M., Predki, P. F., Fields, S., and Huang, J. (2006) A pooling-deconvolution strategy for biological network elucidation. Nature Methods 3, 183–189.

    Article  PubMed  CAS  Google Scholar 

  16. Xin, X., Rual, J. F., Hirozane-Kishikawa, T., Hill, D. E., Vidal, M., Boone, C., and Thierry-Mieg, N. (2009) Shifted Transversal Design smart-pooling for high coverage interactome mapping. Genome Res 19, 1262–9.

    Article  PubMed  CAS  Google Scholar 

  17. Zhong, J., Zhang, H., Stanyon, C. A., Tromp, G., and Finley, R. L., Jr. (2003) A strategy for constructing large protein interaction maps using the yeast two-hybrid system: regulated expression arrays and two-phase mating. Genome Res 13, 2691–9.

    Article  PubMed  CAS  Google Scholar 

  18. Fromont-Racine, M., Mayes, A. E., Brunet-Simon, A., Rain, J. C., Colley, A., Dix, I., Decourty, L., Joly, N., Ricard, F., Beggs, J. D., and Legrain, P. (2000) Genome-wide protein interaction screens reveal functional networks involving Sm-like proteins. Yeast 17, 95–110.

    Article  PubMed  CAS  Google Scholar 

  19. Stellberger, T., Hauser, R., Baiker, A., Pothineni, V. R., Haas, J., and Uetz, P. (2010) Improving the yeast two-hybrid system with permutated fusions proteins: the Varicella Zoster Virus interactome. Proteome Sci 8, 8.

    Article  PubMed  Google Scholar 

  20. Goll, J., Rajagopala, S. V., Shiau, S. C., Wu, H., Lamb, B. T., and Uetz, P. (2008) MPIDB: the microbial protein interaction database. Bioinformatics 24, 1743–4.

    Article  PubMed  CAS  Google Scholar 

  21. Rajagopala, S. V., Hughes, K. T., and Uetz, P. (2009) Benchmarking yeast two-hybrid systems using the interactions of bacterial motility proteins. Proteomics 9, 5296–5302.

    Article  PubMed  CAS  Google Scholar 

  22. Schwartz, H., Alvares, C. P., White, M. B., and Fields, S. (1998) Mutation detection by a two-hybrid assay. Hum Mol Genet 7, 1029–1032.

    Article  PubMed  CAS  Google Scholar 

  23. Vidal, M., and Endoh, H. (1999) Prospects for drug screening using the reverse two-hybrid system. Trends Biotechnol 17, 374–81.

    Article  PubMed  CAS  Google Scholar 

  24. Vidal, M., and Legrain, P. (1999) Yeast forward and reverse ‘n’-hybrid systems. Nucleic Acids Res 27, 919–29.

    Article  PubMed  CAS  Google Scholar 

  25. SenGupta, D. J., Zhang, B., Kraemer, B., Pochart, P., Fields, S., and Wickens, M. (1996) A three-hybrid system to detect RNA–protein interactions in vivo. Proc Natl Acad Sci USA 94, 8496–8501.

    Article  Google Scholar 

  26. Estojak, J., Brent, R., and Golemis, E. A. (1995) Correlation of two-hybrid affinity data with in vitro measurements. Mol Cell Biol 15, 5820–9.

    PubMed  CAS  Google Scholar 

  27. Rain, J. C., Selig, L., De Reuse, H., Battaglia, V., Reverdy, C., Simon, S., Lenzen, G., Petel, F., Wojcik, J., Schachter, V., Chemama, Y., Labigne, A., and Legrain, P. (2001) The protein-protein interaction map of Helicobacter pylori. Nature 409, 211–215.

    Article  PubMed  CAS  Google Scholar 

  28. Raquet, X., Eckert, J. H., Muller, S., and Johnsson, N. (2001) Detection of altered protein conformations in living cells. J Mol Biol 305, 927–38.

    Article  PubMed  CAS  Google Scholar 

  29. Cagney, G., Uetz, P., and Fields, S. (2001) Two-hybrid analysis of the Saccharomyces cerevisiae 26 S proteasome. Physiol Genomics 7, 27–34.

    PubMed  CAS  Google Scholar 

  30. Ito, T., Chiba, T., Ozawa, R., Yoshida, M., Hattori, M., and Sakaki, Y. (2001) A comprehensive two-hybrid analysis to explore the yeast protein interactome. Proc Natl Acad Sci USA 98, 4569–74.

    Article  PubMed  CAS  Google Scholar 

  31. Giot, L., Bader, J. S., Brouwer, C., Chaudhuri, A., Kuang, B., Li, Y., Hao, Y. L., Ooi, C. E., Godwin, B., Vitols, E., Vijayadamodar, G., Pochart, P., Machineni, H., Welsh, M., Kong, Y., Zerhusen, B., Malcolm, R., Varrone, Z., Collis, A., Minto, M., Burgess, S., McDaniel, L., Stimpson, E., Spriggs, F., Williams, J., Neurath, K., Ioime, N., Agee, M., Voss, E., Furtak, K., Renzulli, R., Aanensen, N., Carrolla, S., Bickelhaupt, E., Lazovatsky, Y., DaSilva, A., Zhong, J., Stanyon, C. A., Finley, R. L., Jr., White, K. P., Braverman, M., Jarvie, T., Gold, S., Leach, M., Knight, J., Shimkets, R. A., McKenna, M. P., Chant, J., and Rothberg, J. M. (2003) A protein interaction map of Drosophila melanogaster. Science 302, 1727–36.

