His6

His6, short for hexahistidine tag, is a short peptide tag consisting of six histidine residues (HHHHHH) commonly fused to recombinant proteins to facilitate purification and detection. The tag is encoded in the expression construct and translated as part of the protein, typically added at the N- or C- terminus.

The hexahistidine tag binds selectively to transition metal ions such as Ni2+ or Co2+ immobilized on resin, enabling affinity purification through metal-chelate chromatography (examples include Ni-NTA, Ni-IDA, and TALON resins). Purification is performed under conditions that promote binding, and elution is achieved with imidazole or by lowering pH to disrupt the interaction.

Advantages of the His6 tag include its small size, which minimizes interference with protein folding and function in many cases, broad compatibility with different expression systems, and a simple, cost-effective purification workflow. It is widely used in basic research and biotechnology.

Limitations and variants: the tag can be removed with specific proteases to produce tag-free protein, and in some cases it may affect protein function or stability. Purification can co-purify endogenous histidine-rich proteins, and binding is influenced by buffer composition, salts, detergents, and imidazole concentration. Variants include longer or tandem tags (e.g., His6x, His6-His6) and strategies that place the tag internally or in combination with other affinity tags.

See also: hexahistidine tag; His-tag; Ni-NTA; affinity chromatography.