Expression, Purification, and Characterization of Aspartic Endopeptidases: Plasmodium Plasmepsins and “Short” Recombinant Human Pseudocathepsin

Bret B. Beyer1, Nathan E. Goldfarb1, Ben M. Dunn1

1 University of Florida, Gainesville, Florida
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 21.14
DOI:  10.1002/0471140864.ps2114s32
Online Posting Date:  August, 2004
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Abstract

The unit describes a basic protocols utilized to obtain milligram amounts of enzymatically active, pure recombinant Plasmodium plasmepsins and "short" human pseudocathepsin D. Specific details for the expression and purification of Plasmodium falciparum plasmepsin 2 and "short" human pseudocathepsin D in zymogen form are described in this chapter. The plasmepsin 2 protocols are also applicable to Plasmodium vivax, P. ovale, and P. malariae plasmepsins, as well as P. falciparum plasmepsin 4.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Expression of Aspartic Endopeptidases in E. coli
  • Support Protocol 1: Enzymatic Plasmepsin Chromogenic Assay
  • Support Protocol 2: Titration of Recombinant Plasmepsins
  • Support Protocol 3: Enzymatic Cathepsin D Fluorogenic Assay
  • Support Protocol 4: Titration of Recombinant Cathepsin D
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Expression of Aspartic Endopeptidases in E. coli

