Purification and Characterization of Gingipains

Jan Potempa1, Ky‐Anh Nguyen2

1 Jagiellonian University, Krakow, Poland, 2 Westmead Centre for Oral Health, Sydney, Australia
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 21.20
DOI:  10.1002/0471140864.ps2120s49
Online Posting Date:  August, 2007
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Abstract

Gingipains are cysteine proteases produced in large quantities by Porphyromonas gingivalis which together constitute important virulence factors in the pathogenesis of periodontal disease by that organism. Described is this unit is an efficient procedure for the purification of gingipains from the growth medium of P. gingivalis strain HG66, along with detailed protocols for growth of the organism and basic characterization of the purified proteases. The purification procedure consists of acetone precipitation followed by gel filtration to separate high‐molecular‐mass gingipains (Kgp and HRgpA) from RgpB. Kgp and HRgpA are further separated on Arg‐Sepharose by the virtue of differential elution from the affinity matrix with lysine (Kgp) and arginine (HRgpA) eluant. Obtained from these procedures, the gingipains are stable and can be stored at −80°C for years without loss of activity. Curr. Protoc. Protein Sci. 49:21.20.1‐21.20.27. © 2007 by John Wiley & Sons, Inc.

Keywords: gingipains; purification; Porphyromonas gingivalis; protease

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Maintenance and Growth of P. gingivalis HG66
  • Basic Protocol 2: Purificaton of Gingipains
  • Basic Protocol 3: Gingipain Enzymatic Activity Assay
  • Basic Protocol 4: Titration of Gingipains to Determine Concentration of Active Sites
  • Support Protocol 1: Sds‐Page Analysis of Gingipains
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Maintenance and Growth of P. gingivalis HG66

  Materials
  • P. gingivalis, frozen (strain HG66, isolated in the authors' laboratory, has been submitted to the German Collection of Microorganisms and Cell Cultures; http://www.dsmz.de)
  • Liquid growth medium for P. gingivalis: Enriched Trypticase Soy Broth (eTSB), prereduced in anaerobic atmosphere >4 hr (optimally overnight) before use or prepared in tightly sealed culture flasks if organisms are to be grown outside the anaerobic chamber (see recipe)
  • Gram stain kit (Sigma)
  • Anaerobic blood agar plates (see recipe), prereduced in anaerobic atmosphere >4 hr (optimally overnight) before use
  • p‐Dimethylaminocinnamaldehyde (DMCA) reagent: dissolve 1 g of DMCA (e.g., Sigma) in 99 ml of 10% (v/v) HCl; store in brown bottle at 4°C
  • 60% (v/v) glycerol
  • Liquid N 2
  • Culture tubes
  • Orbital shaker incubator
  • Anaerobic chamber or jars filled with 90% (v/v) N 2/5% (v/v) CO 2/5% (v/v) H 2 gas mixture
  • Whatman no. 1 filter paper, sterile
  • 2‐, 5, 20‐, and 60‐ml syringes
  • 18‐ and 16‐G needles
  • 2‐ml cryovials (e.g., Nunc or Sarstedt)
  • Tube carriers and Dewar flask for liquid N 2
  • Additional reagents and equipment for basic bacteriological techniques including inoculation ( appendix 4A), isolating bacterial colonies by serial dilution ( appendix 4B), monitoring bacterial growth by spectrophotometry ( appendix 4B), and determining gingipain activity ( protocol 3)
NOTE: All reagents and equipment coming into contact with living cells must be sterile, and aseptic techniques should be used throughout.

Basic Protocol 2: Purificaton of Gingipains

  Materials
  • P. gingivalis growing in large‐scale culture ( protocol 1)
  • Acetone (chilled to −20°C)
  • Dithiopyridine disulfide buffer (see recipe)
  • Gel‐filtration buffer (see recipe)
  • Sephadex G‐150 (particle size, 40 to 120 µm) or equivalent gel‐filtration medium such as Superdex‐200 (GE Healthcare)
  • Arginine‐Sepharose (GE Healthcare, cat no. 17‐0542‐01)
  • Affi‐buffer (see recipe)
  • 0.5 M NaCl in Affi‐buffer
  • 750 mM L‐lysine in Affi‐buffer for forming 0 to 750 mM linear gradient in gradient former
  • 100 mM L‐arginine in Affi‐buffer for forming 0 to 100 mM linear gradient in gradient former
  • DE‐52 anion‐exchange resin (Whatman)
  • Ion‐exchange buffer: 50 mM Bis‐Tris, pH 6.5/1 mM CaCl 2
  • 200 mM NaCl in ion‐exchange buffer for forming 0 to 200 mM linear gradient in gradient former
  • Refrigerated centrifuge with Sorvall SCL‐3000 and SS‐34 or equivalent rotors
  • Broad‐bottom 2.8‐liter Pyrex culture flask
  • 2‐liter graduated cylinder
  • Styrofoam box filled with dry ice pellets
  • Dialysis bags, 12,000 to 14,000 MWCO
  • Ultracentrifuge with Beckman 50.2 Ti or equivalent rotor
  • Ultrafiltration concentrator with MWCO 30,000 membrane (PM‐30, Amicon)
  • Chromatography column for gel filtration (Fisher Scientific): 4.8 × 120–cm (cat. no. K420830‐1220) with packing reservoir (cat. no. K420837‐0040) and flow adaptor (cat. no. K420836‐0040); equivalent items also available from VWR Scientific
  • Chromatography columns for affinity and ion‐exchange chromatography (GE Healthcare): two XK16/40 columns (cat. no. 18‐8774‐01) with packing reservoir (cat. no. 18‐8793‐01), or equivalent
  • Liquid chromatography system such as ÄKTAprime plus (GE Healthcare, 11‐0013‐12), or equivalent, including:
    • Gradient former
    • Peristaltic pump
    • Fraction collector
    • Flow UV detector
  • 0.2‐µm syringe filter units (Fisher Scientific)
  • Funnel
  • Spectrophotometer and 1‐cm path length quartz cuvettes
  • Additional reagents and equipment for dialysis ( appendix 3B), assay of gingipain activity ( protocol 3), and spectrophotometric protein assay (unit 3.1)
NOTE: All purification procedures are performed at 4°C and gingipain activity should be determined after each step of purification.

