1 / 23

Protein-Protein Interaction Screens

Protein-Protein Interaction Screens. target. Bacterial Two-Hybrid System. bait. DNA binding protein. RNA polymerase. selectable marker. target sequence. Yeast Two-Hybrid System. activation domain. transcription factor. DNA binding protein. target. Yeast Two-Hybrid System.

marciaw
Télécharger la présentation

Protein-Protein Interaction Screens

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Protein-Protein Interaction Screens

  2. target Bacterial Two-Hybrid System bait DNA binding protein RNA polymerase selectable marker target sequence

  3. Yeast Two-Hybrid System activation domain transcription factor DNA binding protein

  4. target Yeast Two-Hybrid System activation domain transcription factor bait DNA binding protein

  5. target Yeast Two-Hybrid System activation domain bait RNA polymerase DNA binding protein selectable marker transcription factor binding site

  6. His3 as Selectable Marker and 3-AT

  7. 3-amino triazole (3AT) NH3 N N NH

  8. 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

  9. Libraries for 2-Hybrid Screens

  10. gene 1 gene 2 gene 3 gene 4 gene 5 gene 6 Libraries for 2-Hybrid Screens X X X X X

  11. 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)

  12. Library Construction genome target gene PCR primers target gene activation domain/polymerase subunit

  13. Library Construction mRNA target gene (coding region) cDNA cDNA primer fragment cDNA

  14. activation domain/polymerase subunit activation domain/polymerase subunit activation domain/polymerase subunit Library Construction

  15. Library Construction activation domain selectable marker target fragment

  16. Whole-Genome Two-Hybrid Screens • Construction of Bait and Target Libraries Covering Entire Proteome • Automated Screening for Positives

  17. Yeast Two-Hybrid Screen (Uetz et al., Nature 2000)

  18. 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 false-positive (proteins that appear to interact in the assay, but don’t in living cells) and false-negative (proteins that interact in living cells but not in the two-hybrid assay) results?

  19. 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)

  20. 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

  21. Other Methods to ID Protein-Protein Interactions • Co-expression studies • Structure prediction • Purification of Complexes and Mass-Spec

More Related