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...
| Authors: | ; ; ; ; ; ; |
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| Format: | Article |
| Publication date: | 2021 |
| Country: | España |
| Institution: | Universidad de Navarra |
| Repository: | Dadun. Depósito Académico Digital de la Universidad de Navarra |
| Language: | English |
| OAI Identifier: | oai:dadun.unav.edu:10171/63698 |
| Online Access: | https://hdl.handle.net/10171/63698 |
| Access Level: | Open access |
| Keyword: | GDNF Biphasic growth, BHK cells, Semliki Forest Virus Shut-off Parkinson's disease |
| _version_ | 1878732174919729153 |
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| author | Torres-Ortega, P.V. (Pablo Vicente)|||/items/773ce537-c8dd-4be3-b7e5-2f13fb5ad0de |
| 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 |
| author_browse | Ansorena-Artieda, E. (Eduardo)|||/items/eee6c2d6-f73a-48d3-a5f8-fe090b677c82 Ballesteros-Briones, M.C. (María Cristina)|||/items/4ede3e7d-1b82-4945-a24c-a96d8e2dff71 Blanco-Prieto, M.J. (María José)|||/items/93e177db-635f-456f-b672-b79ef8befc40 Garbayo, E. (Elisa)|||/items/c0fa35e4-82c5-44f7-b300-740027106dba Martisova, E. (Eva)|||/items/80cb8569-6236-4f78-b528-c3d5bfc8efde Smerdou-Picazo, C. (Cristian)|||/items/48fd1bd4-6483-45c9-9951-8a1b04873227 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 |
| collection | Dadun. Depósito Académico Digital de la Universidad de Navarra |
| 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. |
| eu_rights_str_mv | openAccess |
| format | article |
| id | ES_3df609c0fca3b51c339f275944da0a73 |
| institution | Universidad de Navarra |
| instname_str | Universidad de Navarra |
| language | eng |
| network_acronym_str | ES |
| network_name_str | España |
| oai_identifier_str | oai:dadun.unav.edu:10171/63698 |
| publishDate | 2021 |
| publishDateSort | 2021 |
| publisher | Elsevier |
| publisherStr | Elsevier |
| reponame_str | Dadun. Depósito Académico Digital de la Universidad de Navarra |
| spelling | Optimization of a GDNF production method based on Semliki Forest virus vector 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 GDNF Biphasic growth, BHK cells, Semliki Forest Virus Shut-off Parkinson's disease 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. Elsevier https://hdl.handle.net/10171/63698 |
| 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 | 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_short | 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 |
| topic | GDNF Biphasic growth, BHK cells, Semliki Forest Virus Shut-off Parkinson's disease |
| topic_facet | GDNF Biphasic growth, BHK cells, Semliki Forest Virus Shut-off Parkinson's disease |
| url | https://hdl.handle.net/10171/63698 |