Performing a Purification of Branched Mannoses, Carbohydrates with Terminal Mannose or Glucose (αMan > αGlc > GlcNAc) with Con A Sepharose 4B

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

Concanavalin A (Con A) is a tetrameric metalloprotein isolated from Canavalia ensiformis (jack bean). Con A binds molecules containing a-D-mannopyranosyl, a-D-glucopyranosyl and sterically related residues. The binding sugar requires the presence of C-3, C-4 and C-5 hydroxyl groups for reaction with Con A. Con A can be used for applications such as:

  • Separation and purification of glycoproteins, polysaccharides and glycolipids.
  • Detection of changes in composition of carbohydrate-containing substances, e.g. during development.
  • Isolation of cell surface glycoproteins from detergent-solubilized membranes.
  • Separation of membrane vesicles into “inside out” and “right side out” fractions.

Purification Options

  Binding capacity/ml medium Maximum operating flow Comments
Con A Sepharose 4B  Porcine thyroglobulin,  20–45 mg 75 cm/h** Supplied as a suspension ready for  column packing.*

* Supplied in acetate buffer solution (0.1 M, pH 6) containing 1 M NaCl, 1 mM CaCl2, 1 mM MgCl2, 1 mM MnCl2, 20% ethanol.

** 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.

Purification Example

Figure 47 shows the purification of a human cell surface alloantigen on Con A Sepharose 4B.

Purification of a cell surface antigen on Con A Sepharose 4B

Fig. 47. Purification of a cell surface antigen on Con A Sepharose 4B. Solid circles represent antigen activity and open circles represent protein profile. Reproduced courtesy of the authors and publishers. 

Reference: A novel heteromorphic human cell surface alloantigen, gp60, defined by a human monoclonal antibody. Schadendorf, D. et al., J. Immunol. 142, 1621 (1989).

Performing a Separation

Binding buffer: 20 mM Tris-HCl, 0.5 M NaCl, 1 mM MnCl2, 1 mM CaCl2, pH 7.4

Elution buffer: 0.1–0.5 M methyl-α-D-glucopyranoside (methyl-α-D-glucoside) or methyl-α-D-mannopyranoside (methyl-α-D-mannoside), 20 mM Tris-HCl, 0.5 M NaCl, pH 7.4

  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 to obtain 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.

Recovery from Con A Sepharose 4B is decreased in the presence of detergents. If the glycoprotein of interest needs the presence of detergent and has affinity for either lentil lectin or wheat germ lectin, the media Lentil Lectin Sepharose 4B or Agarose Wheat Germ Lectin may provide a suitable alternative to improve recovery.

For complex samples containing glycoproteins with different affinities for the lectin, a continuous gradient or step elution may improve resolution. Recovery can sometimes be improved by pausing the flow for some minutes during elution.

Elute tightly bound substances by lowering the pH. Note that elution below pH 4.0 is not recommended and that below pH 5.0 Mn2+ will begin to dissociate from the Con A and the column will need to be reloaded with Mn2+ before reuse.

Cleaning

Wash with 10 column volumes of 0.5 M NaCl, 20 mM Tris-HCl, pH 8.5, followed by 0.5 M NaCl, 20 mM acetate, pH 4.5. Repeat 3 times before re-equilibrating with binding buffer.

Remove strongly bound substances by:

  • washing with 0.1 M borate, pH 6.5 at a low flow rate
  • washing with 20% ethanol or up to 50% ethylene glycol
  • washing with 0.1% Triton X-100 at +37 °C for one minute

Re-equilibrate immediately with 5 column volumes of binding buffer after any of these wash steps.

Media Characteristics

  Ligand density Composition pH stability* Mean particle size
Con A Sepharose 4B 10–16 mg/ml Con A 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

Stable to all commonly used aqueous buffers. Avoid 8 M urea, high concentrations of guanidine hydrochloride, chelating agents such as EDTA, or solutions with pH < 4.0 as these remove the manganese from the lectin or dissociate Con A, resulting in loss of activity.

Storage

Wash media and columns with 20% ethanol in 0.1 M acetate, 1 M NaCl, 1 mM CaCl2, 1 mM MnCl2, 1 mM MgCl2, pH 6 (use approximately 5 column volumes for packed media) and store at +4 to +8 °C.

Materials

     

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