The His-tag (also called 6xHis-tag) is one of the simplest and most widely used purification tags, with six or more consecutive histidine residues. These residues readily coordinate with transition metal ions such as Ni2+ or Co2+ immobilized on beads or a resin for purification. The polyhistidine system of purification of recombinant proteins is successfully used in various expression systems, including bacterial, yeast, plant cell and mammalian cells systems.
His-tagged protein purification requires the His-tag and Ni-NTA interaction, which is based on the selectivity and high affinity of Ni-NTA (nickel nitrilotriacetic acid) resin for proteins containing an affinity tag of e.g. six consecutive Histidine residues. NTA, which has four chelating sites for nickel ions, binds nickel more tightly than metal-chelating purification systems like IDA which have only three sites available for interaction with metal ions.
The his-tag has a high affinity for these metal ions and binds strongly to the IMAC column. Most other proteins in the lysate will not bind to the resin, or bind only weakly.
Histidine-tagged proteins can be purified from a wide range of expression systems under native or denaturing conditions. The main drawback is that the technique often requires optimization to minimize nonspecific binding of host cell proteins.
It is difficult to provide a general protocol for optimal purification for any His-tagged protein, the protocols provided should be used as guidelines and might need to be optimized for a specific target. At first, researchers should decide whether to purify his-tagged protein under native or denaturing conditions. Then the clean lysate under the specific conditions should be prepared and start the batch purification.
Poly-histidine-tag binds to bivalent nickel or cobalt ions chelated by iminodiacetic acid (Ni-IDA) and nitrilotriacetic acid (Ni-NTA) on sepharose resin/agarose, which allows affinity purification of recombinant protein with a poly-his-tag. Cell lysate containing the his-tagged recombinant protein among many other proteins is loaded to a Ni- or Co- affinity column and eluted with 50-3000 mM imidazole, depending on the binding strength of the his-tagged protein. It usually results in relatively pure protein when the recombinant protein is expressed in prokaryotic organisms. Purification of his-tagged protein from higher organisms such as yeasts or other eukaryotes may require a tandem affinity purification using two tags to yield higher purity. Alternatively, immobilized cobalt ions rather than nickel ions generally yields a substantial increase in purity and requires lower imidazole concentrations for elution of the his-tagged protein.
His-tagged fusion protein purification can be used as the only purification step for applications that do not require proteins with extremely high purity. When the highest purity is needed, this technique can be used as the first (capture) step in a multistep purification procedure. It is also suitable for purifying dual-tagged proteins with a histidine-tag at one end and a different tag at the other end.
The his tag usually does not have to be removed. In some cases where it interferes with the function of the target protein or the target protein needs to be in a native state, recombinant proteases are used to easily remove his tag after a second pass over the resin used to purify the protein of interest.
The number of histidine residues in a poly-his-tag can affect the binding of the tagged protein to the affnity resin and elution of the protein off the affinity column. Usually, a hexa-his-tag is sufficient for affinity purification and antibody detection. However, sometime, 7-10-histidines are used to increase binding of the his-tagged protein to the affinity column, possibly due to unexposure of one or more histidine residues to the resin.
In some applications, it is desirable to remove the his-tag, for example, for protein crystallization. To allow cleavage of the tag, a protease cleavage site needs to be engineered between the tag and the protein. An EK cleavage site behind the His-tag (poly-his-EK site-protein structure design) can allow complete removal of the tag and the cleavage site, leaving no additional amino acids after the specific cleavage of the tag, resulting in a native form of the protein. For more information on the cleavage site and tag removal of EK and HRV-3C, please refer to: Enterokinase (EK), HRV-3C (human rhinovirus protease).
Pina AS, et al. (2014) Affinity tags in protein purification and peptide enrichment: An overview. Methods in molecular biology (Clifton, N.J.) 1129: 147-168.
Young CL, et al. (2012) Recombinant protein expression and purification: A comprehensive review of affinity tags and microbial applications. Biotechnol J 7(5): 620-634.