2Markey Cancer Center and Department of Molecular and Biomedical Pharmacology, University of Kentucky, Lexington, KY, USA
3Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, USA
Detection of protein-protein interactions in cells is crucial for understanding the biological functions of proteins, including their roles in signal transduction. However, current methods require specific antibodies both for immunoprecipitation and detection, making them expensive and sometimes unreliable. Here we describe protocols for protein-protein interaction assays that use nonimmune IgG-conjugated Sepharose to precipitate the IgG binding domain (ZZ) fused to the bait protein; the interaction partner is fused to Avitag and biotinylated by BirA so that it can be detected by a one-step blot with Dylight 680 streptavidin to detect the Avitag fusion protein. Since this method does not require specific antibodies and is inexpensive, sensitive, and reliable, it should be useful for detecting protein-protein interactions in cells.
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 protein-protein 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 fluorescent 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 protein-protein 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.
Materials and methods Reagents
Chinese hamster ovary (CHO)–K1 cells were from ATCC (Manassas, VA, USA). DMEM/F-12, FBS, G418, Lipofectamine, and Plus reagents were from Invitrogen(Carlsbad, CA , USA). Dylight 680(DL680)–conjugated streptavidin was from Rockland (Gilbertsville, PA, USA).Biotin and CNBr-activated Sepharose 4B were from Sigma-Aldrich (St. Louis, MO,USA). pET21a-BirA was from Addgene(Boston, MA, USA; deposited by Alice Ting's lab at the Massachusetts Institute of Technology, Cambridge, MA, USA).pEGFP-Git1 and pEGFP-PIPKIγ were provided by Mark Ginsberg (University of California at San Diego, San Diego, CA,USA). pHM6-Tal1–433 was described previously (18). ImmunoPure Immobilized Protein A Plus and DL680 NHS ester were from Pierce (Rockford, IL, USA). Mouse paxillin cDNA was from Open Biosystems (Huntsville, AL, USA).Glutathione Sepharose and pGEX-6X-1vector were from GE Heathcare Biosciences (Piscataway, NJ, USA). Pfu and Quick-Change mutation kit were from Agilent Technologies (Santa Clara, CA, USA). Protease inhibitor cocktail was from Roche Applied Science (Indianapolis, IN, USA). Nonimmune IgG was purified from pre-immune rabbit sera, which was from Genemed Synthesis (San Antonio, TX, USA), and conjugated to CNBr-activated Sepharose 4B according to the manufacturer's protocol.