Genetic Encoding of a Trifunctional Photo‐Cross‐Linker with a Cleavable Alkyl Ester Moiety

Takayama, Masahiro; Tsubota, Tomoya; Yamaguchi, Takao; Chiba, Kosuke; Yoshida, Takumi; Hari, Yoshiyuki; Tian, Yu‐Shi; Takaya, Daisuke; Mori, Asuka; Tsukamoto, Tomohito; Ishimoto, Kenji; Ago, Yukio; Okada, Yoshiaki; Sakamoto, Kensaku; Doi, Takefumi; Fukuzawa, Kaori; Obika, Satoshi; Nakagawa, Shinsaku; Hino, Nobumasa

doi:https://doi.org/10.1002/cbic.202500827

Genetic Encoding of a Trifunctional Photo‐Cross‐Linker with a Cleavable Alkyl Ester Moiety

Takayama, Masahiro; Tsubota, Tomoya; Yamaguchi, Takao et al.

ChemBioChem, 2026, 27(3), e202500827

アクセス数:20件（2026-02-15 20:10 集計）

固定URL: https://hdl.handle.net/11094/103716

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タイトル	Genetic Encoding of a Trifunctional Photo‐Cross‐Linker with a Cleavable Alkyl Ester Moiety
著者	著者名 Takayama, Masahiro
	著者名 Tsubota, Tomoya
	NRID 1000080596601 1000080596601 ORCID 0000-0003-3180-0257 0000-0003-3180-0257 researchmap permalink takao-yamaguchi takao-yamaguchi Scopus Author ID 56322895500 56322895500 著者名 Yamaguchi, Takao
	著者名 Chiba, Kosuke
	著者名 Yoshida, Takumi
	著者名 Hari, Yoshiyuki
	NRID 1000060761252 1000060761252 ORCID 0000-0002-8988-9453 0000-0002-8988-9453 researchmap permalink yushi-tian yushi-tian Scopus Author ID 55480219500 55480219500 著者名 Tian, Yu‐Shi
	NRID 1000050571395 1000050571395 ORCID 0000-0001-6020-8151 0000-0001-6020-8151 researchmap permalink ykgkhakase ykgkhakase 著者名 Takaya, Daisuke
	著者名 Mori, Asuka
	著者名 Tsukamoto, Tomohito
	著者名 Ishimoto, Kenji
	NRID 1000050403027 1000050403027 著者名 Ago, Yukio
	NRID 1000050444500 1000050444500 researchmap permalink magic_roundabout magic_roundabout Scopus Author ID 55721995000 55721995000 著者名 Okada, Yoshiaki
	NRID 1000050240685 1000050240685 著者名 Sakamoto, Kensaku
	NRID 1000000211409 1000000211409 researchmap permalink read0014363 read0014363 Scopus Author ID 55525517900 55525517900 著者名 Doi, Takefumi
	NRID 1000050718244 1000050718244 ORCID 0000-0001-5357-8250 0000-0001-5357-8250 researchmap permalink kaori.fukuzawa kaori.fukuzawa Scopus Author ID 7102943294 7102943294 著者名 Fukuzawa, Kaori
	NRID 1000080243252 1000080243252 ORCID 0000-0002-6842-6812 0000-0002-6842-6812 researchmap permalink read0014368 read0014368 Scopus Author ID 7003800550 7003800550 著者名 Obika, Satoshi
	NRID 1000070207728 1000070207728 researchmap permalink read0042859 read0042859 著者名 Nakagawa, Shinsaku
	NRID 1000090469916 1000090469916 ORCID 0000-0002-7072-1183 0000-0002-7072-1183 researchmap permalink crosslinker crosslinker Scopus Author ID 24175680600 24175680600 著者名 Hino, Nobumasa
キーワード等	genetic code expansion
	noncanonical amino acid
	photo-cross-linking
	protein modifications
	protein-protein interactions
内容	Takayama Masahiro, Tsubota Tomoya, Yamaguchi Takao, et al. Genetic Encoding of a Trifunctional Photo‐Cross‐Linker with a Cleavable Alkyl Ester Moiety. ChemBioChem 27, e202500827 (2026); https://doi.org/10.1002/cbic.202500827.
抄録	Genetically encoded photo-cross-linkable amino acids (PAAs) are powerful tools for analyzing direct protein–protein interactions (PPIs) in mammalian cells. Cleavable PAAs are particularly useful, enabling covalent capture and subsequent release of interacting partners, which facilitates the characterization of interaction interfaces using mass spectrometry. However, the limited options for cleavable linker structures have restricted the design of PAAs. In this study, we genetically encoded a novel trifunctional PAA, DiZAAsu, which contains three distinct chemical groups: diazirine, alkyne, and alkaline-cleavable alkyl ester moieties. An archaeal pyrrolysyl-tRNA synthetase was engineered to incorporate DiZAAsu efficiently into proteins in mammalian cells. We demonstrated the in-cell photoreactive function of diazirine by cross-linking the DiZAAsu-introduced GRB2 protein to its binding partner, SHC. Using the alkyne group for biotinylation, we established a tandem affinity purification strategy that enabled efficient enrichment of the cross–linked complex, thereby reducing nonspecific protein contamination. The alkaline-based cleavage of the ester group in DiZAAsu was also demonstrated, confirming its potential for the dissociation of covalently linked complexes. This system thus expands the design space of multifunctional PAAs and adds alkaline-based dissociation to the limited repertoire of available cleavage strategies.
出版者	Wiley
収録物名	ChemBioChem
巻(号)	27(3)
ページ	e202500827
刊行年月	2026-01-30
言語	英語
ハンドルURL	https://hdl.handle.net/11094/103716
PISSN	14394227
eISSN	14397633
関連情報 (isIdenticalTo)	DOI (出版社版) https://doi.org/10.1002/cbic.202500827
アクセス権	open access
権利情報	This article is licensed under a Creative Commons Attribution 4.0 International License.
出版タイプ	Version of Record
カテゴリ	学術雑誌論文 Journal Article

