Performing a Purification of Detergent-Solubilized Membrane Glycoproteins, Cell Surface Antigens and Viral Glycoproteins with Lentil Lectin Sepharose 4B

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

Lentil lectin binds a-D-glucose and a-D-mannose residues and is an affinity ligand used for the purification of glycoproteins including detergent-solubilized membrane glycoproteins, cell surface antigens and viral glycoproteins. Lentil lectin is the haemagglutinin from the common lentil, Lens culinaris. When compared to Con A, it distinguishes less sharply between glucosyl and mannosyl residues and binds simple sugars less strongly. It also retains its binding ability in the presence of 1% sodium deoxycholate. For these reasons Lentil Lectin Sepharose 4B is useful for the purification of detergent-solubilized membrane proteins, giving high capacities and extremely high recoveries.

Purification Options

  Binding capacity/ml medium Maximum operating flow Comments
Lentil Lectin Sepharose 4B Porcine thyroglobulin, 16–35 mg 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, 1 mM MnCl2, 1 mM CaCl2, pH 7.4.

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

Buffers for soluble glycoproteins:

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

Elution buffer: 0.3 M methyl-α-D-mannopyranoside, 20 mM Tris-HCl, 0.5 M NaCl, pH 7.4

Buffers for detergent-solubilized proteins:

Equilibrate column with the buffer 20 mM Tris-HCl, 0.5 M NaCl, 1 mM MnCl2, 1 mM CaCl2, pH 7.4, to ensure saturation with Mn2+ and Ca2+.

Binding buffer: 20 mM Tris-HCl, 0.5 M NaCl, 0.5% sodium deoxycholate, pH 8.3

Elution buffer: 0.3 M methyl-α-D-mannopyranoside, 20 mM Tris-HCl, 0.5 M NaCl, 0.5% sodium deoxycholate, pH 8.3

  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 using a step or gradient elution.

Below pH 5, excess Mn2+ and Ca2+ (1 mM) are essential to preserve binding activity. It is not necessary to include excess Ca2+ or Mn2+ in buffers if conditions that lead to their  removal from the coupled lectin can be avoided.

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, but not below pH 3. In some cases  strongly bound substances can be eluted with detergent, for example 1.0% deoxycholate.

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
Lentil Lectin Sepharose 4B 2.5 mg/ml Lentil lectin coupled to  Sepharose 4B by CNBr method. Short term 3–10 Long term 3–10 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

To avoid loss of activity of the coupled lectin, avoid solutions having a pH below 3 or above 10, buffers that contain metal chelating agents such as EDTA, or high concentrations of guanidine hydrochloride or urea.

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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