A comprehensive analysis of novel disulfide bond introduction site into the constant domain of human Fab

Abstract Generally, intermolecular disulfide bond contribute to the conformational protein stability. To identify sites where intermolecular disulfide bond can be introduced into the Fab’s constant domain of the therapeutic IgG, Fab mutants were predicted using the MOE software, a molecular simulato...

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Autores principales:	Hitomi Nakamura, Moeka Yoshikawa, Naoko Oda-Ueda, Tadashi Ueda, Takatoshi Ohkuri
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/bd98ac20ab994f19be7387966c045670
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spelling	oai:doaj.org-article:bd98ac20ab994f19be7387966c0456702021-12-02T17:14:30ZA comprehensive analysis of novel disulfide bond introduction site into the constant domain of human Fab10.1038/s41598-021-92225-92045-2322https://doaj.org/article/bd98ac20ab994f19be7387966c0456702021-06-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-92225-9https://doaj.org/toc/2045-2322Abstract Generally, intermolecular disulfide bond contribute to the conformational protein stability. To identify sites where intermolecular disulfide bond can be introduced into the Fab’s constant domain of the therapeutic IgG, Fab mutants were predicted using the MOE software, a molecular simulator, and expressed in Pichia pastoris. SDS-PAGE analysis of the prepared Fab mutants from P. pastoris indicated that among the nine analyzed Fab mutants, the F130C(H):Q124C(L), F174C(H):S176C(L), V177C(H):Q160C(L), F174C(H):S162C(L), F130C(H):S121C(L), and A145C(H):F116C(L) mutants mostly formed intermolecular disulfide bond. All these mutants showed increased thermal stability compared to that of Fab without intermolecular disulfide bond. In the other mutants, the intermolecular disulfide bond could not be completely formed, and the L132C(H):F118C(L) mutant showed only a slight decrease in binding activity and β-helix content, owing to the exertion of adverse intermolecular disulfide bond effects. Thus, our comprehensive analysis reveals that the introduction of intermolecular disulfide bond in the Fab’s constant domain is possible at various locations. These findings provide important insights for accomplishing human Fab stabilization.Hitomi NakamuraMoeka YoshikawaNaoko Oda-UedaTadashi UedaTakatoshi OhkuriNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-10 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Hitomi Nakamura Moeka Yoshikawa Naoko Oda-Ueda Tadashi Ueda Takatoshi Ohkuri A comprehensive analysis of novel disulfide bond introduction site into the constant domain of human Fab
description	Abstract Generally, intermolecular disulfide bond contribute to the conformational protein stability. To identify sites where intermolecular disulfide bond can be introduced into the Fab’s constant domain of the therapeutic IgG, Fab mutants were predicted using the MOE software, a molecular simulator, and expressed in Pichia pastoris. SDS-PAGE analysis of the prepared Fab mutants from P. pastoris indicated that among the nine analyzed Fab mutants, the F130C(H):Q124C(L), F174C(H):S176C(L), V177C(H):Q160C(L), F174C(H):S162C(L), F130C(H):S121C(L), and A145C(H):F116C(L) mutants mostly formed intermolecular disulfide bond. All these mutants showed increased thermal stability compared to that of Fab without intermolecular disulfide bond. In the other mutants, the intermolecular disulfide bond could not be completely formed, and the L132C(H):F118C(L) mutant showed only a slight decrease in binding activity and β-helix content, owing to the exertion of adverse intermolecular disulfide bond effects. Thus, our comprehensive analysis reveals that the introduction of intermolecular disulfide bond in the Fab’s constant domain is possible at various locations. These findings provide important insights for accomplishing human Fab stabilization.
format	article
author	Hitomi Nakamura Moeka Yoshikawa Naoko Oda-Ueda Tadashi Ueda Takatoshi Ohkuri
author_facet	Hitomi Nakamura Moeka Yoshikawa Naoko Oda-Ueda Tadashi Ueda Takatoshi Ohkuri
author_sort	Hitomi Nakamura
title	A comprehensive analysis of novel disulfide bond introduction site into the constant domain of human Fab
title_short	A comprehensive analysis of novel disulfide bond introduction site into the constant domain of human Fab
title_full	A comprehensive analysis of novel disulfide bond introduction site into the constant domain of human Fab
title_fullStr	A comprehensive analysis of novel disulfide bond introduction site into the constant domain of human Fab
title_full_unstemmed	A comprehensive analysis of novel disulfide bond introduction site into the constant domain of human Fab
title_sort	comprehensive analysis of novel disulfide bond introduction site into the constant domain of human fab
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/bd98ac20ab994f19be7387966c045670
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A comprehensive analysis of novel disulfide bond introduction site into the constant domain of human Fab

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