Performing a Purification of Glycoproteins and Polysaccharides with Agarose Wheat Germ Lectin

Extracted from Affinity Chromatography Principles and Methods, GE Healthcare, 2007

Wheat germ lectin can be used for group specific affinity purification of glycoproteins and polysaccharides. This lectin binds N-acetylglucosamine residues and reacts strongly with the chitobiose core of N-linked oligosaccharides. It also has affinity for N-acetylneuraminic acid. Wheat germ lectin is a dimeric, carbohydrate-free protein composed of two identical subunits, each with a molecular weight of approximately Mr 20 000.

Purification Options

  Binding capacity/ml medium Maximum operating flow Comments
Agarose Wheat Germ Lectin No data available. 75 cm/h* Supplied as a suspension  ready for column packing.

* See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm.

Performing a Separation

Binding buffer: 20 mM Tris-HCl, 0.5 M NaCl, pH 7.4

Elution buffer: 0.5 M N-acetylglucosamine, 20 mM Tris-HCl, 0.5 M NaCl, pH 7.4

Agarose Wheat Germ Lectin can be used with detergents, such as 1% deoxycholate or 0.5% Triton X-100.

  1. Pack the column (see Appendix 3, Column packing and preparation) and wash with at least 10 column volumes of binding buffer to remove preservative.
  2. Equilibrate the column with 10 column volumes of binding buffer.
  3. Apply the sample, using a low flow from 15 cm/h, during sample application (flow rate is the most significant factor for maximum binding).
  4. Wash with 5–10 column volumes of binding buffer or until no material appears in the eluent (monitored by UV absorption at A280 nm).
  5. Elute with 5 column volumes of elution buffer.

Use 0–0.5 M N-acetylglucosamine, 20 mM Tris-HCl, 0.5 M NaCl, pH 7.4 with a continuous gradient or step elution to improve resolution of complex samples containing glycoproteins with different affinities for the lectin.

Elute tightly bound substances with 20 mM acetate buffer, pH 4.5 or with an alternative sugar, for example triacetylchitotriose.

Higher concentrations of eluting substances may be necessary and recovery may be improved  by pausing the flow for some minutes during elution.

Cleaning

Wash with 5–10 column volumes of 20 mM Tris-HCl, 1 M NaCl, pH 8.5 and re-equilibrate immediately with binding buffer. Low concentrations of non-ionic detergents in the Tris-HCl buffer can be used if necessary, for example 0.1% Nonidet P-40.

Media Characteristics

  Ligand density Composition pH stability* Mean particle size
Agarose Wheat Germ Lectin 1–2 mg/ml Wheat Germ Lectin coupled to Sepharose 4B by CNBr method Short term 4–9 Long term 4–9 90 µm

* Long term refers to the pH interval over which the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance. Short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures.

Chemical Stability

Avoid exposure to conditions below pH 4.0 as this causes dissociation of the wheat germ lectin dimer.

Storage

Wash media and columns with 20% ethanol (use approximately 5 column volumes for packed media) and store at +4 to +8 °C.

Materials

     

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