Tandem affinity purification

Tandem Affinity Purification (TAP) is a biotechnology technique used in the field of molecular biology to isolate protein complexes from biological samples. This method allows for the purification of protein complexes in a relatively native state, making it invaluable for studying protein-protein interactions, protein function, and the biochemical pathways in which these proteins are involved.

Overview

TAP involves a dual-tagging system where a protein of interest is genetically fused to a TAP tag. This tag typically consists of two affinity tags separated by a protease cleavage site. The most common tags used are the calmodulin binding peptide (CBP) and the immunoglobulin G (IgG) binding domain of Staphylococcus aureus protein A, with a tobacco etch virus (TEV) protease cleavage site in between. The process of TAP can be broken down into several steps: expression of the tagged protein in a host cell, lysis of the cell to release the protein complexes, and sequential affinity purification steps exploiting the dual tags.

Procedure

The TAP procedure starts with the expression of the tagged protein of interest in a suitable host cell, such as yeast, bacteria, or mammalian cells. Once expressed, the cells are lysed to release the protein and any proteins bound to it. The lysate is then subjected to the first affinity purification step, typically using IgG sepharose beads to bind the protein A domain of the TAP tag. After washing away unbound proteins, the bound proteins are released by TEV protease cleavage, which specifically cleaves the site between the two affinity tags.

The eluate from the first purification step is then subjected to a second affinity purification step, this time using calmodulin-binding resin in the presence of calcium ions to bind the CBP tag. After further washing steps to remove non-specifically bound proteins, the target protein complex is finally eluted by chelating the calcium ions, typically using ethylene glycol tetraacetic acid (EGTA).

Applications

Tandem affinity purification has been widely used in the study of protein complexes in various organisms. It is particularly useful for identifying novel protein-protein interactions, understanding the composition of protein complexes, and investigating the roles of proteins within these complexes in cellular processes. TAP has been instrumental in the study of signaling pathways, gene expression regulation, and cellular structure and organization.

Advantages and Limitations

One of the main advantages of TAP is its high specificity and efficiency in purifying protein complexes with minimal contamination. This is largely due to the sequential purification steps that allow for a high degree of purification. However, the method does have limitations, including the potential for the TAP tag to interfere with the normal function of the protein or its interactions. Additionally, the technique requires the generation of genetically modified organisms expressing the tagged protein, which can be time-consuming and technically challenging.

Conclusion

Tandem affinity purification is a powerful tool in the molecular biologist's arsenal, allowing for the detailed study of protein interactions and functions. Despite its limitations, the technique has significantly advanced our understanding of cellular machinery and will continue to be a valuable method for studying complex biological systems.