Expression and purification of a native Thy1-single-chain variable fragment for use in molecular imaging

Abstract Molecular imaging using singlechain variable fragments (scFv) of antibodies targeting cancer specific antigens have been considered a non-immunogenic approach for early diagnosis in the clinic. Usually, production of proteins is performed within Escherichia coli. Recombinant proteins are ei...

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Autores principales:	Natacha Jugniot, Rakesh Bam, Ramasamy Paulmurugan
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/4e661562c8b449a880b468232f766889
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spelling	oai:doaj.org-article:4e661562c8b449a880b468232f7668892021-12-05T12:15:32ZExpression and purification of a native Thy1-single-chain variable fragment for use in molecular imaging10.1038/s41598-021-02445-22045-2322https://doaj.org/article/4e661562c8b449a880b468232f7668892021-11-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-02445-2https://doaj.org/toc/2045-2322Abstract Molecular imaging using singlechain variable fragments (scFv) of antibodies targeting cancer specific antigens have been considered a non-immunogenic approach for early diagnosis in the clinic. Usually, production of proteins is performed within Escherichia coli. Recombinant proteins are either expressed in E. coli cytoplasm as insoluble inclusion bodies, that often need cumbersome denaturation and refolding processes, or secreted toward the periplasm as soluble proteins that highly reduce the overall yield. However, production of active scFvs in their native form, without any heterologous fusion, is required for clinical applications. In this study, we expressed an anti-thymocyte differentiation antigen-scFv (Thy1-scFv) as a fusion protein with a N-terminal sequence including 3 × hexa-histidines, as purification tags, together with a Trx-tag and a S-tag for enhanced-solubility. Our strategy allowed to recover ~ 35% of Thy1-scFv in the soluble cytoplasmic fraction. An enterokinase cleavage site in between Thy1-scFv and the upstream tags was used to regenerate the protein with 97.7 ± 2.3% purity without any tags. Thy1-scFv showed functionality towards its target on flow cytometry assays. Finally, in vivo molecular imaging using Thy1-scFv conjugated to an ultrasound contrast agent (MBThy1-scFv) demonstrated signal enhancement on a transgenic pancreatic ductal adenocarcinoma (PDAC) mouse model (3.1 ± 1.2 a.u.) compared to non-targeted control (0.4 ± 0.4 a.u.) suggesting potential for PDAC early diagnosis. Overall, our strategy facilitates the expression and purification of Thy1-scFv while introducing its ability for diagnostic molecular imaging of pancreatic cancer. The presented methodology could be expanded to other important eukaryotic proteins for various applications, including but not limited to molecular imaging.Natacha JugniotRakesh BamRamasamy PaulmuruganNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-12 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Natacha Jugniot Rakesh Bam Ramasamy Paulmurugan Expression and purification of a native Thy1-single-chain variable fragment for use in molecular imaging
description	Abstract Molecular imaging using singlechain variable fragments (scFv) of antibodies targeting cancer specific antigens have been considered a non-immunogenic approach for early diagnosis in the clinic. Usually, production of proteins is performed within Escherichia coli. Recombinant proteins are either expressed in E. coli cytoplasm as insoluble inclusion bodies, that often need cumbersome denaturation and refolding processes, or secreted toward the periplasm as soluble proteins that highly reduce the overall yield. However, production of active scFvs in their native form, without any heterologous fusion, is required for clinical applications. In this study, we expressed an anti-thymocyte differentiation antigen-scFv (Thy1-scFv) as a fusion protein with a N-terminal sequence including 3 × hexa-histidines, as purification tags, together with a Trx-tag and a S-tag for enhanced-solubility. Our strategy allowed to recover ~ 35% of Thy1-scFv in the soluble cytoplasmic fraction. An enterokinase cleavage site in between Thy1-scFv and the upstream tags was used to regenerate the protein with 97.7 ± 2.3% purity without any tags. Thy1-scFv showed functionality towards its target on flow cytometry assays. Finally, in vivo molecular imaging using Thy1-scFv conjugated to an ultrasound contrast agent (MBThy1-scFv) demonstrated signal enhancement on a transgenic pancreatic ductal adenocarcinoma (PDAC) mouse model (3.1 ± 1.2 a.u.) compared to non-targeted control (0.4 ± 0.4 a.u.) suggesting potential for PDAC early diagnosis. Overall, our strategy facilitates the expression and purification of Thy1-scFv while introducing its ability for diagnostic molecular imaging of pancreatic cancer. The presented methodology could be expanded to other important eukaryotic proteins for various applications, including but not limited to molecular imaging.
format	article
author	Natacha Jugniot Rakesh Bam Ramasamy Paulmurugan
author_facet	Natacha Jugniot Rakesh Bam Ramasamy Paulmurugan
author_sort	Natacha Jugniot
title	Expression and purification of a native Thy1-single-chain variable fragment for use in molecular imaging
title_short	Expression and purification of a native Thy1-single-chain variable fragment for use in molecular imaging
title_full	Expression and purification of a native Thy1-single-chain variable fragment for use in molecular imaging
title_fullStr	Expression and purification of a native Thy1-single-chain variable fragment for use in molecular imaging
title_full_unstemmed	Expression and purification of a native Thy1-single-chain variable fragment for use in molecular imaging
title_sort	expression and purification of a native thy1-single-chain variable fragment for use in molecular imaging
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/4e661562c8b449a880b468232f766889
work_keys_str_mv	AT natachajugniot expressionandpurificationofanativethy1singlechainvariablefragmentforuseinmolecularimaging AT rakeshbam expressionandpurificationofanativethy1singlechainvariablefragmentforuseinmolecularimaging AT ramasamypaulmurugan expressionandpurificationofanativethy1singlechainvariablefragmentforuseinmolecularimaging
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Expression and purification of a native Thy1-single-chain variable fragment for use in molecular imaging

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