Protein-Protein Interaction Screens
Bacterial Two-Hybrid System bait DNA binding protein target sequence target RNA polymerase selectable marker
Yeast Two-Hybrid System transcription factor activation domain DNA binding protein
Yeast Two-Hybrid System transcription factor target activation domain bait DNA binding protein
Yeast Two-Hybrid System bait target DNA binding protein activation domain RNA polymerase selectable marker transcription factor binding site
His 3 as Selectable Marker and 3 -AT
3 -amino triazole (3 AT) NH 3 N N NH
Libraries for 2 -Hybrid Screens • The goal of a two-hybrid experiment is to screen a “library” for proteins that interact with a bait protein of interest • The library is a collection of plasmids that each contain a DIFFERENT TARGET PROTEIN fused to an activation domain or polymerase subunit
Libraries for 2 -Hybrid Screens
Libraries for 2 -Hybrid Screens gene 1 gene 2 X X gene 3 gene 4 gene 5 gene 6 X X X
Libraries for 2 -Hybrid Screens activation domain/polymerase subunit target protein Target protein must be in same reading-frame as activation domain and not contain non-coding sequence (5’ UTR, introns)
Library Construction genome target gene PCR primers target gene activation domain/polymerase subunit
Library Construction m. RNA target gene (coding region) c. DNA primer fragment c. DNA
Library Construction activation domain/polymerase subunit
Library Construction activation domain target fragment selectable marker
Whole-Genome Two-Hybrid Screens • Construction of Bait and Target Libraries Covering Entire Proteome • Automated Screening for Positives
Yeast Two-Hybrid Screen (Uetz et al. , Nature 2000)
Problems with Two-Hybrid Screens While two-hybrid screens can be very useful, they suffer from fairly high false-negative and false-positive rates What are some potential sources of falsepositive (proteins that appear to interact in the assay, but don’t in living cells) and falsenegative (proteins that interact in living cells but not in the two-hybrid assay) results?
Two-Hybrid False Negatives • Target protein not in library • Proteins do not fold properly or interact in the conditions used in the screen (e. g. human proteins in yeast cells) • Proteins only interact in the presence of other proteins • Proteins interact in ways that do not permit activation domain to function (multimerization)
Two-Hybrid False Positives • Non-specific – Bait proteins that activate without target – Target proteins that activate without bait – Target/Bait proteins that are “sticky” and interact with many things • Specific – Interactions between proteins that are never expressed together in living cells – Interactions between proteins that are normally inhibited by the presence of other proteins/conditions
Other Methods to ID Protein-Protein Interactions • Co-expression studies • Structure prediction • Purification of Complexes and Mass-Spec
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