Isolation and Purification of Proteasomes from Primary Cells

Nicholas J. Steers1, Kristina K. Peachman1, Carl R. Alving2, Mangala Rao2

1 Henry M. Jackson Foundation for the Advancement of Military Medicine, Bethesda, Maryland, 2 United States Military HIV Research Program, Walter Reed Army Institute of Research, Silver Spring, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 16.4
DOI:  10.1002/0471142735.im1604s107
Online Posting Date:  November, 2014
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Abstract

Proteasomes play an important role in cell homeostasis and in orchestrating the immune response by systematically degrading foreign proteins and misfolded or damaged host cell proteins. We describe a protocol to purify functionally active proteasomes from human CD4+ T cells and dendritic cells derived from peripheral blood mononuclear cells. The purification is a three‐step process involving ion‐exchange chromatography, ammonium sulfate precipitation, and sucrose density gradient ultracentrifugation. This method can be easily adapted to purify proteasomes from cell lines or from organs. Methods to characterize and visualize the purified proteasomes are also described. © 2014 by John Wiley & Sons, Inc.

Keywords: proteasomes; antigen processing; immunoproteasomes

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Isolation and Purification of Proteasomes from CD4+ T Cells Derived from Human Peripheral Blood Mononuclear Cells (PBMCs)
  • Alternate Protocol 1: Isolation and Purification of Proteasomes from Dendritic Cells or Macrophages Derived from Human PBMCS
  • Basic Protocol 2: Functional Characterization of Purified Proteasomes
  • Alternate Protocol 2: Identification of Proteasome Subunits by Mass Spectrometry
  • Support Protocol 1: Visualization of Proteasomes
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Isolation and Purification of Proteasomes from CD4+ T Cells Derived from Human Peripheral Blood Mononuclear Cells (PBMCs)

  Materials
  • Primary cells: human CD3/CD28/CD134/CD137 activated CD4+ T cells isolated and propagated from PBMC of healthy volunteers (see unit 7.1 and see recipe)
  • Lysis buffer (see recipe)
  • DEAE Sephacel (GE Healthcare)
  • 20 mM Tris·Cl, pH 7.2 ( appendix 2A)
  • Column buffers 1, 2, and 3 (see reciperecipes)
  • Ammonium sulfate (Sigma Aldrich)
  • 20 mM Tris·Cl, pH 7.2 ( appendix 2A) containing 15% (w/v) sucrose (Sigma‐Aldrich)
  • 20 mM Tris·Cl, pH 7.2 ( appendix 2A) containing 45% (w/v) sucrose (Sigma‐Aldrich)
  • 10× SDS‐PAGE reducing sample buffer (available from most molecular biology suppliers)
  • 4% to 20% 12‐well SDS‐PAGE gradient gel (e.g., Life Technologies)
  • Tris‐glycine‐SDS running buffer (Invitrogen)
  • Antibody specific to the α6 subunit of the proteasome complex (Abcam, cat. no. ab3325)
  • Pierce BCA Protein Assay Kit
  • BSA standard: prepare 10 mg/ml BSA in 20 mM Tris·Cl, pH 7.2
  • 50‐ml screw cap polypropylene conical tubes (Falcon)
  • Refrigerated tabletop centrifuge
  • Sorvall RC5B centrifuge with SA‐600 or SS34 rotor (Beckman)
  • 2‐liter glass beaker
  • 2‐liter sidearm flask
  • Sintered glass funnel
  • Vacuum source
  • Glass rod
  • 500‐ml glass bottle or Erlenmeyer flask
  • 50 cm × 1.5 cm glass chromatography columns (BioRad)
  • Biologic LP chromatography system including peristaltic column pump with UV detector and fraction collector (BioRad)
  • Amicon Ultra‐15 centrifugal filter units, 100 K cut off (Millipore)
  • 125‐ml Erlenmeyer flask
  • Magnetic stir plate
  • 50‐ml polycarbonate centrifuge tubes (Nalgene Oak Ridge, cat. no. 3188‐0050)
  • Gradient Master ip gradient maker (Biocomp)
  • 11 mm × 34 mm polyallomer ultracentrifuge tubes (Beckman)
  • Ultracentrifuge (Beckman TL‐100 with TLS55 rotor)
  • Clamp stand
  • 0.5‐ml microcentrifuge tubes
  • XCell SureLock gel rig (Life Technologies)
  • PVDF membranes (BioRad)
  • Additional reagents and equipment for SDS‐PAGE (unit 8.4) and western blotting (unit 8.10)

Alternate Protocol 1: Isolation and Purification of Proteasomes from Dendritic Cells or Macrophages Derived from Human PBMCS

  Additional Materials (also see protocol 1)
  • 15 cm × 0.7 cm glass chromatography column (BioRad)
  • Additional reagents and equipment for isolation of human dendritic cells (unit 7.32) or macrophages (unit 7.6)

