Two‐Hybrid and Related Systems


The two‐hybrid system is an incarnation of the split protein assay. Two‐hybrid assays are usually carried out in vivo, but modifications are possible in vitro. The technique is used to detect interactions between two proteins where the proteins may or may not belong to the same organism. Two‐hybrid assays were originally developed in yeast, but principally they should work in any other eukaryotes. To enable use in bacterial systems, various modifications have been made to the assay by using different split proteins, different types of reporters and different plasmids. Two‐hybrid assays have applications in drug discovery, microbiology, molecular biology and virtually any other study of protein function.

Key Concepts

  • Split protein assays are typically developed from multidomain proteins, which makes their splitting easier and more predictable.
  • Split protein assays make use of proteins that can be split and reconstituted non‐covalently.
  • Two‐hybrid assays can be implemented in bacteria, in eukaryotes or in vitro.
  • Two‐hybrid assays can be used to detect interactions between proteins from distantly related organisms.
  • Protein–protein interactions can be applied to biomarker discovery, to find drug targets, to determining functions of proteins with unknown function, protein–RNA interactions or even protein–small molecule interactions.

Keywords: protein–protein interactions; protein domains; protein function; split protein assay; protein–RNA interaction; drug targets

Figure 1. The two‐hybrid system. (a) In this system, transcription of a reporter gene depends upon reconstitution of two protein domains of a transcriptional activator: a DNA‐binding domain (DBD) and a transcription activation domain. (b) A hybrid protein composed of the DBD and a protein to be screened for protein–protein interactions (protein X) does not activate transcription in the absence of an activation domain. (c) Similarly, a hybrid protein composed of an activation domain and a protein to be screened (protein Y) does not activate transcription in the absence of a DBD. (d) An interaction between protein X and protein Y reconstitutes the transcriptional activator, permitting transcription of the reporter gene.
Figure 2. Variations of the two‐hybrid system. (a) The one‐hybrid assay. This system allows for screening of DNA‐binding proteins such as transcription factors. Working under the assumption that the protein to be screened contains a DNA‐binding domain (DBD), it is expressed as a hybrid protein containing an activation domain (AD). See Reece‐Hoyes and Walhout . (b) A three‐hybrid assay. As with two‐hybrid assays, this method depends upon reconstitution of a transcriptional activator and the resulting transcription of a reporter gene. In the three‐hybrid system, a third interactor (in this case, an RNA molecule) interacts with two proteins to facilitate bringing the DBD and AD together. See Licitra and Liu, . (c) The reverse two‐hybrid assay. With this system, protein interactions enable transcription of a reporter with a lethal or growth‐limiting function. The product of the reporter gene may be lethal to the host. For instance, URA3 can be used as a reporter gene and selected for by uracil‐free media. However, if 5‐fluoro‐orotate (5‐FOA) is added to the media, only cells without Ura3 (i.e. without a PPI) survive because the enzyme Ura3 converts 5‐FOA to the lethal metabolite fluorouracil. See Vidal et al. . (d) Differential two‐hybrid screening. Two different DNA‐binding sites and reporter genes may be used to determine whether a single protein (protein Y and an activation domain) does or does not interact with other screening targets or protein variants (proteins X1 and X2 with DNA‐binding domains). In this example, URA3 is not transcribed but lacZ is transcribed, yielding a Ura‐, Lac+ phenotype. See Grossel et al., . (e) Modification of proteins in two‐hybrid assays. A particular protein may not produce an interaction in its native form and may require post‐translational modification. Co‐expression of the factor required for modification (i.e. a protease) may be necessary to observe an interaction, especially if the host organism does not naturally express the factor. See Osborne, Dalton and Kochan .
Figure 3. Two ‐hybrid results and interactomes. (a) The E. coli interactome. The interactome of an organism is the set of all protein–protein interactions among its proteins. Here, it is displayed as a network, with nodes (grey circles) representing unique proteins and edges (lines between nodes) representing interactions between proteins. All results shown here are from a recent E. coli interactome (Rajagopala et al. ); interactions found using two‐hybrid methods are shown in green, those found in previous studies are shown in grey, and those found in both previous studies and the two‐hybrid screens used by Rajagopala et al. are shown in gold. (b) E. coli PPIs by method. Values shown here are totals of PPI in the IntAct molecular interaction database as of April 2016 except where noted. Counts include all strains of E. coli. ‘Total PPI’ includes all interactions in IntAct, including those derived from spoke expansion models. ‘All affinity‐based methods’ are those annotated under the PSI‐MI ontology as MI:0400 (affinity techniques) or any child method in the ontology, usually using mass spectrometry to identify interaction participants. ‘PPIs from Hu et al. ’ are those defined as physical interactions identified by Hu et al. using tandem affinity purification methods. ‘All PPIs from Rajagopala et al. ’ are those present in the network in part (a). ‘All two‐hybrid methods’ are those annotated as MI:0018 (two hybrid), MI:0397 (two‐hybrid array), MI:0398 (two‐hybrid pooling) or MI:1356 (validated two hybrid). (c) Spoke models for PPIs in complexes. Studies of protein complexes using affinity purification methods, including methods used by Hu et al. in a study of E. coli protein complexes, often produce sets of results involving more than two proteins in a set (as on the left, where each circle is a unique protein). In order to convert these associations to binary interactions, models such as the spoke model are used. This model assumes that, at least, one protein in the set interacts with all others. The resulting interactions are predictions and may not reflect in vivo protein interactions.
Figure 4. Differences in yeast two‐hybrid results when using N‐ and C‐terminal hybrid proteins. Counts of protein–protein interactions found with combinations of N‐ and C‐terminal fusions are shown. For example, 182 interactions were found with N‐terminal bait and prey fusions (blue), of which 115 were only found in this combination. 15 interactions (largest type) were found in all four combinations and are thus considered the most reliable. The box provides summaries of how many interactions (or percentages) were found in one, two, three or four combinations. Reproduced from Stellberger et al. 2010 © Springer under Creative Commons attribution licence 4.0.


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Further Reading

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Sakhawalkar, Neha, Caufield, John Harry, and Uetz, Peter(May 2017) Two‐Hybrid and Related Systems. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0000981.pub2]