Molecular switching system using glycosylphosphatidylinositol to select cells highly expressing recombinant proteins.
Sci Rep
; 7(1): 4033, 2017 06 22.
Article
en En
| MEDLINE
| ID: mdl-28642584
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.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Regulación de la Expresión Génica
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Glicosilfosfatidilinositoles
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Orden Génico
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Proteínas Ligadas a GPI
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Vectores Genéticos
Límite:
Animals
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Humans
Idioma:
En
Revista:
Sci Rep
Año:
2017
Tipo del documento:
Article
País de afiliación:
China
Pais de publicación:
Reino Unido