  Materials
  • E. coli BL21(DE3) cells (Stratagene), growing on LB plates ( appendix 4A), containing plasmepsin gene in pET3a or pTCPSD2 construct of cathepsin D (see appendix 4D for introduction of plasmid DNA into E. coli)
  • LB medium ( appendix 4A)
  • Glycerol (spectrophotometric grade), sterile
  • M9 medium (for cathepsin D; appendix 4A)
  • 50 mg/ml ampicillin stock
  • 1 M isopropyl‐β‐D‐thiogalactopyranoside stock (IPTG; unit 5.2)
  • 6× SDS sample buffer (unit 10.1)
  • 95% ethanol
  • Resuspension buffers A, B, C, and D (see reciperecipes; for plasmepsins) or A2 and B2 (see reciperecipes; for cathepsin D); prechilled
  • DNase I (Sigma)
  • 27% (w/v) sucrose (density, 1.1 g/ml)
  • TE buffer, pH 8.0 ( appendix 2E)
  • Solubilization buffer A (for plasmepsins; see recipe) or solubilization buffer A2 (for cathepsin D; see recipe)
  • Dialysis buffer A (for plasmepsins): 20 mM Tris·Cl, pH 8.0 ( appendix 2E)
  • IEX starting buffer (for plasmepsins): 20 mM Tris·Cl, pH 8.0 ( appendix 2E), filter‐sterilized
  • IEX elution buffer (for plasmepsins): 20 mM Tris·Cl, pH 8.0 ( appendix 2E)/1 M NaCl, filter‐sterilized
  • Superdex 75 prep grade gel filtration resin (optional, for plasmepsins; Amersham Pharmacia Biotech)
  • Rapid dilution buffer (for cathepsin D): 10 mM Tris·Cl, pH 8.7 ( appendix 2E; filter through Whatman no. 4 filter paper to remove particulates that may act as seeds for protein aggregation)
  • Oxidized glutathione (for cathepsin D; Sigma)
  • 95% formic acid (for cathepsin D; Sigma)
  • Pepstatinyl‐agarose affinity resin (for cathepsin D; synthesized by the method of Huang et al., )
  • Pepstatinyl‐agarose equilibration buffer (for cathepsin D; see recipe), 25°C
  • Pepstatinyl‐agarose washing buffer (for cathepsin D; see recipe), 25°C
  • Pepstatinyl‐agarose elution buffer (for cathepsin D; see recipe), 4°C
  • DEAE equilibration buffer (for cathepsin D; see recipe), 4°C
  • DEAE buffer A (for cathepsin D): 10 mM Tris·Cl, pH 8.0 ( appendix 2E)
  • DEAE buffer B (for cathepsin D): 10 mM Tris·Cl, pH 8.0 ( appendix 2E)/80 mM NaCl, pH 8.0
  • 15‐ml capped culture tubes
  • Orbital shaker incubator (preferably with speed up to 275 rpm)
  • 250‐ml baffled bacterial culture flask (or larger flask depending on final culture volume)
  • 2.8‐ or 4‐liter Erlenmeyer flasks
  • 1‐ml syringe with 27‐G needle
  • 0.5‐liter centrifuge bottles
  • Beckman refrigerated centrifuge with JA‐20 rotor (or equivalent)
  • 50‐ml disposable plastic conical screw‐cap centrifuge tubes
  • 30‐ml Corex centrifuge tubes with rubber jackets
  • Dialysis tubing (for plasmepsins), SpectraPor, MWCO 12,000 to 14,000 (smaller MWCO can be used if necessary): prepare as described in manufacturer's instructions and appendix 3B
  • FPLC system (Amersham Pharmacia Biotech) including:
    • 5‐ml HiTrap Q Sepharose HP anion‐exchange column
    • Computer‐controlled linear gradient mixer
    • Peristaltic pump capable of 1.0 to 1.5 ml/min flow rate
    • Fraction collector
  • UV spectrophotometer and 1‐cm path‐length quartz cuvettes
  • 1.5 × 100–cm FPLC column with flow adapter (for plasmepsins, if optional gel‐filtration step is to be performed; available from Bio‐Rad)
  • C 16/20 chromatography column (for cathepsin D; Amersham Pharmacia Biotech)
  • Büchner funnel (for cathepsin D)
  • Whatman no. 4 filter paper (for cathepsin D)
  • Dialysis tubing (for cathepsin D), MWCO 6000 to 8000: prepare as described in manufacturer's instructions and appendix 3B
  • Additional reagents and equipment for transformation ( appendix 4D) and growth of E. coli ( appendix 4B), isolating plasmid DNA ( appendix 4C), DNA sequencing (Ausubel et al., , Chapter ), monitoring bacterial growth by spectrophotometry (unit 5.3), SDS‐PAGE (units 10.1 10.4), lysis of bacteria using a French pressure cell (unit 6.2), dialysis ( 3.NaN), measuring protein concentration by spectrophotometry (unit 3.4), calculating protein concentration (unit 7.2), activity assays for plasmepsins ( 21.14Support Protocols 1 and 21.142), fluorogenic assay for cathpsin D ( 21.14), silver staining of protein gels (unit 10.5), and titration of recombinant cathepsin D ( 21.14)
NOTE: All reagents and equipment coming into contact with live cells must be sterile, and aseptic technique should be used accordingly.

Support Protocol 1: Enzymatic Plasmepsin Chromogenic Assay

  Materials
  • Chromogenic plasmepsin substrate stock (see recipe)
  • 5× plasmepsin assay buffer: 0.5 M sodium formate, pH 4.5, filter‐sterilized
  • Plasmepsin‐containing fraction to be analyzed (see protocol 1)
  • Capless 1.5‐ml microcentrifuge tubes
  • 1‐cm path‐length quartz cuvettes
  • Diode array spectrophotometer with a seven‐cuvette multi‐cell transport thermostatted to 37°C with a circulating water bath

Support Protocol 2: Titration of Recombinant Plasmepsins

  Materials
  • Pepstatin A stock (see recipe)
  • Enzyme Kinetics Module (Version 1.0) of SigmaPlot 2000 (Version 6.10; Sigma)
  • Additional reagents and equipment for enzymatic plasmepsin chromogenic assay ( protocol 2)

