Expression, Purification, and Characterization of Caspases

Jean‐Bernard Denault1, Guy S. Salvesen1

1 The Burnham Institute, La Jolla
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 21.13
DOI:  10.1002/0471140864.ps2113s30
Online Posting Date:  February, 2003
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This unit describes a protocol to obtain milligram amounts of enzymatically active pure recombinant caspases. Specific details for the expression, purification of caspase‐3, ‐6, ‐7, ‐8, ‐9 and ‐10 are discussed along with strategies to obtain particular forms (e.g., the zymogen) of some of them.

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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Expression of Caspases in E. coli
  • Basic Protocol 2: Caspase Enzymatic Assay
  • Support Protocol 1: Titration of Recombinant Caspase
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Expression of Caspases in E. coli

  • Freshly transformed (unit 10.1) BL21(DE3) cells (Stratagene) carrying the appropriate construct, on selective plates
  • 2× TY medium ( appendix 4A) containing 100 µg/ml ampicillin (added from 50 mg/ml ampicillin stock)
  • 1 M 1‐isopropyl‐β‐D‐thiogalactopyranoside (IPTG) stock solution
  • Chelating Sepharose Fast Flow Resin (Amersham Pharmacia Biotech)
  • recipeResuspension buffer (see recipe)
  • 0.1 M NiSO 4, filter‐sterilized
  • recipeWashing buffer (see recipe)
  • recipeElution buffer (see recipe)
  • 15‐ml culture tubes
  • Orbital shaker incubator (with speed up to 275 rpm preferred)
  • Bacterial culture flasks (flask volume depends on the final culture volume)
  • 1‐liter baffled culture flasks
  • Spectrophotometer cuvettes with 1‐cm light path
  • 0.5‐liter centrifuge bottles or tangential flow concentrator system
  • Refrigerated centrifuges with Sorvall SLA‐3000 and SM‐34 or equivalent rotors
  • 50‐ml polypropylene disposable screw‐cap tubes
  • Sonicator with large probe
  • Disposable filter units (e.g., Millipore Stericup 0.45‐µm HV Durapore membrane or equivalent)
  • Chromatography column: e.g., Econo‐Pac 0.7 × 5.0 cm (Bio‐Rad) with funnel and flow adaptor, or equivalent setup
  • Gradient former (e.g., Life Technologies Model 150)
  • Peristaltic pump (able to deliver a flow rate of 1.0 to 1.5 ml/min)
  • Fraction collector
NOTE: All reagents and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.

Basic Protocol 2: Caspase Enzymatic Assay

  • recipe2× caspase buffer (see recipe)
  • Caspase‐containing samples of interest
  • recipeChromogenic or fluorogenic substrate (see recipe and Table 21.13.2)
  • 96‐well flat‐bottom plate (opaque for fluorescent assays and clear for chromogenic assays)
  • Plate reader (colorimetric or fluorometric) ideally with regulated incubator
NOTE: The method below assumes a plate‐reading 96‐well format instrument. If one is not available, then volumes can be increased proportionally for assays in 1‐ml cuvettes.

Support Protocol 1: Titration of Recombinant Caspase

  • recipe10 mM Z‐VAD‐FMK (see recipe)
  • 250 µM pNA‐conjugated caspase substrate (see recipe for recipecaspase substrates) in recipe1× caspase buffer (see recipe for 2× buffer)
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Literature Cited

Literature Cited
   Bury, A. 1981. Analysis of protein and peptide mixtures: Evaluation of three sodium dodecyl sulphate‐polyacrylamide gel electrophoresis buffer systems. J. Chromatog. 213:491‐500.
   Edelhoch, H. 1967. Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry 6:1948‐1954.
   Garcia‐Calvo, M., Peterson, E.P., Rasper, D.M., Vaillancourt, J.P., Zamboni, R., Nicholson, D. W., and Thornberry, N. A. 1999. Purification and catalytic properties of human caspase family members. Cell Death Diff. 6:362‐369.
   Kuida, K., Lippke, J.A., Ku, G., Harding, M.W., Livingston, D.J., Su, M.S.S., and Flavell, R.A. 1995. Altered cytokine export and apoptosis in mice deficient in interleukin‐1‐β converting enzyme. Science 267:2000‐2003.
   Li, P., Allen, H., Bannerjee, S., Franklin, S., Herzog, L., Johnston, C., McDowell, J., Paskind, M., Rodman, L., Salfeld, J., Towne, E., Tracey, D., Wardwell, S., Wei, F.‐Y., Wong, W., Kamen, R., and Seshardi, T. 1995. Mice deficient in IL‐1β‐converting enzyme are defective in production of mature IL‐1β and resistant to endotoxic shock. Cell 80:401‐411.
   Porath, J., Carlsson, J., Olsson, I., and Belfrage, G. 1975. Metal chelate affinity chromatography, a new approach to protein fractionation. Nature 258:598‐599.
   Renatus, M., Stennicke, H.R., Scott, F.L., Liddington, R.C., and Salvesen, G.S. 2001. Dimer formation drives the activation of the cell death protease caspase 9. Proc. Natl. Acad. Sci. U.S.A. 98:14250‐14255.
   Stennicke, H.R. and Salvesen, G.S. 1999. Caspases: Preparation and characterization. Methods 17:313‐319.
   Stennicke, H.R., Renatus, M., Meldal, M., and Salvesen, G.S. 2000. Internally quenched fluorescent peptide substrates disclose the subsite preferences of human caspases 1, 3, 6, 7 and 8. Biochem. J. 350:563‐568.
   Talanian, R.V., Quinlan, C., Trautz, S., Hackett, M.C., Mankovich, J.A., Banach, D., Ghayur, T., Brady, K.D., and Wong, W.W. 1997. Substrate specificities of caspase family proteases. J. Biol. Chem. 272:9677‐9682.
   Thornberry, N.A., Rano, T.A., Peterson, E.P., Rasper, D.M., Timkey, T., Garcia‐Calvo, M., Houtzager, V.M., Nordstrom, P.A., Roy, S., Vaillancourt, J.P., Chapman, K.T., and Nicholson, D.W. 1997. A combinatorial approach defines specificities of members of the caspase family and granzyme B: Functional relationships established for key mediators of apoptosis. J. Biol. Chem. 272:17907‐17911.
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