Purification of green fluorescent protein using fast centrifugal partition chromatography
Autor(a) principal: | |
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Data de Publicação: | 2021 |
Outros Autores: | , , , , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Institucional da UNESP |
Texto Completo: | http://dx.doi.org/10.1016/j.seppur.2020.117648 http://hdl.handle.net/11449/205439 |
Resumo: | The green fluorescent protein (GFP) is a biomolecule used in many biological applications such as biomarkers and biosensors, which require high purity levels. It is usually obtained from recombinant Escherichia coli strains, which also produces other endogenous proteins, demanding multiple purification steps, and consequently, increasing the overall costs to obtain pure GFP. Simpler and cheaper purification methods like Aqueous Biphasic Systems (ABS) were already successfully applied to purify GFP at lab scale. Therefore, the development of automatized industrially compatible purification platforms, such as countercurrent chromatography using ABS, can potentially improve the GFP production. This work studied the continuous purification of the variant enhanced GFP (EGFP) by applying ABS composed of polyethylene glycol (PEG 8000), sodium polyacrylate (NaPA 8000) and sodium sulfate (Na2SO4) as electrolyte. An initial screening was carried by changing the electrolyte content in the ABS. The increase of this condition has demonstrated an increase on the EGFP partition for the PEG-rich phase. The most efficient ABS and, at the same time, with the most appropriate conditions, namely the system composed of 15 wt% PEG 8000 + 4.5 wt% NaPA 8000 + 2.5 wt% Na2SO4 was chosen and applied on the fast centrifugal partition chromatography (FCPC). After optimization, the best operational conditions were identified, i.e. a flow rate of 2.5 mL.min−1 and rotation speed of 2000 rpm at ascending mode, and the best results obtained, meaning a purification of 89.93% and a recovery yield of 82.3%, confirming the potential of FCPC to the continuous purification of EGFP. |
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Purification of green fluorescent protein using fast centrifugal partition chromatographyAqueous biphasic systemsElectrolyteEnhanced green fluorescent proteinFast centrifugal partition chromatographyPurificationThe green fluorescent protein (GFP) is a biomolecule used in many biological applications such as biomarkers and biosensors, which require high purity levels. It is usually obtained from recombinant Escherichia coli strains, which also produces other endogenous proteins, demanding multiple purification steps, and consequently, increasing the overall costs to obtain pure GFP. Simpler and cheaper purification methods like Aqueous Biphasic Systems (ABS) were already successfully applied to purify GFP at lab scale. Therefore, the development of automatized industrially compatible purification platforms, such as countercurrent chromatography using ABS, can potentially improve the GFP production. This work studied the continuous purification of the variant enhanced GFP (EGFP) by applying ABS composed of polyethylene glycol (PEG 8000), sodium polyacrylate (NaPA 8000) and sodium sulfate (Na2SO4) as electrolyte. An initial screening was carried by changing the electrolyte content in the ABS. The increase of this condition has demonstrated an increase on the EGFP partition for the PEG-rich phase. The most efficient ABS and, at the same time, with the most appropriate conditions, namely the system composed of 15 wt% PEG 8000 + 4.5 wt% NaPA 8000 + 2.5 wt% Na2SO4 was chosen and applied on the fast centrifugal partition chromatography (FCPC). After optimization, the best operational conditions were identified, i.e. a flow rate of 2.5 mL.min−1 and rotation speed of 2000 rpm at ascending mode, and the best results obtained, meaning a purification of 89.93% and a recovery yield of 82.3%, confirming the potential of FCPC to the continuous purification of EGFP.CICECO – Aveiro Institute of Materials Department of Chemistry University of AveiroDepartment of Biochemical and Pharmaceutical Technology University of São PauloDepartment of Bioprocesses and Biotechnology School of Pharmaceutical Sciences São Paulo State University (UNESP)Univ Coimbra CIEPQPF Department of Chemical Engineering, Rua Sílvio Lima, Pólo II – Pinhal de MarrocosDepartment of Bioprocesses and Biotechnology School of Pharmaceutical Sciences São Paulo State University (UNESP)University of AveiroUniversidade de São Paulo (USP)Universidade Estadual Paulista (Unesp)CIEPQPFSoares, Bruna P.Santos, João H.P.M.Martins, MargaridaAlmeida, Mafalda R.Santos, Nathalia V. [UNESP]Freire, Mara G.Santos-Ebinuma, Valéria C. [UNESP]Coutinho, João A.P.Pereira, Jorge F.B. [UNESP]Ventura, Sónia P.M.2021-06-25T10:15:20Z2021-06-25T10:15:20Z2021-02-15info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articlehttp://dx.doi.org/10.1016/j.seppur.2020.117648Separation and Purification Technology, v. 257.1873-37941383-5866http://hdl.handle.net/11449/20543910.1016/j.seppur.2020.1176482-s2.0-85095702939Scopusreponame:Repositório Institucional da UNESPinstname:Universidade Estadual Paulista (UNESP)instacron:UNESPengSeparation and Purification Technologyinfo:eu-repo/semantics/openAccess2021-10-23T14:27:06Zoai:repositorio.unesp.br:11449/205439Repositório InstitucionalPUBhttp://repositorio.unesp.br/oai/requestopendoar:29462024-08-05T19:33:07.871063Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP)false |
