Lectin affinity chromatography

Lectin affinity chromatography may be used to purify a range of glycoproteins. Lectins are a group of proteins synthesized by plants, vertebrates and a number of invertebrate species. Especially high levels of lectins are produced by a variety of plant seeds. Plant lectins are often termed

Table 6.4 Some lectins commonly used in immobilized format for the purification of glycoproteins. The sugar specificity is listed, as are the free sugars used to elute the bound glycoprotein

Lectin

Source

Sugar specificity

Eluting sugar

Con A

Jack bean seeds

a-d-Mannose, a-d-glucose

a-d-Methyl mannose

WG A

Wheat germ

N-Acetyl-P-d-glucosamine

N-Acetyl-P-d-glucosamine

PSA

Peas

a-d-Mannose

a-d-Methyl mannose

LEL

Tomato

N-Acetyl-P-d-glucosamine

N-Acetyl-P-d-glucosamine

STL

Potato tubers

N-Acetyl-P-d-glucosamine

N-Acetyl-P-d-glucosamine

PHA

Red kidney bean

N-Acetyl-d-galactosamine

N-Acetyl-d-galactosamine

ELB

Elderberry bark

Sialic acid or N-acetyl-d-galactosamine

Lactose

GNL

Snowdrop bulbs

a-1 ^ 3 Mannose

a-Methyl mannose

AAA

Freshwater eel

a-l-Fucose

l-Fucose

phytohaemagglutinins. All lectins have the ability to bind certain monosaccharides (such as a-D-mannose, a-D-glucose, D-N-acetyl galactosamine), and the sugar specificity for many is known (Table 6.4). Among the best-known and most widely used lectins are concanavalin A (Con A), soybean lectin (SBL) and wheat germ agglutinin (WGA).

Glycoproteins generally bind to lectin affinity columns at pH values close to neutrality. Desorption may be achieved in some cases by alteration of the pH of the eluting buffer. The most common method of desorption, however, involves inclusion of free sugar molecules for which the lectin exhibits a high affinity in this elution buffer, i.e. the inclusion of a competing ligand.

Although lectin affinity chromatography may be utilized to purify a variety of glycoproteins, it has not been widely employed for a number of reasons:

Immobilized antibody

Immobilized antibody

A

Bead support Antigen which was originally Additional ligands in solution employed to stimulate not recognized by the antibody antibody production

Figure 6.15 Principle of immunoaffinity chromatography. Only antigen that is specifically recognized by the immobilized antibody will be retained on the column

• Most lectins are quite expensive.

• Crude protein sources containing one glycoprotein usually contain multiple glycoproteins. In most such instances, lectin-based affinity systems will result in the co-purification of several such glycoproteins.

• Limited application of this approach means that it has little track record, particularly on an industrial scale.

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