TAP-tagging is a protein purification technique that involves creating a fusion protein with a TAP tag to study protein-protein interactions. The TAP tag contains protein A and a calmodulin binding peptide separated by a TEV protease site. This allows two-step affinity purification of associated protein complexes under physiological conditions. TAP-tagging was developed in the 1990s and has been widely used to map protein interaction networks in yeast and identify new protein complexes involved in processes like pre-mRNA splicing.

1. Tandem Affinity Purification
(TAP-Tagging)

2. What is TAP-Tagging
Introduction
• Tandem affinity purification (TAP) is a purification
technique for studying protein-protein interactions. It
involves creating a fusion protein with a designed piece,
the TAP tag, on the end.
• Tandem affinity purification (TAP) tagging is a method to
purify multimeric protein complexes that can be used
under essentially physiological conditions.

3. History
• TAP tagging was invented by a research team working in
the European Molecular Biology Laboratory at late
1990s.
• The first large-scale application of this technique was in
2002,to develop a visual map of the interaction of more
than 230 multi-protein complexes in a yeast cell by
systematically tagging the TAP tag to each protein.

4. Background
• Proteins rarely act alone
• Comprehensive protein interaction studies
thus far:
1. two-hybrid systems (ex vivo)
2. protein chips (in vitro)
3. GST pull-downs (in vivo)
• Nowadays tandem-affinity purification
(TAP) and mass spectrometry is used.

5. Protein-Protein Interactions
Proteins carry out tasks together with other
proteins
• 83% proteins interact with others
• Proteins interact in promoters
• Multi-protein complexes (assemblies)
• Gene regulation
• Biological Pathway

6. Components of TAP-Tag
• Protein A
• Calmodulin Binding Peptide(CBP)
• Tobacco etch virus (TEV) protease
cleavage site

7. Variant Tags
• This tag is also known as the C-terminal TAP tag
because an N-terminal version is also available.
Target
protein

8. TAP-Tag Selection and Design
Optimal tags for protein complex purification
and the analysis of protein interaction should
have the following characteristics:
1. High affinity for the cognate matrix for
quantitative recovery of low-abundance
target proteins in dilute solutions.
2. Highly specific binding to increase the
ratio of specifically to nonspecifically
bound material to the affinity material.

9. 3. Efficient and specific elution allowing
high-level and specific recovery of the target
protein.
4. Mild conditions of elution to preserve
protein interactions and protein complex
structure.

10. TAP-Tagging Strategy
• There are a few methods in which the
fusion protein can be introduced into the
host.
• If the host is yeast, then plasmids will be
used that will eventually translate the
fusion protein within the host.

11. TAP-Tagging Strategy
• The translated fusion protein is retrieved through affinity
selection, together with the other constituents attached
to the new protein, by means of an IgG matrix.

12. TAP-Tagging Strategy
• After washing, TEV protease is introduced to elute the
bound material at the TEV protease cleavage site.

13. TAP-Tagging Strategy
• This eluate is then incubated with calmodulin-coated
beads in the presence of calcium
• This second affinity step is required to remove the TEV
protease as well as traces of contaminants remaining
after the first affinity step.

14. • After washing, the eluate is then released with ethylene
glycol tetraacetic acid (EGTA).SDS-PAGE or Mass
Spectrometry can be used to analyze the eluted protein.

16. Advantages
• An advantage of this method is that there can be real
determination of protein partners quantitatively in vivo
without prior knowledge of complex composition.
• One of the obstacles of studying protein protein
interaction is the contamination of the target protein
especially when we don’t have any prior knowledge of it.
TAP offers an effective, and highly specific means to
purify target protein.After 2 successive affinity
purifications, the chance for contaminants to be retained
in the eluate reduces significantly.

17. Disadvantages
• There is a possibility that a tag added to a
protein might obscure binding of the new
protein to its interacting partners.
• The tag may also affect protein expression
levels.

18. Disadvantages
• The tag may also not be sufficiently
exposed to the affinity beads, hence
skewing the results.
• There may also be a possibility of a
cleavage of the proteins by the TEV
protease.

19. Suitability
• As this method involves at least 2 rounds
of washing, it may not be suitable for
screening transient protein interactions
• But it is a good method for testing stable
protein interactions by controlling the
number of times the protein complex is
purified.

20. Applications
• Tandem affinity purification (TAP) method
combined with LC-MS/MS is the most accurate
and reliable way to study the interaction of
proteins or proteomics in a genome-wide scale.
• Many other proteomic analyses also involve the
use of TAP tag.
• Researchers identified a new protein complex
required for nuclear pre-mRNA retention and
splicing with the help of TAP-Tagging.