Created by researchers in the Stagljar Lab at the University of Toronto, the Yeast Cross and Capture System, a novel “bait” MATa strain assay, is used for assessment of protein–protein interactions. This assay employs a pulldown of differently tagged yeast strain arrays when a specific gene tagged with 6xHIS is mated with a “prey” MATalpha with a specific gene tagged with VSV to generate a diploid organism. The tagged protein and any interacting molecules can then be pulled down via the 6xHIS tag and the VSV can be detected on a western blot with an anti-VSV antibody.
Following diploid growth and cell lysis, extracts are incubated with nickel beads, allowing isolation of the 6xHIS-tagged bait and its associated proteins. Bound proteins are examined by immunoblot analysis for the presence of the bait and prey proteins using anti-V5 and anti-VSV antibodies. If the prey protein binds to the nickel beads in a bait-dependent manner, a PPI is inferred. Conversely, the absence of the prey protein in a pulldown reaction suggests that the two proteins fail to interact.
6xHIS tag on bait Mat-a strain allows for pull-down of interacting protein complexes
Anti-V5 antivody can be used to detect both bait and prey proteins
Anti-VSV antibody can be used to specifically detect prey protein
Amenable to high-throughput screens
Baits are in the BY4741 (MATa ura3Δ0 leu2Δ0 his3Δ1 met15Δ0) background strain.
Preys are in BY4742 (MATα ura3Δ0 leu2Δ0 his3Δ1 lys2Δ0) background strains.
A strain containing the bait ORFX tagged with a V5 epitope and six histidines (6xHIS) is crossed with strains that contain prey ORFY or ORFZ tagged with a V5 epitope followed by a triple VSV tag (3xVSV). Diploids, which express both tagged bait and prey, are grown on selective medium. Protein extracts from the diploids are then incubated with nickel beads (Ni2+ -NTA), allowing isolation of bait (Protein X–6xHIS) and bait-associated prey protein (Protein Y–3xVSV), whereas a non-interacting protein (Protein Z–3xVSV) will not bind. Proteins are then separated by SDS-PAGE, and blots are probed for bait and prey (anti-V5 antibody) and specifically for the prey (anti-VSV antibody) by immunoblot (Suter et al., 2007).
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