Tandem Affinity Purification
Encyclopedia
Tandem affinity purification (TAP) is a technique for studying protein-protein interaction
s. It involves creating a fusion protein
with 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-terminus of the protein under study. The TAP tag consists of calmodulin
binding 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.
, then one of the methods may be the use of plasmids that will eventually translate the 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 translated within the host, the new protein at one end 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 affinity selection, together with the other constituents attached to the new protein, by means of an IgG matrix.
After washing, TEV protease
is introduced to elute the bound material at the TEV protease cleavage
site. 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. After washing, the eluate is then released with ethylene glycol tetraacetic acid (EGTA
).
The native elution, consisting of the new protein and 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
.
without prior knowledge of complex composition. It is also simple to execute and often provides high yield. 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.
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 specificity of the TEV protease.
s. However, it is a good method for testing stable protein interactions and allows various degrees of investigation by controlling the number of times the protein complex is purified.
of yeast by characterizing multiprotein complexes. The study revealed 491 complexes, 257 of them wholly new. The rest were familiar from other research, but now virtually all of them were found to have new components. They drew up a map relating all the protein components functionally in a complex network.
Many other proteomic analyses also involve the use of TAP tag. A research by EMBO (Dziembowski, 2004) identified a new complex required for nuclear pre-mRNA retention and splicing. They have purified a novel trimeric complex composed of 3 other subunits (Snu17p, Bud13p and Pml1p) and find that these subunits are not essential for viability but required for efficient splicing (removal of introns) of pre-mRNA. In 2006, Fleischer et al. systematically identified proteins associated with eukaryotic ribosomal complexes. They used multifaceted mass spectrometry proteomic screens to identify yeast ribosomal complexes and then used TAP tagging to functionally link up all these proteins.
Protein-protein interaction
Protein–protein interactions occur when two or more proteins bind together, often to carry out their biological function. Many of the most important molecular processes in the cell such as DNA replication are carried out by large molecular machines that are built from a large number of protein...
s. It involves creating a fusion protein
Fusion protein
Fusion proteins or chimeric proteins are proteins created through the joining of two or more genes which originally coded for separate proteins. Translation of this fusion gene results in a single polypeptide with functional properties derived from each of the original proteins...
with 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-terminus of the protein under study. The TAP tag consists of calmodulin
Calmodulin
Calmodulin is a calcium-binding protein expressed in all eukaryotic cells...
binding peptide (CBP) from the N-terminal, followed by tobacco etch virus protease (TEV protease) cleavage site and Protein A
Protein A
Protein A is a 40-60 kDa MSCRAMM surface protein originally found in the cell wall of the bacterium Staphylococcus aureus. It is encoded by the spa gene and its regulation is controlled by DNA topology, cellular osmolarity, and a two-component system called ArlS-ArlR. It has found use in...
, 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.
Variant Tags
This tag is also known as the C-terminal TAP tag because an N-terminal version 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.History
TAP tagging was invented by a research team working in the European Molecular Biology Laboratory at late 1990s (Rigaut et al., 1999, Puig et al.,2001) and proposed as a new tool for proteome exploration. It was used by the team to characterize several protein complexes (Rigaut et al., 1999, Caspary et al. 1999, Bouveret et al., 2000, Puig et al., 2001). The first large-scale application of this technique was in 2002, in which the research team worked in collaboration with scientists of the proteomics company Cellzome 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.Process
There are a few methods in which the fusion protein can be introduced into the host. If the host is yeastYeast
Yeasts are eukaryotic micro-organisms classified in the kingdom Fungi, with 1,500 species currently described estimated to be only 1% of all fungal species. Most reproduce asexually by mitosis, and many do so by an asymmetric division process called budding...
, then one of the methods may be the use of plasmids that will eventually translate the 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 translated within the host, the new protein at one end 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 affinity selection, together with the other constituents attached to the new protein, by means of an IgG matrix.
After washing, TEV protease
Protease
A protease is any enzyme that conducts proteolysis, that is, begins protein catabolism by hydrolysis of the peptide bonds that link amino acids together in the polypeptide chain forming the protein....
is introduced to elute the bound material at the TEV protease cleavage
Bond cleavage
Bond cleavage, or scission, is the splitting of chemical bonds.If the two electrons in a cleaved covalent bond are divided between the products, the process is known as homolytic fission and free redicals are generated by homolytic cleavage the process is known as homolytic fission or homolysis...
site. This eluate is then incubated with calmodulin
Calmodulin
Calmodulin is a calcium-binding protein expressed in all eukaryotic cells...
-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. After washing, the eluate is then released with ethylene glycol tetraacetic acid (EGTA
EGTA (chemical)
EGTA is a polyamino carboxylic acid, a chelating agent that is related to the better known EDTA, but with a much higher affinity for calcium than for magnesium ions...
).
The native elution, consisting of the new protein and its interacting protein partners as well as CBP, can now be analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE
SDS-PAGE
SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, describes a collection of related techniques widely used in biochemistry, forensics, genetics and molecular biology to separate proteins according to their electrophoretic mobility...
) or be identified by mass spectrometry
Mass spectrometry
Mass spectrometry is an analytical technique that measures the mass-to-charge ratio of charged particles.It is used for determining masses of particles, for determining the elemental composition of a sample or molecule, and for elucidating the chemical structures of molecules, such as peptides and...
.
Advantages
An advantage of this method is that there can be real determination of protein partners quantitatively in vivoIn vivo
In vivo is experimentation using a whole, living organism as opposed to a partial or dead organism, or an in vitro controlled environment. Animal testing and clinical trials are two forms of in vivo research...
without prior knowledge of complex composition. It is also simple to execute and often provides high yield. 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.
Disadvantages
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 specificity of the TEV protease.
Suitability
As this method involves at least 2 rounds of washing, it may not be suitable for screening transient protein interactions, unlike the yeast two-hybrid method or in vivo crosslinking with photo-reactive amino acid analogPhoto-reactive amino acid analog
Photo-reactive amino acid analogs for in-vivo crosslinking of protein complexes were introduced in 2005 by researchers from the Max Planck Institute. In this method, cells are grown with photoreactive diazirine analogs to leucine and methionine, which are incorporated into proteins...
s. However, it is a good method for testing stable protein interactions and allows various degrees of investigation by controlling the number of times the protein complex is purified.
Applications
In 2002, the TAP tag was first used with mass spectrometry in a large-scale approach to systematically analyse the proteomicsProteomics
Proteomics is the large-scale study of proteins, particularly their structures and functions. Proteins are vital parts of living organisms, as they are the main components of the physiological metabolic pathways of cells. The term "proteomics" was first coined in 1997 to make an analogy with...
of yeast by characterizing multiprotein complexes. The study revealed 491 complexes, 257 of them wholly new. The rest were familiar from other research, but now virtually all of them were found to have new components. They drew up a map relating all the protein components functionally in a complex network.
Many other proteomic analyses also involve the use of TAP tag. A research by EMBO (Dziembowski, 2004) identified a new complex required for nuclear pre-mRNA retention and splicing. They have purified a novel trimeric complex composed of 3 other subunits (Snu17p, Bud13p and Pml1p) and find that these subunits are not essential for viability but required for efficient splicing (removal of introns) of pre-mRNA. In 2006, Fleischer et al. systematically identified proteins associated with eukaryotic ribosomal complexes. They used multifaceted mass spectrometry proteomic screens to identify yeast ribosomal complexes and then used TAP tagging to functionally link up all these proteins.