Proteomics II for Monday

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Proteomics IIfor Monday

• Yeast Two Hybrid
• please study
• Toward a Protein-Protein Map of the Budding Yeast
• (Ito et al., 2000)
• PNAS 97(3), 1143-1147

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Protein-Protein Interactions

• Signal Transduction Pathways,
• Heteromeric Protein Complexes,
• Allosteric Interactions,
• etc.

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Protein-Protein Interactions

• Co-precipitation, affinity chromotography, etc.,
• Protein Chips,
• on the near horizon,
• Phage Display,
• Yeast Two Hybrid (Y2H).

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GAL4 Transcription Activator
GAL4

• BD Binding Domain,
• AD Activation Domain.

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Yeast Two Hybrid Vectors

• ...separate GAL4 Binding Domain and Activation
  Domain,
• ...create chimeric proteins, on expression
  vectors,
• Bait Protein fused to the Binding Domain moiety,
• Target Protein fused to the Activation Domain
  moiety.

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Yeast

• Haploid or diploid viable,
• Can carry vectors,
• Can carry heterogeneous vectors,
• ...transform one haploid yeast with Bait
  plasmid, another with Target plasmid,
• ...mate, look for activation of GAL4 controlled
  marker gene(s).

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Yeast Two Hybrid Vectors
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• ...mate haploid cells, each expressing the
  recombinant proteins,
• one with bait,
• the other(s) with prey.

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No Interaction Bait/Prey

• ...bait binds DNA,
• ...prey does not associate with bait, or
  transcription machinery

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Bait/Prey Interact

• ...bait binds DNA,
• ...prey associates with bait,
• ...activation domain is then in proximity to
  transcriptonal machinery,
• ...reporter gene turned on.

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Variations Exist

• ...choice of BD and AD,
• BD protein is most often the known,
• reduces false positives,
• AD ORFs, cDNA or genomic,
• strength of promoters,
• reporter genes,
• Bait and Target polypeptides can be from varied
  sources, or even synthetic.

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Weaknesses

• False Positives,
• some Baits are sticky, sticks to lots of
  Targets,
• some Targets are sticky, sticks to lots of
  Baits,
• fortuitous activation of marker promoter,
• usually assay for multiple markers,
• False Negatives,
• clone fidelity,
• protein conformation (especially membrane bound
  proteins),
• protein modifications (phosphorylation,
  glycosylation, etc.),
• Artifacts Y2H identified interactions require
  subsequent confirmation.

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Strengths

• Can be used on a single protein-protein
  interaction,
• Scalable,
• Once binary interactions are identified, networks
  can be deduced,
• It works,
• repeatable, bait/prey and prey/bait verification,
• identifies previously discerned interactions,
  plus novel interactions,
• clusters functionally consistent proteins.

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Spliceosome Analysis

• Nature Genetics, 1977, 16 pp 277 - 282,
• Available for photocopy on my office bulletin
  board.

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Strategy

• Bait 10 spliceosome proteins,
• Targets genomic library,
• Selections His and lacZ,
• Sequence identify ORFs,
• 2nd Round use identified preys as bait,
• Repeat.

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Results
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PNAS, 2000 97(3), pp 1143-1147 Assigned,
available online.
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Strategy

• Bait all yeast ORFs,
• Targets all yeast ORFs,
• Matings pooled, random
• Selections Ade His and Ura,
• Sequence identify ORF,
• Build Interaction Maps.