Basic Protocol 2: Functional Characterization of Purified Proteasomes

  Materials
  • Purified proteasomes ( protocol 1 or Alternate Protocol 1)
  • Rehydration buffer (see recipe)
  • Reducing solution (see recipe)
  • Alkylating solution (see recipe)
  • ZOOM Tris‐Glycine (4% to 20%) gradient SDS gel (Invitrogen)
  • Tris‐Glycine SDS Running Buffer (Invitrogen)
  • 0.5% (w/v) agarose (Sigma‐Aldrich) solution
  • Molecular weight ladder: Benchmark pre‐stained protein standard (Invitrogen)
  • Phosphate‐buffered saline (PBS; appendix 2A) containing 0.01% (v/v) Tween 20
  • Monoclonal antibodies (alkaline phosphatase conjugated):
    • Anti‐α6 (c2, ab3325; Abcam)
    • Anti‐β1 (Enzo Life Sciences)
    • Anti‐β2 (Enzo Life Sciences)
    • Anti‐β5 (Enzo Life Sciences)
    • Anti‐β1i (LMP2; Abcam)
    • Anti−β2i (MECL1; Abcam)
    • Anti‐β5i (LMP7; Abcam)
  • NBT/BCIP substrates and solutions (BioRad)
  • GelCode Blue (Pierce)
  • Gel drying solution (Invitrogen)
  • Epoxomicin (Enzo Life Sciences)
  • Proteasome 20S assay kit (Enzo Life Sciences)
  • Chymotrypsin substrate: Suc‐LLVY‐AMC (Enzo Life Sciences)
  • Trypsin substrate: Ac‐RLR‐ AMC (Enzo Life Sciences)
  • Caspase substrate: Z‐LLE‐ AMC (Enzo Life Sciences)
  • ZOOM IPG pH 3‐pH 10 strips (Invitrogen)
  • Forceps
  • ZOOM IPGRunner cassettes (Invitrogen)
  • ZOOM IPGRunner gel rig (Invitrogen)
  • Power pack (BioRad)
  • 15‐ml screw‐cap conical polypropylene tubes (e.g., Corning Falcon)
  • Orbital shaker
  • XCell SureLock gel rig (Invitrogen)
  • PVDF membranes (BioRad)
  • Small glass tray of appropriate size for staining
  • Rocking platform
  • Orbital shaker
  • Gel‐drying kit (Invitrogen) including cellulose membranes
  • Spectramax M2 (Molecular Devices)
  • Flat‐bed scanner and computer running UMAX VistaScan software (http://www.umax.com/)
  • 96‐well V‐bottom fluorescence plate (Enzo Life Sciences)
  • Fluorescence microplate reader M2 (Molecular Devices)
  • Additional reagents and equipment for SDS‐PAGE (unit 8.4) and western blotting (unit 8.10)

Alternate Protocol 2: Identification of Proteasome Subunits by Mass Spectrometry

  Materials
  • GelCode Blue‐stained gel ( protocol 3, step 30)
  • In‐Gel Trypsin Digestion kit (Pierce)
  • 20 mM Tris·Cl, pH 7.2 ( appendix 2A)
  • Acetonitrile containing 0.1% formic acid, mass spectrometry grade (Thomas Scientific)
  • Distilled H 2O containing 0.1% formic acid, mass spectrometry grade (Thomas Scientific)
  • Scalpel or razor blade
  • LCMS‐IT‐TOF mass spectrometer (Shimadzu)
  • Shim‐Pack XR‐ODS II column (2.0 mm ×150 mm; Shimadzu)
  • UFLC (Shimadzu)
  • Mascot distiller and data base (Matrix Science)

Support Protocol 1: Visualization of Proteasomes

  Materials
  • Purified proteasomes ( protocol 1 or Alternate Protocol 1)
  • Tris buffer (see recipe)
  • 1.0% (v/v) glutaraldehyde/4% (v/v) formaldehyde (Electron Microscopy Sciences) made in distilled water
  • 2% uranyl acetate (Electron Microscopy Sciences) made in distilled water (filter through 0.22‐μm filter that has been pre‐rinsed with distilled H 2O; store in the dark at 4°C)
  • 300‐mesh nickel grid with Formar carbon coating (Electron Microscopy Sciences)
  • 75‐mm petri dish lid
  • No. 1 Whatman filter paper
  • Phillips CM100 Transmission Electron Microscope (FEI)
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Figures

Videos

Literature Cited

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Key Reference
  Tenzer, S., Stoltze, L., Schonfisch, B., Dengjel, J., Muller, M., Stevanovic, S., Rammensee, H.G., and Schild, H. 2004. Quantitative analysis of prion‐protein degradation by constitutive and immuno‐20S proteasomes indicates differences correlated with disease susceptibility. J. Immunol. 172:1083‐1091.
  Describes a method for purifying proteasomes from B cell lines that produce either immunoproteasomes or constitutive proteasomes.
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