Purification of SPARC/Osteonectin

E. Helene Sage1

1 The Hope Heart Institute, Seattle, Washington
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 10.11
DOI:  10.1002/0471143030.cb1011s17
Online Posting Date:  February, 2003
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Abstract

SPARC is a matricellular protein that regulates cell adhesion, extracelluolar matrix production, growth factor activity and cell cycle. This unit describes the purification of SPARC, also termed osteonectin and BM/40, from cultured mammalian cells. Additional information is presented on the purification of recombinant SPARC (rSPARC) from E. coli and from Sf9 cells, as well as its isolation from blood platelets. Assays for the activity of SPARC, de‚Äźadhesion and inhibition of cellular proliferation in vitro, are described. The expression of SPARC during remodeling and repair tissue in response to injury identifies it as a therapeutic target for the treatment of fibrotic disease, certain cancers and other disorders in which regulation of angiogenesis is a key factor.

     
 
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Table of Contents

  • Basic Protocol 1: Purification of SPARC from PYS‐2 Cells
  • Alternate Protocol 1: Purification of rSPARC from E. coli
  • Alternate Protocol 2: Purification of rSPARC from Insect (Sf9) Cells
  • Alternate Protocol 3: Purification of SPARC/Osteonectin from Tissues
  • Assays for the Evaluation of SPARC Activity
  • Support Protocol 1: Proliferation Assay
  • Support Protocol 2: De‐adhesion Assay
  • Support Protocol 3: Endotoxin Assay
  • Reagents and Solutions
  • Commentary
     
 
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Materials

Basic Protocol 1: Purification of SPARC from PYS‐2 Cells

  Materials
  • 50% to 70% confluent PYS‐2 cells (see recipe)
  • DMEM (serum‐free; appendix 2A)
  • 1100 Ci/mmol (12.5 Ci/ml) [trans35S]methionine/cysteine (ICN; optional)
  • DMEM minus methionine and cysteine (optional)
  • 0.2 M PMSF stock solution (see recipe)
  • N‐Ethylmaleimide (NEM)
  • Ammonium sulfate, ultrapure
  • DEAE buffer, 4°C (see recipe)
  • NaCl
  • ∼2 × 20–cm DEAE column (see recipe)
  • S‐200 buffer (see recipe)
  • Scintillation fluid (optional)
  • Sephacryl molecular‐sieve column (see recipe)
  • 0.05 M acetic acid
  • Plastic pipets
  • 50‐ml polycarbonate high‐speed centrifuge tubes
  • Low‐speed GPKR (Beckman) centrifuge with swinging bucket rotor
  • High‐speed refrigerated centrifuge with GSA (Sorvall) or JA‐17 rotors (Beckman) or equivalent
  • 12,000‐ to 14,000‐MWCO dialysis tubing (Spectrapor) or equivalent, prewashed with DEAE buffer
  • Dialysis clips (optional)
  • Standard gradient maker (e.g., Amersham Biosciences)
  • Peristaltic pump
  • Fraction collector
  • Lyophilizer
  • 50 or 250 ml centrifuge tubes
  • Additional reagents and equipment for SDS‐PAGE (unit 6.1) with autoradiography (unit 6.3), if appropriate, and determination of protein concentration by spectroscopy ( appendix 3B)
CAUTION: When working with radioactivity, take appropriate precautions to avoid contamination of the experimenter and surroundings. Carry out the experiments and dispose of wastes in appropriately designated area, following guidelines provided by the local radiation safety officer (also see appendix 1D).

Alternate Protocol 1: Purification of rSPARC from E. coli

  • LB medium with appropriate selective reagents ( appendix 2A)
  • E. coli strain transfected with SPARC expression vector (Bassuk et al., )
  • Inducing agent (e.g., IPTG; appendix 3A)
  • 10 mM sodium phosphate, pH 7.0 ( appendix 2A)/10% (v/v) glycerol
  • 90 mM sodium phosphate buffer (pH 7.8)/10% (v/v) glycerol/0.2 mM AEBSF, 4°C (see recipe), with and without 0.5 M NaCl
  • DEAE‐Sepharose Fast Flow anion‐exchange resin (Amersham Biosciences): equilibrate in 90 mM sodium phosphate buffer (pH 7.8)/10% (v/v) glycerol/0.2 mM AEBSF and allow to settle
  • 5 M NaCl ( appendix 2A)
  • 0.2 M AEBSF stock solution (see recipe)
  • Nickel/nitrilotriacetic acid (Ni‐NTA) metal‐chelate affinity resin (Qiagen)
  • 50 mM sodium phosphate (pH 5.3, 6.0, and 7.8)/0.5 M NaCl/10% (v/v) glycerol (see recipe)
  • 1.6 × 60–cm Superdex 70 column (see recipe)
  • 50 mM Tris⋅Cl (pH 8.0)/0.15 M NaCl (see recipe)
  • 1× PBS ( appendix 2A) containing 1 to 4 mM Ca2+ (optional)
  • French press
  • 2 × 20– and 1 × 10–cm chromatography columns
  • Flow cell coupled to a UV monitor set at 280 nm
  • Chart recorder
  • Conductivity meter (optional)
  • Disposable 10‐ml gel‐filtration column, sterile (optional)
  • Additional reagents and equipment for transfecting SPARC expression vector ( appendix 3A) and for SDS‐PAGE on minigels (unit 6.1)

