Optimization of a GDNF production method based on Semliki Forest virus vector
Human glial cell line-derived neurotrophic factor (hGDNF) is the most potent dopaminergic factor described so far, and it is therefore considered a promising drug for Parkinson’s disease (PD) treatment. However, the production of therapeutic proteins with a high degree of purity and a specific glyco...
| Autores: | , , , , , , |
|---|---|
| Tipo de recurso: | artículo |
| Fecha de publicación: | 2021 |
| País: | España |
| Institución: | Universidad de Navarra |
| Repositorio: | Dadun. Depósito Académico Digital de la Universidad de Navarra |
| Idioma: | inglés |
| OAI Identifier: | oai:dadun.unav.edu:10171/63698 |
| Acceso en línea: | https://hdl.handle.net/10171/63698 |
| Access Level: | acceso abierto |
| Palabra clave: | GDNF Biphasic growth, BHK cells, Semliki Forest Virus Shut-off Parkinson's disease |
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Optimization of a GDNF production method based on Semliki Forest virus vectorTorres-Ortega, P.V. (Pablo Vicente)|||/items/773ce537-c8dd-4be3-b7e5-2f13fb5ad0deSmerdou-Picazo, C. (Cristian)|||/items/48fd1bd4-6483-45c9-9951-8a1b04873227Ansorena-Artieda, E. (Eduardo)|||/items/eee6c2d6-f73a-48d3-a5f8-fe090b677c82Ballesteros-Briones, M.C. (María Cristina)|||/items/4ede3e7d-1b82-4945-a24c-a96d8e2dff71Martisova, E. (Eva)|||/items/80cb8569-6236-4f78-b528-c3d5bfc8efdeGarbayo, E. (Elisa)|||/items/c0fa35e4-82c5-44f7-b300-740027106dbaBlanco-Prieto, M.J. (María José)|||/items/93e177db-635f-456f-b672-b79ef8befc40GDNFBiphasic growth, BHK cells, Semliki ForestVirusShut-offParkinson's diseaseHuman glial cell line-derived neurotrophic factor (hGDNF) is the most potent dopaminergic factor described so far, and it is therefore considered a promising drug for Parkinson’s disease (PD) treatment. However, the production of therapeutic proteins with a high degree of purity and a specific glycosylation pattern is a major challenge that hinders its commercialization. Although a variety of systems can be used for protein production, only a small number of them are suitable to produce clinical-grade proteins. Specifically, the baby hamster kidney cell line (BHK-21) has shown to be an effective system for the expression of high levels of hGDNF, with appropriate post-translational modifications and protein folding. This system, which is based on the electroporation of BHK-21 cells using a Semliki Forest virus (SFV) as expression vector, induces a strong shut-off of host cell protein synthesis that simplify the purification process. However, SFV vector exhibits a temperature dependent cytopathic effect on host cells, which could limit hGDNF expression. The aim of this study was to improve the expression and purification of hGDNF using a biphasic temperature cultivation protocol that would decrease the cytopathic effect induced by SFV. Here we show that an increase in the temperature from 33◦C to 37◦C during the “shut-off period”, produced a significant improvement in cell survival and hGDNF expression. Inconsonance, this protocol led to the production of almost 3-fold more hGDNF when compared to the previously described methods. Therefore, a “recovery period” at 37◦C before cells are exposed at 33◦C is crucial to maintain cell viability and increase hGDNF expression. The protocol described constitutes an efficient and highly scalable method to produce highly pure hGDNF.ElsevierDadun. Depósito Académico Digital Universidad de Navarra20222022-06-2820212021-01-0120212021-01-01journal articlehttp://purl.org/coar/resource_type/c_6501info:eu-repo/semantics/articleapplication/pdfhttps://hdl.handle.net/10171/63698reponame:Dadun. Depósito Académico Digital de la Universidad de Navarrainstname:Universidad de NavarraInglésengopen accesshttp://purl.org/coar/access_right/c_abf2info:eu-repo/semantics/openAccessoai:dadun.unav.edu:10171/636982026-06-21T12:47:57Z |
| dc.title.none.fl_str_mv |
Optimization of a GDNF production method based on Semliki Forest virus vector |
| title |
Optimization of a GDNF production method based on Semliki Forest virus vector |
| spellingShingle |
Optimization of a GDNF production method based on Semliki Forest virus vector Torres-Ortega, P.V. (Pablo Vicente)|||/items/773ce537-c8dd-4be3-b7e5-2f13fb5ad0de GDNF Biphasic growth, BHK cells, Semliki Forest Virus Shut-off Parkinson's disease |
| title_short |
Optimization of a GDNF production method based on Semliki Forest virus vector |
| title_full |
Optimization of a GDNF production method based on Semliki Forest virus vector |
| title_fullStr |
Optimization of a GDNF production method based on Semliki Forest virus vector |
| title_full_unstemmed |
Optimization of a GDNF production method based on Semliki Forest virus vector |
| title_sort |
Optimization of a GDNF production method based on Semliki Forest virus vector |
| dc.creator.none.fl_str_mv |
Torres-Ortega, P.V. (Pablo Vicente)|||/items/773ce537-c8dd-4be3-b7e5-2f13fb5ad0de Smerdou-Picazo, C. (Cristian)|||/items/48fd1bd4-6483-45c9-9951-8a1b04873227 Ansorena-Artieda, E. (Eduardo)|||/items/eee6c2d6-f73a-48d3-a5f8-fe090b677c82 Ballesteros-Briones, M.C. (María Cristina)|||/items/4ede3e7d-1b82-4945-a24c-a96d8e2dff71 Martisova, E. (Eva)|||/items/80cb8569-6236-4f78-b528-c3d5bfc8efde Garbayo, E. (Elisa)|||/items/c0fa35e4-82c5-44f7-b300-740027106dba Blanco-Prieto, M.J. (María José)|||/items/93e177db-635f-456f-b672-b79ef8befc40 |
