Identification of dynamin as an interactor of rice GIGANTEA by tandem affinity purification (TAP)

GIGANTEA (GI), CONSTANS (CO) and FLOWERING LOCUS T (FT) regulatephotoperiodic flowering in Arabidopsis. In rice, OsGI, Hd1 andHd3a were identified as orthologs of GI, CO and FT, respectively,and are also important regulators of flowering. Although GIhas roles in both flowering and the circadian clock, our understandingof its biochemical functions is still limited. In this study,we purified novel OsGI-interacting proteins by using the tandemaffinity purification (TAP) method. The TAP method has beenused effectively in a number of model species to isolate proteinsthat interact with proteins of interest. However, in plants,the TAP method has been used in only a few studies, and no novelproteins have previously been isolated by this method. We generatedtransgenic rice plants and cell cultures expressing a TAP-taggedversion of OsGI. After a two-step purification procedure, theinteracting proteins were analyzed by mass spectrometry. Sevenproteins, including dynamin, were identified as OsGI-interactingproteins. The interaction of OsGI with dynamin was verifiedby co-immunoprecipitation using a myc-tagged version of OsGI.Moreover, an analysis of Arabidopsis dynamin mutants indicatedthat although the flowering times of the mutants were not differentfrom those of wild-type plants, an aerial rosette phenotypewas observed in the mutants. We also found that OsGI is presentin both the nucleus and the cytosol by Western blot analysisand by transient assays. These results indicate that the TAPmethod is effective for the isolation of novel proteins thatinteract with target proteins in plants.     

Boosting tandem affinity purification of plant protein complexes

Protein-interaction mapping based on the tandem affinity purification (TAP) approach has been successfully established for several systems, such as yeast and mammalian cells. However, relatively few protein complex purifications have been reported for plants. Here, we highlight solutions for the pitfalls and propose a major breakthrough in the quest for a better TAP tag in plants.     

The tandem affinity purification technology: an overview

Tandem affinity purification (TAP) is a methodology for the isolation of protein complexes from endogenous sources. It involves incorporation of a dual-affinity tag into the protein of interest and introduction of the construct into desired cell lines or organisms. Using the two affinity handles, the protein complex assembled under physiological conditions, which contains the tagged target protein and its interacting partners, can be isolated by a sequential purification scheme. Compared with single-step purification, TAP greatly reduces non-specific background and isolates protein complexes with higher purity. TAP-based protein retrieval plus mass spectrometry-based analysis has become a standard approach for identification and characterization of multi-protein complexes. The present article gives an overview of the TAP method, with a focus on its key feature—the dual-affinity tag. In addition, the application of this technology in various systems is briefly discussed.     

An efficient tandem affinity purification procedure for interaction proteomics in mammalian cells

Tandem affinity purification (TAP) is a generic two-step affinity purification protocol that enables the isolation of protein complexes under close-to-physiological conditions for subsequent analysis by mass spectrometry. Although TAP was instrumental in elucidating the yeast cellular machinery, in mammalian cells the method suffers from a low overall yield. We designed several dual-affinity tags optimized for use in mammalian cells and compared the efficiency of each tag to the conventional TAP tag. A tag based on protein G and the streptavidin-binding peptide (GS-TAP) resulted in a tenfold increase in protein-complex yield and improved the specificity of the procedure. This allows purification of protein complexes that were hitherto not amenable to TAP and use of less starting material, leading to higher success rates and enabling systematic interaction proteomics projects. Using the well-characterized Ku70-Ku80 protein complex as an example, we identified both core elements as well as new candidate effectors.     

