Brief Introduction about Tandem Affinity Purification

Posted by Ellen Burns on August 4th, 2021

The study of protein interactions is an important means to interpret gene functions. The high-resolution mass spectrometry technology developed in recent years provides a powerful tool for the identification of protein complexes. Therefore, the limiting factor for determining protein interactions is not protein identification. It is the purification of protein complexes. Traditional purification methods (such as affinity chromatography or co-immunoprecipitation) are difficult to obtain protein complexes close to the natural state, and the experimental results may have false positives.

Tandem affinity purification (TAP) is a technology that can quickly study protein interactions in the body. After two-step specific affinity purification, proteins that actually interact with target proteins under physiological conditions can be quickly obtained. The TAP method was first used in yeast. It has been rapidly developed due to its versatility, high efficiency, high purity and low false positives. It has been successfully applied to the study of interactions among many other organisms (mammals, plants, etc.).

Background

The emergence of powerful and sensitive high-throughput mass spectrometry (MS) technology can detect peptide molecules in the range below femtomole. It is often used to identify interacting protein complexes or complex subunits, which greatly promotes the development of biochemical purification methods for cell proteome. However, it is usually difficult to obtain a sufficient amount of purified protein complexes, which has become a rate-limiting step for mass spectrometry applications in this area. In 1999, Rigaut et al. jointly proposed a new method for the separation of complex proteins—tandem affinity purification (TAP), which has the advantages of both standard affinity purification and co-immunoprecipitation biochemical methods and provides a new way for the isolation and identification of protein complexes. Tandem affinity purification combined with protein mass spectrometry (MS) identification technology can be used for biochemical purification and the analysis of bimolecular and macromolecular protein complexes

Advantages of TAP technology

l  It can truly reflect the behavior of the protein in the organism in the physiological state of the cell or close to the physiological state;

l  After two elutions, the amount of non-specific protein is reduced, and there are fewer false positives;

l  Suitable for large-scale protein interaction research;

l  It can reflect complex protein correlations. In addition to identifying direct binding proteins, indirect binding proteins can also be detected, and even small molecules other than proteins can be captured.

l  There are various types of TAP tags, which can be selected and designed according to research needs. The technology is widely applicable and has strong practicability;

Limitations of TAP technology

l  The TAP tag itself may have an impact on the structure of the protein;

l  The TAP tag may affect the expression level of the protein (it is helpful to solve this problem by changing the position of the tag);

l  Due to the different affinities between the components, the loosely bound components in the complex may be lost during the purification and rinsing process;

TAP technology development

 

With the development of TAP technology, more and more tags are available for different tandem combinations. Common TAP tags include FLAG tag, two IgG binding domains of Staphylococcus aureus protein A (ProtA), Strep tag, His tag, and Calmodulin Protein binding peptide (calmodulin2binding peptide, CBP) and chitin2binding domain (CBD) etc. When selecting tandem tags, consideration should be given to factors such as purification recovery rate, purity, impact on the structure and biology of the fusion protein, and cost.

There is a restriction site between the amphiphilic purification tag, and tobacco etch virus restriction point (TEV) is the most common site, because TEV protease is highly specific, basically does not contain other cellular protein recognition sites, and the probability of using TEV protease to cut off related proteins is very low.

Like it? Share it!


Ellen Burns

About the Author

Ellen Burns
Joined: November 1st, 2019
Articles Posted: 31

More by this author