Polyhistidinecontaining

Polyhistidine-containing refers to proteins or peptides that include a sequence of histidine residues, most commonly a polyhistidine tag used to aid purification and detection of recombinant proteins. The prevalent form is a hexahistidine tag (6xHis), typically placed at the N- or C-terminus, though longer variants (8xHis, 10xHis, or more) are also used to modulate binding strength.

Purification of polyhistidine-containing proteins relies on immobilized metal affinity chromatography (IMAC). The histidine residues coordinate with divalent metal ions, such as Ni2+ or Co2+, attached to chelating resins (for example, Ni-NTA). Binding occurs under mild conditions and elution is usually achieved with imidazole or through lowering metal affinity. This approach enables selective enrichment of the tagged protein from complex mixtures and can be compatible with various expression systems.

Design considerations include the tag’s position (N- or C-terminal is most common), the potential need for a flexible linker, and whether the tag will affect protein folding or activity. Tags can facilitate detection using anti-His antibodies or mass spectrometry, and they are often removable via protease cleavage if the native protein sequence must be preserved for downstream applications. Tag removal may require additional purification steps to separate the cleaved tag and protease.

Limitations include potential co-purification of endogenous histidine-rich proteins, risk of altered solubility or function, and possible immunogenicity in certain applications. In cases where the tag interferes with function, alternative affinity tags (such as Strep-tag or FLAG) or tagless purification strategies may be preferred.