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Tandem Affinity Purification of Proteins

Arthur Günzl¹, Bernd Schimanski²

¹Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, Connecticut
²Institute of Cell Biology, University of Bern, Bern, Switzerland

Publication Name:

Current Protocols in Protein Science

Unit Number:

Unit 19.19

DOI:

10.1002/0471140864.ps1919s55

Online Posting Date:

February, 2009

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Abstract

Tandem affinity purification (TAP) is a very efficient method to isolate proteins, protein complexes, or ribonucleoprotein particles from crude extracts. The method depends on the expression of one protein component fused N- or C-terminally to a TAP tag in the organism of interest. The TAP tag is a composite tag consisting of two different epitope domains and a protease cleavage site, and it facilitates the purification of the tagged protein in two consecutive, high-affinity chromatography steps. Combined, the two steps are typically so efficient that a protein complex can be purified virtually to homogeneity without the need for protein overexpression. If the tag does not interfere with protein function, TAP is likely to yield an intact protein complex because all steps of the procedure are carried out under nondenaturing conditions. In this unit, a TAP procedure is detailed which employs a novel epitope combination termed PTP. Curr. Protoc. Protein Sci. 55:19.19.1-19.19.16. © 2009 by John Wiley & Sons, Inc.

Keywords: tandem affinity purification; TAP; PTP tag; protein A domain; protein C epitope; tobacco etch virus (TEV) protease; IgG chromatography; anti-protein C immunoaffinity chromatography

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Introduction
Strategic Planning
Basic Protocol 1: Tandem Affinity Purification of PTP-Tagged Proteins from Crude Cell-Free Extract
Basic Protocol 2: Concentrating Tandem Affinity–Purified Proteins and Preparation of an SDS Sample
Alternate Protocol 1: Concentrating the Final TAP Eluate in the Absence of SDS
Basic Protocol 3: Analysis of Tandem Affinity Purification by Immunoblotting
Basic Protocol 4: Analysis of Tandem Affinity Purification by SDS-PAGE
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: Tandem Affinity Purification of PTP-Tagged Proteins from Crude Cell-Free Extract

Materials

IgG Sepharose 6 Fast Flow matrix (GE Healthcare)
PA-150 buffer (see recipe)
Complete Mini, EDTA-free protease inhibitor cocktail tablets (Roche)
Cell extract for analysis (see Strategic Planning; buffer used should resemble PA-150 buffer, see recipe, in pH and salt conditions)
10% (v/v) Tween 20 (Sigma-Aldrich)
Liquid nitrogen
TEV protease buffer (see recipe)
PC-150 buffer (see recipe)
AcTEV protease (Invitrogen)
Anti-Protein C (anti-ProtC) affinity matrix (Roche)
1 M CaCl₂ stock solution
EGTA elution buffer (see recipe)
Protein C peptide (EDQVDPRLIDGK; synthesized in-house)
Peptide elution buffer (see recipe)

0.8 × 4–cm Poly-Prep chromatography columns (Bio-Rad)
Adjustable stand to hold columns
End-over-end rotator
Scalpel

NOTE: Unless otherwise specified, all steps should be carried out at 4°C. All buffers should be cooled on ice prior to use.

Basic Protocol 2: Concentrating Tandem Affinity–Purified Proteins and Preparation of an SDS Sample

Materials

Solution of TAP-purified protein (EGTA eluate; Basic Protocol 1, step 14a)
StrataClean resin (Stratagene)
1× SDS gel loading buffer (see recipe)

Vacuum concentrator (e.g., SpeedVac evaporator)
End-over-end rotator
Minifuge (Harvard Apparatus; or other microcentrifuge)
100°C water bath

Alternate Protocol 1: Concentrating the Final TAP Eluate in the Absence of SDS

Additional Materials (also see Basic Protocol 2)

10% (v/v) Tween 20 (Sigma-Aldrich)
Dialysis buffer (see recipe)

1-liter glass beaker
Vacuum concentrator (e.g., SpeedVac evaporator)
Standard floating device for microcentrifuge tubes
Mini dialysis units (Slide-A-Lyzer, 3500 MWCO, Pierce)

Basic Protocol 3: Analysis of Tandem Affinity Purification by Immunoblotting

Materials

TAP fractions (Basic Protocol 1)
4× SDS gel loading buffer (see recipe)
Monoclonal anti-ProtC antibody HPC4 (Roche)
Anti-mouse IgG antibody, peroxidase conjugated (Vector Laboratories)
BM chemiluminescent blotting substrate (Roche)

Additional reagents and equipment for SDS-PAGE (unit 10.1), immunoblotting (units 10.7), and immunoblot detection (unit 10.10)

Basic Protocol 4: Analysis of Tandem Affinity Purification by SDS-PAGE

Materials

TAP fractions (Basic Protocol 1)
1× SDS gel loading buffer (see recipe)
GelCode blue stain reagent (Pierce)

Additional reagents and equipment for collecting proteins with StrataClean resin beads (Basic Protocol 2, steps 2 to 5) and SDS-PAGE (unit 10.1)

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Figures

Figure 19.19.1

Tandem affinity purification through PTP tagging. Top, the PTP tag drawn to scale. Bottom, outline of the purification procedure. As indicated at the top, black, dark gray, light gray, and open boxes represent the ProtC epitope, the TEV protease cleavage site, the tandem ProtA domains, and spacer regions, respectively. The tagged protein is indicated by a striped rectangle.

View Image
Figure 19.19.2

Immunoblot monitoring of the tandem affinity purification of a PTP-tagged trypanosome transcription factor (TF-PTP). The protein was detected with the monoclonal anti-ProtC antibody in crude extract (Inp), flowthrough of IgG column (FT-IgG), TEV protease eluate (TEV elu), flowthrough of the anti-ProtC column (FT-ProtC), and the final EGTA eluate (EGTA elu). Values of x indicate relative volumes of each fraction analyzed. Marker sizes in kDa are indicated on the left. Note that after TEV protease cleavage, the protein size is reduced by ~15 kDa as indicated on the right (TF-PTP =>TF-P).

View Image
Figure 19.19.3

SDS-PAGE and Coomassie blue staining of TF-PTP purification (modified from Schimanski et al., 2005). Proteins from the final eluate (EGTA elu) of a standard PTP purification (Basic Protocol 1) were collected and released in SDS loading buffer (Basic Protocol 2) and separated on a 12.5% SDS-PAGE gel. For comparison, aliquots of crude extract (Inp) and TEV protease eluate (TEV elu) were co-analyzed. Percent values specify relative volumes of each fraction analyzed. Six major protein bands were detected in the final eluate, all of which were subsequently identified by mass spectrometry as subunits (S1 to S6) of a transcription factor complex. Comparison with the input material and the TEV protease eluate showed that the most abundant proteins in these fractions had a size of ~55 kDa and were later identified as tubulins. Accordingly, tubulins were minor contaminants together with IgG heavy chain from the anti-ProtC column in the 55-kDa range (cont.). T marks the TEV protease (~27 kDa) in the TEV protease eluate.

