Screening Peptide/Protein Libraries Fused to the λ Repressor DNA-Binding Domain in E. coli Cells

  • Leonardo Mariño-RamÍrez
  • Lisa Campbell
  • James C. Hu
Part of the Methods in Molecular Biology™ book series (MIMB, volume 205)


The use of λ repressor fusions to study protein-protein interactions in E. coli was first described by Hu and others (1). Since then, the repressor system has been employed by several laboratories to screen genomic (2, 3, 4, 5) and cDNA libraries (6) for homotypic or heterotypic interactions. λ repressor consists of distinct and separable domains: the N-terminal domain which has DNA binding activity and the C-terminal domain which mediates dimerization. The repressor fusion system is based on reconstituting the activity of the repressor by replacing the C-terminal domain with aheterologous oligomerization domain. The interaction is detected when the C-terminal domain forms a dimer (or higher order oligomer) with itself (homotypic interaction) or with a different domain from other fusion (heterotypic interaction) (see Fig. 1).
Fig. 1.

The rationale of λ repressor fusions. Repressor fusions are used to detect protein-protein interactions in vivo. Protein or peptide targets are fused to the λ repressor DNA binding domain; these fusions can be evaluated for repressor activity using direct selection with λ phage, or a variety of reporter genes suitable for library screening. (B) Active repressor fusions can be reconstituted when a dimeric peptide/protein is placed at the C terminus. The fusions are able to bind λ operators in the promoter and the reporter or phage genes are repressed. In this example the fusion is dimeric but a higher order oligomer can also reconstitute the activity of the repressor. (C) Heterodimers can also reconstitute the activity of the λ repressor. In this example, a target peptide (C1) is encoded in a first plasmid and a peptide library is introduced in the cell by transformation. One of the library encoded peptides (C2) is able to form a heterodimer with the target peptide reconstituting the activity of λ repressor.