| author |
Torres-Ortega, P.V. (Pablo Vicente)|||/items/773ce537-c8dd-4be3-b7e5-2f13fb5ad0de |
| author_facet |
Torres-Ortega, P.V. (Pablo Vicente)|||/items/773ce537-c8dd-4be3-b7e5-2f13fb5ad0de Smerdou-Picazo, C. (Cristian)|||/items/48fd1bd4-6483-45c9-9951-8a1b04873227 Ansorena-Artieda, E. (Eduardo)|||/items/eee6c2d6-f73a-48d3-a5f8-fe090b677c82 Ballesteros-Briones, M.C. (María Cristina)|||/items/4ede3e7d-1b82-4945-a24c-a96d8e2dff71 Martisova, E. (Eva)|||/items/80cb8569-6236-4f78-b528-c3d5bfc8efde Garbayo, E. (Elisa)|||/items/c0fa35e4-82c5-44f7-b300-740027106dba Blanco-Prieto, M.J. (María José)|||/items/93e177db-635f-456f-b672-b79ef8befc40 |
| author_role |
author |
| author2 |
Smerdou-Picazo, C. (Cristian)|||/items/48fd1bd4-6483-45c9-9951-8a1b04873227 Ansorena-Artieda, E. (Eduardo)|||/items/eee6c2d6-f73a-48d3-a5f8-fe090b677c82 Ballesteros-Briones, M.C. (María Cristina)|||/items/4ede3e7d-1b82-4945-a24c-a96d8e2dff71 Martisova, E. (Eva)|||/items/80cb8569-6236-4f78-b528-c3d5bfc8efde Garbayo, E. (Elisa)|||/items/c0fa35e4-82c5-44f7-b300-740027106dba Blanco-Prieto, M.J. (María José)|||/items/93e177db-635f-456f-b672-b79ef8befc40 |
| author2_role |
author author author author author author |
| dc.contributor.none.fl_str_mv |
Dadun. Depósito Académico Digital Universidad de Navarra |
| dc.subject.none.fl_str_mv |
GDNF Biphasic growth, BHK cells, Semliki Forest Virus Shut-off Parkinson's disease |
| topic |
GDNF Biphasic growth, BHK cells, Semliki Forest Virus Shut-off Parkinson's disease |
| description |
Human glial cell line-derived neurotrophic factor (hGDNF) is the most potent dopaminergic factor described so far, and it is therefore considered a promising drug for Parkinson’s disease (PD) treatment. However, the production of therapeutic proteins with a high degree of purity and a specific glycosylation pattern is a major challenge that hinders its commercialization. Although a variety of systems can be used for protein production, only a small number of them are suitable to produce clinical-grade proteins. Specifically, the baby hamster kidney cell line (BHK-21) has shown to be an effective system for the expression of high levels of hGDNF, with appropriate post-translational modifications and protein folding. This system, which is based on the electroporation of BHK-21 cells using a Semliki Forest virus (SFV) as expression vector, induces a strong shut-off of host cell protein synthesis that simplify the purification process. However, SFV vector exhibits a temperature dependent cytopathic effect on host cells, which could limit hGDNF expression. The aim of this study was to improve the expression and purification of hGDNF using a biphasic temperature cultivation protocol that would decrease the cytopathic effect induced by SFV. Here we show that an increase in the temperature from 33◦C to 37◦C during the “shut-off period”, produced a significant improvement in cell survival and hGDNF expression. Inconsonance, this protocol led to the production of almost 3-fold more hGDNF when compared to the previously described methods. Therefore, a “recovery period” at 37◦C before cells are exposed at 33◦C is crucial to maintain cell viability and increase hGDNF expression. The protocol described constitutes an efficient and highly scalable method to produce highly pure hGDNF. |
| publishDate |
2021 |
| dc.date.none.fl_str_mv |
2021 2021-01-01 2021 2021-01-01 2022 2022-06-28 |
| dc.type.none.fl_str_mv |
journal article http://purl.org/coar/resource_type/c_6501 |
| dc.type.openaire.fl_str_mv |
info:eu-repo/semantics/article |
| format |
article |
| dc.identifier.none.fl_str_mv |
https://hdl.handle.net/10171/63698 |
| url |
https://hdl.handle.net/10171/63698 |
| dc.language.none.fl_str_mv |
Inglés eng |
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Inglés |
| language |
eng |
| dc.rights.none.fl_str_mv |
open access http://purl.org/coar/access_right/c_abf2 |
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info:eu-repo/semantics/openAccess |
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open access http://purl.org/coar/access_right/c_abf2 |
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openAccess |
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application/pdf |
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Elsevier |
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Elsevier |
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reponame:Dadun. Depósito Académico Digital de la Universidad de Navarra instname:Universidad de Navarra |
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Universidad de Navarra |
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Dadun. Depósito Académico Digital de la Universidad de Navarra |
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Dadun. Depósito Académico Digital de la Universidad de Navarra |
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