Over‐Expression and Purification of Active Serine Proteases and Their Variants from Escherichia coli Inclusion Bodies

Marina A. A. Parry1

1 Actelion Pharmaceuticals Ltd., Allschwil
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 21.11
DOI:  10.1002/0471140864.ps2111s27
Online Posting Date:  May, 2002
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Abstract

This unit describes the over‐expression and purification of active serine proteases and their variants from E. coli inclusion bodies. The strategy includes the folding and purification of a stable zymogen precursor protein, and its later activation with the appropriate convertase to the less stable but active protease. A test to follow the presence of activity in the samples, together with an active‐site titration protocol to determine the number of active sites per mole of total protein are provided. It should be emphasized that although most of the protocols described are applied to a specific example, they are fairly representative of the methods and approaches generally used for laboratory‐scale preparation of other recombinant serine proteases. The critical steps and how this template protocol can be adapted for the purification of other serine proteases are described.

     
 
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Table of Contents

  • Basic Protocol 1: Over‐Expression and Purification of Microplasminogen and its Variants from Escherichia Coli Inclusion Bodies
  • Basic Protocol 2: Activation of the Zymogen Microplasminogen and Purification of the Catalytically Active Microplasmin
  • Support Protocol 1: Assay to Determine the Activity of the Recombinant Microplasmin
  • Support Protocol 2: Assay to Determine the Active‐Site Concentration
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Over‐Expression and Purification of Microplasminogen and its Variants from Escherichia Coli Inclusion Bodies

  Materials
  • recipe2× TY kan/amp medium (see recipe), 37°C
  • Glycerol stock of cells harboring the construct of interest
  • recipe500 mM (250×) isopropyl‐β‐D‐thiogalactopyranosidase (IPTG; see recipe)
  • recipeLysis buffer (see recipe)
  • recipeLysozyme stock (see recipe)
  • 1 M MgCl 2 stock ( appendix 2E)
  • recipe0.2 mg/ml DNase I stock (see recipe)
  • recipeWash buffer 1 (see recipe)
  • recipeWash buffer 2 (see recipe)
  • recipeDenaturing buffer (see recipe), freshly made
  • 1 M HCl
  • recipeDialysis buffer 1 (see recipe), 4°C
  • recipeRefolding buffer (see recipe)
  • recipeDialysis buffer 2 (see recipe), 4°C
  • 15‐ml culture tubes
  • 1, 2‐, and 3‐liter baffled culture flasks
  • French press (Spectronic Instruments) or sonicator (Sonics & Materials)
  • Tissue homogenizer (e.g., Potter‐Elvejhem, Braun Biotech)
  • End‐over‐end rotator
  • pH‐indicator paper 0 to 14 or 4 to 7 (Merck)
  • Dialysis tubing, ∼3.2‐cm diameter (Spectrum Laboratories)
  • 0.4‐µm nitrocellulose membrane cut to fit vacuum filtration apparatus (Schleicher & Schuell) or 0.7‐µm glass microfiber filters (Whatman), optional
  • Tangential flow ultrafiltration equipment with membrane (Pall Filtron)
  • Additional reagents and equipment for absorbance measurements (OD 600 and OD 280; unit 3.1), centrifugation (unit 4.1), dialysis ( appendix 3B), chromatography (Chapter 8), and SDS‐PAGE (unit 10.1)

Basic Protocol 2: Activation of the Zymogen Microplasminogen and Purification of the Catalytically Active Microplasmin

  Materials
  • Urokinase plasminogen activator (uPA; Sigma)
  • CNBr‐activated Sepharose 4B (Amersham Pharmacia)
  • Recombinant microplasminogen protein (see protocol 1)
  • 5 M NaCl ( appendix 2E)
  • 87% glycerol
  • recipeUrokinase‐Sepharose storage buffer (see recipe), optional
  • Benzamidine‐Sepharose column (Sigma)
  • Wash buffer for benzamidine‐Sepharose column (see recipe)
  • Elution buffer 1 or 2 for benzamidine‐Sepharose column (see reciperecipe)
  • recipeRegeneration buffer for benzamidine‐Sepharose column (see recipe)
  • End‐over‐end rotator
  • Additional reagents and equipment for absorbance measurements (unit 3.1), centrifugation (unit 4.1), dialysis ( appendix 3B), chromatography (Chapter 8), and SDS‐PAGE (unit 10.1)

Support Protocol 1: Assay to Determine the Activity of the Recombinant Microplasmin

  Materials
  • S‐2251 chromogenic substrate (Chromogenix)
  • recipeAssay buffer (see recipe)
  • 1.5‐ml plastic cuvettes (Amersham Pharmacia)
  • Additional reagents and equipment for absorbance measurements (unit 3.1)

Support Protocol 2: Assay to Determine the Active‐Site Concentration

  Materials
  • Substrate‐buffer mix (see protocol 3, steps to )
  • 0.5 µM protease inhibitor (bovine pancreatic trypsin inhibitor, BPTI)
  • 0.1 M acetic acid, pH 3.0
  • 1.5‐ml plastic cuvettes
  • Additional reagents and equipment for amino acid analysis (unit 11.9), determination of protein concentration (unit 3.1), Lowry or Bradford methods (unit 3.4)
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Figures

