FN3 Domain Displaying Double Epitopes: A Cost-Effective Strategy for Producing Substitute Antigens

Construction of substitute antigens based on alternative scaffold proteins is a promising strategy in bioassay technology. In this study, we proposed a strategy for constructing substitute antigens derived from 10th human fibronectin type III (FN3) using two peptide epitopes of terminal pro-brain na...

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Autores principales:	Yao Ruan, Shuangying Chao, Xuejun Hu, Longzhen Lu, Yue Lin, Qian Wang, Yang Zheng, Junming Li, Ning Ding
Formato:	article
Lenguaje:	EN
Publicado:	Frontiers Media S.A. 2021
Materias:	FN3 substitute antigen epitope NT-ProBNP Escherichia coli Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/486a3731d4d74b4ca6ce544c1527f2be
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spelling	oai:doaj.org-article:486a3731d4d74b4ca6ce544c1527f2be2021-11-08T09:33:58ZFN3 Domain Displaying Double Epitopes: A Cost-Effective Strategy for Producing Substitute Antigens2296-889X10.3389/fmolb.2021.742617https://doaj.org/article/486a3731d4d74b4ca6ce544c1527f2be2021-11-01T00:00:00Zhttps://www.frontiersin.org/articles/10.3389/fmolb.2021.742617/fullhttps://doaj.org/toc/2296-889XConstruction of substitute antigens based on alternative scaffold proteins is a promising strategy in bioassay technology. In this study, we proposed a strategy for constructing substitute antigens derived from 10th human fibronectin type III (FN3) using two peptide epitopes of terminal pro-brain natriuretic peptide (NT-proBNP) as an example. The base sequences encoding the two antigenic epitopes of NT-proBNP were recombined into the FG loop region and the C-terminus of FN3, fused by 4 GS or polyN linker. The fusion proteins (named FN3-epitopes-4GS and FN3-epitopes-polyN, respectively) were expressed and purified cost-effectively using an Escherichia coli expression system. The immunoreactivity of recombinant substitutes was preliminarily confirmed by western blot analysis using epitope-specific antibodies. The sandwich enzyme-linked immunosorbent assay demonstrated that either FN3-epitopes-polyN or FN3-epitopes-4GS was highly sensitive, and FN3-epitopes-polyN exhibited better kinetics to specific antibodies than FN3-epitopes-4GS, showing a linear dose-response relationship in the concentration range of 0.06–12.85 ng/ml, which suggest that the polyN linker was more suitable for constructing the FN3-based substitute antigens compared to the 4 GS linker. Furthermore, the serum stability test and differential scanning calorimetry analysis showed that the recombinant FN3-epitopes-polyN maintained the original stability of FN3. Therefore, it was confirmed that FN3 could be engineered to construct a stable biomacromolecular substitute for displaying double epitopes of antigen proteins, such as NT-proBNP. In summary, a cost-effective strategy to produce NT-proBNP substitute antigens with good immunoreactivity and physicochemical stability was established in this work, which may provide potential uses for the production of other substitute antigens in the future.Yao RuanYao RuanYao RuanShuangying ChaoXuejun HuLongzhen LuYue LinQian WangYang ZhengJunming LiNing DingFrontiers Media S.A.articleFN3substitute antigenepitopeNT-ProBNPEscherichia coliBiology (General)QH301-705.5ENFrontiers in Molecular Biosciences, Vol 8 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	FN3 substitute antigen epitope NT-ProBNP Escherichia coli Biology (General) QH301-705.5
spellingShingle	FN3 substitute antigen epitope NT-ProBNP Escherichia coli Biology (General) QH301-705.5 Yao Ruan Yao Ruan Yao Ruan Shuangying Chao Xuejun Hu Longzhen Lu Yue Lin Qian Wang Yang Zheng Junming Li Ning Ding FN3 Domain Displaying Double Epitopes: A Cost-Effective Strategy for Producing Substitute Antigens
description	Construction of substitute antigens based on alternative scaffold proteins is a promising strategy in bioassay technology. In this study, we proposed a strategy for constructing substitute antigens derived from 10th human fibronectin type III (FN3) using two peptide epitopes of terminal pro-brain natriuretic peptide (NT-proBNP) as an example. The base sequences encoding the two antigenic epitopes of NT-proBNP were recombined into the FG loop region and the C-terminus of FN3, fused by 4 GS or polyN linker. The fusion proteins (named FN3-epitopes-4GS and FN3-epitopes-polyN, respectively) were expressed and purified cost-effectively using an Escherichia coli expression system. The immunoreactivity of recombinant substitutes was preliminarily confirmed by western blot analysis using epitope-specific antibodies. The sandwich enzyme-linked immunosorbent assay demonstrated that either FN3-epitopes-polyN or FN3-epitopes-4GS was highly sensitive, and FN3-epitopes-polyN exhibited better kinetics to specific antibodies than FN3-epitopes-4GS, showing a linear dose-response relationship in the concentration range of 0.06–12.85 ng/ml, which suggest that the polyN linker was more suitable for constructing the FN3-based substitute antigens compared to the 4 GS linker. Furthermore, the serum stability test and differential scanning calorimetry analysis showed that the recombinant FN3-epitopes-polyN maintained the original stability of FN3. Therefore, it was confirmed that FN3 could be engineered to construct a stable biomacromolecular substitute for displaying double epitopes of antigen proteins, such as NT-proBNP. In summary, a cost-effective strategy to produce NT-proBNP substitute antigens with good immunoreactivity and physicochemical stability was established in this work, which may provide potential uses for the production of other substitute antigens in the future.
format	article
author	Yao Ruan Yao Ruan Yao Ruan Shuangying Chao Xuejun Hu Longzhen Lu Yue Lin Qian Wang Yang Zheng Junming Li Ning Ding
author_facet	Yao Ruan Yao Ruan Yao Ruan Shuangying Chao Xuejun Hu Longzhen Lu Yue Lin Qian Wang Yang Zheng Junming Li Ning Ding
author_sort	Yao Ruan
title	FN3 Domain Displaying Double Epitopes: A Cost-Effective Strategy for Producing Substitute Antigens
title_short	FN3 Domain Displaying Double Epitopes: A Cost-Effective Strategy for Producing Substitute Antigens
title_full	FN3 Domain Displaying Double Epitopes: A Cost-Effective Strategy for Producing Substitute Antigens
title_fullStr	FN3 Domain Displaying Double Epitopes: A Cost-Effective Strategy for Producing Substitute Antigens
title_full_unstemmed	FN3 Domain Displaying Double Epitopes: A Cost-Effective Strategy for Producing Substitute Antigens
title_sort	fn3 domain displaying double epitopes: a cost-effective strategy for producing substitute antigens
publisher	Frontiers Media S.A.
publishDate	2021
url	https://doaj.org/article/486a3731d4d74b4ca6ce544c1527f2be
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FN3 Domain Displaying Double Epitopes: A Cost-Effective Strategy for Producing Substitute Antigens

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