Alternate Protocol 2: Purification of rSPARC from Insect (Sf9) Cells

  Materials
  • Sf9 cells (Invitrogen) infected with baculoviral SPARC expression vector, grown in serum‐free Sf‐900 II medium (Invitrogen)
  • 200 mM MOPS, pH 6.5 (see recipe)
  • 10 N NaOH ( appendix 2A)
  • Q‐Sepharose Fast Flow column (see recipe)
  • 200 and 400 mM LiCl/20 mM MOPS, pH 6.5 (see recipe)
  • 0.1 N acetic acid, 4°C: 0.6 ml glacial acetic acid in 100 ml H 2O
  • Hanks' buffered saline solution (see recipe)
  • 50‐ml conical tube
  • 0.22‐µm filter bottle
  • AktaPrime automated liquid chromatography system (Amersham Biosciences) or equivalent conventional model
  • 10,000‐NMWL Ultrafree‐15 (Millipore) or Centricon Plus‐80 (Amicon) centrifugal filter device
  • 0.22‐µm sterile syringe‐driven filter

Alternate Protocol 3: Purification of SPARC/Osteonectin from Tissues

  Materials
  • Platelet‐rich plasma or platelet suspension, or informed, nonsmoking, aspirin‐free, consenting adult blood donors
  • 0.156 M citrate containing 0.1 M dextrose and 5.0 µM prostaglandin E 1 (Sigma; optional)
  • 0.02 M Tris⋅Cl, pH 7.6/0.15 and 1.0 M NaCl (see recipe)
  • Thrombin
  • Sepharose 4B–AON IgG column (see recipe)
  • 3.0 M NaSCN/0.02 M Tris⋅Cl (pH 7.6)/0.15 M NaCl (see recipe)
  • 0.05 M NH 4HCO 3
  • 19‐G butterfly needles
  • 50 or 250‐ml plastic centrifuge bottles with caps
  • 12,000 to 14,000‐MWCO dialysis tubing
  • Lyophilizer
  • Additional reagents and equipment for thrombin activation of platelets (Kelm and Mann, ) and SDS‐PAGE (unit 6.1; also see protocol 1, step 22)

Support Protocol 1: Proliferation Assay

  • Bovine aortic endothelial (BAE) cells
  • DMEM/0% and 10% (w/v) FBS ( appendix 2A)
  • Purified SPARC (see protocol 1 or Alternate Protocol protocol 21 to protocol 43) and appropriate control buffer
  • 6.71 Ci/mmol (1 mCi/ml) [methyl‐3H]thymidine (PerkinElmer)
  • 10% (w/v) trichloroacetic acid (TCA), ice cold
  • 95% (v/v) ethanol
  • 0.4 N NaOH
  • Glacial acetic acid
  • Scintillation fluid
  • 24‐well tissue culture plate
  • 15‐ml conical tube
  • Radioactivity warning tape
  • Additional reagents and equipment for trypsinizing cells (unit 1.1)
CAUTION: When working with radioactivity, take appropriate precautions to avoid contamination of the experimenter and surroundings. Carry out the experiments and dispose of wastes in appropriately designated area, following guidelines provided by the local radiation safety officer (also see appendix 1D).

Support Protocol 2: De‐adhesion Assay

  • One 100‐mm dish of nearly‐confluent bovine aortic endothelial (BAE) cells, passaged not greater than ten times, grown in DMEM/10% FBS containing appropriate antibiotics
  • DMEM/2% and 10% FBS ( appendix 2A)
  • Purified SPARC (see protocol 1 and Alternate Protocols protocol 21 to protocol 43) and appropriate control buffer
  • 12‐well tissue culture dishes
  • Phase‐contrast microscope (unit 4.1)
  • Additional reagents and equipment for trypsinizing cells (unit 1.1)

Support Protocol 3: Endotoxin Assay

  Materials
  • Purified SPARC (see protocol 1 and Aletenate Protocols protocol 21 to protocol 43) and appropriate buffer
  • Limulus Amoebocyte Lysate (LAL) Pyrochrome kit (Associates of Cape Cod) for the Detection and Quantification of Gram‐Negative Bacterial Endotoxin:
  •  Pyrochrome LAL reagent
  •  Pyrochrome Reconstitution buffer
  •  Control Standard Endotoxin (CSE)
  • 50% (v/v) glacial acetic acid
  • Nonpyrogenic 96‐well tissue culture plate
  • Microtiter plate reader
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Figures

Videos

Literature Cited

Literature Cited
   Bassuk, J.A., Baneyx, F., Vernon, R.B., Funk, S.E., and Sage, E.H. 1996a. Expression of biologically active human SPARC in E. coli Arch. Biochem. Biophys. 325:8‐19.
   Bassuk, J.A., Braun, L.P., Motamed, K., Baneyx, F., and Sage, E.H. 1996b. Renaturation of secreted protein acidic and rich in cysteine (SPARC) expressed in Escherichia coli requires isomerization of disulfide bonds for recovery of biological activity. Intl. J. Biochem. Cell Biol. 28:1031‐1043.
   Bornstein, P. and Sage, E.H. 2002. Matricellular proteins: Extracellular modulators of cell function. Curr. Opin. Cell Biol. 64:608‐616.
   Bradshaw, A.D. and Sage, E.H. 2001. SPARC, a matricellular protein that functions in cellular differentiation and tissue response to injury. J. Clin. Invest. 107:1049‐1054.
   Bradshaw, A.D., Bassuk, J.A., Francki, A., and Sage, E.H. 2000. Expression and purification of recombinant human SPARC produced by baculovirus. Mol. Cell Biol. Res. Comm. 3:345‐351.
   Brekken, R.A. and Sage, E.H. 2000. SPARC, a matricellular protein: At the crossroads of cell‐matrix communication. Matrix Biol. 19:569‐580.
   Kelm, R.J. and Mann, K.G. 1990. Human platelet osteonectin: Release, surface expression, and partial characterization. Blood. 75:1105‐1113.
   Kelm, R.J. and Mann, K.G. 1991. The collagen binding specificity of bone and platelet osteonectin is related to differences in glycosylation. J. Biol. Chem. 266:9632‐9639.
   Lane, T.F. and Sage, E.H. 1994. The biology of SPARC, a protein that modulates cell‐matrix interactions. FASEB J. 8:163‐173.
   Sage, E.H., Vernon, R.B., Funk, S.E., Everitt, E.A., and Angello, J. 1989. SPARC, a secreted protein associated with cellular proliferation, inhibits cell spreading in vitro and exhibits Ca+2 dependent binding to the extracellular matrix. J. Cell. Biol. 109:341‐356.
   Sage, E.H. and Bornstein, P. 1995. Matrix components produced by endothelial cells: Type VIII collagen, SPARC, and thrombospondin. In Extracellular Matrix: A Practical Approach. (M.A. Haralson and J. R. Hassell, eds.) pp.131‐160. Oxford University Press, Oxford.
   Sasaki, T., Miosge, N., and Timpl, R. 1999. Immunochemical and tissue analysis of protease‐generated neoepitopes of BM‐40 (osteonectin, SPARC) which are correlated to a higher affinity binding to collagens. Matrix Biology. 18:499‐508.
   Termine, J.D., Kleinman, H.K., Whitson, S.W., Conn, K.M., McGarvey, M.L., and Martin, G.R. 1981. Osteonectin, a bone‐specific protein linking mineral to collagen. Cell 26:99‐105.
   Yost, J.C., Bell, A., Seale, R., and Sage, E.H. 1994. Purification of biologically active SPARC expressed in Saccharomyces cerevisiae. Arch. Biochem. Biophys. 314:50‐63.
Key References
   Lane and Sage, 1994. See above.
  This review of SPARC provides useful summaries of its location/abundance in tissues, sequence homologies, and physical characteristics.
   Brekken and Sage, 2000. See above.
  An up‐to‐date review of the structure and biology of SPARC.
   Reed, M., Puolakkainen, P.A., Lane, T.F., Dickerson, D., Bornstein, P. and Sage, E.H. 1993. Differential expression of SPARC and thrombospondin‐1 in wound repair: Immunolocalization and in situ hybridization. J. Histochem. Cytochem. 41:1467‐1477.
  A useful reference for immunostaining and in situ hybridization protocols for the detection of SPARC.
Acknowledgement
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