What is a Polyhistidine or Histidine Affinity Tag

Polyhistidine is a common and ubiquitous affinity tag used in expression vectors or protein expression and purification reagents. Many researchers use the polyhistidine tag with six histidines also known as the His-tag or His₆ tag. However, the tag's length can vary between two (2) to ten (10) histidine residues per tag. Histidine readily forms coordination bonds with immobilized metal ions. The fusion of a tag to peptides or proteins allows efficient purification using affinity chromatography. Also, in-vivo tagged proteins enable functional imaging of protein locations in cells or tissue. Selecting sequences of gene fragments and clonal genes allow the design of desired fusion proteins using DNA sequences originating from natural or mutated proteins.

Since the purification of specific proteins from native sources is often very challenging, researchers take advantage of heterologous expression systems. In this approach, cloning genes into artificial vectors allow the expression of selected tagged proteins within easily cultured cells, such as Escherichia coli (bacteria), Pichia pastoris (yeast), and several insect and mammalian cells. The fusion of the protein of interest with a carrier protein or a peptide and protein tag can increase solubility, change the localization, allow affinity purification, or allow immunological assays.

 His-Tags

Immobilized metal affinity chromatography (IMAC) based on polyhistidine tracts' affinity for divalent cations allows purification of His-tag fusion proteins. Typically, resins with the immobilized nickel cation, Ni2+, chelate complex is utilized for affinity purification. The immobilized nickel ion, Ni2+, is most commonly used combined with the His-tag, but immobilized Co2+, Cu2+, Ni2+, Zn2+, Ca2+, and Fe3+ can all be used as well for the purification of polyhistidine fusion proteins.

IMAC allows the purification of recombinant proteins containing the tag as a fusion protein, either on the n-terminal or c-terminal end. The resin or matrix most widely used for IMAC is Ni(II)-nitrilotriacetic acid (Ni-NTA). However, other resins that also allow the immobilization of transition metal ions include iminodiacetic acid agarose and carboxy-methyl-aspartate agarose.

The chelating agent nitrilotriacetic acid (NTA) with the formula N(CH₂CO₂H)₃ forms coordination complexes with metal ions, known as chelates, such as Ca²⁺, Co²⁺, Cu²⁺, Fe³⁺, Ni²⁺ and Zi²⁺.

Figure 1: Chelation of metal ions by NTA.