Two-hybrid system for detecting protein-protein interactions

BIOC/MCB 568 -- Fall 2010
John W. Little--University of Arizona

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The two-hybrid system is a useful way to detect proteins that interact with a protein you are studying. In general, it is used primarily for initial identification of interacting proteins, not for detailed characterization of the interaction. See here for more detail.

This system is based on the modular organization of many transcription factors (see figure). Many such proteins have two or more discrete structural and functional units, or domains (see here for caution about this way of thinking). The first protein to be used for this was the yeast protein GAL4; many later studies use the DNA-binding domain of the E. coli protein LexA. GAL4 has a DNA-binding domain (or DBD) and an activation domain (or AD). The structure of GAL4 complexed to its specific site (PDB file) is only of the first 65 amino acids, and comprises a minimal DBD; usually residues 1-100 or so are used.

When GAL4 binds to its cognate binding site, the activation domain is brought close to the promoter, allowing the activation domain to interact with the transcription machinery and resulting in activation of transcription. Typically a reporter gene, often lacZ, is used. Hence, there are standard reporter constructs, with variable numbers of binding sites and a reporter gene.

Now consider how these elements can be used to detect protein-protein interactions. Two types of hybrids are made:

DBD Hybrid: This hybrid contains the DBD fused to a protein of interest (often termed the "bait"). This fusion protein can bind to the DNA, but cannot activate transcription because the bait does not contain an activation function (if it does, this procedure will not work).

AD Hybrid: This hybrid contains the AD fused to another protein (often termed the "prey"). Usually, a recombinant DNA "library" is prepared in which genes for many different proteins are fused to the AD. Then both hybrid proteins are expressed in the same cell. Those expressing the reporter gene are identified and purified for further characterization.

Typically, libraries will contain large numbers of different clones (>106 different ones); a few of them will be able to interact with the bait. These few can then be recognized by their ability to turn on the reporter gene.



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BIOC/MCB 568 -- University of Arizona
Last modified August 18, 2010
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