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An efficient protein complexpurification method forfunctional proteomics inhigher eukaryotesDaniel Forler*, Thomas Köcher*, Michaela Rode*, Mark Gentzel, Elisa Izaurralde, and Matthias Wilm†

Published online 16 December 2002; doi:10.1038/nbt773

The ensemble of expressed proteins in a given cell is organized inmultiprotein complexes1,2. The identification of the individual com-ponents of these complexes is essential for their functional char-acterization. The introduction of the ‘tandem affinity purification’(TAP) methodology substantially improved the purification andsystematic genome-wide characterization of protein complexes inyeast1,3,4. The use of this approach in higher eukaryotic cells haslagged behind its use in yeast because the tagged proteins arenormally expressed in the presence of the untagged endogenousversion, which may compete for incorporation into multiproteincomplexes. Here we describe a strategy in which the TAPapproach is combined with double-stranded RNA interference(RNAi)5,6 to avoid competition from corresponding endogenousproteins while isolating and characterizing protein complexesfrom higher eukaryotic cells. This strategy allows the determina-tion of the functionality of the tagged protein and increases thespecificity and the efficiency of the purification.

The TAP method is a protein tag–based affinity purification tech-nique originally developed in yeast3,4. The application of thismethodology on a genome-wide scale has led to the systematicstudy of the functional organization of the yeast proteome with thepurification of >589 multiprotein assemblies1. Although much ofthe information obtained from yeast can be extrapolated to highereukaryotes, the greater complexity of higher eukaryotic genomes,together with differences in post-translational modificationsbetween yeast and metazoa, necessitates the use of homologoussystems for the functional characterization of higher eukaryoticproteomes. In yeast, the endogenous protein is replaced by thetagged version by means of homologous recombination in haploidcells. To reproduce this feature in higher eukaryotic cells, we havedeveloped the so-called iTAP strategy, in which the TAP approach3,4

is combined with suppression of the corresponding endogenousprotein by RNAi5,6.

The TAP tag consists of two immunoglobulin-binding domainsof protein A from Staphylococcus aureus (zz-tag), a cleavage site forthe tobacco etch virus (TEV) protease, and the calmodulin-bindingpeptide (CBP)3,4. Plasmids expressing TAP-tagged proteins are sta-bly transfected into Drosophila melanogaster (Dm) Schneider cells(S2 cells). We chose these cells because of the ease with which poly-clonal lines expressing the tagged proteins can be established aswell as the efficiency with which endogenous genes can be silencedby RNAi5,6.

European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, D-69012Heidelberg, Germany. *These three authors contributed equally to this work.†Corresponding author (wilm@embl-heidelberg.de).

microtiter wells and detected by using an anti-HA antibody (Babco,Richmond, CA) as a primary and an anti-mouse-HRP conjugate (Sigma, St.Louis, MO) as a secondary antibody. The ELISA was developed using the per-oxidase substrate 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)diammonium salt (ABTS), and the signal (absorbance) was measured at 405 nm(OD405). The background signal was defined as the OD405 of cell lysatesobtained from cells treated exactly as just described but omitting theBGBT. To determine the efficiency of the labeling with BGBT, lysates ofcells were either directly analyzed by ELISA as just described or incubatedwith an oligonucleotide containing 0.04 µM BGBT and then analyzed byELISA as described.

Covalent labeling of hAGT in CHO cells. CHO cells deficient in AGT weretransfected with a vector encoding either W160hAGT-NLS3 or W160hAGT-ECFP-NLS3. After 24 h of transient expression, cells grown on 0.18-mm-thick glassslides were transferred to a perfusion chamber and incubated with BGFL(5 µM) for 5 min. Cells were washed three times with PBS buffer to removeexcess substrate. A Zeiss LSM510 laser scanning confocal microscope wasused for fluorescence measurements. Detection of fluorescein or ECFP sig-nals was achieved by appropriate filters. Scanning speed and laser intensitywere adjusted to avoid photobleaching of the fluorescent probes and damageor morphological changes to the cells.

Note: Supplementary information is available on the Nature Biotechnologywebsite.

AcknowledgmentsFunding of this work was provided by the Swiss Science Foundation, theEuropean Community, and the EPFL. S.G. was supported by a fellowship fromthe Boehringer Ingelheim Foundation. Nils Johnsson is acknowledged for help-ful discussions and advice, Maik Kindermann for help with the synthesis ofBGBT, Stefan Pitsch for the gift of 6-chloroguanine, Robert Damoiseaux for thesynthesis of oligonucleotide containing BGBT, and Bernd Kaina for the CHOcell line.

Conflicting interests statementThe authors declare that they have no competing financial interests.

Received 3 June 2002; accepted 2 October 2002

1. Tsien, R.Y. The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544(1998).

2. Fields, S. & Song, O. A novel genetic system to detect protein–protein interactions.Nature 340, 245–246 (1989).

3. Johnsson, N. & Varshavsky, A. Split ubiquitin as a sensor of protein interactions invivo. Proc. Natl. Acad. Sci. USA 91, 10340–10344 (1994).

4. Farinas, J. & Verkman, A.S. Receptor-mediated targeting of fluorescent probes inliving cells. J. Biol. Chem. 274, 7603–7606 (1999).

5. Wu, M.M. et al. Organelle pH studies using targeted avidin and fluorescein–biotin.Chem. Biol. 7, 197–209 (2000).

6. Griffin, B.A., Adams, S.R. & Tsien, R.Y. Specific covalent labeling of recombinantprotein molecules inside live cells. Science 281, 269–272 (1998).

7. Adams, S.R. et al. New biarsenical ligands and tetracysteine motifs for proteinlabeling in vitro and in vivo: synthesis and biological applications. J. Am. Chem.Soc. 124, 6063–6076 (2002).

8. Gaietta, G. et al. Multicolor and electron microscopic imaging of connexin traffick-ing. Science 296, 503–507 (2002).

9. Cronan, J.E., Jr. Biotination of proteins in vivo. A post-translational modification tolabel, purify and study proteins. J. Biol. Chem. 265, 10327–10333 (1990).

10. Smith, P.A. et al. A plasmid expression system for quantitative in vivo biotinylationof thioredoxin fusion proteins in Escherichia coli. Nucleic Acids Res. 26,1414–1420 (1998).

11. Pegg, A.E. Repair of O6-alkylguanine by alkyltransferases. Mutat. Res. 462,83–100 (2000).

12. Damoiseaux, R., Keppler, A. & Johnsson, K. Synthesis and applications of chemi-cal probes for human O6-alkylguanine-DNA alkyltransferase. ChemBioChem 2,285–287 (2001).

13. Xu-Welliver, M., Leitao, J., Kanugula, S., Meehan, W.J. & Pegg, A.E. Role of codon160 in the sensitivity of human O6-alkylguanine-DNA alkyltransferase to O6-ben-zylguanine. Biochemical Pharmacol. 58, 1279–1285 (1999).

14. Kaina, B., Fritz, G., Mitra, S. & Coquerelle, T. Transfection and expression ofhuman O6-methylguanine-DNA methyltransferase (MGMT) cDNA in Chinesehamster cells: the role of MGMT in protection against the genotoxic effects of alky-lating agents. Carcinogenesis 12, 1857–1867 (1991).

15. Kalderon, D., Roberts, B.L., Richardson, W.D. & Smith, A.E. A short amino acidsequence able to specify nuclear location. Cell 39, 499–509 (1984).

16. Belanich, M. et al. Intracellular localization and intercellular heterogeneity of thehuman DNA repair protein O6-methylguanine-DNA methyltransferase. CancerChemother. Pharmacol. 37, 547–555 (1996).

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Protein complexes containing the tagged protein are purifiedessentially as described before3,4. Briefly, lysates of cells expressingthe TAP-tagged protein are first passed over an IgG-Sepharose col-umn to which the zz-tag binds with high affinity. The retained pro-tein complexes are cleaved from the column by digestion with theTEV protease and then bound to calmodulin-coated beads for fur-ther purification. Several days before the purification, the expres-sion of the corresponding untagged endogenous protein is sup-pressed by RNAi6. The TAP-tagged protein is expressed from a het-erologous cDNA to prevent its simultaneous depletion. We havedemonstrated the feasibility of this approach with the purificationof protein complexes consisting of a TAP-tagged human (Hs) pro-tein bound to its Dm partners. In all cases, the similarity betweenthe human and Dm proteins was >50%. Furthermore, becausebinding partners of the tagged proteins are reported in the litera-ture, their detection in the purified complexes was taken as an indi-cation of the specificity of the selection.

Figure 1. Selection of the Dm exosome. (A) S2 cells were stablytransfected with plasmids expressing the TAP tag alone or N-terminallyfused to HsRrp4. Endogenous DmRrp4 was depleted by RNAi asindicated. DmRrp4 dsRNA corresponds to a cDNA fragment comprisingresidues 1–700. Cells were lysed 5 d after depletion, and proteins boundto TAP-HsRrp4 were purified. Eluates from calmodulin beads wereseparated by 12% SDS-PAGE and silver stained. Lane 1, proteins purifiedfrom lysates of cells expressing the TAP tag alone. Lane 2, proteinspurified from S2 cells expressing TAP-HsRrp4. Lane 3, proteins purifiedfrom S2 cells expressing TAP-HsRrp4 but depleted of endogenousDmRrp4. Numbers at left are molecular masses (kDa).(B) Cells described in (A) were treated again with DmRrp4 dsRNA on day7. Proteins bound to TAP-HsRrp4 were purified on day 18 and analyzedas described in (A). Lane 1, proteins purified from lysates of cellsexpressing the TAP tag alone. Lane 2, proteins purified from S2 cellsexpressing TAP-HsRrp4 but depleted of endogenous DmRrp4. Allproteins were identified by mass spectrometry; the corresponding genenames in FlyBase (http://flybase.bio.indiana.edu) are indicated. Asterisksindicate the position of contaminant proteins. Black squares indicateadditional proteins detected on day 18 but not day 5. DmRrp45 is partiallydegraded on the preparation on day 5. (C) Western blot analysis ofsamples collected 6, 12, and 18 d after the first addition of DmRrp4dsRNA. In lanes 1–3, dilutions of the sample isolated on day 0 wereloaded. DmY14 served as loading control.

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To validate our approach directly, we purified the Dm exosomefrom S2 cells expressing human TAP-tagged Rrp4 (HsRrp4 is 56%identical to DmRrp4). The exosome is a multienzyme complex of3′- to 5′-exoribonucleases7,8. The stable core of the yeast exosomeconsists of ten highly conserved proteins, namely Rrp4, Csl4, Mtr3,and Rrp40–Rrp46 (refs. 7, 8). Homologues of these proteins are pre-sent in higher eukaryotes and are thought to be organized in a com-parable complex7,8.

Cells expressing TAP-HsRrp4 were treated with DmRrp4dsRNA on day 0 and day 7, and protein complexes were purifiedon day 5 and day 18. In parallel, cells expressing either the TAP tag

Figure 2. Selection of a tetrameric complex involved in mRNA nuclearexport. (A) S2 cells expressing TAP-Hsp15 were treated with a dsRNAcorresponding to full-length Dmp15 cDNA. Cells were lysed 5 d aftertransfection, and proteins bound to TAP-Hsp15 were purified andanalyzed as described in Figure 1A. Lane 1, proteins purified fromlysates of cells expressing the TAP tag alone. Lane 2, proteins purifiedfrom S2 cells expressing TAP-Hsp15. Lane 3, proteins purified from S2cells expressing TAP-Hsp15 but depleted of endogenous p15. Theidentity of the selected proteins is indicated on the right. Asterisksindicate the position of proteins whose binding is nonspecific. Numbersat left are molecular masses (kDa). (B) Cells from the same experimentas in (A) were analyzed by western blotting with antibodies raised torecombinant Dmp15 (ref. 11). Dmp15 was efficiently depleted (lane 6),whereas the expression levels of TAP-Hsp15 increased. In lanes 1–4,dilutions of the sample isolated on day 0 were loaded to assess theefficiency of the depletion. Tubulin served as a loading control.

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alone or TAP-HsRrp4 (but in which the endogenous protein wasnot depleted) were subjected to the same purification procedure.Analysis of samples eluted from the calmodulin beads revealedthat 5 days after the first addition of dsRNA, TAP-HsRrp4 co-puri-fies with seven proteins (Fig. 1A, lane 3). These were identified asDmRrp40–42, DmRrp44–46, and a putative homologue of Mtr3(Fig. 1A, lane 3). In contrast, in the presence of the endogenousprotein only three partners were selected (Fig. 1A, lane 2).Notably, when the purification was carried out on day 18, theyields were substantially increased and three additional proteinswere detected (Fig. 1B, lane 2), including DmCsl4. Western blotanalysis revealed that the expression of TAP-HsRrp4 increasedsubstantially when cells were transfected twice with DmRrp4dsRNA (Fig. 1C, lane 6 versus lane 3), suggesting that consecutiveknockdowns allow for the enrichment of cells expressing thetagged protein. In summary, the iTAP strategy increased both theyields and the specificity of the purification, leading to the identi-fication of the core components of the Dm exosome.

An example of a large complex is the heterodimeric nuclearexport receptor NXF1-p15. This receptor mediates the export of

mRNA to the cytoplasm and consists of a largesubunit, NXF1 (∼ 70 kDa), and a small subunit,p15 (∼ 15 kDa)9. p15 is conserved in metazoabut not in Saccharomyces cerevisiae9,10, so itsbinding partners cannot be identified in yeast.Human p15 has 61% similarity (38% identity)to Dmp15 and has been shown to interact withDmNXF1 in vitro11. Proteins bound to p15 were purified from a cell population express-ing TAP-Hsp15, in which the expression ofendogenous Dmp15 was suppressed by RNAi.The efficiency of the depletion was analyzed bywestern blotting (Fig. 2B).

In the absence of endogenous p15, TAP-tagged p15 co-purifies with three proteins,which were identified as DmRanBP2 (the largestnucleoporin in higher eukaryotes), DmNXF1,and DmRanGAP1 (Fig. 2A, lane 3). These pro-teins were selected only when the expression ofthe untagged p15 was suppressed (Fig. 2A, lane 3versus lane 2). Moreover, the expression of thetagged p15 increased after depletion of theendogenous protein (Fig. 2A, lane 3 versus lane2 and Fig. 2B, lane 6 versus lane 5). These obser-vations indicate that TAP-Hsp15 is efficientlyincorporated into endogenous complexes, andthereby stabilized, only when expression ofDmp15 is silenced. The selection of Dm/NXF1,Dm/RanGAP1, and Dm/RanBP2 validates thepurification procedure, because these interac-tions have been reported before12,13.

Two additional bands of ∼ 53–54 kDa wereselected with TAP-Hsp15 independently ofwhether or not endogenous p15 was depleted(Fig. 2A, lanes 2 and 3, asterisks). The yields ofthese proteins did not increase with increasinglevels of TAP-Hsp15, suggesting that their bind-ing is nonspecific. Indeed, these proteins werenot consistently detected in independent purifi-cations, indicating that the iTAP strategy alsopermits discrimination between specific andnonspecific binding.

The implementation of the iTAP techniquealso allows us to control whether the tagged pro-

teins are functional, as illustrated with mago nashi (MGN), a nuclearprotein implicated in multiple post-transcriptional steps of geneexpression14. MGN dimerizes with Y14, and both are highly con-served in metazoa14,15. However, no obvious MGN or Y14 homo-logues are encoded by the S. cerevisiae genome.

DmMGN-Y14 dimers are essential for growth, and their deple-tion inhibits cell proliferation15. We expected that if TAP-HsMGNwere functional, it should rescue growth of cells depleted ofDmMGN (HsMGN is 89% identical to DmMGN15). EndogenousMGN was depleted by RNAi from wild-type cells or from cellsexpressing TAP-HsMGN. No surviving wild-type cells were detect-ed 19 days after transfecting MGN dsRNA (Fig. 3A). In contrast,cells expressing TAP-HsMGN survived and started to grow expo-nentially ∼ 15 days after transfection (Fig. 3A, in red). Western blotanalyses indicated that DmMGN was efficiently depleted (Fig. 3B,lanes 3–6 and 9–11). Thus, TAP-HsMGN fully supports cell prolif-eration in the absence of endogenous MGN.

The reduced growth rate of cells expressing TAP-MGN withinthe first 15 days after transfection with MGN dsRNA reflects thepolyclonal nature of this cell line, and suggests that only cells

Figure 3. Selection of proteins associating with MGN-Y14 dimers. (A) Wild-type (wt) cells, or cellsexpressing TAP-HsMGN were transfected with a dsRNA corresponding to the full-length DmMGNcDNA. Cell numbers were determined every 3 d up to 21 d after transfection. Data are given as theincrease in the population relative to that used for transfection on day 0. Wild-type cells did notsurvive MGN depletion (in black), whereas cells expressing TAP-HsMGN survived and started togrow exponentially 15 d after transfection (in red). (B) Cells from the same experiment as in (A) were analyzed by western blotting with antibodies raised against recombinant DmMGN andY14 (ref. 15). In wild-type cells the expression levels of both DmMGN and Y14 were stronglyreduced (lanes 9–11). In cells expressing TAP-HsMGN, DmMGN was efficiently depleted whereasY14 expression was rescued (lanes 3–6). In lanes 1–3 and 7–9, dilutions of the sample isolated onday 0 were loaded to assess the efficiency of the depletion. DmNXF1 served as loading control.(C) Proteins purified from lysates of cells expressing either the TAP tag alone (lane 1) or TAP-HsMGN (lanes 2, 3) were analyzed by 14% SDS-PAGE followed by silver staining. In lane 3,expression of endogenous MGN was silenced. The identity of the selected proteins is indicated.The DmPYM cDNA sequence has been submitted to the EMBO database under accession no.AJ459405. The asterisk indicates the position of actin, which is a major contaminant protein.Numbers at left are molecular masses (kDa). (D) Proteins purified from lysates of cells expressingeither the TAP tag alone (lane 1) or TAP-DmMGN (lane 2).

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expressing the tagged protein at levels that can sustain growth sur-vive and proliferate. Thus, for essential proteins, the iTAP strategypermits the selection of cells expressing the tagged version at levelsthat restore viability. Consistently, western blot analysis of samplescollected on day 21 confirmed that the cell population is enrichedin cells expressing TAP-HsMGN (Fig. 3B, lane 6 versus lane 3). Anadditional indication that HsMGN is functional is the observationthat, whereas in wild-type cells depletion of DmMGN leads to asubstantial decrease of the endogenous levels of Y14 (Fig. 3B, lanes9–11), in cells expressing HsMGN, Y14 expression levels were res-cued (Fig. 3B, lanes 3–6). This indicates that the expression of thefunctional tagged protein protects other components of the com-plex from degradation, thus reflecting the true in vivo assembly ofthe complex.

Proteins associated with TAP-HsMGN were purified from thedepleted cell population on day 21. In the absence of the endoge-nous form, TAP-HsMGN stoichiometrically co-purifies with Y14.An additional protein with an apparent molecular weight of 29 kDawas specifically selected. This protein was named PYM (partner ofY14 and mago) and corresponds to the uncharacterized product ofthe wibg gene. In contrast to the complexes described above, thetrimeric MGN-Y14-PYM complex could be purified despite thepresence of endogenous MGN, but its depletion increases the yieldsof the purification (Fig. 3C, lane 2 versus lane 3). Similarly, Y14 andPYM are selected with TAP-DmMGN in the presence of theuntagged form, but in this case a five- to sixfold larger number ofcells were required to isolate equivalent amounts of complex (Fig. 3D). The elucidation of the biological significance of PYMinteraction with the MGN-Y14 dimers requires further studies.

