Download TAP(Tandem Affinity Purification)

TAP(Tandem Affinity Purification)
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Billy Baader
Genetics 677
Protein-protein interactions
Protein Identification
20,000+ genes in humans
Millions of proteins
Understanding human biological
processes
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Protein Identification
Other available methods
-2D gel analysis
-Labeling methods
-Antibodies
-Peptide tagging
-Mass Spectrometry
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What are some of the problems with these
methods?
Problems with Classical Methods
Requires large amounts of protein
Limitations in the number of testable samples
Purification
Contamination
Time
How TAP works
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TAP compared to other methods
Flag Tag
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– Small peptide tag
Natural protein levels vs. overexpressed
proteins
Yeast Two Hybrid
– Low level of overlap
– Assays protein interactions
Protein internal structure
• Desire to understand molecular mechanisms
Some 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 yeast
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Method for purification
TAP
1. High affinity purification
2. Elution
3. Second affinity purification
Separate with gel electrophoresis
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Digest with trypsin
Analyze with mass spectrometry
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TAP tags
TAP cassette created through PCR
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Insertion at the C-terminus of a selected
yeast ORF by homologous
recombination
Examined 1,739 genes
Homologous Recombination of TAP tag
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Proteins purified from different
organelles
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Efficiency
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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
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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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Protein Network
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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
Ortholog Comparison Results
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Results
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• 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’
Advantages of TAP
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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
Possibilities
Gavin 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
processes
Drug discovery and usage may be greatly enhanced through this
knowledge
Identification of a vast number of proteins and protein complexes
Other techniques may be used to understand the function of these
proteins
A 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?
Questions
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