Tandem affinity purification

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: two-hybrid systems (ex vivo) protein chips (in vitro) 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: High affinity for the cognate matrix for quantitative recovery of low-abundance target proteins in dilute solutions. 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.

15. 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.

16. 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.

17. 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.

18. 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.

19. 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.