Chapter 12: Analysis of Protein-Protein Interactions

When studying protein of unknown function, common approach is to do bioinformatics, and then consider how strong is the interaction, does it require other components, specific conditions

  • determine subcellular localization and search for interactors,

Pull Down Assay using Epitope Tagged Proteins

  • Affinity column for epitope tag on a protein or an extract invitro
  • Tag added to protein to tether or link the protein to column, protein extracts prepared from cells that express the linked protein normally are applied to the column to see if other proteins will bind
  • Interactors identified by SDS PAGE and autoradiography or mass spectrometry (sequence amino acid and blast to identify)
  • Controls: Test by applying extract to empty column (interactions with substrate, or non specific binding)

Easy, fast, cheap but strong interaction to be detected, if interactor rare needs radioactive or fluorescence detection

Co-Immunoprecipitation

  • Detects interactors in vivo to see what other proteins precipitate with protein of interest that binds antibody
  • Sensitive specific antibody required for protein of interest
  • Cells expressing protein are lysed and incubated with primary antibody, secondary antibody added
  • One method has secondary antibody attached to magnetic beads to collect by centrifuge
  • Prepare extract with interaction of interest, incubate with primary and secondary antibody, precipitate antibody and bound complex, analyze proteins by SDS PAGE/mass spec/ W Blot
  • Controls: Is antibody specific (test mutants)

Easy, fast, requires strong interaction but uses a lot of antibody, if no antibody buy antibody for tag and tag protein

Yeast Two-Hybrid Assay

Targeted (suspect specific proteins)

Untargeted (screen expression cDNA for proteins interacting with protein of interest)

Relies on transcription factors, each has DNA binding domain recognizing specific cis element and a transcription activation site, each on a different polypeptide so when they interact it creates a functional transcription factor

  • Coding sequence of protein is fused to coding sequence of DNA binding domain (bait)
  • Coding sequence of protein fused to coding sequence of transcription activator domain (prey)
  • Promoter gene in yeast cell will produce colour when proteins interact/bind
  • 3 constructs in 1 yeast cell bait, prey, Gal 4 responsive promoter (lac z produced blue)
  • Make clones in yeast vectors and grow each strain, make extracts and take samples for W Blot to validate expression, do lac z quantitative assay and sequence the clones that give positive signal through sanger (not high volume because this is a targeted approach to a sequence)
  • Controls: Reverse bait and prey, empty bait and prey, mutate bait and prey

In vivo method, detect weaker interactions, use to screen for possible interactors using cDNA in prey & done in a large scale, mutations can identify essential regions, have the clone at the end but only detects bi-molecular interactions, tag can interfere with normal function, lengthy process, prone to false results (non-specific interaction)

Bimolecular Fluorescence Complementation BIFC

Clone C terminus and N terminus of YFP fused to each putative interactor and transformed into host, use microscopy to detect fluorescence produced by interaction brining YFP together

Controls: Have YN and YC without anything attached and see if light produced (random connect), switch proteins

Detects protein in vivo in tissue and you can see subcellular localization, often possible to look at interaction after transfection, specific mutation identifies essential region but tag can interfere with function, less quantitative and hard to find negative control, best done with native promoter over expression can lead to mis-localization

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