Modification, Development, Application and Prospects of Tandem Affinity Purification Method

Abstract

Since the completion of genome sequences of several organisms, attention has been focused on the analysis of the function and functional network of proteins. Most cell type-specific functions and phenotypes are mediated and regulated by the activities of multiprotein complexes as well as other types of protein–protein interactions and posttranslational modifications. Accordingly, the formation and function of macromolecular protein complexes support the whole of cell processes. Consequently, analysis of the variations of protein complex composition in different cell and tissue types is essential to understand the relationship between gene products and cellular functions in diverse physiological contexts (Alberts, 1998; Cusick et al., 2005). With the development of research strategies, many large-scaleprotein-protein interaction studies have been performed in model organisms, especially the budding yeast Saccharomyces cervisiae. Genome-wide yeast two-hybrid screens (Fromont-Racine et al., 1997; Ito et al., 2001; Uetz et al., 2000) and protein chip-based methods (Zhu et al., 2001) allow broader insight into the interaction networks and afford the possibility of highthroughput analysis of function and functional network of proteins. While the former approach provides information relating to interactions between two proteins, typically of binary nature, and has the potential for false-positive and false-negative results, the latter approach is time consuming and labor intensive. These defects may limit their application in large scale protein complex purification. A novel protein complex purification strategy, named tandem affinity purification (TAP) (Puig et al., 2001; Rigaut et al., 1999), in cooperation 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.