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Bacterial Pathogenesis pp 319–329Cite as

Generating and Purifying Fab Fragments from Human and Mouse IgG Using the Bacterial Enzymes IdeS, SpeB and Kgp

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1535))

Abstract

Fab fragments are valuable research tools in various areas of science including applications in imaging, binding studies, removal of Fc-mediated effector functions, mass spectrometry, infection biology, and many others. The enzymatic tools for the generation of Fab fragments have been discovered through basic research within the field of molecular bacterial pathogenesis. Today, these enzymes are widely applied as research tools and in this chapter, we describe methodologies based on bacterial enzymes to generate Fab fragments from both human and mouse IgG. For all human IgG subclasses, the IdeS enzyme from Streptococcus pyogenes has been applied to generate F(ab′)2 fragments that subsequently can be reduced under mild conditions to generate a homogenous pool of Fab′ fragments. The enzyme Kgp from Porphyromonas gingivalis has been applied to generate intact Fab fragments from human IgG1 and the Fab fragments can be purified using a CH1-specific affinity resin. The SpeB protease, also from S. pyogenes, is able to digest mouse IgGs and has been applied to digest antibodies and Fab fragments can be purified on light chain affinity resins. In this chapter, we describe methodologies that can be used to obtain Fab fragments from human and mouse IgG using bacterial proteases.

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References

  1. Liu H, May K (2012) Disulfide bond structures of IgG molecules: structural variations, chemical modifications and possible impacts to stability and biological function. MAbs 4:17–23

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Sjögren J, Collin M (2014) Bacterial glycosidases in pathogenesis and glycoengineering. Future Microbiol 9:1039–1051

    Article  PubMed  Google Scholar 

  3. Collin M, Olsén A (2003) Extracellular enzymes with immunomodulating activities: variations on a theme in Streptococcus pyogenes. Infect Immun 71:2983–2992

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Walker MJ, Barnett TC, Mcarthur JD et al (2014) Disease manifestations and pathogenic mechanisms of group a Streptococcus. Clin Microbiol Rev 27:264–301

    Article  PubMed  PubMed Central  Google Scholar 

  5. Nizet V (2007) Understanding how leading bacterial pathogens subvert innate immunity to reveal novel therapeutic targets. J Allergy Clin Immunol 120:13

    Article  CAS  PubMed  Google Scholar 

  6. von Pawel-Rammingen U, Johansson BP, Björck L (2002) IdeS, a novel streptococcal cysteine proteinase with unique specificity for immunoglobulin G. EMBO J 21:1607–1615

    Article  Google Scholar 

  7. Abe N, Kadowaki T, Okamoto K et al (1998) Biochemical and functional properties of lysine-specific cysteine proteinase (Lys-gingipain) as a virulence factor of Porphyromonas gingivalis in periodontal disease. J Biochem 123:305–312

    Article  CAS  PubMed  Google Scholar 

  8. Collin M, Olsén A (2001) Effect of SpeB and EndoS from Streptococcus pyogenes on human immunoglobulins. Infect Immun 69:7187–7189

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Vincents B, von Pawel-Rammingen U, Björck L et al (2004) Enzymatic characterization of the streptococcal endopeptidase, IdeS, reveals that it is a cysteine protease with strict specificity for IgG cleavage due to exosite binding. Biochemistry 43:15540–15549

    Article  CAS  PubMed  Google Scholar 

  10. Sjögren J, Olsson F, Beck A (2016) Rapid and improved characterization of therapeutic antibodies and antibody related products using IdeS digestion and subunit analysis, The Analyst 141(11):3114–3125

    Google Scholar 

  11. An Y, Zhang Y, Mueller H-M et al (2014) A new tool for monoclonal antibody analysis: application of IdeS proteolysis in IgG domain-specific characterization. MAbs 6:879–893

    Article  PubMed  PubMed Central  Google Scholar 

  12. Beck A, Debaene F, Diemer H et al (2015) Cutting-edge mass spectrometry characterization of originator, biosimilar and biobetter antibodies. J Mass Spectrom 50:285–297

    Article  CAS  PubMed  Google Scholar 

  13. Beck A, Terral G, Debaene F et al (2016) Cutting-edge mass spectrometry methods for the multi-level structural characterization of antibody-drug conjugates. Expert Rev Proteomics 13:157–183

    Article  CAS  PubMed  Google Scholar 

  14. Johansson BP, Shannon O, Björck L (2008) IdeS: a bacterial proteolytic enzyme with therapeutic potential. PLoS One 3:e1692

    Article  PubMed  PubMed Central  Google Scholar 

  15. Winstedt L, Järnum S, Nordahl EA et al (2015) Complete removal of extracellular IgG antibodies in a randomized dose-escalation phase I study with the bacterial enzyme IdeS - a novel therapeutic opportunity. PLoS One 10:e0132011

    Article  PubMed  PubMed Central  Google Scholar 

  16. Mysak J, Podzimek S, Sommerova P et al (2014) Porphyromonas gingivalis: major periodontopathic pathogen overview. J Immunol Res 2014:1–8

    Article  Google Scholar 

  17. Vincents B, Guentsch A, Kostolowska D et al (2011) Cleavage of IgG1 and IgG3 by gingipain K from Porphyromonas gingivalis may compromise host defense in progressive periodontitis. FASEB J 25:3741–3750

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. de Diego I, Veillard F, Sztukowska MN et al (2014) Structure and mechanism of cysteine peptidase gingipain K (Kgp), a major virulence factor of Porphyromonas gingivalis in periodontitis. J Biol Chem 289:32291–32302

    Article  PubMed  PubMed Central  Google Scholar 

  19. Shen A, Bogyo M (2012) Chapter 523 Gingipain K, Elsevier Ltd. Handbook of Proteolytic Enzymes, (2012) 2337-2344. doi:10.1016/B978-0-12-382219-2.00521-4

  20. Elliott SD (1945) A proteolytic enzyme produced by group A streptococci with special reference to its effect on the type-specific m antigen. J Exp Med 81:573–592

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Elliott SD (1950) The crystallization and serological differentiation of a streptococcal proteinase and its precursor. J Exp Med 92:201–218

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Olsson F, Norgren M, Mejare M et al (2013) Initial characterization of a recombinantly modified SpeB from Streptococcus pyogenes for antibody subunit domain generation. Poster presented at CASSS Mass Spectrometry Conference, Boston, MA, USA

    Google Scholar 

  23. Persson H, Vindebro R, von Pawel-Rammingen U (2013) The streptococcal cysteine protease SpeB is not a natural immunoglobulin-cleaving enzyme. Infect Immun 81:2236–2241

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Collin M, Svensson MD, Sjöholm AG et al (2002) EndoS and SpeB from Streptococcus pyogenes inhibit immunoglobulin-mediated opsonophagocytosis. Infect Immun 70:6646–6651

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Nelson DC, Garbe J, Collin M (2011) Cysteine proteinase SpeB from Streptococcus pyogenes - a potent modifier of immunologically important host and bacterial proteins. Biol Chem 392:1077–1088

    Article  CAS  PubMed  Google Scholar 

  26. Eriksson A, Norgren M (2003) Cleavage of antigen-bound immunoglobulin G by SpeB contributes to streptococcal persistence in opsonizing blood. Infect Immun 71:211–217

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Singh R, Whitesides GM (1993) Thiol—disulfide interchange. In: Patai S, Rappoport Z (eds) Sulphur-containing functional groups (1993). John Wiley & Sons, Inc., Chichester, UK

    Google Scholar 

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Author information

Authors and Affiliations

  1. Genovis AB, Box 790, 22007, Lund, Sweden

    Jonathan Sjögren Ph.D., Linda Andersson M.Sc., Malin Mejàre Ph.D. & Fredrik Olsson M.Sc.

Authors
  1. Jonathan Sjögren Ph.D.
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  2. Linda Andersson M.Sc.
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  3. Malin Mejàre Ph.D.
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  4. Fredrik Olsson M.Sc.
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Corresponding author

Correspondence to Jonathan Sjögren Ph.D. .

Editor information

Editors and Affiliations

  1. Department of Clinical Sciences, Lund University, Division of Infection Medicine, Lund, Sweden

    Pontus Nordenfelt

  2. Department of Clinical Sciences, Lund University, Division of Infection Medicine, Lund, Sweden

    Mattias Collin

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Sjögren, J., Andersson, L., Mejàre, M., Olsson, F. (2017). Generating and Purifying Fab Fragments from Human and Mouse IgG Using the Bacterial Enzymes IdeS, SpeB and Kgp. In: Nordenfelt, P., Collin, M. (eds) Bacterial Pathogenesis. Methods in Molecular Biology, vol 1535. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6673-8_21

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  • DOI: https://doi.org/10.1007/978-1-4939-6673-8_21

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6671-4

  • Online ISBN: 978-1-4939-6673-8

  • eBook Packages: Springer Protocols

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