Two‐Hybrid Systems to Measure Protein–Protein Interactions

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Abstract

Abstract To understand how proteins function to control cellular processes, their interactions with other proteins must be identified and characterised. The yeast two‐hybrid system is a simple and efficient assay for protein interactions. In a yeast two‐hybrid assay, the two proteins to be tested are expressed in a yeast nucleus with each protein fused to one‐half of a transcription activator. If the two‐hybrid proteins interact, the transcription activator is reconstituted and turns on reporter genes that can be easily detected. This assay has been used to identify tens of thousands of protein interactions, to map protein interaction domains and to characterise mutant variants of proteins. A variety of related assays have been developed, all based on the ability of two interacting hybrid proteins to activate a reporter system. These assays along with the original yeast two‐hybrid assay contribute to the characterisation of the protein interactions – or protein interactome – for humans and a wide range of other organisms. Key Concepts The function of most proteins involves interacting with one or more other proteins. A binary interaction is a direct physical interaction between two proteins. Understanding a protein's function requires charting its binary interactions. The interactome is all of the protein interactions for a particular cell or an entire organism. Two‐hybrid assays detect binary protein interactions by expressing the two test proteins in cells as hybrids fused to protein moieties that when brought into proximity via the protein interaction produce a detectable signal. In a yeast two‐hybrid assay, the two proteins to be tested for interaction are fused to the two halves of a transcription factor in yeast. Two‐hybrid assays, like all protein interaction assays, can produce false positives, which are interactions that are detected in the assay even though they do not occur under normal conditions in vivo . Two‐hybrid and other protein interaction assays can also result in missed interactions or false negatives. Use of multiple different protein interaction assays can reduce the number of false negatives and provide cross‐validation to rule out false positives.

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