Yeast Cross & Capture strain - Bait MATa
Opis
Developed by Dr. Stagljar of University of Toronto, the Dharmacon™ Cross-and-Capture™ Collection assay is a novel method that permits rapid analysis of protein-protein interactions (PPIs). This system uses differentially tagged ORF arrays in the two haploid yeast (Saccharomyces cerevisiae) mating types MATa and MATα. In MATa cells, “bait” ORFs are tagged at the 3' end with a sequence encoding six histidines (6xHIS), while “prey” ORFs in MATα cells are tagged with a sequence encoding a triple VSV tag (3xVSV). Both tags also contain a V5 epitope to allow identification of both bait and prey proteins. To examine a particular PPI, a bait strain is crossed with a prey strain to generate diploid yeast expressing the desired bait- and prey-tagged proteins. 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 ant-V5 and anti-VSV antibodies. If the prey protein binds to the nickel beads in a bait-dependent manner, a PPI is inferred (Figure 1A). Conversely, the absence of the prey protein in a pulldown reaction suggests that the two proteins fail to interact (Figure 1B).
In total 506 yeast ORF-baits and a same number of ORF-preys constitute the Dharmacon Cross and Capture Collection. The ORF composition consists in 258 ORFs encoding proteins involved in DNA repair, replication and recombination (Saccharomyces Genome Database, http://www.yeastgenome.org),as well as 248 ORFs encoding proteins of unknown function that were assigned to the nucleus based on their localization patterns (Huh et al. 2003).
To generate bait and prey strains, PCR products containing the desired tags and the Kanr cassette were produced from bait- and prey-specific plasmids and transformed into MATa and MATα strains, respectively (Figure 2). Confirmation of the successful tagging of all 506 ORFs was achieved by colony PCR via sequencing of the ORF/tag junction and by immunoblotting.
Figure 1.
Cross-and-Capture Assay.
A., B. 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) A., whereas a non-interacting protein (Protein Z–3xVSV) will not bind B. 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).
Figure 2.
Generation and verification of tagged protein arrays. To tag ORFX as bait (V5–6xHIS) and prey (V5–3xVSV), a set of primers is used that anneal to identical binding sites within the template plasmids and have flanking sequence homologous to ORFX. PCR products generated from the bait and prey templates are transformed into a- and α-cells, respectively.
Homologous recombination occurs between the variable portion of the 5’ primer (light blue) and the 3’ terminus of the ORF, and between the variable portion of the 3’ primer (red) and the 3’ UTR) of ORFX. Transformants are selected on G418 plates, and colony PCR is performed to verify integration of the Kanr downstream of the desired ORF. Abbreviations: TEF, translational elongation factor; TEFp, TEF promoter; TEFt, TEF terminator: Kanr, kanamycin resistance; loxp, site for CRE specific homologous recombination (Suter et al, 2007