Special Considerations for Glycoproteins and Their Purification

Hudson H. Freeze1

1 La Jolla Cancer Research Foundation, La Jolla, California
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 17.1
DOI:  10.1002/0471142727.mb1701s22
Online Posting Date:  May, 2001
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Abstract

This unit begins by describing some properties of glycoproteins (e.g., subcellular location and solubility) that may be useful in determining which purification techniques to try. This discussion is followed by two protocols describing preparative glycoprotein purification using lectin‐affinity chromatography, as well as an outline for a small‐scale pilot procedure designed to check lectin binding and elution conditions. Lectins are often used for purifying glycoproteins because, in contrast to conventional purification procedures (e.g., gel filtration and ion‐exchange chromatography) that exploit general physical properties of glycoproteins, lectins recognize specific three‐dimensional structures created by a cluster of sugar residues. Conventional purification procedures are generally tried before applying lectin‐affinity chromatography.

     
 
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Table of Contents

  • SECTION I: Special Considerations of Glycoconjugates and Their Purification
  • Special Considerations in the Purification of Glycoproteins
  • 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
     
 
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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
  • 0.5 M αMM in recipecolumn buffer
  • Protein sample in recipecolumn buffer
  • Glass wool
  • 1.5 × 30–cm glass or disposable chromatographic column
  • Additional reagents and equipment for degassing solutions (unit 10.12), phenol–sulfuric acid assay for sugars (unit 17.9), and specific assay for detecting the protein of interest
NOTE: This procedure should be carried out 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

  Additional Materials
  • 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

  Additional Materials
  • 5 mg/ml wheat germ agglutinin (WGA)–agarose (E‐Y Laboratories, Pharmacia Biotech, Sigma)
  • Phosphate‐buffered saline (PBS; appendix 22)
  • 0.1 M N‐acetylglucosamine (GlcNAc) in PBS
  • Protein sample in PBS
  • 1.0 × 10–cm glass or disposable column
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Figures

Videos

Literature Cited

Literature Cited
   Gibbons, R.A. 1972. Physico‐chemical methods for the determination of the purity, molecular size and shape of glycoproteins. In Glycoproteins: Their Composition, Structure and Function (A. Gottschalk, ed.) pp. 31‐140. Elsevier Science Publishing, New York.
   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.
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.
   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.
   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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