View Image

Literature Cited

Literature Cited
	Brandenburg, J., Schimanski, B., Nogoceke, E., Nguyen, T.N., Padovan, J.C., Chait, B.T., Cross, G.A.C.M., and Günzl, A. 2007. A novel protein complex is essential for class I transcription from the VSG expression site promoter in Trypanosoma brucei. EMBO J. 26:4856-4866.
	Cox, D.M., Du, M., Guo, X., Siu, K.W., and McDermott, J.C. 2002. Tandem affinity purification of protein complexes from mammalian cells. Biotechniques 33:267-268.
	Dougherty, W.G., Cary, S.M., and Parks, T.D. 1989. Molecular genetic analysis of a plant virus polyprotein cleavage site: A model. Virology 171:356-364.
	Drakas, R., Prisco, M., and Baserga, R. 2005. A modified tandem affinity purification tag technique for the purification of protein complexes in mammalian cells. Proteomics 5:132-137.
	Estevez, A.M., Kempf, T., and Clayton, C. 2001. The exosome of Trypanosoma brucei. EMBO J. 20:3831-3839.
	Forler, D., Kocher, T., Rode, M., Gentzel, M., Izaurralde, E., and Wilm, W. 2003. An efficient protein complex purification method for functional proteomics in higher eukaryotes. Nat. Biotechnol. 21:89-92.
	Gavin, A.C., Aloy, P., Grandi, P., Krause, R., Boesche, M., Marzioch, M., Rau, C., Jensen, L.J., Bastuck, S., Dumpelfeld, B., Edelmann, A., Heurtier, M.A., Hoffman, V., Hoefert, C., Klein, K., Hudak, M., Michon, A.M., Schelder, M., Schirle, M., Remor, M., Rudi, T., Hooper, S., Bauer, A., Bouwmeester, T., Casari, G., Drewes, G., Neubauer, G., Rick, J.M., Kuster, B., Bork, P., Russell, R.B., and Superti-Furga, G. 2006. Proteome survey reveals modularity of the yeast cell machinery. Nature 440:631-636.
	Gloeckner, C.J., Boldt, K., Schumacher, A., Roepman, R., and Ueffing, M. 2007. A novel tandem affinity purification strategy for the efficient isolation and characterisation of native protein complexes. Proteomics 7:4228-4234.
	Kamil, J.P. and Coen, D.M. 2007. Human cytomegalovirus protein kinase UL97 forms a complex with the tegument phosphoprotein pp65. J. Virol. 81:10659-10668.
	Krogan, N.J., Cagney, G., Yu, H., Zhong, G., Guo, X., Ignatchenko, A., Li, J., Pu, S., Datta, N., Tikuisis, A.P., Punna, T., Peregrin-Alvarez, J.M., Shales, M., Zhang, X., Davey, M., Robinson, M.D., Paccanaro, A., Bray, J.E., Sheung, A., Beattie, B., Richards, D.P., Canadien, V., Lalev, A., Mena, F., Wong, P., Starostine, A., Canete, M.M., Vlasblom, J., Wu, S., Orsi, C., Collins, S.R., Chandran, S., Haw, R., Rilstone, J.J., Gandi, K., Thompson, N.J., Musso, G., St. Onge, P., Ghanny, S., Lam, M.H., Butland, G., Altaf-Ul, A.M., Kanaya, S., Shilatifard, A., O'Shea, E., Weissman, J.S., Ingles, C.J., Hughes, T.R., Parkinson, J., Gerstein, M., Wodak, S.J., Emili, A., and Greenblatt, J.F. 2006. Global landscape of protein complexes in the yeast Saccharomyces cerevisiae. Nature 440:637-643.
	Puig, O., Caspary, F., Rigaut, G., Rutz, B., Bouveret, E., Bragado-Nilsson, E., Wilm, M., and Séraphin, B. 2001. The tandem affinity purification (TAP) method: A general procedure of protein complex purification. Methods 24:218-229.
	Rigaut, G., Shevchenko, A., Rutz, B., Wilm, M., Mann, M., and Séraphin, B. 1999. A generic protein purification method for protein complex characterization and proteome exploration. Nat. Biotechnol. 17:1030-1032.
	Rivas, S., Romeis, T., and Jones, J.D. 2002. The Cf-9 disease resistance protein is present in an approximately 420-kilodalton heteromultimeric membrane-associated complex at one molecule per complex. Plant Cell 14:689-702.
	Schimanski, B., Nguyen, T.N., and Günzl, A. 2005. Highly efficient tandem affinity purification of trypanosome protein complexes based on a novel epitope combination. Eukaryot. Cell 4:1942-1950.
	Swaffield, J.C., Melcher, K., and Johnston, S.A. 1996. Correction: A highly conserved ATPase protein as a mediator between acidic activation domains and the TATA-binding protein. Nature 379:658.
	Takebe, S., Witola, W.H., Schimanski, B., Günzl, A., and Ben Mamoun, C. 2007. Purification of components of the translation elongation factor complex of Plasmodium falciparum by tandem affinity purification. Eukaryot. Cell 6:584-591.
	Tsai, A. and Carstens, R.P. 2006. An optimized protocol for protein purification in cultured mammalian cells using a tandem affinity purification approach. Nat. Protocols 1:2820-2827.
	Yang, W., Steen, H., and Freeman, M.R. 2008. Proteomic approaches to the analysis of multiprotein signaling complexes. Proteomics 8:832-851.
	Ziegler, J., Vogt, T., Miersch, O., and Strack, D. 1997. Concentration of dilute protein solutions prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Anal. Biochem. 250:257-260.