Videos

Literature Cited

Literature Cited
   Bachman, F. 1994. Fibrinolysis. In Haemostasis and Thrombosis. (A.L.M. Bloom, C.D. Forbes, D.P. Thomas, and E.G.D. Tuddenham, eds.), pp.549‐625. Churchill Livingstone, London.
   Bode, W. and Schwager, P. 1975. The refined crystal structure of bovine beta‐trypsin at 1.8 A resolution. II. Crystallographic refinement, calcium binding site, benzamidine binding site and active site at pH 7.0. J. Mol. Biol. 98:693‐717.
   Bode, W., Engh, R., Hopfner, K.‐P., Huber, R., and Kopetzki, E. July 1999. Chimeric serine proteases. EP‐00927764.
   Castellino, F.J. and Powell, J.R. 1981. Human plasminogen. Methods Enzymol. 80:365‐378.
   Chapman, H.A. 1997. Plasminogen activators, integrins, and the coordinated regulation of cell adhesion and migration. Curr. Opin. Cell Biol. 9:714‐724.
   Davie, E.W., Fujikawa, K., and Kisiel, W. 1991. The coagulation cascade: Initiation, maintenance, and regulation. Biochemistry 30:10363‐10370.
   Hedstrom, L., Szilagyi, L., and Rutter, W.J. 1992. Converting trypsin to chymotrypsin: The role of surface loops. Science 255:1249‐1253.
   Hopfner, K.P., Brandstetter, H., Karcher, A., Kopetzki, E., Huber, R., Engh, R.A., and Bode, W. 1997. Converting blood coagulation factor IXa into factor Xa: Dramatic increase in amidolytic activity identifies important active site determinants. Embo. J. 16:6626‐6635.
   Jenne, D., Wilharm, E., Huber, R., Bode, W., and Parry, M., Procedure to prepare active serine proteases and their inactive variants. International patent filed.
   Ke, S.H., Coombs, G.S., Tachias, K., Navre, M., Corey, D.R., and Madison, E.L. 1997. Distinguishing the specificities of closely related proteases. Role of P3 in substrate and inhibitor discrimination between tissue‐type plasminogen activator and urokinase. J. Biol. Chem. 272:16603‐16609.
   Kopetzki, E. and Hopfner, K.‐P. December 1997. Recombinant blood‐coagulation proteases. WO‐09747737.
   Kopetzki, E., Rudolph, R., and Grossmann, A. 1993. Recombinant core streptavidin. U.S, patent US 5,489,528 and European patent EP612,325.
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   Madison, E.L. and Sambrook, J.E. 1993. Probing structure‐function relationships of tissue‐type plasminogen activator by oligonucleotide‐mediated site‐specific mutagenesis. Methods Enzymol. 223:249‐271.
   Murakami, M., Towatari, T., Ohuchi, M., Shiota, M., Akao, M., Okumura, Y., Parry, M.A., and Kido, H. 2001. Mini‐plasmin found in the epithelial cells of bronchioles triggers infection by broad‐spectrum influenza A viruses and Sendai virus. Eur. J. Biochem. 268:2847‐2855.
   Oberg, K., Chrunyk, B.A., Wetzel, R., and Fink, A.L. 1994. Nativelike secondary structure in interleukin‐1 beta inclusion bodies by attenuated total reflectance FTIR. Biochemistry 33:2628‐2634.
   Parry, M.A., Fernandez‐Catalan, C., Bergner, A., Huber, R., Hopfner, K.P., Schlott, B., Guhrs, K.H., and Bode, W. 1998. The ternary microplasmin‐staphylokinase‐microplasmin complex is a proteinase‐cofactor‐substrate complex in action. Nat. Struct. Biol. 5:917‐923.
   Parry, M.A., Zhang, X.C., and Bode, I. 2000. Molecular mechanisms of plasminogen activation: Bacterial cofactors provide clues. Trends Biochem. Sci. 25:53‐59.
   Seckler, R. and Jaenicke, R. 1992. Protein folding and protein refolding. FASEB J. 6:2545‐2552.
   Sottrup‐Jensen, L., Zajdel, M., Claeys, H., Petersen, T.E., and Magnusson, S. 1975. Amino‐acid sequence of activation cleavage site in plasminogen: Homology with “pro” part of prothrombin. Proc. Natl. Acad. Sci. U.S.A. 72:2577‐2581.
   Wang, X., Lin, X., Loy, J.A., Tang, J., and Zhang, X.C. 1998. Crystal structure of the catalytic domain of human plasmin complexed with streptokinase. Science 281:1662‐1665.
   Wang, X., Terzyan, S., Tang, J., Loy, J.A., Lin, X., and Zhang, X.C. 2000. Human plasminogen catalytic domain undergoes an unusual conformational change upon activation. J. Mol. Biol. 295:903‐914.
   Wilharm, E., Parry, M.A., Friebel, R., Tschesche, H., Matschiner, G., Sommerhoff, C.P., and Jenne, D.E. 1999. Generation of catalytically active granzyme K from Escherichia coli inclusion bodies and identification of efficient granzyme K inhibitors in human plasma. J. Biol. Chem. 274:27331‐27337.
   Wu, H.L., Shi, G.Y., and Bender, M.L. 1987. Preparation and purification of microplasmin. Proc. Natl. Acad. Sci. U.S.A. 84:8292‐8295.
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