Chloramphenicol Acetyl Transferase Green Fluorescent Protein Reporter Nonsense Suppression High Order Oligomer Amber Mutation 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Hu, J. C., O’shea, E. K., Kim, P. S., and Sauer, R. T. (1990) Sequence requirements for coiled-coils: analysis with λ repressor-GCN4 leucine zipper fusions. Science 250, 1400–1403.PubMedCrossRefGoogle Scholar
  2. 2.
    Park, S. H. and Raines, R. T. (2000) Genetic selection for dissociative inhibitors of designated protein-protein interactions. Nat. Biotechnol. 18, 847–851.PubMedCrossRefGoogle Scholar
  3. 3.
    Zhang, Z., Murphy, A., Hu, J. C., and Kodadek, T. (1999) Genetic selection of short peptides that support protein oligomerization in vivo. Curr. Biol. 9, 417–420.PubMedCrossRefGoogle Scholar
  4. 4.
    Jappelli, R. and Brenner, S. (1999) A genetic screen to identify sequences that mediate protein oligomerization in Escherichia coli. Biochem. Biophys. Res. Commun. 266, 243–247.CrossRefGoogle Scholar
  5. 5.
    Zhang, Z., Zhu, W., and Kodadek, T. (2000) Selection and application of peptide-binding peptides. Nat. Biotechnol. 18, 71–74.PubMedCrossRefGoogle Scholar
  6. 6.
    Bunker, C. A. and Kingston, R. E. (1995) Identification of a cDNA for SSRP1, an HMG-box protein, by interaction with the c-Myc oncoprotein in a novel bacterial expression screen. Nucleic Acids Res. 23, 269–276.PubMedCrossRefGoogle Scholar
  7. 7.
    Mariño-RamÍrez, L. and Hu, J. C. (2001) Using λ repressor fusions to isolate and characterize self-assembling domains, in Protein-Protein Interactions: A Laboratory Manual, (ed.), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 375–393.Google Scholar
  8. 8.
    Cairns, M., Green, A., White, P., Johnston, P., and Brenner, S. (1997) A novel bacterial vector system for monitoring protein-protein interactions in the cAMP-dependent protein kinase complex. 185, 5–9.Google Scholar
  9. 9.
    Jappelli, R. and Brenner, S. (1998) Changes in the periplasmic linker and in the expression level affect the activity of ToxR and λ-ToxR fusion proteins in Escherichia coli. FEBSLett. 423, 371–375.Google Scholar
  10. 10.
    Edgerton, M. D. and Jones, A. M. (1992) Localization of protein-protein interactions between subunits of phytochrome. The Plant Cell 4, 161–171.PubMedCrossRefGoogle Scholar
  11. 11.
    Miller, J. H. (1972) Experiments in molecular genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar
  12. 12.
    Jaroszeski, M. J. and Radcliff, G. (1999) Fundamentals of flow cytometry. Mol. Biotechnol. 11, 37–53.PubMedCrossRefGoogle Scholar
  13. 13.
    Radcliff, G. and Jaroszeski, M. J. (1998) Basics of flow cytometry. Methods Mol. Biol. 91, 1–24.PubMedGoogle Scholar
  14. 14.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning, a laboratory manual 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar
  15. 15.
    Cowell, I. G. and Austin, C. A., eds. (1996) Methods in Molecular Biology. Vol. 69: cDNA Library Protocols. Humana Press, Totowa, NJ.Google Scholar
  16. 16.
    James, P., Halladay, J., and Craig, E. A. (1996) Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics 144, 1425–1436.PubMedGoogle Scholar
  17. 17.
    Hu, J., Newell, N., Tidor, B., and Sauer, R. (1993) Probing the roles of residues at the e and g positions of the GCN4 leucine zipper by combinatorial mutagenesis. Protein Science 2, 1072–1084.PubMedCrossRefGoogle Scholar
  18. 18.
    Zeng, X. and Hu, J. C. (1997) Detection of tetramerization domains in vivo by cooperative DNA binding to tandem lambda operator sites. Gene 185, 245–249.PubMedCrossRefGoogle Scholar
  19. 19.
    Cormack, B. P., Valdivia, R. H., and Falkow, S. (1996) FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173, 33–38.PubMedCrossRefGoogle Scholar
  20. 20.
    Siegele, D. A., Campbell, L., and Hu, J. C. (2000) Green fluorescent protein as a reporter of transcriptional activity in a prokaryotic system. Methods Enzymol. 305, 499–513.PubMedCrossRefGoogle Scholar
  21. 21.
    Zagursky, R. J. and Berman, M. L. (1984) Cloning vectors that yield high levels of single-stranded DNA for rapid DNA sequencing. Gene 27, 183–191.PubMedCrossRefGoogle Scholar
  22. 22.
    Vershon, A. K., Bowie, J. U., Karplus, T. M., and Sauer, R. T. (1986) Isolation and analysis of Arc repressor mutants: evidence for an unusual mechanism of DNA binding. Proteins: Structure Function and Genetics 1, 302–311.CrossRefGoogle Scholar
  23. 23.
    Meyer, B. J., Maurer, R., and Ptashne, M. (1980) Gene regulation at the right operator (OR) of bacteriophage lambda. II. OR1, OR2, and OR3: their roles in mediating the effects of repressor and cro. J. Mol. Biol. 139, 163–194.PubMedCrossRefGoogle Scholar
  24. 24.
    Beckett, D., Burz, D. S., Ackers, G. K., and Sauer, R. T. (1993) Isolation of lambda repressor mutants with defects in cooperative operator binding. Biochemistry 32, 9073–9079.PubMedCrossRefGoogle Scholar
  25. 25.
    Hays, L. B., Chen, Y. S., and Hu, J. C. (2000) Two-hybrid system for characterization of protein-protein interactions in E. coli. Biotechniques 29, 288–290, 292–294, 296.Google Scholar
  26. 26.
    Hu, J. C. and Gross, C. A. (1988) Mutations in rpoD that increase expression of genes in the mal regulon of Escherichia coli K-12. J. Mol. Biol. 203, 15–27.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2003

Authors and Affiliations

  • Leonardo Mariño-RamÍrez
    • 1
  • Lisa Campbell
    • 1
  • James C. Hu
    • 1
  1. 1.Center for Macromolecular Design, Department of Biochemistry and BiophysicsTexas A&M UniversityCollege Station

Personalised recommendations