Native purification of protein and RNA-protein complexes using a novel affinity procedure

Genetic studies in invertebrate model organisms such as Drosophila melanogaster have been a fundament of cell and developmental biology for more than one century. It is mainly the lack of an efficient purification strategy which has hampered biochemical and proteomic analyses of gene products. We describe a novel affinity-tag, termed TagIt-epitope specifically designed for affinity-purifications of multiprotein complexes from Drosophila. TagIt-fusion proteins can be efficiently purified using a monoclonal antibody and eluted under native conditions by competition with synthetic peptide encompassing the epitope. We demonstrate that this tag is suitable for the purification of proteinaceous assemblies such as the PRMT5-complex and RNA-protein complexes such as snoRNPs from Drosophila Schneider2 cells. Furthermore, we describe a novel approach by which this tag can be used to affinity-purify RNA-binding proteins from cell extracts. Therefore, the TagIt-technique or modifications thereof will be of great value in analyzing macromolecular complexes in Drosophila and also other invertebrates by biochemical means. In addition, RNA-peptide hybrid molecules may become a novel tool to purify RNA binding proteins.     

A modified mammalian tandem affinity purification procedure to prepare functional polycystin-2 channel

The tandem affinity purification (TAP) procedure was initially developed as a tool for rapid purification of native protein complexes expressed at their natural levels in yeast cells. This purification procedure was also applied to study interactions between soluble proteins in mammalian cells. In order to apply this procedure to mammalian membrane proteins, we created a modified TAP tag expression vector and fused with the PKD2 gene, encoding a membrane cation channel protein, polycystin-2, mutated in 15% of autosomal dominant polycystic kidney disease. We generated epithelial Madin-Darby canine kidney cell line stably expressing TAP-tagged polycystin-2, improved the subsequent steps for membrane protein release and stability, and succeeded in purifying this protein. Using patch clamp electrophysiology, we detected specific polycystin-2 channel activities when the purified protein was reconstituted into a lipid bilayer system. Thus, this modified TAP procedure provides a powerful alternative to functionally characterize membrane proteins, such as ion channels, transporters and receptors, using cell-free system derived from mammalian cells.     

Tandem affinity purification and identification of protein complex components

As with the budding yeast Saccharomyces cerevisiae, the completion of the Schizosaccharomyces pombe genome sequence has opened new opportunities to investigate the functional organization of a eukaryotic cell. These include analysis of gene expression patterns, comprehensive gene knockout and synthetic lethal screens, global protein localization analysis, and direct protein interaction mapping. We describe here the tandem affinity purification or TAP approach combined with DALPC mass spectrometry to identify components of protein complexes as we have applied it to S. pombe. This approach can theoretically be applied to the entire proteome as has been done in S. cerevisiae to gain insight into functional protein assemblies and to elucidate functions of uncharacterized proteins.     

A protein network for phospholipid synthesis uncovered by a variant of the tandem affinity purification method in Escherichia coli

In prokaryotes, acyl carrier protein (ACP) is a cofactor central to a myriad of syntheses, including fatty acid and phospholipid synthesis. To fulfill its function, ACP must therefore interact with a multitude of different enzymes, which includes the thioesterase YbgC. We found a specific interaction between ACP and YbgC whose thioesterase activity has been demonstrated in vitro on acyl-CoA derivatives, but whose physiological function in bacteria remains unknown. Therefore, YbgC could be a thioesterase active on some specific acyl-ACPs. We then assigned a function to the ACP/YbgC pair by employing a proteomic approach derived from tandem affinity purification, the split tag method. This technique allowed us to purify proteins interacting with ACP and YbgC proteins at the same time. Interactions with PlsB, a sn-glycerol-3-phosphate acyltransferase and PssA, a phosphatidylserine synthase, were identified and validated, showing that YbgC is involved in phospholipid metabolism. Furthermore, using an in vivo bacterial two-hybrid interaction analysis, we showed for the first time that enzymes of the phospholipid synthesis pathway form a complex in the inner membrane. Taken together, these results describe an integrated protein network that could be involved in the coordination of phospholipid metabolism.     

