Screening Peptide/Protein Libraries Fused to the Repressor DNA-Binding Domain in E. coli Cells
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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 ).
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 (C
2
) is able to form a heterodimer with the target peptide reconstituting the activity of λ repressor.
