Review
The tandem affinity purification method: An efficient system for protein complex purification and protein interaction identification

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Abstract

Isolation and identification of protein partners in multi-protein complexes are important in gaining further insights into the cellular roles of proteins and determining the possible mechanisms by which proteins have an effect in the molecular environment. The tandem affinity purification (TAP) method was originally developed in yeast for the purification of protein complexes and identification of protein–protein interactions. With modifications to this method and many variations in the original tag made over the past few years, the TAP system could be applied in mammalian, plant, bacteria and other systems for protein complex analysis. In this review, we describe the application of the TAP method in various organisms, the modification in the tag, the disadvantages, the developments and the future prospects of the TAP method.

Introduction

Recently, a large number of studies have focused on proteins because it was realized that the intact genome sequence information was not enough to explain and predict cellular mechanisms. Proteins carry out and regulate the majority of cellular activities and generally interact with neighboring proteins and form multi-protein complexes in a time- and space-dependent manner [1], or in response to intra- and intercellular signals. Within a protein complex, each individual protein has a significant role within the whole function of the complex and this function may rely on association with other proteins. This combination of proteins may provide regulation of protein activity through conformational transformation or post-translational modification. It is increasingly clear that functional research of single proteins in a complex organization may yield a better understanding of their functions [2].

Genome-wide yeast two-hybrid screens [3], [4], [5] and protein chip-based methods [6] allow broader insight into the interaction networks. However, the yeast two-hybrid system only produces binary interactions and has the potential for false–positive and false–negative results. As for protein chips, the task of purifying and spotting proteins is time consuming and labor intensive. These defects may limit their application in large scale protein complex purification.

A generic protein complex purification strategy, named tandem affinity purification (TAP)1 [7], [8], in combination with mass spectrometry allows identification of interaction partners and purification of protein complexes. This strategy was originally developed in yeast and has been tested in many cells and organisms.

Section snippets

Overview of the TAP tag and the TAP method

The TAP method requires fusing a TAP tag to the target protein. The TAP tag is composed of two IgG-binding units of protein A of Staphylococcus aureus (ProtA) and a calmodulin-binding domain (CBP), with a cleavage site for the tobacco etch virus (TEV) protease inserted between them [7]. In addition to the C-terminal TAP tag, an N-terminal TAP tag [8], which is a reverse orientation of the C-terminal TAP tag, was also generated (Fig. 1A).

The TAP method involves the fusion of the TAP tag to

Application of the TAP method

With the development of the TAP approach over the past decade, this method has been employed in the analysis of protein–protein interactions and protein complexes in many different organisms, including yeast, mammals, plants, Drosophila and bacteria (Table 1) [9].

Diversity of TAP tags

Although the TAP system was originally developed in yeast, it has been proven to successfully work in a broad range of organisms. The classic ProtA-TEV-CBP tag is unable to provide high efficiency for all given protein complexes. Thus several variations of the TAP tag based on other affinity tags have been developed that offer advantages in specific cases (Fig. 1B). The properties of these basic affinity tags [59], [60], [61], [62] are summarized in Table 2 to highlight the advantages and

Problems and future prospects

The TAP method has been successfully used for purification and identification of protein complexes and complex components both in prokaryotic and eukaryotic organisms. However, in a practical situation, some inherent shortcomings of the method have been uncovered. In a systematic analysis of the yeast proteome, Gavin et al. [10] found that they were unable to isolate and identify interacting proteins in 22% of purified tagged proteins and were unable to purify all of the tagged proteins. They

Acknowledgments

This work has been supported by the National Outstanding Youth Foundation of China (30625008); the major project of cultivating new varieties of Transgenic organisms (2009ZX08009-029B); the National High Technology Research and Development Program (863 Program) (2007AA021401); the National Basic Research Program of China (973 Program) (2007CB108902).

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