Versatile screening for binary protein-protein interactions by yeast two-hybrid mating.
Identification of binary protein-protein interactions is a crucial step in determining the molecular context and functional pathways of proteins. State-of-the-art proteomics techniques provide high-throughput information on the content of proteomes and protein complexes, but give little information about transient interactions, about the binary protein pairs, or about the interacting epitopes. A powerful method to reveal this information is the yeast two-hybrid system. We have employed an optimized GAL4-based yeast two-hybrid system to dissect the photoreceptor cilium-associated protein complex around the retinitis pigmentosa GTPase regulator (RPGR) in mammalian photoreceptors. This enabled us to identify associating protein partners that, similar to RPGR, were also associated with a heterogeneous group of inherited retinal degenerations arising from ciliary defects. We describe how to generate high content pretransformed cDNA libraries, and perform an efficient yeast mating screen for protein-protein interactions with any bait protein of interest.
Letteboer
Stef J F
SJ
Department of Human Genetics Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
Roepman
Ronald
R
D017930:Genes, Reporter; D006801:Humans; D025941:Protein Interaction Mapping / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques / Q000295:instrumentation; D015003:Yeasts / Q000235:genetics / Q000378:metabolism
D011993:Recombinant Fusion Proteins