Taken together, these examples show that the iTAP strategy(that is, the combination of TAP-tagged protein expression withRNAi) introduces into higher eukaryotic cells two of the threecritical features of the TAP method in yeast: (i) the enzymaticallycleavable tag-based affinity purification and (ii) the replacementof the original protein by a tagged version in the cell proteome.The third feature, the use of the native promoter to drive theexpression of the tagged protein, is still absent. However, establish-ing stably transfected cell lines allows the selection of only thosecells in which the expression levels of the tagged protein have no deleterious effects. The strategy presented here isof broad interest, because any tag-based affinity purification sys-tem in higher eukaryotes should benefit from the simultaneoussuppression of the endogenous protein by RNAi in a similar man-ner. The recent development of vectors for expression of shortinterfering RNA duplexes (siRNAs) within cells to silence theexpression of endogenous proteins will certainly extend the reachof the approach described here to mammalian tissue culture cells(reviewed in ref. 16). We anticipate that the TAP methodology,combined with simultaneous knockdowns, will be of exceptionalvalue in elucidating the functional network of protein–proteininteractions in cellular processes.

Experimental protocolPlasmids. A pBS-derived vector (pBSactTAP) allowing the expression of TAP-tagged proteins in S2 cells was generated by inserting the TAP-tag cDNA down-stream of the Dm actin promoter. Plasmids encoding TAP-tagged fusions ofhuman HsRrp4, p15, and MGN were generated by in-frame insertion of the cor-responding cDNAs downstream of the TAP-tag cassette.Additional informationis available at http://www.narrador.embl-heidelberg.de/GroupPages/PageLink/activities/iTAP.html.

D. melanogaster cell culture and RNA interference. S2 cells (AmericanType Culture Collection, Manassas, VA) were propagated and treated withdsRNAs as described before6,11. Stable cell lines expressing TAP-tagged pro-teins were established by co-transfecting the corresponding pBSactTAP

constructs together with a plasmid encoding puromycin acetyltransferaseat a 5:1 ratio (pBS-PURO17). Cells were transfected with Lipofectin(Invitrogen, Carlsbad, CA) and selected in medium containing 10 µg/mlpuromycin. Pools of cells expressing the TAP-tag fusions were tested bywestern blotting. Protein lysates for western blot analysis were prepared asdescribed previously11.

TAP-tag selections. Cytoplasmic extracts from S2 cells expressing TAP-taggedproteins were prepared according to Dignam et al. (1983)18. Nuclei were lysedin buffer B (20 mM HEPES, pH 7.9, 20% glycerol, 200 mM KCl, 0.5 mMEDTA, 0.5% Nonidet P-40, 0.5 mM dithiothreitol, and protease inhibitors).Starting from cytoplasmic or nuclear extracts or a mixture of both, depend-ing on the fractionation of the tagged protein, protein complexes were purified essentially as described before3,4. About 3–10 × 108 cells were usedper selection.

Mass spectrometry. Silver-stained protein bands were digested in-gel withtrypsin as previously described19. Tryptic peptides were sequenced by nano-electrospray tandem mass spectrometry on an API III triple-quadrupolemass spectrometer (PE Sciex, Toronto, Ontario, Canada)20. Proteins wereidentified by searching peptide sequence tags against a comprehensive nonre-dundant protein database containing 840,000 proteins using PeptideSearchversion 3.0 (ref. 21).

AcknowledgmentsThis study was supported by the European Molecular Biology Organization(EMBO) and by the Bundesministerium für Bildung und Forschung (BMBF),BioFuture grant no 0311862.

Competing interests statementThe authors declare that they have no competing financial interests.

Received 3 September 2002; accepted 12 November 2002

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