Generic protocols for in vitro GST pulldown assays and protein-protein interaction assays

Protein-protein interactions contribute to a variety of biological processes, including signal transduction, tissue integrity, and force generation (1,2). An inexpensive and reliable method to measure protein-protein interactions would be of considerable utility. Currently, co-immunoprecipitations (Co-IP) are routinely used to detect protein-protein interactions in cells, where one antibody is used to isolate the bait proteins and another antibody is used to detect the interaction. Since antibodies are expensive reagents, qualities of antibodies change from lot to lot, and manufacturer claims may not be true (3), this method is neither economical nor reliable. Thus, a technique that does not depend on specific antibodies would be useful for proteinprotein interactions assays. The biotin/avidin (or streptavidin) system has numerous applications in modern biological studies because the interaction of biotin with avidin is one of the highest affinity interactions known in nature. Recently, the applications of this system have been greatly promoted by the discovery of BirA, an Escherichia coli biotin ligase that specifically conjugates biotin to a 15-aminoacid Avitag (GLNDIFEAQKIEWHE) (4,5). BirA-mediated biotinylation of Avitag fusion proteins has been used in protein purification (6), detection (7,8), and fluorescence imaging (9–12). Thus, BirA-mediated biotinylation, which can be easily detected by f luorescent streptavidin blot (designated as the Avitag-BirA system), has great potential application in detecting interacting Avitag fusion proteins in protein-protein interaction assays without using specific antibodies. For protein-protein interaction assays in cells, a method is needed to isolate bait proteins. The ZZ domain, a synthetic IgG binding protein derived from tandem repeats of the B domain of protein A, was successfully used to replace protein A in antibody purification (13,14). It was also engineered to fuse with many different proteins and expressed as ZZ-tagged fusion proteins in diversified cell types, ranging from bacterium to mammalian cells (15–17). To date, no reports have suggested that the ZZ domain impairs the function of proteins fused to it, and ZZ fusion proteins can be easily purified by using IgG-Sepharose. Therefore, we proposed a novel method for proteinprotein interaction assays in cells, in which inexpensive, nonimmune rabbit IgG-conjugated Sepharose beads can be used to precipitate the ZZ domain fusion protein (as bait); subsequently, fluorescent streptavidin can be used to detect the interacting Avitag protein that was biotinylated by BirA. In this study, we have examined whether the Avitag-BirA system is useful for in vitro GST pulldown assays and whether the Avitag-BirA system, in combination with the ZZ domain purification technique (designated as the AviZZ system), can be used for protein-protein interaction assays in cells. Generic protocols for in vitro GST pulldown assays and protein-protein interaction assays in cells are schematically depicted in Figure 1, A and B, respectively.