Abstract
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 (2–5) 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).
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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
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.
Park, S. H. and Raines, R. T. (2000) Genetic selection for dissociative inhibitors of designated protein-protein interactions. Nat. Biotechnol. 18, 847–851.
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.
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.
Zhang, Z., Zhu, W., and Kodadek, T. (2000) Selection and application of peptide-binding peptides. Nat. Biotechnol. 18, 71–74.
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.
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.
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.
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.
Edgerton, M. D. and Jones, A. M. (1992) Localization of protein-protein interactions between subunits of phytochrome. The Plant Cell 4, 161–171.
Miller, J. H. (1972) Experiments in molecular genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Jaroszeski, M. J. and Radcliff, G. (1999) Fundamentals of flow cytometry. Mol. Biotechnol. 11, 37–53.
Radcliff, G. and Jaroszeski, M. J. (1998) Basics of flow cytometry. Methods Mol. Biol. 91, 1–24.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning, a laboratory manual 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Cowell, I. G. and Austin, C. A., eds. (1996) Methods in Molecular Biology. Vol. 69: cDNA Library Protocols. Humana Press, Totowa, NJ.
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.
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.
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.
Cormack, B. P., Valdivia, R. H., and Falkow, S. (1996) FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173, 33–38.
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.
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.
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.
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.
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.
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.
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.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2003 Humana Press Inc.
About this protocol
Cite this protocol
Mariño-RamÍrez, L., Campbell, L., Hu, J.C. (2003). Screening Peptide/Protein Libraries Fused to the λ Repressor DNA-Binding Domain in E. coli Cells. In: Vaillancourt, P.E. (eds) E. coliGene Expression Protocols. Methods in Molecular Biology™, vol 205. Humana Press. https://doi.org/10.1385/1-59259-301-1:235
Download citation
DOI: https://doi.org/10.1385/1-59259-301-1:235
Publisher Name: Humana Press
Print ISBN: 978-1-58829-008-3
Online ISBN: 978-1-59259-301-9
eBook Packages: Springer Protocols