Skip to main content
SpringerLink
Log in

Screening Arrayed Libraries with DNA and Protein Baits to Identify Interacting Proteins

  • Protocol
  • First Online:
Two-Hybrid Systems

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1794))

Abstract

Molecular interactions are an integral part of the regulatory mechanisms controlling gene expression. The yeast one- and two-hybrid systems (Y1H/Y2H) have been widely used by many laboratories to detect DNA–protein (Y1H) and protein–protein interactions (Y2H). The development of efficient cloning systems have promoted the generation of large open reading frame (ORF) clone collections (libraries) for several organisms. Functional analyses of such large collections require the establishment of adequate protocols. Here, we describe a simple straightforward procedure for high-throughput screenings of arrayed libraries with DNA or protein baits that can be carried out by one person with minimal labor and not requiring robotics. The protocol can also be scaled up or down and is compatible with several library formats. Procedures to make yeast stocks for long-term storage (tube and microplate formats) are also provided.

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 139.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 179.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 219.99
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

References

  1. Brent R, Ptashne M (1985) A eukaryotic transcriptional activator bearing the DNA specificity of a prokaryotic repressor. Cell 43:729–736

    Article  CAS  PubMed  Google Scholar 

  2. Ma J, Ptashne M (1998) Converting a eukaryotic transcriptional inhibitor into an activator. Cell 55:443–446

    Article  Google Scholar 

  3. Fields S, Song O (1989) A novel genetic system to detect protein–protein interactions. Nature 340:245–246

    Article  CAS  PubMed  Google Scholar 

  4. Wilson TE, Fahrner TJ, Johnston M, Milbrandt J (1991) Identification of the DNA binding site for NGFIB by genetic selection in yeast. Science 252:1296–1300

    Article  CAS  PubMed  Google Scholar 

  5. Li JJ, Herskowitz I (1993) Isolation of the ORC6, a component of the yeast origin recognition complex by a one-hybrid system. Science 262:1870–1874

    Article  CAS  PubMed  Google Scholar 

  6. Wang MM, Reed RR (1993) Molecular cloning of the olfactory neuronal transcription factor Olf-1 by genetic selection in yeast. Nature 364:121–126

    Article  CAS  PubMed  Google Scholar 

  7. Dowell SJ, Romanowski P, Diffley JF (1994) Interaction of Dbf4, the Cdc7 protein kinase regulatory subunit, with yeast replication origins in vivo. Science 265:1243–1246

    Article  CAS  PubMed  Google Scholar 

  8. Inouye C, Remondelli P, Karin M, Elledge S (1994) Isolation of a cDNA encoding a metal response element binding protein using a novel expression cloning procedure: the one hybrid system. DNA Cell Biol 13:731–742

    Article  CAS  PubMed  Google Scholar 

  9. Rezwan M, Auerbach D (2012) Yeast “N”-hybrid systems for protein-protein and drug-protein interaction discovery. Methods 57(4):423–429

    Article  CAS  PubMed  Google Scholar 

  10. Ferro E, Trabalzini L (2013) The yeast two-hybrid and related methods as powerful tools to study plant cell signalling. Plant Mol Biol 83(4–5):287–301

    Article  CAS  PubMed  Google Scholar 

  11. Ji X, Wang L, Nie X, He L, Zang D, Liu Y, Zhang B, Wang Y (2014) A novel method to identify the DNA motifs recognized by a defined transcription factor. Plant Mol Biol 86:367–380

    Article  CAS  PubMed  Google Scholar 

  12. Ota K, Feng SY, Ito T (2014) Detecting protein-DNA interactions using a modified yeast one-hybrid system. Methods Mol Biol 1164:39–50

    Article  CAS  PubMed  Google Scholar 

  13. Mallick J, Jansen G, Wu C, Whiteway M (2016) SRYTH: a new yeast two-hybrid method. Methods Mol Biol 1356:31–41

    Article  CAS  PubMed  Google Scholar 

  14. Snider J, Stagljar I (2016) Membrane yeast two-hybrid (MYTH) mapping of full-length membrane protein interactions. Cold Spring Harb Protoc. https://doi.org/10.1101/pdb.top077560

  15. Reece-Hoyes JS, Walhout AJ (2012) Yeast one-hybrid assays: a historical and technical perspective. Methods 57(4):441–447

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Mehla J, Caufield JH, Uetz P (2015) The yeast two-hybrid system: a tool for mapping protein-protein interactions. Cold Spring Harb Protoc 5:425–430

    Google Scholar 

  17. Paz-Ares J (2002) REGIA, an EU project on functional genomics of transcription factors from Arabidopsis thaliana. Comp Funct Genomics 3:102–108

