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7.25J: Two-Hybrid Analysis

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    9508
    • Boundless
    • Boundless

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    Learning Objectives
    • Design a two-hybrid experiment

    Understanding how proteins are physically connected reveals clues about their structure, function, and makes them an ideal target for drug therapy. Several methodologies exist to study the interaction of proteins in vivo. The most widely employed tools are the yeast two-hybrid system. The yeast two-hybrid screening system is an effective and quick tool for the in vivo study of protein–protein interaction both in prokaryotes and eukaryotes. The method consists of splitting a yeast transcription factor into its binding domain and activation domain, fusing the binding domain to one protein of interest (the bait) and the activation domain to another protein of interest (the prey), and reconstituting the activity of the transcription factor by bringing the two domains back into physical proximity. In the absence of an interaction the domains remain distant, preventing a detectable output. If the two proteins do interact the bait recruits the prey to a specific cellular location where it can stimulate a detectable output (e.g., gene activation). This experimental approach measures direct physical interaction between proteins and is called a binary method. Datasets obtained from such tools are further analyzed using computational methods to draw a map of protein connectivity and achieve system level understanding of a microorganism.

    image
    Figure: Two-hybrid technique: Overview of two-hybrid assay, checking for interactions between two proteins, called here Bait and Prey.

    One limitation of classic yeast two-hybrid screens is that they are limited to soluble proteins. It is therefore impossible to use them to study the protein–protein interactions between insoluble integral membrane proteins. The split- ubiquitin system provides a method for overcoming this limitation. In the split-ubiquitin system, two integral membrane proteins to be studied are fused to two different ubiquitin moieties: a C-terminal ubiquitin moiety (“Cub”, residues 35–76) and an N-terminal ubiquitin moiety (“Nub”, residues 1–34). These fused proteins are called the bait and prey, respectively. In addition to being fused to an integral membrane protein, the Cub moiety is also fused to a transcription factor (TF) that can be cleaved off by ubiquitin specific proteases. Upon bait–prey interaction, Nub and Cub-moieties assemble, reconstituting the split-ubiquitin. The reconstituted split-ubiquitin molecule is recognized by ubiquitin specific proteases, which cleave off the reporter protein, allowing it to induce the transcription of reporter genes.

    Summary

    A key part of gene functional analysis and potential drug target discovery is an understanding of how proteins interact within the cell. Commercially available products facilitate the characterization of these interactions in yeast systems. The basic format involves the creation of two hybrid molecules, one in which a “bait” protein is fused with a transcription factor, and one in which a “prey” protein is fused with a related transcription factor. If the bait and prey proteins indeed interact, then the two factors fused to these two proteins are also brought into proximity with each other.

    Key Terms

    • computational: Of or relating to computation.
    • ubiquitin: A small regulatory protein sequence that directs proteins to specific compartments within the cell. Specifically, a ubiquitin tag directs the protein to a proteasome, which destroys and recycles the components.
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