Purification of the Eukaryotic 20S Proteasome

Sherwin Wilk1, Wei‐Er Chen1

1 Mount Sinai School of Medicine, New York
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 21.6
DOI:  10.1002/0471140864.ps2106s25
Online Posting Date:  November, 2001
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Abstract

The 20S proteasome is the catalytic core of the major extralysosomal proteolytic system of the cell. Combination of the 20S proteasome with a complex of regulatory proteins forms the 26S proteasome, which in turn is responsible for the recognition and degradation of ubiquitin‐protein conjugates. As described in this unit, the constitutive form of the 20S proteasome can be conveniently purified as a stable and homogeneous preparation from bovine pituitaries. A support protocol details an enzyme assay used in evaluating proteasomal activity. The 20S proteasome is the catalytic core of the major extralysosomal proteolytic system of the cell.

     
 
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Table of Contents

  • Basic Protocol 1: Purification of the 20S Proteasome from Bovine Pituitaries
  • Support Protocol 1: Enzyme Assay to Analyze Proteasome‐Catalyzed Cleavage
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Purification of the 20S Proteasome from Bovine Pituitaries

  Materials
  • Frozen bovine pituitaries (Pel‐Freez)
  • recipe10 mM Tris‐EDTA buffer, pH 7.5 (see recipe)
  • Ammonium sulfate, ultrapure or enzyme grade
  • Toyopearl DEAE‐650M anion‐exchange resin (Rohm and Haas)
  • Eluting buffer: recipe300 mM Tris‐EDTA buffer, pH 7.5 (see recipe)
  • Nitrogen source
  • Ultrogel AcA22 gel‐filtration resin (BioSepra)
  • AcA22 buffer: recipe10 mM Tris‐EDTA buffer, pH 7.5 (see recipe), containing 0.1 M NaCl
  • Phenyl‐Sepharose 6 Fast Flow (high sub; Amersham Pharmacia Biotech)
  • Starting buffer: recipe10 mM Tris‐EDTA buffer, pH 7.5 (see recipe), containing 1 M ammonium sulfate
  • Blender
  • Refrigerated centrifuge
  • Glass rod
  • Dialysis tubing (16 mm diameter, 3500 MWCO; soak in distilled water and bring to boil, then decant and repeat two more times)
  • 2.5‐cm inner diameter glass chromatography column (25 cm length) for ion‐exchange chromatography
  • Peristaltic pump
  • Amicon 50‐ml ultrafiltration cell and PM 10 ultrafiltration membranes
  • 5 × 50–cm glass chromatography column for gel‐filtration chromatography
  • 1‐cm diameter glass chromatography column (25 cm length) for phenyl‐Sepharose chromatography
  • Additional reagents and equipment for dialysis ( appendix 3B), assaying proteasome enzymatic activity (see protocol 2), and SDS‐PAGE (unit 10.1)
NOTE: Since it takes about 2 days to equilibrate the Ultrogel AcA22 gel filtration column (see step ), it should be prepared before starting the purification protocol. Also be aware that the phenyl‐Sepharose column (step ) must be equilibrated overnight; therefore the experimental timetable must be arranged accordingly.

Support Protocol 1: Enzyme Assay to Analyze Proteasome‐Catalyzed Cleavage

  Materials
  • 0.05 M Tris⋅Cl, pH 7.5 ( appendix 2E)
  • Substrate: 10 mM N‐benzyloxycarbonyl‐Gly‐Gly‐Leu‐p‐nitroanilide (Z‐GGL‐pNA; Sigma) in DMSO
  • Enzyme (e.g., fraction from chromatographic purification of 20S proteasome; see protocol 1)
  • 10% (w/v) trichloroacetic acid (TCA)
  • 0.1% (w/v) NaNO 2
  • 0.5% (w/v) ammonium sulfamate
  • 0.05% N‐(1‐naphthyl)ethylenediamine⋅2HCl (NNEDA; see recipe)
  • 10 mM p‐nitroaniline stock in methanol (prepare fresh)
  • Shaking incubator
  • Tabletop centrifuge
CAUTION: Wear gloves when handling trichloroacetic acid. Store the NaNO 2 and NNEDA solutions in dark bottles. Store the stock substrate solutions in the freezer..
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Figures

Videos

Literature Cited

Literature Cited
   Arendt, C.S. and Hochstrasser, M. 1997. Identification of the yeast 20S proteasome catalytic centers and subunit interactions required for active‐site formation. Proc. Natl. Acad. Sci. U.S.A. 94:7156‐7161.
   Baumeister, W., Walz, J., Zuhl, F., and Seemuller, E. 1998. The proteasome: Paradigm of a self‐compartmentalizing protease. Cell 92:367‐380.
   Eleuteri, A.M., Kohanski, R.A., Cardozo, C., and Orlowski, M. 1997. Bovine spleen multicatalytic proteinase complex (proteasome). Replacement of X, Y, and Z subunits by LMP7, LMP2 and MECL1 and changes in properties and specificity. J. Biol. Chem. 272:11824‐11831.
   Groll, M., Ditzel, L., Lowe, J., Stock, D., Bochtler, B., Bartunik, H., and Huber, R. 1997. Structure of 20S proteasome from the yeast at 2.4 Åresolution. Nature 386:463‐471.
   Hendil, K.B. and Uerkvitz, W. 1991. The human multicatalytic proteinase: Affinity purification using a monoclonal antibody. J. Biochem. Biophys. Methods 22:159‐165.
   Orlowski, M. and Wilk, S. 1981. A multicatalytical protease complex that forms enkephalin and enkephalin containing peptides. Biochem. Biophys. Res. Comm. 101:814‐822.
   Orlowski, M. and Wilk, S. 2000. Catalytic activities of the 20S proteasome, a multicatalytic proteinase complex. Arch. Biochem. Biophys. 383:1‐16.
   Seemuller, E., Lupas, A., Stock, D., Lowe, J., Huber, R., and Baumeister, W. 1995. Proteasome from Thermoplasma acidophilum: A threonine protease. Science 268:579‐582.
   Seol, J.H., Park, S.C., Ha, D.B., Chung, C.H., Tanaka, K., and Ichihara, A. 1989. Na+, K+‐specific inhibition of protein and peptide hydrolyses by proteasomes from human hepatoma tissues. FEBS Lett. 247:197‐200.
   Tanaka, K., Tanahashi, N., Tsurumi, C., Yokota, K.‐Y., and Shimbara, N. 1997. Proteasomes and antigen processing. Adv. Immunol. 64:1‐38.
   Wagner, B.J. and Margolis, J.W. 1995. Age‐dependent association of isolated bovine lens multicatalytic proteinase complex (proteasome) with heat shock protein 90, an endogenous inhibitor. Arch. Biochem. Biophys. 323:455‐462.
   Wilk, S. and Orlowski, M. 1980. Cation‐sensitive neutral endopeptidase: Isolation and purification of the bovine pituitary enzyme. J. Neurochem. 35:1172‐1182.
   Wilk, S. and Orlowski, M. 1983. Evidence that pituitary cation‐sensitive neutral endopeptidase is a multicatalytic protease complex. J. Neurochem. 40:842‐849.
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