    Article  PubMed  CAS  Google Scholar 

  32. Li, S., Armstrong, C. M., Bertin, N., Ge, H., Milstein, S., Boxem, M., Vidalain, P. O., Han, J. D., Chesneau, A., Hao, T., Goldberg, D. S., Li, N., Martinez, M., Rual, J. F., Lamesch, P., Xu, L., Tewari, M., Wong, S. L., Zhang, L. V., Berriz, G. F., Jacotot, L., Vaglio, P., Reboul, J., Hirozane-Kishikawa, T., Li, Q., Gabel, H. W., Elewa, A., Baumgartner, B., Rose, D. J., Yu, H., Bosak, S., Sequerra, R., Fraser, A., Mango, S. E., Saxton, W. M., Strome, S., Van Den Heuvel, S., Piano, F., Vandenhaute, J., Sardet, C., Gerstein, M., Doucette-Stamm, L., Gunsalus, K. C., Harper, J. W., Cusick, M. E., Roth, F. P., Hill, D. E., and Vidal, M. (2004) A map of the interactome network of the metazoan C. elegans. Science 303, 540–3.

    Article  PubMed  CAS  Google Scholar 

  33. Rual, J. F., Venkatesan, K., Hao, T., Hirozane-Kishikawa, T., Dricot, A., Li, N., Berriz, G. F., Gibbons, F. D., Dreze, M., Ayivi-Guedehoussou, N., Klitgord, N., Simon, C., Boxem, M., Milstein, S., Rosenberg, J., Goldberg, D. S., Zhang, L. V., Wong, S. L., Franklin, G., Li, S., Albala, J. S., Lim, J., Fraughton, C., Llamosas, E., Cevik, S., Bex, C., Lamesch, P., Sikorski, R. S., Vandenhaute, J., Zoghbi, H. Y., Smolyar, A., Bosak, S., Sequerra, R., Doucette-Stamm, L., Cusick, M. E., Hill, D. E., Roth, F. P., and Vidal, M. (2005) Towards a proteome-scale map of the human protein-protein interaction network. Nature 437, 1173–1178.

    Google Scholar 

  34. Stelzl, U., Worm, U., Lalowski, M., Haenig, C., Brembeck, F. H., Goehler, H., Stroedicke, M., Zenkner, M., Schoenherr, A., Koeppen, S., Timm, J., Mintzlaff, S., Abraham, C., Bock, N., Kietzmann, S., Goedde, A., Toksoz, E., Droege, A., Krobitsch, S., Korn, B., Birchmeier, W., Lehrach, H., and Wanker, E. E. (2005) A human protein-protein interaction network: A resource for annotating the proteome. Cell 122, 957–968.

    Article  PubMed  CAS  Google Scholar 

  35. Parrish, J. R., Yu, J., Liu, G., Hines, J. A., Chan, J. E., Mangiola, B. A., Zhang, H., Pacifico, S., Fotouhi, F., Dirita, V. J., Ideker, T., Andrews, P., and Finley, R. L., Jr. (2007) A proteome-wide protein interaction map for Campylobacter jejuni. Genome Biol 8, R130.

    Article  PubMed  Google Scholar 

  36. Titz, B., Rajagopala, S. V., Goll, J., Hauser, R., McKevitt, M. T., Palzkill, T., and Uetz, P. (2008) The binary protein interactome of Treponema pallidum – the syphilis spirochete. PLoS ONE 3, e2292.

    Article  PubMed  Google Scholar 

  37. Rajagopala, S. V., Titz, B., and Uetz, P. (2007) Array-based yeast two-hybrid screening for protein-protein interactions. In: Yeast Gene Analysis, Second Edition, 2nd Edn., Stark, M., Stansfelid. I., (Eds), 2007, Elsevier Amsterdem, 36, 139–163.

    Google Scholar 

Download references

Acknowledgments

Work on this paper was supported by NIH grant RO1GM79710, the European Union (HEALTH-F3-2009-223101), and by the Landesstiftung Baden-Württemberg.

Author information

Authors and Affiliations

  1. Karlsruhe Institute of Technology, Karlsruhe, Germany

    Roman Häuser & Thorsten Stellberger

  2. J Craig Venter Institute (JCVI), Rockville, MD, USA

    Seesandra V. Rajagopala

  3. Center for the Study of Biological Complexity Virginia Commonwealth University, Richmond, VA, USA

    Peter Uetz

Authors
  1. Roman Häuser
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Thorsten Stellberger
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Seesandra V. Rajagopala
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Peter Uetz
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Peter Uetz .

Editor information

Editors and Affiliations

  1. Molekulare Medizin (MDC), Max-Delbrück-Centrum für, Robert-Rössle-Str. 10, Berlin-Buch, 13125, Germany

    Bernhard Suter

  2. Max-Delbrück-Centrum-Für Molekulare Medi, Robert-Rössle-Strabe 10, Berlin, 13125, Berlin, Germany

    Erich E. Wanker

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Häuser, R., Stellberger, T., Rajagopala, S.V., Uetz, P. (2012). Matrix-Based Yeast Two-Hybrid Screen Strategies and Comparison of Systems. In: Suter, B., Wanker, E. (eds) Two Hybrid Technologies. Methods in Molecular Biology, vol 812. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-455-1_1

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-455-1_1

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-454-4

  • Online ISBN: 978-1-61779-455-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation