Purification of Recombinant Proteins and Study of Protein Interaction by Epitope Tagging

Ning Zhang1, Jin‐Long Chen1

1 Tularik, Inc., South San Francisco, California
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 10.15
DOI:  10.1002/0471142727.mb1015s41
Online Posting Date:  May, 2001
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Abstract

A protein molecule can be engineered to include a short stretch of residues corresponding to an epitope to facilitate its subsequent biochemical and immunological analysis; a technique often referred to as “epitope tagging.” This unit presents a protocol for small‐scale immunoprecipitation of epitope‐tagged recombinant proteins expressed in transiently transfected mammalian cells. The immunoprecipitant can then be analyzed by SDS‐PAGE. An immunoprecipitation protocol is also provided that has been optimized for use with a baculovirus overexpression system. An describes how multisubunit complexes can be assembled by starting with a core protein affixed to beads via an epitope tag, and adding the other members of the complex in a stepwise manner.

     
 
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Table of Contents

  • Specialized Applications
  • Basic Protocol 1: Immunoprecipitation of Epitope‐Tagged Recombinant Proteins
  • Basic Protocol 2: Immunoprecipitation of Epitope‐Tagged Recombinant Proteins from a Baculovirus Overexpression System
  • Alternate Protocol 1: Stepwise Assembly of Protein Complexes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Tables
     
 
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Materials

Basic Protocol 1: Immunoprecipitation of Epitope‐Tagged Recombinant Proteins

  Materials
  • Transfected cells (see Chapter 9), adherent (on a 100‐mm plate) or nonadherent, expressing epitope‐tagged protein of interest
  • PBS ( appendix 22), ice cold, with and without 0.02% NaN 3
  • recipeLysis buffer (see recipe)
  • Protein A– or protein G–conjugated agarose beads (Pharmacia Biotech)
  • Normal mouse IgG
  • Epitope‐specific antibody (see Table 10.15.1)
  • 1.5× SDS sample buffer (prepare as a dilution of 2× SDS sample buffer; see unit 10.2)
  • 10‐ and 15‐ml centrifuge tubes
  • Additional reagents and equipment for SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE; unit 10.2)

Basic Protocol 2: Immunoprecipitation of Epitope‐Tagged Recombinant Proteins from a Baculovirus Overexpression System

  Materials
  • Baculovirus‐infected Sf 9 cells (unit 16.11), expressing epitope‐tagged protein of interest
  • PBS ( appendix 22), ice cold
  • recipeBinding buffer (see recipe), prepared as indicated and with an additional 60 mM KCl
  • 50% (v/v) slurry of anti‐epitope beads
  • Normal mouse IgG
  • recipeEpitope elution buffer (see recipe)

Alternate Protocol 1: Stepwise Assembly of Protein Complexes

  • Baculovirus‐infected Sf9 cell lines (unit 16.11), each expressing an additional epitope‐tagged protein of interest (protein II and subsequent complex proteins)
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Figures

Videos

Literature Cited

   Brown, P.M., Tagari, P., Rowan, K.R., Yu, V.L., O'Neill, G.P., Middaugh, C.R., Sanyal, G., Ford‐Hutchinson, A.W., and Nicholson, D.W. 1995. Epitope‐labeled soluble human interleukin 5 (IL‐5) receptors. J. Biol. Chem. 270:29236‐29243.
   Burke, T.W. and Kadonaga, J.T. 1996. Drosophila TFIID binds to a conserved downstream basal promoter element that is present in many TATA‐box‐deficient promoters. Genes & Dev. 10:711‐724.
   Canfield, V.A., Norbeck, L., and Levenson, R. 1996. Localization of cytoplasmic and extracellular domains of Na,K‐ATPase by epitope tag insertion. Biochemistry 31:14165‐14172.
   Chubet, R.G. and Brizzard, B.L. 1996. Vectors for expression and secretion of FLAG epitope‐tagged proteins in mammalian cells. BioTechniques 20:136‐141.
   Dear, T.N., Hainzl, T., Follo, M., Nehls, M., Wilmore, H., Matena, K., and Boehm, T. 1997. Identification of interaction partners for thebasic‐helix‐loop‐helix protein E47. Oncogene 14:891‐898.
   Harlow, E.D. and Lane, D. 1988. Immunoaffinity purification. In Antibodies: A Laboratory Manual. pp. 522‐523. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Molloy, S.S., Thomas, L., Van Slyke, J.K., Stenberg, P.E., and Thomas, G. 1994. Intracellular trafficking and activation of the furin proprotein convertase: Localization of the TGN and recycling from the cell surface. EMBO J. 13:18‐33.
   Murray, P.J., Watowich, S.S., Lodish, H.F., Young, R.A., and Hilton, D.J. 1995. Epitope tagging of the human endoplasmic reticulum HSP70 protein, BiP, to facilitate analysis of BiP‐substrate interactions. Anal. Biochem. 229:170‐179.
   Nakajima, K. and Yaoita, Y. 1997. Construction of multiple‐epitope tag sequence by PCR for sensitive Western blot analysis. Nucl. Acids Res. 25:2231‐2232.
   Pathak, R. and Imperiali, B. 1997. A dual affinity tag on the 64‐kDa Nlt1p subunit allows the rapid characterization of mutant yeast oligosaccharyl transferase complexes. Arch. Biochem. Biophys. 338:1‐6.
   Pati, U.K. 1992. Novel vectors for expression of cDNA encoding epitope‐tagged proteins in mammalian cells. Gene 114:285‐288.
   Sells, M.A. and Chernoff, J. 1995. Epitope‐tag vectors for eukaryotic protein production. Gene 152:187‐189.
   Thibault, C., Sganga, M.W., and Miles, M.F. 1997. Interaction of phosducin‐like protein with G protein βγ subunits. J. Biol. Chem. 272:12253‐12256.
   Witzgall, R., O'Leary, E., and Bonventre, J.V. 1994. A mammalian expression vector for the expression of GAL4 fusion proteins with an epitope tag and histidine tail. Anal. Biochem. 223:291‐298.
   Zhou, Q., Lieberman, P.M., Boyer, T.G., and Berk, A.J. 1992. Holo‐TFIID supports transcriptional stimulation by diverse activators and from a TATA‐less promoter. Genes & Dev. 6:1964‐1974.
Key References
   Chen, J.‐L. and Tjian, R. 1996. Reconstitution of TATA‐binding protein‐associated factor/TATA‐binding protein complexes for in vitro transcription. Methods Enzymol. 273:208‐217.
   Chiang, C.‐M. and Roeder, R.G. 1993. Expression and purification of general transcription factors by FLAG epitope tagging and peptide elution. Pept. Res. 6:62‐64.
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