Lectin Affinity Chromatography

Hudson H. Freeze1

1 La Jolla Cancer Research Foundation, La Jolla
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 9.1
DOI:  10.1002/0471140864.ps0901s00
Online Posting Date:  May, 2001
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Abstract

This unit describes the use of lectins for preparative glycoprotein purification. Con A‐Sepharose and WGA‐agarose are used for convenience and availability. Instructions are given for a small‐scale pilot procedure to test for lectin binding and to determine elution conditions. There are many variations on the basic procedure in the literature, but all use the same principles: bind the protein to immobilized lectin through its sugar chain, wash away unbound protein, and elute bound protein with a simple sugar that resembles the sugar ligand of the bound protein.

     
 
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Table of Contents

  • Basic Protocol 1: Con A–Sepharose Affinity Chromatography
  • Support Protocol 1: Pilot Study to Determine Lectin Binding and Elution Conditions
  • Alternate Protocol 1: Wheat Germ Agglutinin (WGA)–Agarose Affinity Chromatography
  • Reagents and Solutions
  • Commentary
  • Tables
     
 
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Materials

Basic Protocol 1: Con A–Sepharose Affinity Chromatography

  Materials
  • 10 mg/ml Con A–Sepharose (Pharmacia Biotech or Sigma)
  • recipeColumn buffer (see recipe)
  • 0.5 M αMM in recipecolumn buffer
  • Protein sample in recipecolumn buffer
  • 1.5 × 30–cm glass or disposable chromatographic column
  • Glass wool
NOTE: Carry out this procedure at room temperature if the protein to be isolated will tolerate this condition. If not, carry it out in a cold room, and prechill all solutions to maintain temperature.

Support Protocol 1: Pilot Study to Determine Lectin Binding and Elution Conditions

  • Sepharose 4B (Pharmacia Biotech) or other beaded gel to fill space in the tubes
  • αMM: 0, 0.1, 0.2, 0.4, 0.8, 1.0, and 1.5 M concentrations, in recipecolumn buffer

Alternate Protocol 1: Wheat Germ Agglutinin (WGA)–Agarose Affinity Chromatography

  • 5 mg/ml wheat germ agglutinin (WGA)–agarose (E‐Y Labs, Pharmacia Biotech, or Sigma)
  • PBS ( appendix 2E)
  • 0.1 M N‐acetyl‐D‐glucosamine (GlcNAc) in PBS
  • Protein sample in PBS
  • 1.0 × 10–cm glass or disposable column (or other column whose length is 10 times its diameter)
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Figures

Videos

Literature Cited

Literature Cited
   Cummings, R.D. 1994. Use of lectins in analysis of glycoconjugates. Methods Enzymol. 230:66‐86.
   Ketcham, C.M. and Kornfeld, S. 1992. Purification of UDP‐N‐acetylglucosamine: Glycoprotein N‐acetylglucosamine‐1‐phosphotransferase from Acanthamoeba castellanii and identification of a subunit of the enzyme. J. Biol. Chem. 267:11645‐11653.
   Li, M. Jourdian, G. W. 1991. Isolation and characterization of the two glycosylation isoforms of low molecular weight mannose‐6‐phosphate receptor from bovine testis. J. Biol. Chem. 266:17621‐17630.
   Manzi, A.E. 1993. Phenol–sulfuric acid assay for hexoses and pentoses. In Current Protocols in Molecular Biology (F.A. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 17.9.1‐17.9.2. John Wiley & Sons, New York.
Key References
   Beeley, J.G. 1984. Glycoprotein and proteoglycan techniques. In Laboratory Techniques in Biochemistry and Molecular Biology, pp.29‐99. Elsevier Science Publishing, New York.
  Good discussions about general properties of glycoconjugates.
   Cummings, R.D. 1994. See above.
  Most current description of using lectins to characterize sugar chain structures. Lots of details on procedures to analyze free sugar chains rather than glycoproteins.
   Dulaney, J.T. 1979. Binding interactions of glycoproteins with lectins. Mol. Cell. Biochem. 21:43‐62.
  Lists many references and conditions used for lectins in protein purification.
   Li, M. and Jourdian, G.W. 1991. See above.
  Excellent example of separation of various glycoforms using lectin–affinity chromatography.
   Montreuil, J., Bouquelet, S., Debary, H., Fournat, B., Spik, G., and Strecker, G. 1986. Glycoproteins. In Carbohydrate Analysis: A Practical Approach (M.F. Chaplin and J.F. Kennedy, eds.) pp. 166‐173. IRL Press, Washington, D.C.
  Lists several conditions for selected lectin‐affinity purifications of proteins.
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