Molecular switching system using glycosylphosphatidylinositol to select cells highly expressing recombinant proteins

Abstract Although many pharmaceutical proteins are produced in mammalian cells, there remains a challenge to select cell lines that express recombinant proteins with high productivity. Since most biopharmaceutical proteins are secreted by cells into the medium, it is difficult to select cell lines t...

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Autores principales:	Emmanuel Matabaro, Zeng’an He, Yi-Shi Liu, Hui-Jie Zhang, Xiao-Dong Gao, Morihisa Fujita
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/4f584acd06094ce5b04b57b53a29be8f
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spelling	oai:doaj.org-article:4f584acd06094ce5b04b57b53a29be8f2021-12-02T12:30:43ZMolecular switching system using glycosylphosphatidylinositol to select cells highly expressing recombinant proteins10.1038/s41598-017-04330-32045-2322https://doaj.org/article/4f584acd06094ce5b04b57b53a29be8f2017-06-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-04330-3https://doaj.org/toc/2045-2322Abstract Although many pharmaceutical proteins are produced in mammalian cells, there remains a challenge to select cell lines that express recombinant proteins with high productivity. Since most biopharmaceutical proteins are secreted by cells into the medium, it is difficult to select cell lines that produce large amounts of the target protein. To address this issue, a new protein expression system using the glycosylphosphatidylinositol (GPI)-anchor was developed. PGAP2 is involved in processing GPI-anchored proteins (GPI-APs) during transport. In PGAP2 mutant cells, most GPI-APs are secreted into the medium. Here, we established a HEK293 cell line where endogenous PGAP2 was knocked out and exogenous PGAP2 was inserted with a piggyBac transposon in the genome. Using these cells, human lysosomal acid lipase (LIPA) and α-galactosidase A (GLA) were expressed as GPI-anchored forms (LIPA-GPI and GLA-GPI) and cells expressing high levels of LIPA-GPI or GLA-GPI on the cell surface were enriched. Removal of the PGAP2 gene by piggyBac transposase or FLP recombinase converted LIPA-GPI and GLA-GPI from membrane-bound to the secreted forms. Thus, cells expressing LIPA or GLA in large amounts could be enriched using this approach. The GPI-based molecular switching system is an efficient approach to isolate cells expressing recombinant proteins with high productivity.Emmanuel MatabaroZeng’an HeYi-Shi LiuHui-Jie ZhangXiao-Dong GaoMorihisa FujitaNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-13 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Emmanuel Matabaro Zeng’an He Yi-Shi Liu Hui-Jie Zhang Xiao-Dong Gao Morihisa Fujita Molecular switching system using glycosylphosphatidylinositol to select cells highly expressing recombinant proteins
description	Abstract Although many pharmaceutical proteins are produced in mammalian cells, there remains a challenge to select cell lines that express recombinant proteins with high productivity. Since most biopharmaceutical proteins are secreted by cells into the medium, it is difficult to select cell lines that produce large amounts of the target protein. To address this issue, a new protein expression system using the glycosylphosphatidylinositol (GPI)-anchor was developed. PGAP2 is involved in processing GPI-anchored proteins (GPI-APs) during transport. In PGAP2 mutant cells, most GPI-APs are secreted into the medium. Here, we established a HEK293 cell line where endogenous PGAP2 was knocked out and exogenous PGAP2 was inserted with a piggyBac transposon in the genome. Using these cells, human lysosomal acid lipase (LIPA) and α-galactosidase A (GLA) were expressed as GPI-anchored forms (LIPA-GPI and GLA-GPI) and cells expressing high levels of LIPA-GPI or GLA-GPI on the cell surface were enriched. Removal of the PGAP2 gene by piggyBac transposase or FLP recombinase converted LIPA-GPI and GLA-GPI from membrane-bound to the secreted forms. Thus, cells expressing LIPA or GLA in large amounts could be enriched using this approach. The GPI-based molecular switching system is an efficient approach to isolate cells expressing recombinant proteins with high productivity.
format	article
author	Emmanuel Matabaro Zeng’an He Yi-Shi Liu Hui-Jie Zhang Xiao-Dong Gao Morihisa Fujita
author_facet	Emmanuel Matabaro Zeng’an He Yi-Shi Liu Hui-Jie Zhang Xiao-Dong Gao Morihisa Fujita
author_sort	Emmanuel Matabaro
title	Molecular switching system using glycosylphosphatidylinositol to select cells highly expressing recombinant proteins
title_short	Molecular switching system using glycosylphosphatidylinositol to select cells highly expressing recombinant proteins
title_full	Molecular switching system using glycosylphosphatidylinositol to select cells highly expressing recombinant proteins
title_fullStr	Molecular switching system using glycosylphosphatidylinositol to select cells highly expressing recombinant proteins
title_full_unstemmed	Molecular switching system using glycosylphosphatidylinositol to select cells highly expressing recombinant proteins
title_sort	molecular switching system using glycosylphosphatidylinositol to select cells highly expressing recombinant proteins
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/4f584acd06094ce5b04b57b53a29be8f
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Molecular switching system using glycosylphosphatidylinositol to select cells highly expressing recombinant proteins

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