Expression and Purification of Epitope‐Tagged Multisubunit Protein Complexes from Mammalian Cells

Shwu‐Yuan Wu1, Cheng‐Ming Chiang1

1 Case Western Reserve University School of Medicine, Cleveland, Ohio
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 16.13
DOI:  10.1002/0471142727.mb1613s60
Online Posting Date:  November, 2002
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Abstract

Biochemical characterization and functional studies of mammalian proteins are often hampered by the availability of the purified protein, in particular, when the functional entity is present as a multisubunit protein complex in the cell. To overcome the difficulties in the purification of multisubunit protein complexes from mammalian cells, one may create stable cell lines containing epitope‐tagged protein. The first protocol in this unit describes the procedures involved in the establishment of a stable cell line constitutively expressing the FLAG‐tagged protein by retrovirus‐mediated gene transfer and immunoaffinity purification of the epitope‐tagged multisubunit protein complex. The next protocol outlines the steps involved in the establishment of an inducible cell line conditionally expressing the FLAG‐tagged protein by a tetracycline‐regulated system, and the one‐step immunoaffinity purification of the multisubunit protein complex. An alternate protocol provides an excellent example for the purification of different forms of human RNA polymerase II complexes, achieved simply by choosing the appropriate starting material and by varying wash conditions. The isolation of various human TATA‐binding protein (TBP)‐containing complexes is described in a support protocol, and is a good example of combining the P11 column and immunoaffinity purification. These protocols, collectively, illustrate a powerful methodology in applying epitope tagging and stable cell line approaches for the purification of multisubunit protein complexes from mammalian cells.

     
 
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Table of Contents

  • Basic Protocol 1: Purification of Multisubunit Protein Complexes from Clonal Cell Lines Constitutively Expressing a Flag‐Tagged Protein
  • Basic Protocol 2: Purification of Multisubunit Protein Complexes from Clonal Cell Lines Conditionally Expressing the Flag‐Tagged Protein
  • Alternate Protocol 1: Purification of Multiple Forms of Epitope‐Tagged Protein Complexes by Varying the Starting Material and Wash Conditions
  • Support Protocol 1: Purification of Multiple Forms of Epitope‐Tagged Protein Complexes Following a P11 Ion‐Exchange Chromatographic Column
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Purification of Multisubunit Protein Complexes from Clonal Cell Lines Constitutively Expressing a Flag‐Tagged Protein

  Materials
  • FLAG‐tagged protein‐coding sequence
  • Retroviral vector (e.g., pBabe neo, Morgenstern and Land, ; also see units 9.9 9.14)
  • 2× HEPES‐buffered saline (HBS), pH 7.12 (unit 16.14)
  • 0.25 M CaCl 2
  • Retrovirus‐packaging cells (e.g., ψ CRIP, Danos and Mulligan, )
  • Dulbecco's modified Eagle's medium (DMEM) with and without 10% calf serum
  • 15% (v/v) glycerol/DMEM
  • DMEM with 10% fetal bovine serum (FBS; appendix 3F)
  • 2.5 mg/ml polybrene stock solution
  • 50% confluent HeLa cells
  • Drug‐containing selection medium (e.g., 1 mg/ml of G418 (total weight), 0.2 mg/ml of hygromycin, or 0.5 µg/ml of puromycin; see unit 9.5)
  • Trypsin‐EDTA ( appendix 3F)
  • 1× PBS ( appendix 22)
  • 1× SDS‐PAGE protein sample buffer (unit 10.2)
  • Anti‐FLAG M2 monoclonal antibody (and/or anti‐protein antibodies; Sigma)
  • Joklik's medium (Sigma) with 10% calf serum and 5% calf serum
  • Anti‐FLAG M2 monoclonal antibody‐conjugated beads (Sigma)
  • recipeBC100 (see recipe)
  • recipeBC300 (see recipe)/0.1% Nonidet P‐40
  • FLAG peptide (100 mg/ml in water)
  • Liquid nitrogen
  • 5‐ml round‐bottom Falcon tube
  • 60‐mm and 100‐mm tissue culture plates
  • 15‐ml sterile tube
  • 0.45‐µm cellulose acetate syringe filter
  • 24‐well tissue culture plate
  • 1.5‐ml and 0.5‐ml microcentrifuge tubes
  • 250‐ml, 500‐ml, 1‐liter, 3‐liter, and 12‐liter spinner flasks
  • End‐over‐end tube rotator
  • Rotor (e.g., Sorvall H‐6000A or equivalent)
  • Microcentrifuge spin column
  • Additional reagents and equipment for use of retroviral vectors (units 9.9 9.14), preparation of nuclear extracts (unit 12.1), and immunoblotting (unit 10.8)

