The most commonly used tag for collecting large amounts of highly purified protein is a poly-histidine tag (His-tag). It comprises 6 – 14 histidines and is typically fused to the N- or C-terminal end of a target protein. In some cases, the tag can also be inserted into an exposed loop of the target protein. The imidazole side chains of a His-tag can engage inreversible coordinative bonds to certain transition metal ions, such as Ni2+, Co2+, or Zn2+, respectively.
Ni2+ shows the highest affinity and selectivity for His-tags and is therefore preferred. Using certain chelators covalently linked to a matrix, Ni2+ can be immobilized in a way that still allows the Ni2+ ion to interact with h istidine side chains. When His-tagged proteins are applied to this Ni2+ matrix, they specifically bind to the resin, while most untagged proteins do not. Bound proteins can be released from the matrix using mild conditions. Imidazole competes for coordination sites on Ni2+ displacing His-tagged proteins from the matrix. Alternatively, lowering pH will protonate His-tags and hence also elute the His-tagged proteins.
His-tagged protein should form a considerably stronger bond to the Ni-chelate matrix than any endogenous histidine-containing protein of the expression host. Relative binding strength depends on how many histidines bind simultaneously to the matrix (avidity effect).
Longer His-tags produce stronger binding and better separation of the target from potentially contaminating host proteins. The classic His-tag has six consecutive histidines. Tags with 10 to 14 histidines purify better. Compared to commonly used affinity tags, such as Glutathione S-Transferase (GST) or maltosebinding protein (MBP), even the longest His-tags are small. Most importantly, His-tagged proteins can be purified on nickel chelate matrices under native as well as denaturing conditions.
Due to their hydrophilic and flexible nature, His-tags can often increase the solubility of target proteins and only rarely interfere with protein’s function. This unique combination of features allows using His-tags as a versatile tool for a wide range of protein purification purposes.