Question

what is the main purpose of induce E. coli to express a His6-tagged recombinant form of green fluorescent protein and then to purify rGFP using Ni2+-agarose

affinity chromatography?

Answer 1

The green fluorescent protein is a recombinant protein which has the capacity of fluorescence. It is generally used in protein expression system to visually tag the expressed protein. The histidine tagging is used for the purification of the expressed recombinant protein. The six or more histidine residues will get fused to the N or C terminal of the protein.

For the purification using Ni2+ agarose affinity chromatography, the histidine tag will be readily bind to the transition metals like Ni2+ which is immobilized to the column resin. When the his tag rGFP pass through such Ni2+ affinity column, the protein will bind to it and will get eluted by using elution buffer for the purification.