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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: Ruan, Yao, Chao, Shuangying, Hu, Xuejun, Lu, Longzhen, Lin, Yue, Wang, Qian, Zheng, Yang, Li, Junming, Ding, Ning
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8607273/
https://www.ncbi.nlm.nih.gov/pubmed/34820421
http://dx.doi.org/10.3389/fmolb.2021.742617
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author Ruan, Yao
Chao, Shuangying
Hu, Xuejun
Lu, Longzhen
Lin, Yue
Wang, Qian
Zheng, Yang
Li, Junming
Ding, Ning
author_facet Ruan, Yao
Chao, Shuangying
Hu, Xuejun
Lu, Longzhen
Lin, Yue
Wang, Qian
Zheng, Yang
Li, Junming
Ding, Ning
author_sort Ruan, Yao
collection PubMed
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.
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spelling pubmed-86072732021-11-23 FN3 Domain Displaying Double Epitopes: A Cost-Effective Strategy for Producing Substitute Antigens Ruan, Yao Chao, Shuangying Hu, Xuejun Lu, Longzhen Lin, Yue Wang, Qian Zheng, Yang Li, Junming Ding, Ning Front Mol Biosci Molecular Biosciences 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. Frontiers Media S.A. 2021-11-04 /pmc/articles/PMC8607273/ /pubmed/34820421 http://dx.doi.org/10.3389/fmolb.2021.742617 Text en Copyright © 2021 Ruan, Chao, Hu, Lu, Lin, Wang, Zheng, Li and Ding. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Molecular Biosciences
Ruan, Yao
Chao, Shuangying
Hu, Xuejun
Lu, Longzhen
Lin, Yue
Wang, Qian
Zheng, Yang
Li, Junming
Ding, Ning
FN3 Domain Displaying Double Epitopes: A Cost-Effective Strategy for Producing Substitute Antigens
title 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_short 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
topic Molecular Biosciences
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8607273/
https://www.ncbi.nlm.nih.gov/pubmed/34820421
http://dx.doi.org/10.3389/fmolb.2021.742617
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