You have isolated a protein of interest tagged with the GST

You have isolated a protein of interest tagged with the GST domain. At this point, the fusion protein is bound to the glutathione Sepharose 4B resin and the unbound proteins have been washed away. Describe, in detail, two different techniques that you could use to isolate the protein without the affinity tag using only thrombin, glutathione Sepharose 4B, and reduced glutathione.

Solution

Chromatographic purification can be used to isolate protein by using affinity tags. The process includes pGEX gene fusion construction, Expression of GST fusion protein, Affinity purification of GST fusion protein, Enzymatic cleavage to remove GST affinity tag, Removal of GST affinity tag after protease cleavage in solution, Further purification of cleaved recombinant protein.