Experiments on protein purification by lectin affinity chromatography

Summary

Lectins are proteins that reversibly bind carbohydrates. Because most lectins have at least two carbohydrate binding sites per molecule, they can precipitate glycoproteins and agglutinate cells. Lectins can also be used as affinity ligands for glycoproteins, and glycoproteins with monosaccharides can be separated by mild protein desorption conditions. Although lectin affinity folding is not highly selective, it has undoubtedly become a common method of protein purification. It is often used as one of a series of purification steps. Lectin affinity columns can also be used to remove glycoproteins as contaminants. Source: Handbook of Protein Technology

Operation method

Protein purification by cutin A affinity columns

Principle

Lectins used for affinity chromatography should be selected on the basis of their binding specificity and tightness. For example, cutin A (Con A) binds to glucose or mannose contained in glycoproteins, whereas maltodextrin binds only to proteins with N-acetylglucosamine. While cytosolic glycoproteins are often bound to maltodextrin, soluble glycoproteins are usually purified using Con A or lentil lectin affinity columns. Since in many cases the structure and identity of the purified glycoproteins are not known, it is often useful and necessary to screen some of the lectins for their binding status to the target protein. Lectin kits for this purpose are also available. Lectin affinity chromatography is relatively easy to perform. A number of methods are available to solidify the lectin to the medium. Cyanogen bromide coupling is a commonly used method. Each ml of media material couples the lectin. The presence of appropriate divalent ions (0.1 mmol/L) and protective sugars (5%, w/v) during coupling is critical. Protective sugars protect the binding site by making it less accessible during the coupling process. Many lectin affinity columns are also commercially available. The following procedure describes the purification of proteins on a cutin A affinity column as an example. The procedure for other lectin affinity columns is essentially similar, as long as they are replaced with a suitable eluting sugar. Relatively long and thin chromatographic columns have better resolution, and short and thick columns have faster flow rates for small volume elutions. Small test columns (e.g., those packed in Pasteur pipettes) or chromatographic tests performed in small centrifuge tubes can be used to estimate the binding capacity of the column.

Materials and Instruments

Glycoprotein Sample Solution
Methyl-α-D-mannose NaCl NaN3 Tris-HCl NaCl Mncl2 CaCl2 Octreotide
Chromatography column

Move

1. Prepare a cutin A affinity column. 2;

2. equilibrate the loaded column with 10 times the column volume of chromatography buffer (20 mmol/L, pH 8.0 Tris-HCl, 0.15 mol/L NaCl, 1 mmol/L _MnCl2, 1 mmol/L _CaCl2, 30 mmol/L octreotide);

3. Dialyze the glycoprotein sample solution against dialysis buffer or dilute 1:1 with the buffer, and then centrifuge or filter to remove the precipitate;

4. the adjusted glycoprotein sample solution is loaded onto the affinity column;

5. Wash the column with Chromatography Buffer until the _A280 value returns to baseline;

6. Elute with 5x column volume of Chromatography Buffer containing 10 mmol/L methyl-α-D-mannose. 7. Continue to elute with 5x column volume of Chromatography Buffer containing 10 mmol/L methyl-α-D-mannose;

7. continue to elute with 5x column volume of 20, 50, 100, 250 and 500 mmol/L Methyl-α-D-Mannose Chromatography Buffer;

8. Detect the target protein fraction. The pooled target protein fractions are dialyzed to remove sugars. 9;

9. the column is washed with chromatography buffer containing 1 mol/L NaCl to obtain regeneration;

10. when ready for storage, wash the column with 0.02% _NaN3 to prevent contamination.

Caveat

Some lectins require certain divalent ions for their structural integration. Because lectin affinity chromatography often involves membrane proteins, detergents are often required to keep these proteins soluble. Although lectins are fairly tolerant of nonionic detergents, ionic detergents reduce their specific binding.

For more product details, please visit Aladdin Scientific website.

https://www.aladdinsci.com/