Murine C4-binding protein: a rapid purification method by affinity chromatography

A simple, 3-step method was described for purification of murine C4 binding protein (C4-bp), a recently recognized serum protein that functions as one of the regulatory proteins of the complement system. The method consists of 1) affinity chromatography using TNBS-BGG-conjugated Sepharose beads, 2) gel filtration on a Sepharose 6B column, and 3) heparin-Sepharose chromatography. By this method, milligram quantities of C4-bp can be easily purified by more than 500-fold from EDTA-serum of various mouse strains, and the whole purification process can be completed within 1 wk. The overall yield of C4-bp is about 15%. The C4-bp thus prepared is homogeneous as judged by SDS-polyacrylamide gel electrophoresis and immunelectrophoresis. The purified mouse C4-bp showed physicochemical properties very similar to those described for human C4-bp. Like human C4-bp, mouse C4-bp is composed of several apparently identical subunits of the m.w. of 80,000. However unlike the human counterpart, the subunits of mouse C4-bp are not linked by disulfide bonds but are connected by non-covalent forces that can be disrupted by SDS. The purified mouse C4-bp retained binding affinity for C4 and showed unaltered antigenicity. Immunization of rabbits with the purified mouse C4-bp resulted in the production of potent and monospecific antisera.     

A modified tandem affinity purification tag technique for the purification of protein complexes in mammalian cells

The tandem affinity purification (TAP) tag technique has been used with success to identify under nondenaturing conditions protein complexes in yeast. The technique can be used in mammalian cells, but we found that the original technique does not yield enough recovery for the identification of proteins when mammalian cells growing in monolayer have to be used. We present here a modified TAP tag technique that allows sufficient recovery of proteins from mouse fibroblasts growing in monolayer cultures. The recovery allows protein identification by mass spectrometry.     

Deciphering protein complexes and protein interaction networks by tandem affinity purification and mass spectrometry: analytical perspective

We employed a combination of tandem affinity purification and mass spectrometry for deciphering protein complexes and the protein interaction network in budding yeast. 53 genes were epitope-tagged, and their interaction partners were isolated by two-step immunoaffinity chromatography from whole cell lysates. 38 baits pulled down a total of 220 interaction partners, which are members of 19 functionally distinct protein complexes. We identified four proteins shared between complexes of different functionality thus charting segments of a protein interaction network. Concordance with the results of genome-wide two-hybrid screening was poor (14% of identified interactors overlapped) suggesting that the two approaches may provide complementary views on physical interactions within the proteome.     

Control of an affinity purification procedure using a thermal biosensor

Lactate dehydrogenase (LDH) was recovered from a solution by affinity binding to an N(6)-(6-aminohexyl)-AMP-Sepharose gel. An enzyme thermistor unit was employed to continously measure the activity of the unbound LDH. The enzyme activity signal from the enzyme thermistor was used in a PID controller to regulate the addition of AMP-Sepharose gel to the LDH solution. In another type of experiment, a desktop computer was utilized to control the addition of the adsorbent. Both systems worked satisfactorily, and enabled a rapid and accurate assessment of correct addition of adsorbent.     

New partners of acyl carrier protein detected in Escherichia coli by tandem affinity purification

We report the first use of tandem affinity purification (TAP) in a prokaryote to purify native protein complexes, and demonstrate its reliability and power. We purified the acyl carrier protein (ACP) of Escherichia coli, a protein involved in a myriad of metabolic pathways. Besides the identification of several known partners of ACP, we rediscovered ACP/MukB and ACP/IscS interactions already detected but previously disregarded as due to contamination. Here, we demonstrate the specificity of these interactions and characterize them. This suggests that ACP is involved in additional previously unsuspected pathways. Furthermore, this study shows how the TAP method can be simply used in prokaryotes such as E. coli to identify new partners in protein-protein interactions under physiological conditions and thereby uncover novel protein functions.     