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Gong W, Shen YP, Ma LG, Pan Y, Du YL, Wang DH, Yang JY, Hu LD, Liu XF, Dong CX, Ma L, Chen YH, Yang XY, Gao Y, Zhu D, Tan X, Mu JY, Zhang DB, Liu YL, Dinesh-Kumar SP, Li Y, Wang XP, Gu HY, Qu LJ, Bai SN, Lu YT, Li JY, Zhao JD, Zuo J, Huang H, Deng XW, Zhu YX (2004) Genome-wide ORFeome cloning and analysis of Arabidopsis transcription factor genes. Plant Physiol 135:773–782

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Mitsuda N, Ikeda M, Takada S, Takiguchi Y, Kondou Y, Yoshizumi T, Fujita M, Shinozaki K, Matsui M, Ohme-Takagi M (2010) Efficient yeast one-/two-hybrid screening using a library composed only of transcription factors in Arabidopsis thaliana. Plant Cell Physiol 51:2145–2151

    Article  CAS  PubMed  Google Scholar 

  20. Arabidopsis Interactome Mapping Consortium (2011) Evidence for network evolution in an Arabidopsis interactome map. Science 333(6042):601–607

    Article  CAS  PubMed Central  Google Scholar 

  21. Brady SM, Zhang L, Megraw M, Martinez NJ, Jiang E, Yi CS, Liu W, Zeng A, Taylor-Teeples M, Kim D, Ahnert S, Ohler U, Ware D, Walhout AJ, Benfey PN (2011) A stele-enriched gene regulatory network in the Arabidopsis root. Mol Syst Biol 7:459

    Article  PubMed  PubMed Central  Google Scholar 

  22. Castrillo G, Turck F, Leveugle M, Lecharny A, Carbonero P, Coupland G, Paz-Ares J, Oñate-Sánchez L (2011) Speeding cis-trans regulation discovery by phylogenomic analyses coupled with screenings of an arrayed library of Arabidopsis transcription factors. PLoS One 6:e21524

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Gaudinier A, Zhang L, Reece-Hoyes JS, Taylor-Teeples M, Pu L, Liu Z, Breton G, Pruneda-Paz JL, Kim D, Kay SA, Walhout AJ, Ware D, Brady SM (2011) Enhanced Y1H assays for Arabidopsis. Nat Methods 8(12):1053–1055

    Article  CAS  PubMed  Google Scholar 

  24. Ou B, Yin KQ, Liu SN, Yang Y, Gu T, Wing Hui JM, Zhang L, Miao J, Kondou Y, Matsui M, Gu HY, Qu LJ (2011) A high-throughput screening system for Arabidopsis transcription factors and its application to Med25-dependent transcriptional regulation. Mol Plant 4:546–555

    Article  CAS  PubMed  Google Scholar 

  25. Burdo B, Gray J, Goetting-Minesky MP, Wittler B, Hunt M, Li T, Velliquette D, Thomas J, Gentzel I, dos Santos Brito M, Mejía-Guerra MK, Connolly LN, Qaisi D, Li W, Casas MI, Doseff AI, Grotewold E (2014) The maize TFome-development of a transcription factor open reading frame collection for functional genomics. Plant J 80:356–366

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Pruneda-Paz JL, Breton G, Nagel DH, Kang SE, Bonaldi K, Doherty CJ, Ravelo S, Galli M, Ecker JR, Kay SA (2014) A genome-scale resource for the functional characterization of Arabidopsis transcription factors. Cell Rep 8:622–632

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Taylor-Teeples M, Lin L, de Lucas M, Turco G, Toal TW, Gaudinier A, Young NF, Trabucco GM, Veling MT, Lamothe R, Handakumbura PP, Xiong G, Wang C, Corwin J, Tsoukalas A, Zhang L, Ware D, Pauly M, Kliebenstein DJ, Dehesh K, Tagkopoulos I, Breton G, Pruneda-Paz JL, Ahnert SE, Kay SA, Hazen SP, Brady SM (2015) An Arabidopsis gene regulatory network for secondary cell wall synthesis. Nature 517(7536):571–575

    Article  CAS  PubMed  Google Scholar 

  28. Liu J, Wilson TE, Milbrandt J, Johnston M (1993) Identifying DNA-binding sites and analyzing DNA-binding domains using a yeast selection system. Methods 5:125–137

    Article  CAS  Google Scholar 

  29. Harper JW, Adami GR, Wei N, Keyomarsi K, Elledge SJ (1993) The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell 75:805–816

    Article  CAS  PubMed  Google Scholar 

  30. James P, Halladay J, Craig EA (1996) Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics 144:1425–1436

    PubMed  PubMed Central  CAS  Google Scholar 

  31. Elble R (1992) A simple and efficient procedure for transformation of yeasts. Biotechniques 13(1):18–20

    PubMed  CAS  Google Scholar 

  32. Dobi KC, Winston F (2007) Analysis of transcriptional activation at a distance in Saccharomyces cerevisiae. Mol Cell Biol 27(15):5575–5586

