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. 2000 Jun 20;97(13):7382-7.
doi: 10.1073/pnas.110149297.

A bacterial two-hybrid selection system for studying protein-DNA and protein-protein interactions

J K Joung  1 , E I Ramm, C O Pabo
Affiliations

A bacterial two-hybrid selection system for studying protein-DNA and protein-protein interactions

J K Joung et al. Proc Natl Acad Sci U S A. .

Abstract

We have developed a bacterial "two-hybrid" system that readily allows selection from libraries larger than 10(8) in size. Our bacterial system may be used to study either protein-DNA or protein-protein interactions, and it offers a number of potentially significant advantages over existing yeast-based one-hybrid and two-hybrid methods. We tested our system by selecting zinc finger variants (from a large randomized library) that bind tightly and specifically to desired DNA target sites. Our method allows sequence-specific zinc fingers to be isolated in a single selection step, and thus it should be more rapid than phage display strategies that typically require multiple enrichment/amplification cycles. Given the large library sizes our bacterial-based selection system can handle, this method should provide a powerful tool for identifying and optimizing protein-DNA and protein-protein interactions.

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Figures

Figure 1
Figure 1
(A) Transcriptional activation in a previously described E. coli-based genetic screen [developed by Hochschild and coworkers (8, 10)] for studying protein–DNA and protein–protein interactions. (B) Modified reporter template for our E. coli-based genetic selection system. (C) Model for transcriptional activation of the Pzif promoter by fusion proteins Gal11P-Zif123 and αGal4. ZF1, ZF2, and ZF3 are the three zinc fingers of the Zif268 protein. (Although Gal11P-Zif123 efficiently activates our Pzif promoter, we note that the spacing between the Zif268-binding site and the transcription start site has not yet been optimized.)
Figure 2
Figure 2
An E. coli-based selection system for identifying zinc finger variants from large randomized libraries. (Left) A selection strain cell bearing a randomized zinc finger (white oval) that is unable to bind the target DNA subsite of interest (black box). This candidate fails to activate transcription of the weak promoter controlling HIS3 expression and therefore cells expressing this candidate fail to grow on HIS-selective medium. (Right) A library candidate bearing a particular zinc finger (one member of the randomized library) (black oval) that can bind the target DNA site. This candidate can activate HIS3 expression and therefore cells expressing this candidate grow on HIS-selective medium.
Figure 3
Figure 3
Recognition helix sequences of fingers isolated by our selection. For candidates that were isolated multiple times (as judged by nucleotide sequence), the number of clones obtained is shown in parentheses. The consensus sequence(s) of fingers selected by phage display for each target subsite also are shown (6). +, positively charged residue; _, no discernible preference; *, candidates with a 2-bp deletion downstream of the sequence encoding the recognition helix; and arrows illustrate a few of the most plausible potential base contacts.
See this image and copyright information in PMC

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