Question

Ni-NTA resin was used to purify His6-tagged fusion protein Y. What method is used to desorb the fusion protein Y attached to the Ni-NTA resin?

Answer 1

Recombinant proteins which have histidine tags can be purified using immobilized metal ion chromatography (IMAC). The His-tag can be placed on either the N- or C-terminus. Optimal binding and, therefore, purification efficiency is achieved when the His-tag is freely accessible to metal ion support.

Histidine tags have strong affinity for metal ions (e.g. Co2+, Ni2+, Cu2+, and Zn2+). One of the first support materials used immobilized iminodiacetic acid which can bind metal ions and allow for the coordination complex with the His-tagged protein. One difficulty with iminodiacetic acid supports is the potential for metal ion leaching leading to a decreased protein yield. Modern support materials, including nickel-nitrilotriacetic acid (Ni-NTA) and cobalt-carboxymethylasparate (Co-CMA), show limited leaching and, therefore, result in more efficient protein purifications. Once the tagged protein is bound by the immobilized chelating agent, it can be eluted by introducing a competing agent for the chelating group (imidazole) or an additional metal chelating agent (EDTA).

One advantage of using His-tags for protein purification includes the small size of the affinity ligand. Due to the small size, it has minimal effects on the folding of the protein. In addition, if the His-tag is placed on the N-terminal end of the protein, it can easily be removed using an endoprotease. Another advantage of using His-tag purification methods is that polyhistidine tags can bind proteins under both native and denaturing conditions. The use of denaturing conditions becomes important when proteins are found in inclusion bodies and must be denatured so they can be solubilized. Disadvantages of using His-tag protein purification include potential degradation of the Histag, dimer and tetramer formation, and coelution of other histidine-containing proteins. First, when a few histidine residues are proteolytically degraded, the affinity of the tagged protein is greatly reduced leading to a decrease in the protein yield. Second, once a protein has a His-tag added to its structure, it has the potential to form dimers and tetramers in the presence of metal ions. While this is often not a large problem, it can lead to inaccurate molecular mass estimates of the tagged protein. A third disadvantage of protein purification using His-tags is coelution of proteins that naturally have two or more adjacent histidine residues.