Advertisement

Direct Conjugation of Bacterial Polysaccharides to Proteins by Squaric Acid Chemistry

  • Peng Xu
  • Pavol KováčEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1954)

Abstract

Bacterial polysaccharides that contain one amino group can be conjugated using squaric acid chemistry directly to a protein carrier. The conjugation is a two-step process consisting of labeling the polysaccharide with a squarate group and a reaction of the squarate formed with protein. The intermediate squarate derivative and the product glycoconjugate can be easily purified using centrifugal filtration devices. This method is experimentally simple and affords glycoconjugates with predictable carbohydrate–protein ratio (carbohydrate content), high conjugation efficiency, and excellent yield.

Key words

Bacterial polysaccharides O antigens O-specific polysaccharides Squaric acid chemistry Conjugation Glycoconjugate 

References

  1. 1.
    Dick JW, Beurret M (1989) Glycoconjugates of bacterial carbohydrate antigens. In: Cruse JM, Lewis JRE (eds) Conjugate vaccines, Contributions to microbiology and immunology, vol 10. Karger, Switzerland, pp 48–114Google Scholar
  2. 2.
    Tietze LF, Schroter C, Gabius S et al (1991) Conjugation of p-aminophenyl glycosides with squaric acid diester to a carrier protein and the use of neoglycoprotein in the histochemical detection of lectins. Bioconjug Chem 2:148–153CrossRefGoogle Scholar
  3. 3.
    Astronomo RD, Lee HK, Scanlan CN et al (2008) A glycoconjugate antigen based on the recognition motif of a broadly neutralizing human immunodeficiency virus antibody, 2g12, is immunogenic but elicits antibodies unable to bind to the self glycans of gp120. J Virol 82:6359–6368CrossRefGoogle Scholar
  4. 4.
    Hou S-J, Saksena R, Kováč P (2008) Preparation of glycoconjugates by dialkyl squarate chemistry revisited. Carbohydr Res 343:196–210CrossRefGoogle Scholar
  5. 5.
    Xu P, Alam MM, Kalsy A et al (2011) Simple, direct conjugation of bacterial o-sp-core antigens to proteins: development of cholera conjugate vaccines. Bioconjug Chem 22:2179–2185CrossRefGoogle Scholar
  6. 6.
    Udenfriend S, Stein S, Bohlen P, Dairman W, Leimgruber W, Weigele M (1972) Fluorescamine: a reagent for assay of amino acids, peptides, proteins, and primary amines in the picomole range. Science 178:871–872CrossRefGoogle Scholar
  7. 7.
    Chernyak A, Ogawa Y, Kováč P (2001) Conjugating oligosaccharides to proteins by squaric acid diester chemistry: rapid monitoring of the progress of conjugation, and recovery of the unused ligand. Carbohydr Res 330:479–486CrossRefGoogle Scholar
  8. 8.
    Saksena R, Chernyak A, Karavanov A et al (2003) Conjugating low molecular mass carbohydrates to proteins 1. Monitoring the progress of conjugation. Methods Enzymol 362:125–139CrossRefGoogle Scholar
  9. 9.
    Pluskal MG (2000) Microscale sample preparation. Nat Biotechnol 18:104–105CrossRefGoogle Scholar
  10. 10.
    Zhang Y, Sinaiko AR, Nelsestuen GL (2012) Glycoproteins and glycosylation: Apolipoprotein c3 glycoforms by top-down MALDI-TOF mass spectrometry. Methods Mol Biol 909:141–150PubMedGoogle Scholar
  11. 11.
    Xu P, Kelly M, Vann WF et al (2017) Conjugate vaccines from bacterial antigens by squaric acid chemistry: a closer look. Chembiochem 18:799–815CrossRefGoogle Scholar
  12. 12.
    Cohen S, Cohen S (1966) Preparation and reactions of derivatives of squaric acid. Alkoxy-, hydroxy-, and aminocyclobutenediones. J Am Chem Soc 88:1533–1536CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Laboratory of Bioorganic ChemistryNational Institutes of HealthBethesdaUSA

Personalised recommendations