Basic Protocol 3: Gingipain Enzymatic Activity Assay

  Materials
  • 2× gingipain assay buffer (see recipe)
  • Gingipain‐containing samples of interest
  • Substrate solutions (store up to 1 year at –20°C):
    • Rgp substrate: 10 mM Bz‐L‐arginine‐pNA (L‐BAPNA; Sigma) in DMSO
    • Kgp substrate: 10 mM Ac‐Lys‐pNA (Bachem) in DMSO
  • p‐Nitroaniline (pNA; Sigma)
  • Dimethylsulfoxide (DMSO)
  • Gingipain enzymes (purified as in protocol 2)
  • 96‐well flat‐bottom clear microtiter plate
  • Spectrophotometer equipped with colorimetric plate reader and, ideally, with temperature‐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.

Basic Protocol 4: Titration of Gingipains to Determine Concentration of Active Sites

  • Activation buffer (A‐buffer; see recipe)
  • Reaction buffer (R‐buffer; see recipe)
  • 10 mM FFR‐CMK (see recipe)
  • 10 mM Z‐FK‐BMK (see recipe)
  • 1 mM HCl
  • Inhibition buffer (I‐buffer; see recipe)
  • A‐buffer (see recipe) supplemented with 200 µM Ac‐LVK‐ald (add from 10 mM Ac‐LVK‐ald stock, see recipe), for Rgp assay
  • R‐buffer (see recipe) supplemented with 100 µM AcLVK‐ald (add from 10 mM Ac‐LVK‐ald stock, see recipe), for Rgp assay
  • I‐buffer (see recipe) supplemented with 100 µM AcLVK‐ald (add from 10 mM Ac‐LVK‐ald stock, see recipe), for Rgp assay
  • A‐buffer (see recipe) supplemented with 50 µM leupeptin (add from 10 mM leupeptin stock, see recipe), for Kgp assay
  • R‐buffer (see recipe) supplemented with 25 µM leupeptin (add from 10 mM leupeptin stock, see recipe), for Kgp assay
  • I‐buffer (see recipe) supplemented with 25 µM leupeptin (add from 10 mM leupeptin stock, see recipe), for Kgp assay
  • Additional reagents and equipment for colorimetric protein assay (BCA assay; unit 3.4)

Support Protocol 1: Sds‐Page Analysis of Gingipains

  Materials
  • Gingipain sample for analysis
  • 200 mM tosyl‐L‐lysine chloromethylketone; (TLCK; Sigma or Calbiochem) in DMSO
  • 5× nonreducing sample buffer (see recipe)
  • Reducing reagent: 10% (v/v) 2‐mercaptoethanol (prepare fresh)
  • 100°C water bath
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1)
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Figures

Videos

Literature Cited

   Ally, N., Whisstock, J.C., Sieprawska‐Lupa, M., Potempa, J., Le Bonniec, B.F., Travis, J., and Pike, R.N. 2003. Characterization of the specificity of arginine‐specific gingipains from Porphyromonas gingivalis reveals active site differences between different forms of the enzymes. Biochemistry 42:11693‐11700.
   Banbula, A., Mak, P., Travis, J., and Potempa, J. 2001. Arginine‐specific proteinase from Porphyromonas gingivalis as a convenient tool in protein chemistry. Biol. Chem. 382:1399‐1404.
   Barrett, A.J., and Rawlings, N.D. 2001. Evolutionary lines of cysteine peptidases. Biol Chem. 382:727‐733.
   Chen, Z., Potempa, J., Polanowski, A., Renvert, S., Wikstrom, M., and Travis, J. 1991. Stimulation of proteinase and amidase activities in Porphyromonas (Bacteroides) gingivalis by amino acids and dipeptides. Infect. Immun. 59:2846‐2850.
   Ciborowski, P., Nishikata, M., Allen, R.D., and Lantz, M.S. 1994. Purification and characterization of two forms of a high‐molecular‐weight cysteine proteinase (porphypain) from Porphyromonas gingivalis. J. Bacteriol. 176:4549‐4557.
   Mikolajczyk‐Pawlinska, J., Boatright, K.M., Stennicke, H.R., Nazif, T., Potempa, J., Bogyo, M., and Salvesen, G.S. 2003. Sequential autolytic processing activates the zymogen of Arg‐gingipain. J. Biol. Chem. 278:10458‐10464.
   O'Brien‐Simpson, N.M., Veith, P.D., Dashper, S.G., and Reynolds, E.C. 2003. Porphyromonas gingivalis gingipains: The molecular teeth of a microbial vampire. Curr. Protein Pept. Sci. 4:409‐426.
   Pike, R., McGraw, W., Potempa, J., and Travis, J. 1994. Lysine‐ and arginine‐specific proteinases from Porphyromonas gingivalis: Isolation and evidence for the existence of complexes with hemagglutinins. J. Biol. Chem. 269:406‐411.
   Potempa, J., Pike, R., and Travis, J. 1995a. The multiple forms of trypsin‐like activity present in various strains of Porphyromonas gingivalis are due to the presence of either Arg‐gingipain or Lys‐gingipain. Infect. Immun. 63:1176‐1182.
   Potempa, J., Pavloff, N., and Travis, J. 1995b. Porphyromonas gingivalis: A proteinase/gene accounting audit. Trends Microbiol. 3:430‐434.
   Potempa, J., Pike, R., and Travis, J. 1997. Titration and mapping of the active site of cysteine proteinases from Porphyromonas gingivalis (Gingipains) using peptidyl chloromethanes. Biol. Chem. 378:223‐230.
   Potempa, J., Mikolajczyk‐Pawlinska, J., Brassell, D., Nelson, D., Thogersen, I.B., Enghild, J.J., and Travis, J. 1998. Comparative properties of two cysteine proteinases (gingipains R), the products of two related but individual genes of Porphyromonas gingivalis. J. Biol. Chem. 273:21648‐21657.
   Potempa, J., Sroka, J., Imamura, T., and Travis, J. 2003. Gingipains, the major cysteine proteinase and virulence factors of Porphyromonas gingivalis: Structure, function and assembly of multidomain protein complexes. Curr. Protein Pept. Sci. 4:397‐407.
   Rangarajan, M., Aduse‐Opoku, J., Slaney, J.M., Young, K.A., and Curtis, M.A. 1997. The prpR1 and prR2 arginine‐specific protease genes of Porphyromonas gingivalis W50 produce five biochemically distinct enzymes. Mol. Microbiol. 23:955‐965.
   Sato, K., Sakai, E., Veith, P.D., Shoji, M., Kikuchi, Y., Yukitake, H., Ohara, N., Naito, M., Okamoto, K., Reynolds, E.C., and Nakayama, K. 2005. Identification of a new membrane‐associated protein that influences transport/maturation of gingipains and adhesins of Porphyromonas gingivalis. J. Biol. Chem. 280:8668‐8677.
   Scott, C.F., Whitaker, E.J., Hammond, B.F., and Colman, R.W. 1993. Purification and characterization of a potent 70‐kDa thiol lysyl‐proteinase (Lys‐gingivain) from Porphyromonas gingivalis that cleaves kininogens and fibrinogen. J. Biol. Chem. 268:7935‐7942.
   Shoji, M., Ratnayake, D.B., Shi, Y., Kadowaki, T., Yamamoto, K., Yoshimura, F., Akamine, A., Curtis, M.A., and Nakayama, K. 2002. Construction and characterization of a nonpigmented mutant of Porphyromonas gingivalis: Cell surface polysaccharide as an anchorage for gingipains. Microbiology 148:1183‐1191.
  Shi, Y., Ratnayake, D.B., Okamoto, K., Abe, N., Yamamoto, K., and Nakayama, K. 1999. Genetic analyses of proteolysis, hemoglobin binding, and hemagglutination of Porphyromonas gingivalis: Construction of mutants with a combination of rgpA, rgpB, kgp, and hagA. J. Biol. Chem. 274:17955‐17960.
   Wikstrom, M., Potempa, J., Polanowski, A., Travis, J., and Renvert, S. 1994. Detection of Porphyromonas gingivalis in gingival exudate by dipeptide enhanced trypsin‐like activity. J. Periodontol. 65:47‐55.
Internet Resources
   http://www1.amershambiosciences.com/aptrix/upp00919.nsf/(FileDownload)?OpenAgent&docid=6EEE47990D9F933EC1256F90000DD697&file=18102218AI.pdf
  Gel Filtration: Principles and Methods, from Amersham Biosciences.
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