Tandem Affinity Purification
The Tandem Affinity Purification (TAP) is a technique for studying
protein-protein interactions. It involves creating a fusion proteinwith a designed piece, the TAP tag, on the end. The protein of interest with the TAP tag first binds to beads coated with IgG, the TAP tag is then broken apart by an enzyme, and finally a different part of the TAP tag binds reversibly to beads of a different type. After the protein of interest has been washed through two affinity columns, it can be examined for binding partners.
The TAP method involves the fusion of the TAP tag to the
C-terminusof the protein under study. The TAP tag consists of calmodulinbinding peptide (CBP) from the N-terminal, followed by tobacco etch virus protease(TEV protease) cleavage site and Protein A, which binds tightly to IgG. The relative order of the modules of the tag is important because Protein A needs to be at the extreme end of the fusion protein so that the entire complex can be retrieved using an IgG matrix.
This tag is also known as the C-terminal TAP tag because an
N-terminalversion is also available. However, the method to be described assumes the use of a C-terminal tag, although the principle behind the method is still the same.
There are a few methods in which the fusion protein can be introduced into the host. If the host is
yeast, then one of the methods may be the use of plasmidsthat will eventually translatethe fusion protein within the host. Whichever method that is being used, it is preferable to maintain expression of the fusion protein as close as possible to its natural level.
Once the fusion protein is transcribed within the host, the new protein at one of the fusion protein would be able to interact with other proteins. Subsequently, the fusion protein is retrieved from the host by breaking the cells and retrieving the fusion protein through
affinityselection, together with the other constituents attached to the new protein, by means of an IgG matrix.
After washing, TEV
proteaseis introduced to elute the bound material at the TEV protease cleavagesite. 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 cite journal| author=Puig, O., et al.| title=The Tandem Affinity Purification (TAP) Method: A General Procedure of Protein Complex Purification|journal=Methods|date=2001| volume=24|pmid=11403571| pages= 218–229| doi=10.1006/meth.2001.1183] . After washing, the eluate is then released with ethylene glycol tetraacetic acid ( EGTA).
The native elution consisting of the new protein, its interacting protein partners as well as CBP can now be analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (
SDS-PAGE) or be identified by mass spectrometry.
An advantage of this method is that there can be real determination of protein partners quantitatively
in vivowithout prior knowledge of complex composition. It is also simple to execute and often provides high yield .
However, there is also the possibility that a tag added to a protein might obscure binding of the new protein to its interacting partners. In addition, the tag may also affect protein expression levels. On the other hand, 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, although this is unlikely to be frequent given the high
specificityof the TEV protease [cite journal|author=Dougherty, W.G., S.M. Cary, and T.D. Parks| journal=Virology| date=1989| volume=171| pmid=2669323|title=Molecular genetic analysis of a plant virus polyprotein cleavage site: a model| pages=356–364|doi=10.1016/0042-6822(89)90603-X] .
As this method involves at least 2 rounds of washing, it may not be suitable for screening
transient protein interactions, unlike the yeast two-hybridmethod. However, it is a good method for testing permanent interactions and allows various degrees of investigation by controlling the number of times the protein complex is purified.
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