Functional production of a soluble and secreted single-chain antibody by a bacterial secretion system.

Single-chain variable fragments (scFvs) serve as an alternative to full-length monoclonal antibodies used in research and therapeutic and diagnostic applications. However, when recombinant scFvs are overexpressed in bacteria, they often form inclusion bodies and exhibit loss of function. To overcome...

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Autores principales:	Chiu-Min Cheng, Shey-Cherng Tzou, Ya-Han Zhuang, Chien-Chiao Huang, Chien-Han Kao, Kuang-Wen Liao, Ta-Chun Cheng, Chih-Hung Chuang, Yuan-Chin Hsieh, Ming-Hong Tai, Tian-Lu Cheng
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2014
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/fadea00e77d54de7841f016df53dba0a
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spelling	oai:doaj.org-article:fadea00e77d54de7841f016df53dba0a2021-11-18T08:19:31ZFunctional production of a soluble and secreted single-chain antibody by a bacterial secretion system.1932-620310.1371/journal.pone.0097367https://doaj.org/article/fadea00e77d54de7841f016df53dba0a2014-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24824752/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203Single-chain variable fragments (scFvs) serve as an alternative to full-length monoclonal antibodies used in research and therapeutic and diagnostic applications. However, when recombinant scFvs are overexpressed in bacteria, they often form inclusion bodies and exhibit loss of function. To overcome this problem, we developed an scFv secretion system in which scFv was fused with osmotically inducible protein Y (osmY), a bacterial secretory carrier protein, for efficient protein secretion. Anti-EGFR scFv (αEGFR) was fused with osmY (N- and C-termini) and periplasmic leader sequence (pelB) to generate αEGFR-osmY, osmY-αEGFR, and pelB-αEGFR (control), respectively. In comparison with the control, both the osmY-fused αEGFR scFvs were soluble and secreted into the LB medium. Furthermore, the yield of soluble αEGFR-osmY was 20-fold higher, and the amount of secreted protein was 250-fold higher than that of osmY-αEGFR. In addition, the antigen-binding activity of both the osmY-fused αEGFRs was 2-fold higher than that of the refolded pelB-αEGFR from inclusion bodies. Similar results were observed with αTAG72-osmY and αHer2-osmY. These results suggest that the N-terminus of osmY fused with scFv produces a high yield of soluble, functional, and secreted scFv, and the osmY-based bacterial secretion system may be used for the large-scale industrial production of low-cost αEGFR protein.Chiu-Min ChengShey-Cherng TzouYa-Han ZhuangChien-Chiao HuangChien-Han KaoKuang-Wen LiaoTa-Chun ChengChih-Hung ChuangYuan-Chin HsiehMing-Hong TaiTian-Lu ChengPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 9, Iss 5, p e97367 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Chiu-Min Cheng Shey-Cherng Tzou Ya-Han Zhuang Chien-Chiao Huang Chien-Han Kao Kuang-Wen Liao Ta-Chun Cheng Chih-Hung Chuang Yuan-Chin Hsieh Ming-Hong Tai Tian-Lu Cheng Functional production of a soluble and secreted single-chain antibody by a bacterial secretion system.
description	Single-chain variable fragments (scFvs) serve as an alternative to full-length monoclonal antibodies used in research and therapeutic and diagnostic applications. However, when recombinant scFvs are overexpressed in bacteria, they often form inclusion bodies and exhibit loss of function. To overcome this problem, we developed an scFv secretion system in which scFv was fused with osmotically inducible protein Y (osmY), a bacterial secretory carrier protein, for efficient protein secretion. Anti-EGFR scFv (αEGFR) was fused with osmY (N- and C-termini) and periplasmic leader sequence (pelB) to generate αEGFR-osmY, osmY-αEGFR, and pelB-αEGFR (control), respectively. In comparison with the control, both the osmY-fused αEGFR scFvs were soluble and secreted into the LB medium. Furthermore, the yield of soluble αEGFR-osmY was 20-fold higher, and the amount of secreted protein was 250-fold higher than that of osmY-αEGFR. In addition, the antigen-binding activity of both the osmY-fused αEGFRs was 2-fold higher than that of the refolded pelB-αEGFR from inclusion bodies. Similar results were observed with αTAG72-osmY and αHer2-osmY. These results suggest that the N-terminus of osmY fused with scFv produces a high yield of soluble, functional, and secreted scFv, and the osmY-based bacterial secretion system may be used for the large-scale industrial production of low-cost αEGFR protein.
format	article
author	Chiu-Min Cheng Shey-Cherng Tzou Ya-Han Zhuang Chien-Chiao Huang Chien-Han Kao Kuang-Wen Liao Ta-Chun Cheng Chih-Hung Chuang Yuan-Chin Hsieh Ming-Hong Tai Tian-Lu Cheng
author_facet	Chiu-Min Cheng Shey-Cherng Tzou Ya-Han Zhuang Chien-Chiao Huang Chien-Han Kao Kuang-Wen Liao Ta-Chun Cheng Chih-Hung Chuang Yuan-Chin Hsieh Ming-Hong Tai Tian-Lu Cheng
author_sort	Chiu-Min Cheng
title	Functional production of a soluble and secreted single-chain antibody by a bacterial secretion system.
title_short	Functional production of a soluble and secreted single-chain antibody by a bacterial secretion system.
title_full	Functional production of a soluble and secreted single-chain antibody by a bacterial secretion system.
title_fullStr	Functional production of a soluble and secreted single-chain antibody by a bacterial secretion system.
title_full_unstemmed	Functional production of a soluble and secreted single-chain antibody by a bacterial secretion system.
title_sort	functional production of a soluble and secreted single-chain antibody by a bacterial secretion system.
publisher	Public Library of Science (PLoS)
publishDate	2014
url	https://doaj.org/article/fadea00e77d54de7841f016df53dba0a
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Functional production of a soluble and secreted single-chain antibody by a bacterial secretion system.

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