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Aronheim A, Zandi E, Hennemann H, Elledge SJ, Karin M (1997) Isolation of an AP-1 repressor by a novel method for detecting protein-protein interactions. Mol Cell Biol 17:3094–3102

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Stagljar I, Korostensky C, Johnsson N, te Heesen S (1998) A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo. Proc Natl Acad Sci U S A 95:5187–5192

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Smirnov MN, Smirnov VN, Budowsky EI, Inge-Vechtomov SG, Serebrjakov NG (1967) Red pigment of adenine-deficient yeast Saccharomyces cerevisiae. Biochem Biophys Res Commun 27(3):299–304

    Article  CAS  PubMed  Google Scholar 

  36. Weisman LS, Bacallao R, Wickner W (1987) Multiple methods of visualizing the yeast vacuole permit evaluation of its morphology and inheritance during the cell cycle. J Cell Biol 105(4):1539–1547

    Article  CAS  PubMed  Google Scholar 

  37. Rueda-Romero P, Barrero-Sicilia C, Gómez-Cadenas A, Carbonero P, Oñate-Sánchez L (2012) Arabidopsis thaliana DOF6 negatively affects germination in non-after-ripened seeds and interacts with TCP14. J Exp Bot 63:1937–1949

    Article  CAS  PubMed  Google Scholar 

  38. Iglesias-Fernández R, Barrero-Sicilia C, Carrillo-Barral N, Oñate-Sánchez L, Carbonero P (2013) Arabidopsis thaliana bZIP44: a transcription factor affecting seed germination and expression of the mannanase encoding gene AtMAN7. Plant J 74:767–780

    Article  CAS  PubMed  Google Scholar 

  39. Iglesias-Fernández R, Wozny D, Iriondo-de Hond M, Oñate-Sánchez L, Carbonero P, Barrero-Sicilia C (2014) The AtCathB3 gene, encoding a cathepsin B-like protease, is expressed during germination of Arabidopsis thaliana and transcriptionally repressed by the basic leucine zipperP protein GBF1. J Exp Bot 65:2009–2021

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Marín-de la Rosa N, Sotillo B, Mizckolczi P, Gibbs DJ, Vicente J, Carbonero P, Oñate-Sánchez L, Holdsworth MJ, Bhalerao R, Alabadí D, Blázquez MA (2014) Large-scale identification of gibberellin-related transcription factors defines Group VII ERFs as functional DELLA partners. Plant Physiol 166:1022–1032

    Article  CAS  PubMed  Google Scholar 

  41. Ballester P, Navarrete-Gomez M, Carbonero P, Oñate-Sánchez L, Ferrándiz C (2015) Leaf expansion in Arabidopsis is controlled by a TCP-NGA regulatory module likely conserved in distantly related species. Physiol Plant 155:21–32

    Article  CAS  PubMed  Google Scholar 

  42. Rombolá-Caldentey B, Rueda-Romero P, Iglesias-Fernández R, Carbonero P, Oñate-Sánchez L (2014) Arabidopsis DELLA and two HD-ZIP transcription factors regulate GA signalling in the epidermis through the L1-box cis-element. Plant Cell 26:2905–2919

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Rajagopala SV, Hughes KT, Uetz P (2009) Benchmarking yeast two-hybrid systems using the interactions of bacterial motility proteins. Proteomics 9:5296–5302

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

The work in L.O.-S. lab is supported by MINECO grants BIO2013-46076-R and BIO2016-77840-R. We thank to all people that contributed to the development of the Arabidopsis TF library [22] as well as all the labs that have used this resource, thus helping to ascertain its potential. We apologize to those authors whose publications are not cited here due to space limitations.

Author information

Authors and Affiliations

  1. Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM)–Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Campus Montegancedo UPM, Madrid, Spain

    Rocío Sánchez-Montesino & Luis Oñate-Sánchez

Authors
  1. Rocío Sánchez-Montesino
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Luis Oñate-Sánchez
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Luis Oñate-Sánchez .

Editor information

Editors and Affiliations

  1. Centro de Biotecnología y Genómica de Plantas, Universidad Politécnica de Madrid (UPM)–Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Campus Montegancedo UPM, Madrid, Spain

    Luis Oñate-Sánchez

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Sánchez-Montesino, R., Oñate-Sánchez, L. (2018). Screening Arrayed Libraries with DNA and Protein Baits to Identify Interacting Proteins. In: Oñate-Sánchez, L. (eds) Two-Hybrid Systems. Methods in Molecular Biology, vol 1794. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7871-7_9

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7871-7_9

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7870-0

  • Online ISBN: 978-1-4939-7871-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation