Download - TAP(Tandem Affinity Purification)
TAP(Tandem Affinity Purification)
Billy Baader
Genetics 677
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Protein-protein interactions
Protein Identification
20,000+ genes in humans
Millions of proteins
Understanding human biological processes
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Protein IdentificationOther available methods-2D gel analysis-Labeling methods-Antibodies-Peptide tagging-Mass Spectrometry
What are some of the problems with these methods?
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Problems with Classical Methods
Requires large amounts of protein
Limitations in the number of testable samples
Purification
Contamination
Time
TAP compared to other methods
Flag Tag– Small peptide tag
Natural protein levels vs. overexpressed proteins
Yeast Two Hybrid– Low level of overlap– Assays protein interactions
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Protein internal structure
• Desire to understand molecular mechanismsSome proteins lack obvious enzymatic activity
Once proteins are discovered we would like to know how they work and interact
Possibilities-Crystillization-Electron Microscopy-Two Hybrid Assay-Chemical Cross-Linking
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Functional organization of the yeast proteome by systematic analysis of protein complexes
Gavin et al.
Practical application of TAP and mass spectrometry on S. cerevisiae
Emphasizes the potential for a massive amount of information to be obtained through TAP
Potential of protein knowledge
“Whenever it has been possible to retrieve and analyze particular cellular protein complexes under physiological conditions, the insight gained from the analysis has been fundamental for the biological understanding of their function”
i.e. spliceosome, cyclosome, proteasome
Examined 25% of ORFs in yeastQuickTime™ and a
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Method for purificationTAP1. High affinity purification2. Elution3. Second affinity purification
Separate with gel electrophoresis
Digest with trypsin
Analyze with mass spectrometry
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TAP tags
TAP cassette created through PCR
Insertion at the C-terminus of a selected yeast ORF by homologous recombination
Examined 1,739 genes
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Homologous Recombination of TAP tag
Proteins purified from different organelles
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Examining the data
Technical Bias against proteins below 15kDa
Possiblity of using different entry points to purify protein complexes
Comparison to literature
70% reproducability
Polyadenylation machinery
• Responsible for eukaryotic mRNA cleavage and polyadenylation
• Single entry point used: Pta1
• 12 of 13 known interactors and 7 new components
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Reproducibility using various entry points
Polyadenylation machinery
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Protein complex networks
Utilized an algorithm to automatically generate map
Links are between complexes sharing at least one protein
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Orthologs
Examined the hypothesis that orthologous gene products are responsible for essential cellular activities
Orthologous complexes interact preferentially with other orthologous complexes
Nonorthologous complexes do not interact at as well with the orthologous complexes
The same relation is present between essential and non-essential complexes
Human/Yeast Orthologs
• Arp2/3– Cytoskeleton-associated complex
• Ccr4-Not– Involved in control of gene expression
• TRAPP– Transport protein particle– associated with Golgi body
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Ortholog Comparison Results
Results
• Huge increase in number of proteome components
• TAP was responsible for a efficient identificaiton of low-abundance proteins as well as large complexes
• Differences in the aspects of protein interaction detected through TAP compared with Y2H
• Orthologous complexes appear to represent the building blocks of a ‘core proteome’
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Advantages of TAP
• Simplicity
• Cleaner, more intact complexes
• Higher yield
• Low false negative rates
• Tags show little protein alteration
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TAP vs. Y2H
• Analyzes complexes and can create protein network maps
• Analyzes more of the proteome
• Works in membrane proteins
• Works in many organisms
• Analyzes binary interaction between proteins
• Works with transient protein interactions
• Performed in vivo
These methods yield different information and should be used complementarily
PossibilitiesGavin et al. believe that there methods are one of the most efficient
and unambiguous routes towards the assignment of gene identity and function
Easy to analyze large amounts of protein complexes and assess there relation to each other
Increase in understanding of biological systems and their processesDrug discovery and usage may be greatly enhanced through this
knowledgeIdentification of a vast number of proteins and protein complexesOther techniques may be used to understand the function of these
proteinsA more complete understanding of the proteomes can hopefully be
developed
Questions
The review, when talking about TAP, said that it has been used to purify membrane bound protein complexes. I don't know a lot about protein purification, but I have always heard that purifying membrane proteins is notoriously difficult. Can you explain what make TAP better suited for this than other methods?
Questions
In the Review paper table 1 compares Flag and TAP; why is the fraction of successful purification (both with and without interacting proteins) higher for Flag? The other statistics in the table seem to make Flag a poor alternative, however, the fraction of successful purifications would seem to be an important percentage to raise. What is being done to improve this?