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Two-Hybrid Systems to Measure Protein–Protein Interactions

  1. Russell L Finley Jr.1,
  2. Dumrong Mairiang2

Published Online: 17 FEB 2014

DOI: 10.1002/9780470015902.a0005980.pub2

eLS

eLS

How to Cite

Finley, R. L. and Mairiang, D. 2014. Two-Hybrid Systems to Measure Protein–Protein Interactions. eLS. .

Author Information

  1. 1

    Wayne State University School of Medicine, Detroit, Michigan, USA

  2. 2

    Mahidol University, Bangkok, Thailand

  1. Based in part on the previous version of this eLS article ‘Yeast Two-hybrid System and Related Methodology’ (2005) by Russell L Finley Jr.

Publication History

  1. Published Online: 17 FEB 2014

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–protein interactions. In a yeast two-hybrid assay, two proteins 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 easily detectable reporter genes. 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 two-hybrid assay are contributing to the characterisation of the protein interactions – or protein interactome – for humans and several model organisms.

Key Concepts:

  • The role that most proteins play in cells involves interacting with one or more proteins.

  • A binary interaction is a physical interaction between two proteins.

  • Understanding a protein's function requires charting its binary interactions.

  • Interactome is a term used to refer to all of the protein interactions for a particular cell or an entire organism.

  • Two-hybrid assays are assays for binary protein interactions, where two test proteins are expressed in cells as hybrids fused to protein moieties that when brought into proximity via the protein interaction produces 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, which activates reporter genes if the proteins interact.

  • In a protein complementation assay, the two proteins are fused to separate halves of a reporter protein like an enzyme, which will be reconstituted if the two halves are brought into close proximity via the protein–protein interaction.

  • False positives are interactions that are detected in the assay even though they do not occur under normal conditions in vivo.

  • 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.

Keywords:

  • two-hybrid;
  • yeast;
  • protein interaction;
  • interactome;
  • networks;
  • complementation