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Publication Abstract from PubMed

The polyhistidine tag (His-tag) is one of the most widely used peptide tags for the purification of recombinant proteins, owing to its compatibility with immobilized metal affinity chromatography. While numerous anti-His-tag antibodies are commercially available, their quantitative affinity data and structural insights are limited. Here, we present a detailed physicochemical and structural characterization of a novel anti-His-tag antibody, HisMab-1. Isothermal titration calorimetry showed that the Fab fragment of HisMab-1 binds to a hexahistidine peptide in an enthalpy-driven manner, with a dissociation constant (K(D)) of approximately 30 nM at a neutral pH. The crystal structure of the HisMab-1-hexahistidine peptide complex at 2.39-A resolution revealed that HisMab-1 primarily recognizes the first, second, fourth, and fifth histidine residues of the peptide through multiple interactions, including hydrogen bonding and pi-pi stacking, which collectively contribute to the high specificity of the antibody. Notably, HisMab-1 also binds to a His-tag embedded within a conformationally constrained beta-hairpin loop without reducing affinity, highlighting its structural adaptability. These findings establish HisMab-1 as a high-affinity, high-specificity, structurally validated anti-His-tag antibody with broad potential in diverse protein engineering and structural biology applications.

Functional and Structural Characterization of a Novel Anti-His-tag Antibody, HisMab-1.,Hitomi N, Hoshi S, Kaneko MK, Kato R, Iwasaki K, Takagi J, Kato Y, Harada-Hikita A, Arimori T J Mol Biol. 2025 Dec 3;438(2):169574. doi: 10.1016/j.jmb.2025.169574. PMID:41349762^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.