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

Shwu‐Yuan Wu¹, Cheng‐Ming Chiang¹

¹Case 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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Unit Introduction
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: Purification of Multiple Forms of Epitope-Tagged Protein Complexes by Varying the Starting Material and Wash Conditions
Support Protocol: Purification of Multiple Forms of Epitope-Tagged Protein Complexes Following a P11 Ion-Exchange Chromatographic Column
Reagents and Solutions
Commentary
Bibliography
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, 1990; also see UNITS 9.9-9.14)
2× HEPES-buffered saline (HBS), pH 7.12 (UNIT 16.14)
0.25 M CaCl₂
Retrovirus-packaging cells (e.g., _CRIP, Danos and Mulligan, 1988)
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 2)
1× SDS-PAGE protein sample buffer (UNIT 10.2A)
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)
BC100 (see recipe)
BC300 (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, 1998; 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, 1992)
DMEM with 10% FBS (APPENDIX 3F)
G418 sulfate
Trypsin-EDTA (APPENDIX 3F)
1 M BES buffer, pH 7.2 (see recipe)
Hygromycin B
Tetracycline
1× SDS-PAGE protein sample buffer (UNIT 10.2A)
1× PBS (APPENDIX 2)
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 recipes)
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: Purification of Multiple Forms of Epitope-Tagged Protein Complexes by Varying the Starting Material and Wash Conditions

Additional Materials (also see Basic Protocols 1 and 2)

10 M urea
BC850 (see recipe)
Buffer B (see recipe)
Buffer D (see recipe)
FLAG peptide elution buffer (see recipe)
Ammonium sulfate

Sonicator
Rotor (e.g., Bechman 45-Ti or equivalent)

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

Additional Materials (also see Basic Protocol 1)

P11 ion-exchange resin
Various BC buffers (BC100, BC300, BC500, BC850, and BC1200; see recipes)

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

Figure 16.13.1

Purification of human RNA polymerase II complexes. The hRPB9-3 cells (Wu and Chiang, 1998), derived from human HeLa cells that conditionally express the FLAG-tagged RPB9 subunit of human RNA polymerase II, are first separated into cytoplasmic S100, nuclear extract, and nuclear pellet. Immunoaffinity purification is then performed with S100 or nuclear extract under either low salt (100 mM KCl-containing buffer) or high salt (850 mM KCl and 1.0 M urea-containing buffer) wash conditions, which results in the purification of human RNA polymerase II (pol II) holoenzyme and pol II, respectively. The pol II holoenzyme complex purified from S100 or nuclear extract contains pol II, a subset of general transcription factors (TFIIB, TFIIE, TFIIF, and TFIIH), SRBs, histone acetyltransferase GCN5, and chromatin remodeling factor SWI/SNF (Wu and Chiang, 1998; Wu et al., 1999). Additional forms of pol II complexes with different phosphorylation status on the largest subunit (RPB1) of pol II can also be purified from the nuclear pellet under the high salt wash condition. The IIO form of pol II contains the highly phosphorylated carboxy-terminal domain (CTD), indicated by a tail, in RPB1, whereas the IIA and IIB forms of pol II have non-phosphorylated or truncated CTD in RPB1, respectively.

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Figure 16.13.2

Purification of different FLAG-tagged TBP complexes. Nuclear extract prepared from 3-10 (Chiang et al., 1993), which is a HeLa-derived cell line constitutively expressing FLAG-tagged TBP, is loaded onto a P11 ion-exchange column. Proteins bound to the P11 resin are sequentially eluted off the column by step elutions with 0.1, 0.3, 0.5, and 0.85 M KCl-containing buffer. The FLAG-tagged TBP complexes are mainly detected at the 0.3 and 0.85 M KCl fractions, which are then used for immunoaffinity purification by anti-FLAG M2 monoclonal antibody-conjugated beads to isolate TFIIIB and TFIID complexes, respectively.

View Image

Literature Cited

Literature Cited
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	Chiang, C.-M. and Roeder, R.G. 1993. Expression and purification of general transcription factors by FLAG epitope-tagging and peptide elution. Peptide Res. 6:62-64.
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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.