Question

Describe the two roles of glutathione in the affinity chromatography procedure.

Also, indicate in which chromatography fraction(s) you expect the GST-­??EGFP protein to be collected. Explain.

Answer 1

Glutathione is a tripeptide found in cell which important in measuring the redox state of cells. Glutathione-S-transferase is an enzyme that binds to Glutathione. In GST fusion affinity chromatography the protein that is required to be purified is tagged (fused) with GST on one end ( GST fusion protein). This fusion protein is then resolved in a chromatography column where Glutathione is immobilized in agarose beads. The GST fusion protein binds to immobilized glutathione on the affinity column. This bound GST fusion protein is then eluted by adding elution buffer containing glutathione ( GST has more affinity towards free glutathione in the elution buffer than the immobilized glutathione.)

So the two roles of glutathione here is to bind and separate the GST fusion protein and then to elute it.