Identification and isolation of Dictyostelium microtubule-associated protein interactors by tandem affinity purification

Tandem affinity purification (TAP) is a method originally established in yeast to isolate highly purified protein complexes in a very gentle and efficient way. In this work, we have modified TAP for Dictyostelium applications and have proved it as a useful method to specifically isolate and identify microtubule-associated protein (MAP) complexes. MAPs are known to interact with other proteins to fulfill their complex functions in balancing the dynamic instability of microtubules as well as anchoring microtubules at the cell cortex, controlling mitosis at the centrosome and guiding transport along them. DdEB1 and the Dictyostelium member of the XMAP215 protein family, DdCP224, are known to be part of complexes at the microtubule tips as well as at the centrosome. Employing TAP and mass spectrometry we were able to prove an interaction between EB1 and the DdCP224. Additionally, among other interactions that remain to be confirmed by other methods, an interaction between DdCP224 and a TACC-family protein could be shown for the first time in Dictyostelium and was confirmed by colocalization and co-immunoprecipitation analyses.     

Purification of components of the translation elongation factor complex of Plasmodium falciparum by tandem affinity purification

Plasmodium falciparum is the causative agent of severe human malaria, responsible for over 2 million deaths annually. Of the 5,300 polypeptides predicted to control the parasite life cycle in mosquitoes and humans, 60% are of unknown function. A major challenge of malaria postgenomic biology is to understand how the 5,300 predicted proteins coexist and interact to perform the essential tasks that define the complex life cycle of the parasite. One approach to assign function to these proteins is by identifying their physiological partners. Here we describe the use of tandem affinity purification (TAP) and mass spectrometry for identification of native protein interactions and purification of protein complexes in P. falciparum. Transgenic parasites were generated which express the translation elongation factor PfEF-1β harboring a C-terminal PTP tag which consists of the protein C epitope, a tobacco etch virus protease cleavage site, and two protein A domains. Purification of PfEF-1β-PTP from crude extracts followed by mass spectrometric analysis revealed, in addition to the tagged protein itself, the presence of the native PfEF-1β, the G-protein PfEF-1α, and two new proteins that we named PfEF-1γ and PfEF-1δ based on their homology to other eukaryotic γ and δ translation elongation factor subunits. These data, which constitute the first application of TAP for purification of a protein complex under native conditions in P. falciparum, revealed that the translation elongation complex in this organism contains at least two subunits of PfEF-1β. The success of this approach will set the stage for a systematic analysis of protein interactions in this important human pathogen.     

Coupling tandem affinity purification and quantitative tyrosine iodination to determine subunit stoichiometry of protein complexes

Rapid protein purification methodologies, such as strategies involving the tandem affinity purification module, have resulted in the identification of a tremendous number of multisubunit protein complexes. Furthermore, in this modern genomic age, mass spectrometry methods are often coupled with affinity purification to identify the genes that encode each protein subunit. However, simple methodologies to determine the stoichiometry of individual subunits within a multisubunit complex have not received much attention. In this article we describe a procedure to rapidly and efficiently determine the stoichiometry of subunits within multisubunit complexes using a combination of tandem affinity purification and quantitative 125I labeling of subunit tyrosines.     

Interaction between heparin and human pregnancy-associated plasma protein A (PAPP-A): a simple purification procedure

Human pregnancy-associated plasma protein A (PAPP-A) binds to heparin-Sepharose. This affinity chromatography preceded by molecular sieve chromatography provides a simple two-step purification procedure of PAPP-A from late pregnancy plasma. One hundred percent of the applied PAPP-A was recovered, with more than 40% being electrophoretically homogeneous after the two procedures. The remaining PAPP-A could be purified by negative affinity chromatography on anti-total human serum immobilized on agarose.     

Metal chelate affinity precipitation: a new approach to protein purification

Metal chelate affinity precipitation of proteins, a method combining metal–protein interaction and affinity precipitation is being discussed as a selective separation process for proteins. The technique utilizes a flexible soluble–insoluble thermo-responsive polymer with a covalently linked ligand loaded with metal ions. The affinity binding of the target protein varies with different metal ions. Copolymers of N-isopropylacrylamide with 1-vinylimidazole loaded with Cu(II) ions are designed as a potential carriers for affinity purification and proved to be successful for purification of protein inhibitors from a variety of cereals. This revised version was published online in July 2006 with corrections to the Cover Date.