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Yeast two-hybrid (Y2H) system

 

tech yeast two hybrid system fig 1
Figure 1. Protein–protein interaction (PPI) between retinoblastoma protein and large T antigen protein. (a) A cell of a reporter strain of a yeast two-hybrid (Y2H) system that has been cotransformed with a plasmid that expresses Gal4(1-147)-Rb and a plasmid that expresses Gal4(768-881)-LTP has an active reporter gene in its genome—data acquired and diagram adapted from NAR 23:1152, Oxford Journals[4]; Rb: Retinoblastoma protein (301-918); LTP (for Large T antigen Peptide): SV40 Large T Antigen (residues 103-115). Coordinates from PDB ID: 1GH6[5] were used to generate representations of: (b) Retinoblastoma protein (residues 378-772 minus residues 578-644); (c) SV40 Large T Antigen (residues 103-115); and (d) Retinoblastoma protein (residues 378-772 minus residues 578-644) and SV40 Large T Antigen (residues 103-115).

 

 

 Yeast two-hybrid (Y2H) assays

The yeast two-hybrid system/assay[1,2], which has already been used to study protein–protein interactions for over 25 years, and its derivatives, for example, the adenylate cyclase-based bacterial two-hybrid (B2H) system[3], rely on two specific protein domains, which on this webpage are referred to as System Domain 1 (SD1) and System Domain 2 (SD2). If these two domains are in close proximity of each other, then a specific reporter gene can become active, which then becomes visible to the researcher that conducts the assay (Figure 1a—in this case SD1 is Gal4(1-147) and SD2 is Gal4(768-881); Table 1, Assays 1a and 1b). In a yeast two-hybrid assay one of the proteins of the assayed PPI is referred to as BAIT and one is referred to as PREY—in case of Figure 1, BAIT is Rb and PREY is LTP. Custom-made plasmids are used to express SD1-BAIT and PREY-SD2 in the cells of a reporter strain—these two fusion proteins are the two hybrids of the 'yeast two-hybrid system'; note: in hybrids of most Y2H systems SD1 and SD2 are located N-terminally (with regard to the BAIT/PREY), however, placing them C-terminally may be possible. If BAIT and PREY physically bind to each other, then SD1 and SD2 are in close proximity of each other, and this can lead to activation of a specific reporter (Table 1, Assay 2a). Reporter activity of (control) Assays 2b and 2c (Table 1) should be determined to monitor potential false-positive reporter activity caused by (1) BAIT has activity as SD2; (2) PREY has activity as SD1; (3) BAIT physically binds to SD2; and (4) PREY physically binds to SD1.

 

Table 1. The yeast two-hybrid system & the bacterial two-hybrid system briefly explained (Assays 1-3); and an hypothetical example of the mapping of a protein–protein interaction domain using two-hybrid technology (Assays 5-8). See also the text on this webpage.
tech yeast two hybrid system table 1
(1) The indicated fusion proteins and separate protein domains are expressed from appropriate plasmids in cells of a yeast or bacterial two-hybrid system reporter strain.
(2) The xxxx represents a chain of n amino acid residues (n means a certain number and differs per system).

 

Yeast two-hybrid screen

In the yeast two-hybrid system a cell of a yeast reporter strain that has been cotransformed with a plasmid that expresses SD1-BAIT (or BAIT-SD1) and a plasmid that expresses PREY-SD2 (or SD2-PREY) is referred to as a cotransformant. In a typical yeast two-hybrid screen a large number of cotransformants are generated and subsequently grown on appropriate agar plates. In every cotransformant potential protein–protein interaction between a known BAIT and a PREY from a library is tested. On agar plates a cotransformant (a single cell) can grow to a colony—consisting of a large number of clones of the original cell. The dots on the illustrated agar plate (Figure 2) represent such colonies. In the PPI identification screen that is depicted in Figure 2, an active reporter leads to blue coloring of the corresponding colony. Thus in the three blue colored colonies (Figure 2) potentially physical binding between BAIT and PREY occurs (Table 1, Assay 3a), and in most or all of the white colored colonies BAIT–PREY binding (with strong enough affinity) does not occur (Table 1, Assay 3b). Next steps would be (1) isolation of PREY encoding DNA from cells of the three blue colored colonies; (2) use this DNA to conduct control assays (similar as Table 1, Assays 2b and 2c); and (3) identify the PREYs through sequencing of the corresponding DNA. Thus the yeast two-hybrid system can be used to screen for (novel) protein–protein interactions. Physical binding between a protein of interest (the bait) and proteins of a library (the preys) can be detected. When using sophisticated strategies/systems, relatively easy millions of "library PREYs" can be screened for BAIT-binding activity.

 

 tech yeast two hybrid system fig 2
Figure 2. A yeast two-hybrid screen. Two-hybrid technology can be used to identify novel protein–protein interactions. See the text (in particular Table 1 Assays 3a and 3b) for explanation of the Y2H screen that is illustrated in this picture.

 

Confirming protein–protein interactions

The yeast two-hybrid system can be used to attempt to confirm a putative protein–protein interaction. For example, if (yeast two-hybrid) Assay 4a is positive and (control) Assays 4b and 4c are negative (Table 1), then (hypothetical) Protein_X–Protein_Y interaction is confirmed—DNA molecules that encode hypothetical Protein_X and hypothetical Protein_Y are used as input for the assays.

 

Mapping protein–protein interaction domains

Using the yeast two-hybrid system it is possible to demonstrate binding between Rb protein and LTP (a linear peptide of 13 amino acid residues) experimentally (Figure 1a—related tertiary structures are shown in Figures 1b-d)—this showed that yeast two-hybrid assays can be used to map short protein–protein interaction domains (PPIDs) experimentally. To identify a region of Protein_Y (see the confirming PPIs section above) that contains a PPID with PPI activity towards Protein_X, fragments of Protein_Y can be tested for Protein_X-binding activity (Table 1, Assays 5a-5c—in this case Protein_Y consists of a chain of 600 amino acid residues; the N-terminal 50% (Assay 5a) / middle 50% (Assay 5b) / C-terminal 50% (Assay 5c) of Protein_Y is used as PREY). In this hypothetical example, positive Assays 5a and 5b (Table 1) suggest that Protein_Y(151-300) contains a PPID that is involved in binding between Protein_X and Protein_Y—it is less likely that Protein_Y(1-150) and Protein_Y(301-450) both contain a PPID with such PPI activity. Fragments of this putative positive region can be tested in similar assays and by repeating this procedure several times, a short Protein_X-binding fragment can be systematically identified (Assays 6-8).

 

Footnotes

[1] Fields, S. & Song, O. (1989), 'A novel genetic system to detect protein-protein interactions.', Nature 340, 245--246.
[2] Vidal, M. & Fields, S. (2014), 'The yeast two-hybrid assay: still finding connections after 25 years.', Nature methods 11, 1203--1206.
[3] Karimova, G.; Gauliard, E.; Davi, M.; Ouellette, S. P. & Ladant, D. (2017), 'Protein-Protein Interaction: Bacterial Two-Hybrid.', Methods in molecular biology (Clifton, N.J.) 1615, 159--176.
[4] Yang, M.; Wu, Z. & Fields, S. (1995), 'Protein-peptide interactions analyzed with the yeast two-hybrid system.', Nucleic acids research 23, 1152--1156.
[5] Kim, H. Y.; Ahn, B. Y. & Cho, Y. (2001), 'Structural basis for the inactivation of retinoblastoma tumor suppressor by SV40 large T antigen.', The EMBO journal 20, 295--304.

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