A Novel Screen Identifying Ternary Binding Partners Of Known Interacting Proteins

Many biological processes depend on oligomeric protein-protein interactions (PPI). However, state-of-the-art in vivo PPI techniques focus on analysing binary interactions (i.e. the Split-Ubiquitin System (SUS)1). The SUS can also be used to analyse binding of three proteins in the so-called SUS Bridge Assay (SUB)2 – see Figure A. Nevertheless, this assay makes an unbiased screening approach cumbersome and its low efficiency restricts the identification of meaningful candidates.
Here, we present the first screening system in yeast that permits high-throughput screening of cDNA libraries for ternary binding partners of a known interaction couple. This is achieved by a unique combination of SUB and yeast mating. Technical prerequisites are special “2in1”-vectors3, which allow simultaneous transformation of “Bait I” and “Bait II” on a single plasmid in yeast of one mating type and the cDNA library in the other – see Figure B.

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