資源タイプ	学術雑誌論文
ローカル資源タイプ	学術雑誌論文
DCMI資源タイプ	text
DCTERMS.bibliographicCitation	ChemBioChem. 2026, 27(3), p. e202500827
DC.title	Genetic Encoding of a Trifunctional Photo‐Cross‐Linker with a Cleavable Alkyl Ester Moiety
DC.creator	Takayama, Masahiro
	Tsubota, Tomoya
	Yamaguchi, Takao
	Chiba, Kosuke
	Yoshida, Takumi
	Hari, Yoshiyuki
	Tian, Yu‐Shi
	Takaya, Daisuke
	Mori, Asuka
	Tsukamoto, Tomohito
	Ishimoto, Kenji
	Ago, Yukio
	Okada, Yoshiaki
	Sakamoto, Kensaku
	Doi, Takefumi
	Fukuzawa, Kaori
	Obika, Satoshi
	Nakagawa, Shinsaku
	Hino, Nobumasa
DC.publisher	Wiley
DC.language' scheme='DCTERMS.RFC1766	英語
DC.type' scheme='DCTERMS.DCMIType	text
DCTERMS.issued' scheme='DCTERMS.W3CDTF	2026-01-30
DC.identifier	https://doi.org/10.1002/cbic.202500827
DC.subject	genetic code expansion
	noncanonical amino acid
	photo-cross-linking
	protein modifications
	protein-protein interactions
DC.description	Takayama Masahiro, Tsubota Tomoya, Yamaguchi Takao, et al. Genetic Encoding of a Trifunctional Photo‐Cross‐Linker with a Cleavable Alkyl Ester Moiety. ChemBioChem 27, e202500827 (2026); https://doi.org/10.1002/cbic.202500827.
DCTERMS.abstract	Genetically encoded photo-cross-linkable amino acids (PAAs) are powerful tools for analyzing direct protein–protein interactions (PPIs) in mammalian cells. Cleavable PAAs are particularly useful, enabling covalent capture and subsequent release of interacting partners, which facilitates the characterization of interaction interfaces using mass spectrometry. However, the limited options for cleavable linker structures have restricted the design of PAAs. In this study, we genetically encoded a novel trifunctional PAA, DiZAAsu, which contains three distinct chemical groups: diazirine, alkyne, and alkaline-cleavable alkyl ester moieties. An archaeal pyrrolysyl-tRNA synthetase was engineered to incorporate DiZAAsu efficiently into proteins in mammalian cells. We demonstrated the in-cell photoreactive function of diazirine by cross-linking the DiZAAsu-introduced GRB2 protein to its binding partner, SHC. Using the alkyne group for biotinylation, we established a tandem affinity purification strategy that enabled efficient enrichment of the cross–linked complex, thereby reducing nonspecific protein contamination. The alkaline-based cleavage of the ester group in DiZAAsu was also demonstrated, confirming its potential for the dissociation of covalently linked complexes. This system thus expands the design space of multifunctional PAAs and adds alkaline-based dissociation to the limited repertoire of available cleavage strategies.
DC.rights	This article is licensed under a Creative Commons Attribution 4.0 International License.
DC.format	103716
citation_title	Genetic Encoding of a Trifunctional Photo‐Cross‐Linker with a Cleavable Alkyl Ester Moiety
citation_author	Takayama, Masahiro
	Tsubota, Tomoya
	Yamaguchi, Takao
	Chiba, Kosuke
	Yoshida, Takumi
	Hari, Yoshiyuki
	Tian, Yu‐Shi
	Takaya, Daisuke
	Mori, Asuka
	Tsukamoto, Tomohito
	Ishimoto, Kenji
	Ago, Yukio
	Okada, Yoshiaki
	Sakamoto, Kensaku
	Doi, Takefumi
	Fukuzawa, Kaori
	Obika, Satoshi
	Nakagawa, Shinsaku
	Hino, Nobumasa
citation_publisher	Wiley
citation_language	英語
citation_date	2026-01-30
citation_journal_title	ChemBioChem
citation_volume	27
citation_issue	3
citation_firstpage	e202500827
citation_doi	https://doi.org/10.1002/cbic.202500827
citation_public_url	https://hdl.handle.net/11094/103716

Genetic Encoding of a Trifunctional Photo‐Cross‐Linker with a Cleavable Alkyl Ester Moiety

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