Skip to main content

Preparation of Affinity Adsorbents and Purification of Lectins from Natural Sources

  • Protocol
  • First Online:
Lectins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1200))

  • 3486 Accesses

Abstract

Lectins are purified by affinity chromatography to take advantage of their carbohydrate-specific interactions. Highly efficient affinity adsorbents are powerful tools to obtain homogeneous lectins with distinct specificities. Here, we describe three methods to prepare affinity adsorbents by immobilizing carbohydrates or glycoconjugates on agarose gel beads. Because the ligands are immobilized via a stable and nonionic linkage under mild conditions, the adsorbents possess high binding capacity for lectins with low nonspecific adsorption and can withstand repeated use. The procedures require neither specialized techniques and apparatus nor highly toxic compounds. Using these adsorbents, many plant and animal lectins can be purified in a few steps.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Roth J (2011) Lectins for histochemical demonstration of glycans. Histochem Cell Biol 136:117–130

    Article  PubMed  CAS  Google Scholar 

  2. Lowe CR, Dean PDG (1974) Affinity chromatography. Wiley, London

    Google Scholar 

  3. Tercero JC, Diaz-Maurino T (1988) Affinity chromatography of fibrinogen on Lens culinaris agglutinin immobilized on CNBr-activated sepharose: study of the active groups involved in nonspecific adsorption. Anal Biochem 174:128–136

    Article  PubMed  CAS  Google Scholar 

  4. Matsumoto I, Mizuno Y, Seno N (1979) Activation of Sepharose with epichlorohydrin and subsequent immobilization of ligand for affinity adsorbent. J Biochem 85:1091–1098

    PubMed  CAS  Google Scholar 

  5. Matsumoto I, Kitagaki H, Akai Y, Ito Y, Seno N (1981) Derivatization of epoxy-activated agarose with various carbohydrates for the preparation of stable and high-capacity affinity adsorbents: their use for affinity chromatography of carbohydrate-binding proteins. Anal Biochem 116:103–110

    Article  PubMed  CAS  Google Scholar 

  6. Kojima K, Ogawa HK, Seno N, Matsumoto I (1992) Affinity purification and affinity characterization of carbohydrate-binding proteins in bovine kidney. J Chromatogr 597:323–330

    Article  PubMed  CAS  Google Scholar 

  7. Kitagaki H, Seno N, Yamaguchi H, Matsumoto I (1985) Isolation and characterization of a lectin from the fruit of Clerodendron trichotomum. J Biochem 97:791–799

    PubMed  CAS  Google Scholar 

  8. Ueno M, Ogawa H, Matsumoto I, Seno N (1991) A novel mannose-specific and sugar specifically aggregatable lectin from the bark of the Japanese pagoda tree (Sophora japonica). J Biol Chem 266:3146–3153

    PubMed  CAS  Google Scholar 

  9. Ina C, Sano K, Yamamoto-Takahashi M, Matsushita-Oikawa H, Takekawa H, Takehara Y, Ueda H, Ogawa H (2005) Screening for and purification of novel self-aggregatable lectins reveal a new functional lectin group in the bark of leguminous trees. Biochim Biophys Acta 1726:21–27

    Article  PubMed  CAS  Google Scholar 

  10. Nakagawa K, Nakamura K, Haishima Y, Yamagami M, Saito K, Sakagami H, Ogawa H (2009) Pseudoproteoglycan (pseudoPG) probes that simulate PG macromolecular structure for screening and isolation of PG-binding proteins. Glycoconj J 26:1007–1017

    Article  PubMed  CAS  Google Scholar 

  11. Matsumoto H, Natsume A, Ueda H, Saitoh T, Ogawa H (2001) Screening of a unique lectin from 16 cultivable mushrooms with hybrid glycoprotein and neoproteoglycan probes and purification of a novel N-acetylglucosamine-specific lectin from Oudemansiella platyphylla fruiting body. Biochim Biophys Acta 1526:37–43

    Article  PubMed  CAS  Google Scholar 

  12. Ito Y, Yamasaki Y, Seno N, Matsumoto I (1986) Preparation of high capacity affinity adsorbents using new hydrazino-carriers and their use for low and high performance affinity chromatography of lectins. J Biochem 99:1267–1272

    PubMed  CAS  Google Scholar 

  13. Kanamori A, Seno N, Matsumoto I (1986) Preparation of high-capacity affinity adsorbents using formyl carriers and their use for low-and high-performance affinity chromatography of trypsin-family proteases. J Chromatogr 363:231–242

    Article  CAS  Google Scholar 

  14. Matsumoto I, Seno N, Golovtchenko-Matsumoto AM, Osawa T (1980) Amination and subsequent derivatization of epoxy-activated agarose for the preparation of new affinity adsorbents. J Biochem 87:535–540

    PubMed  CAS  Google Scholar 

  15. Ueda H, Kojima K, Saitoh T, Ogawa H (1999) Interaction of a lectin from Psathyrella velutina mushroom with N-acetylneuraminic acid. FEBS Lett 448:75–80

    Article  PubMed  CAS  Google Scholar 

  16. Sasaki H, Hayashi A, Kitagaki-Ogawa H, Matsumoto I, Seno N (1987) Improved method for the immobilization of heparin. J Chromatogr 400:123–132

    Article  PubMed  CAS  Google Scholar 

  17. Kitagaki-Ogawa H, Yatohgo T, Izumi M, Hayashi M, Kashiwagi H, Matsumoto I, Seno N (1990) Diversities in animal vitronectins. Differences in molecular weight, immunoreactivity and carbohydrate chains. Biochim Biophys Acta 1033:49–56

    Article  PubMed  CAS  Google Scholar 

  18. Asanuma-Date K, Hirano Y, Le N, Sano K, Kawasaki N, Hashii N, Hiruta Y, Nakayama K, Umemura M, Ishikawa K, Sakagami H, Ogawa H (2012) Functional regulation of sugar assimilation by N-glycan-specific interaction of pancreatic alpha-amylase with glycoproteins of duodenal brush border membrane. J Biol Chem 287:23104–23118

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  19. Ueda H, Matsumoto H, Takahashi N, Ogawa H (2002) Psathyrella velutina mushroom lectin exhibits high affinity toward sialoglycoproteins possessing terminal N-acetylneuraminic acid alpha 2,3-linked to penultimate galactose residues of trisialyl N-glycans. Comparison with other sialic acid-specific lectins. J Biol Chem 277:24916–24925

    Article  PubMed  CAS  Google Scholar 

  20. Agrawal BB, Goldstein IJ (1965) Specific binding of concanavalin A to cross-linked dextran gels. Biochem J 96:23contd–25contd

    PubMed  CAS  PubMed Central  Google Scholar 

  21. LeVine D, Kaplan MJ, Greenaway PJ (1972) The purification and characterization of wheat-germ agglutinin. Biochem J 129:847–856

    PubMed  CAS  PubMed Central  Google Scholar 

Download references

Acknowledgement

We thank Prof. Isamu Matsumoto for supervision of development of preparation of the adsorbents.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Haruko Ogawa .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2014 Springer Science+Business Media New York

About this protocol

Cite this protocol

Date, K., Ogawa, H. (2014). Preparation of Affinity Adsorbents and Purification of Lectins from Natural Sources. In: Hirabayashi, J. (eds) Lectins. Methods in Molecular Biology, vol 1200. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1292-6_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-1292-6_5

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1291-9

  • Online ISBN: 978-1-4939-1292-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics