Functional and Structural Characterization of a Novel Anti-His-tag Antibody, HisMab-1

Hitomi, Natsuki; Hoshi, Satowa; Kaneko, Mika K.; Kato, Ryuichi; Iwasaki, Kenji; Takagi, Junichi; Kato, Yukinari; Harada-Hikita, Ayaka; Arimori, Takao

doi:https://doi.org/10.1016/j.jmb.2025.169574

Functional and Structural Characterization of a Novel Anti-His-tag Antibody, HisMab-1

Hitomi, Natsuki; Hoshi, Satowa; Kaneko, Mika K. et al.

Journal of Molecular Biology, 2026, 438(2), 169574

Number of Access:45（2026-02-01 13:31 Counts）

Identifier to cite or link to this item: https://hdl.handle.net/11094/103579

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Title	Functional and Structural Characterization of a Novel Anti-His-tag Antibody, HisMab-1
Creator	CreatorName Hitomi, Natsuki
	CreatorName Hoshi, Satowa
	NRID 1000000323163 1000000323163 CreatorName Kaneko, Mika K.
	NRID 1000050240833 1000050240833 CreatorName Kato, Ryuichi
	NRID 1000020342751 1000020342751 CreatorName Iwasaki, Kenji
	NRID 1000090212000 1000090212000 researchmap permalink read0105692 read0105692 Scopus Author ID 7102173538 7102173538 CreatorName Takagi, Junichi
	NRID 1000000571811 1000000571811 CreatorName Kato, Yukinari
	NRID 1000000800476 1000000800476 CreatorName Harada-Hikita, Ayaka
	NRID 1000080582064 1000080582064 ORCID 0000-0002-6063-5572 0000-0002-6063-5572 researchmap permalink t_arimori t_arimori Scopus Author ID 35336011300 35336011300 CreatorName Arimori, Takao
Subject	antibody
	isothermal titration calorimetry
	polyhistidine tag
	surface plasmon resonance
	X-ray crystallography
Description	Hitomi N., Hoshi S., Kaneko M.K., et al. Functional and Structural Characterization of a Novel Anti-His-tag Antibody, HisMab-1. Journal of Molecular Biology 438, 169574 (2026); https://doi.org/10.1016/j.jmb.2025.169574.
Abstract	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 (KD) of ∼30 nM at a neutral pH. The crystal structure of the HisMab-1–hexahistidine peptide complex at 2.39-Å 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 π–π 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 β-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.
Publisher	Academic Press
Source Title	Journal of Molecular Biology
Volume (Issue)	438(2)
Page	169574
Date of Issued	2026-01-15
Language	English
Handle URL	https://hdl.handle.net/11094/103579
PISSN	00222836
eISSN	10898638
Relation.isIdenticalTo	DOI (Publisher Version) https://doi.org/10.1016/j.jmb.2025.169574
Access Rights	open access
CopyRight	This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
oaire:version	Version of Record
Category	学術雑誌論文 Journal Article

Resource Type	journal article
Local Type	Journal Article
DCMI Type	Text
DCTERMS.bibliographicCitation	Journal of Molecular Biology. 2026, 438(2), p. 169574
DC.title	Functional and Structural Characterization of a Novel Anti-His-tag Antibody, HisMab-1
DC.creator	Hitomi, Natsuki
	Hoshi, Satowa
	Kaneko, Mika K.
	Kato, Ryuichi
	Iwasaki, Kenji
	Takagi, Junichi
	Kato, Yukinari
	Harada-Hikita, Ayaka
	Arimori, Takao
DC.publisher	Academic Press
DC.language' scheme='DCTERMS.RFC1766	English
DC.type' scheme='DCTERMS.DCMIType	Text
DCTERMS.issued' scheme='DCTERMS.W3CDTF	2026-01-15
DC.identifier	https://doi.org/10.1016/j.jmb.2025.169574
DC.subject	antibody
	isothermal titration calorimetry
	polyhistidine tag
	surface plasmon resonance
	X-ray crystallography
DC.description	Hitomi N., Hoshi S., Kaneko M.K., et al. Functional and Structural Characterization of a Novel Anti-His-tag Antibody, HisMab-1. Journal of Molecular Biology 438, 169574 (2026); https://doi.org/10.1016/j.jmb.2025.169574.
DCTERMS.abstract	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 (KD) of ∼30 nM at a neutral pH. The crystal structure of the HisMab-1–hexahistidine peptide complex at 2.39-Å 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 π–π 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 β-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.
DC.rights	This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
DC.format	103579
citation_title	Functional and Structural Characterization of a Novel Anti-His-tag Antibody, HisMab-1
citation_author	Hitomi, Natsuki
	Hoshi, Satowa
	Kaneko, Mika K.
	Kato, Ryuichi
	Iwasaki, Kenji
	Takagi, Junichi
	Kato, Yukinari
	Harada-Hikita, Ayaka
	Arimori, Takao
citation_publisher	Academic Press
citation_language	English
citation_date	2026-01-15
citation_journal_title	Journal of Molecular Biology
citation_volume	438
citation_issue	2
citation_firstpage	169574
citation_doi	https://doi.org/10.1016/j.jmb.2025.169574
citation_public_url	https://hdl.handle.net/11094/103579

Functional and Structural Characterization of a Novel Anti-His-tag Antibody, HisMab-1

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