Question

does nickle agarose affinity chromatography allow the his 6 tag to purify rGFP? please explain

Answer 1

Nickel columns are used for immobilized metal affinity chromatography (IMAC) for the purification of recombinant proteins with a polyhistidine tag on either terminus. The most common tag is a hexahistidine tag (6xHis tag or His6 tag). Vectors with longer or shorter histidine tags are also used, and some recombinant proteins have tandem 6xHis tags.

A nickel column is the most common column type used for IMAC, although cobalt, copper and zinc are also used to charge the column. Generally nickel resin gives the highest yield. A recombinant protein with a 6xHis tag has a high affinity for nickel, whereas most other proteins will either bind with low affinity, or not at all.

Nonspecific binding due to electrostatic attraction to the nickel beads can be minimized by addition of NaCl to the load buffer. After application of the sample, using a wash buffer with a low concentration of imidazole elutes any proteins that are weakly bound to the nickel column. Additionally, low concentrations of a nonionic detergent or glycerol can reduce hydrophobic interactions.

Recombinant protein is usually eluted from an Ni column with a high concentration of imidazole. Other elution methods include lowering pH, so that the histidines become protonated and no longer have affinity for the nickel resin, or using strong chelating agents such as EDTA and EGTA. The use of a strong chelating agent results in the nickel being stripped from the column and the elution of protein-nickel complexes.

An advantage of using an IMAC column for recombinant protein purification is that the nickel-histidine reaction is not dependent on secondary structure. Therefore a nickel column can be used for purification of denatured protein. A further potential benefit of using a nickel column is that a protein can be renatured while still bound to the nickel resin. Elution with imidazole retains the renatured state.