Aim:

To purify an overexpressed protein (with 6×His tag) using Ni-NTA affinity chromatography

Introduction:

Affinity chromatography encompasses the techniques wherein separation is achieved based on specific binding of the protein to their ligands or receptors. Some of the receptors or ligands, such as antigenic epitopes and the active sites of enzymes, are highly specific to the protein of interest and affinity chromatography takes advantage of these biospecific interactions for purifying a protein . This approach of purification should, in principle, purify a protein in a single step.

Although receptor-ligand interactions are natural properties of the proteins and enzymes, it is sometimes convenient to engineer the proteins making them amenable for specific interactions. This has become popular for the purification of the recombinant proteins. The cDNA sequences are engineered so that the expressed proteins have the desired binding site at one of its termini. All forms of affinity chromatography usually include the following steps:

1. 1. Choosing an appropriate ligand.

2.  Immobilization of the ligand onto the matrix gel.

3. Loading the sample (protein mixture) on the matrix gel.

4. Washing to remove the substances that are bound non-specifically.

5. Elution of the protein of interest.

A variety of protein interactions are utilized for purifying a protein using affinity chromatography; some of them are listed in table 25.1.

Table 25.1 Some of the routinely using affinity chromatographic techniques