Support Protocol 3: Enzymatic Cathepsin D Fluorogenic Assay

  Materials
  • Cathepsin D assay buffer: 1 M sodium formate, pH 3.7
  • Cathepsin D–containing fraction to be analyzed (see protocol 1)
  • 100 µM fluorogenic cathepsin D substrate stock (see recipe)
  • 96‐well round‐bottom microtiter plates
  • Fluorescence multi‐well plate reader with regulated incubator
  • Multichannel pipettor

Support Protocol 4: Titration of Recombinant Cathepsin D

  Materials
  • Pepstatin A stock (see recipe)
  • Enzyme Kinetics Module (Version 1.0) of SigmaPlot 2000 (Version 6.10; Sigma)
  • Additional reagents and equipment for enzymatic cathepsin D fluorogenic assay ( protocol 2)
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Figures

Videos

Literature Cited

Literature Cited
   Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (eds.). 2003. Current Protocols in Molecular Biology. John Wiley & Sons, New York.
   Beyer, B.M. and Dunn, B.M. 1996. Self‐activation of recombinant human lysosomal procathepsin D at a newly engineered cleavage junction, “short” pseudocathepsin D. J. Biol. Chem. 271:15590‐15596.
   Bieth, J.G. 1995. Theoretical and practical aspects of proteinase inhibition kinetics. Methods Enzymol. 248:59‐84.
   Conner, G.E. and Udey, J.A. 1990. Expression and refolding of recombinant human fibroblast procathepsin D. DNA Cell Biol. 9:1‐9.
   Dunn, B.M., Scarborough, P.E., Davenport, R., and Swietnicki, W. 1994. Analysis of proteinase specificity by studies of peptide substrates: The use of UV and fluorescence spectroscopy to quantitate rates of enzymatic cleavage. Methods Mol. Biol. 36:225‐243.
   Grunberg‐Manago, M. 1999. Messenger RNA stability and its role in control of gene expression in bacteria and phages. Annu. Rev. Genet. 33:193‐227.
   Gulnik, S.V., Afonina, E.I., Gustchina, E., Yu, B., Silva, A.M., Kim, Y., and Erickson, J.W. 2002. Utility of (His)6 tag for purification and refolding of proplasmepsin‐2 and mutants with altered activation properties. Protein Expr. Purif. 24:412‐419.
   Huang, J.S., Huang, S.S., and Tang, J. 1979. Cathepsin D isozymes from porcine spleens: Large scale purification and polypeptide chain arrangements. J. Biol. Chem. 254:11405‐11417.
   Lopez, P.J., Marchand, I., Joyce, S.A., and Dreyfus, M. 1999. The C‐terminal half of RNase E, which organizes the Escherichia coli degradosome, participates in mRNA degradation but not rRNA processing in vivo. Mol. Microbiol. 33:188‐199.
   Morrison, J.F. 1969. Kinetics of the reversible inhibition of enzyme‐catalysed reactions by tight‐binding inhibitors. Biochim. Biophys. Acta. 185:269‐286.
   Taylor, G., Hoare, M., Gray, D., and Marston, F. 1986. Biotechnology 4:553‐557.
   Westling, J., Yowell, C.A., Majer, P., Erickson, J.W., Dame, J.B., and Dunn, B.M. 1997. Plasmodium falciparum, P. vivax, and P. malariae: A comparison of the active site properties of plasmepsins cloned and expressed from three different species of the malaria parasite. Exp. Parasitol. 87:185‐193.
   Westling, J., Cipullo, P., Hung, S.H., Saft, H., Dame, J.B., and Dunn, B.M. 1999. Active site specificity of plasmepsin II. Protein Sci. 8:2001‐2009.
   Yasuda, Y., Kageyama, T., Akamine, A., Shibata, M., Kominami, E., Uchiyama, Y., and Yamamoto, K. 1999. Characterization of new fluorogenic substrates for the rapid and sensitive assay of cathepsin E and cathepsin D. J. Biochem. (Tokyo) 125:1137‐1143.
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