Basic Protocol 2: Purification of Multisubunit Protein Complexes from Clonal Cell Lines Conditionally Expressing the Flag‐Tagged Protein

  Materials
  • Protein‐coding sequence
  • Tetracycline‐regulated expression plasmid (e.g., pTetCMV‐F°(S), Wu and Chiang, ; unit 16.14)
  • Sheared calf thymus DNA
  • SacI‐linearized plasmid containing a drug‐selection marker (e.g., pREP4, Invitrogen)
  • Cell line expressing a tetracycline‐controlled transactivator (e.g., HtTA‐1, Gossen and Bujard, )
  • DMEM with 10% FBS ( appendix 3F)
  • G418 sulfate
  • Trypsin‐EDTA ( appendix 3F)
  • recipe1 M BES buffer, pH 7.2 (see recipe)
  • Hygromycin B
  • Tetracycline
  • 1× SDS‐PAGE protein sample buffer (unit 10.2)
  • 1× PBS ( appendix 22)
  • Anti‐FLAG M2 monoclonal antibody and/or anti‐protein antibodies
  • Joklik's medium (Sigma) with 10% calf serum
  • Joklik's medium with 5% calf serum
  • HeLa cells
  • Anti‐FLAG M2‐conjugated beads
  • BC100 and BC300 (see reciperecipes)
  • 10% Nonidet P‐40
  • FLAG peptide
  • Liquid nitrogen
  • 0.4‐cm cuvettes for electroporation
  • 24‐well, 60‐mm, and 100‐mm tissue culture plates
  • 15‐ml and 50‐ml Falcon tubes
  • Rotor (e.g., Sorvall H‐6000A or equivalent)
  • Electroporator (e.g., Bio‐Rad Gene Pulser II)
  • Disposable glass pipet
  • 1.5‐ml and 0.5‐ml microcentrifuge tubes
  • 250‐ml, 500‐ml, 1‐liter, 3‐liter, and 12‐liter spinner flasks
  • 250‐ml sterile conical centrifuge tubes
  • Microcentrifuge spin column
  • Additional reagents and equipment for tissue culture ( appendix 3F) and immunoblotting (unit 10.8)

Alternate Protocol 1: Purification of Multiple Forms of Epitope‐Tagged Protein Complexes by Varying the Starting Material and Wash Conditions

  • 10 M urea
  • recipeBC850 (see recipe)
  • recipeBuffer B (see recipe)
  • recipeBuffer D (see recipe)
  • recipeFLAG peptide elution buffer (see recipe)
  • Ammonium sulfate
  • Sonicator
  • Rotor (e.g., Bechman 45‐Ti or equivalent)

Support Protocol 1: Purification of Multiple Forms of Epitope‐Tagged Protein Complexes Following a P11 Ion‐Exchange Chromatographic Column

  • P11 ion‐exchange resin
  • Various BC buffers (BC100, BC300, BC500, BC850, and BC1200; see reciperecipes)
  • Chromatographic column (∼100 ml capacity)
  • Flow adapter
  • Chart recorder
  • Fraction collector
  • Dialysis tubing (MWCO 12,000; Sigma)
  • Additional reagents and equipment for dialysis ( appendix 3C)
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Figures

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Literature Cited

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Key References
   Chiang et al., 1993. See above.
  This paper describes the purification of human TFIID and TFIIIB complexes from a stable cell line constitutively expressing FLAG‐tagged TBP.
   Chiang and Roeder, 1993. See above.
  This paper documents the development of the FLAG peptide elution method for purification of recombinant FLAG‐tagged protein from bacteria.
   Kershnar, et al., 1998. See above.
  This paper is the first documentation detailing the procedures for biochemical purification of multisubunit protein complexes from an inducible mammalian expression system.
   Wu and Chiang, 1996. See above.
  This paper describes the construction of tetracycline‐regulated FLAG‐tagged expression plasmids, and the isolation and characterization of the inducible cell lines.
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