Tandem Affinity Purification (TAP) Service
Protein-protein interactions are essential for a wide range of biological processes, including signal transduction, gene regulation, and cellular metabolism. Understanding these interactions and the resulting protein complexes is crucial for gaining insight into the underlying mechanisms of various biological phenomena. Tandem Affinity Purification (TAP) is a powerful technique used to isolate protein complexes from endogenous sources. TAP is based on the incorporation of a dual-affinity tag into the protein of interest and the introduction of the construct into desired cell lines or organisms.
Tandem-affinity purification (Huber et al., 2003).
The TAP method involves two consecutive affinity purification steps, which allows for the isolation of protein complexes with high purity and specificity. The dual-affinity tag consists of two components, which are fused to the N- or C-terminus of the protein of interest. The first component is a protein A (ProtA) domain, which binds to immunoglobulin G (IgG) with high affinity. The second component is a calmodulin-binding peptide (CBP), which binds to calmodulin with high affinity in the presence of calcium ions.
The TAP method is relatively simple and can be applied to various systems. It allows the purification of protein complexes even when their composition or function is not known beforehand. Starting from a relatively small number of cells, active macromolecular complexes can be isolated and used for multiple applications. The technique can be modified to purify complexes containing two given components or to subtract undesired complexes.
Schematic representation of TAP (Bürckstümmer et al., 2006)
TAP has become a powerful tool for the identification and characterization of multi-protein complexes involved in various biological processes. Compared with single-step purification methods, TAP greatly reduces non-specific background and isolates protein complexes with higher purity, which simplifies subsequent identification and validation of the isolated proteins as true interacting partners.
The TAP method has several advantages over other protein purification methods. Firstly, it is a rapid and efficient method for the purification of protein complexes. The method requires fusion of the TAP tag, either N- or C-terminally, to the target protein of interest. Starting from a relatively small number of cells, active macromolecular complexes can be isolated and used for multiple applications. Variations of the method to specifically purify complexes containing two given components or to subtract undesired complexes can easily be implemented.
Secondly, TAP is a very versatile technique that can be adapted to various organisms. The method was initially developed in yeast but has been successfully adapted to other systems, including mammalian cells and bacteria. The simplicity, high yield, and wide applicability of the TAP method make it a very useful procedure for protein purification and proteome exploration.
Thirdly, TAP coupled with mass spectrometry-based analysis has become a standard approach for identification and characterization of multi-protein complexes. The use of TAP coupled with mass spectrometry enables the identification of not only the target protein but also its interacting partners. This information is invaluable for understanding the composition and function of protein complexes in various biological processes.
One key aspect of the TAP method is the use of a TAP vector, which is a plasmid DNA construct that contains the genetic information necessary for expression of the dual-affinity tag in cells. The TAP vector usually contains a promoter region that controls the expression of the tagged protein of interest, as well as antibiotic resistance genes to allow for selection of cells that have taken up the plasmid.
The dual-affinity tag typically consists of two components: a protein A domain and a calmodulin-binding peptide (CBP). The protein A domain binds to immunoglobulin G (IgG), which is coupled to an affinity resin. The CBP domain binds to calmodulin, which is coupled to a resin via calcium ions. This dual-affinity tag enables efficient and highly specific purification of the protein complex.
To use the TAP vector, researchers first clone the gene of interest into the plasmid DNA. This can be done using restriction enzymes to cut the plasmid and insert the gene, or by using modern DNA assembly techniques such as Gibson assembly or Golden Gate cloning. The resulting plasmid is then transfected into cells, where it is expressed and incorporated into the protein complex.
Advantages of using TAP vectors include the ability to purify protein complexes from endogenous sources, which provides a more accurate representation of the in vivo protein-protein interactions. Additionally, the sequential purification scheme greatly reduces non-specific background and isolates protein complexes with higher purity. TAP vectors are also highly adaptable, and variations of the method can be used to specifically purify complexes containing two given components or to subtract undesired complexes.