dc.title.none.fl_str_mv |
Purification of green fluorescent protein using fast centrifugal partition chromatography |
title |
Purification of green fluorescent protein using fast centrifugal partition chromatography |
spellingShingle |
Purification of green fluorescent protein using fast centrifugal partition chromatography Soares, Bruna P. Aqueous biphasic systems Electrolyte Enhanced green fluorescent protein Fast centrifugal partition chromatography Purification |
title_short |
Purification of green fluorescent protein using fast centrifugal partition chromatography |
title_full |
Purification of green fluorescent protein using fast centrifugal partition chromatography |
title_fullStr |
Purification of green fluorescent protein using fast centrifugal partition chromatography |
title_full_unstemmed |
Purification of green fluorescent protein using fast centrifugal partition chromatography |
title_sort |
Purification of green fluorescent protein using fast centrifugal partition chromatography |
author |
Soares, Bruna P. |
author_facet |
Soares, Bruna P. Santos, João H.P.M. Martins, Margarida Almeida, Mafalda R. Santos, Nathalia V. [UNESP] Freire, Mara G. Santos-Ebinuma, Valéria C. [UNESP] Coutinho, João A.P. Pereira, Jorge F.B. [UNESP] Ventura, Sónia P.M. |
author_role |
author |
author2 |
Santos, João H.P.M. Martins, Margarida Almeida, Mafalda R. Santos, Nathalia V. [UNESP] Freire, Mara G. Santos-Ebinuma, Valéria C. [UNESP] Coutinho, João A.P. Pereira, Jorge F.B. [UNESP] Ventura, Sónia P.M. |
author2_role |
author author author author author author author author author |
dc.contributor.none.fl_str_mv |
University of Aveiro Universidade de São Paulo (USP) Universidade Estadual Paulista (Unesp) CIEPQPF |
dc.contributor.author.fl_str_mv |
Soares, Bruna P. Santos, João H.P.M. Martins, Margarida Almeida, Mafalda R. Santos, Nathalia V. [UNESP] Freire, Mara G. Santos-Ebinuma, Valéria C. [UNESP] Coutinho, João A.P. Pereira, Jorge F.B. [UNESP] Ventura, Sónia P.M. |
dc.subject.por.fl_str_mv |
Aqueous biphasic systems Electrolyte Enhanced green fluorescent protein Fast centrifugal partition chromatography Purification |
topic |
Aqueous biphasic systems Electrolyte Enhanced green fluorescent protein Fast centrifugal partition chromatography Purification |
description |
The green fluorescent protein (GFP) is a biomolecule used in many biological applications such as biomarkers and biosensors, which require high purity levels. It is usually obtained from recombinant Escherichia coli strains, which also produces other endogenous proteins, demanding multiple purification steps, and consequently, increasing the overall costs to obtain pure GFP. Simpler and cheaper purification methods like Aqueous Biphasic Systems (ABS) were already successfully applied to purify GFP at lab scale. Therefore, the development of automatized industrially compatible purification platforms, such as countercurrent chromatography using ABS, can potentially improve the GFP production. This work studied the continuous purification of the variant enhanced GFP (EGFP) by applying ABS composed of polyethylene glycol (PEG 8000), sodium polyacrylate (NaPA 8000) and sodium sulfate (Na2SO4) as electrolyte. An initial screening was carried by changing the electrolyte content in the ABS. The increase of this condition has demonstrated an increase on the EGFP partition for the PEG-rich phase. The most efficient ABS and, at the same time, with the most appropriate conditions, namely the system composed of 15 wt% PEG 8000 + 4.5 wt% NaPA 8000 + 2.5 wt% Na2SO4 was chosen and applied on the fast centrifugal partition chromatography (FCPC). After optimization, the best operational conditions were identified, i.e. a flow rate of 2.5 mL.min−1 and rotation speed of 2000 rpm at ascending mode, and the best results obtained, meaning a purification of 89.93% and a recovery yield of 82.3%, confirming the potential of FCPC to the continuous purification of EGFP. |
publishDate |
2021 |
dc.date.none.fl_str_mv |
2021-06-25T10:15:20Z 2021-06-25T10:15:20Z 2021-02-15 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://dx.doi.org/10.1016/j.seppur.2020.117648 Separation and Purification Technology, v. 257. 1873-3794 1383-5866 http://hdl.handle.net/11449/205439 10.1016/j.seppur.2020.117648 2-s2.0-85095702939 |
url |
http://dx.doi.org/10.1016/j.seppur.2020.117648 http://hdl.handle.net/11449/205439 |
identifier_str_mv |
Separation and Purification Technology, v. 257. 1873-3794 1383-5866 10.1016/j.seppur.2020.117648 2-s2.0-85095702939 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
Separation and Purification Technology |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.source.none.fl_str_mv |
Scopus reponame:Repositório Institucional da UNESP instname:Universidade Estadual Paulista (UNESP) instacron:UNESP |
instname_str |
Universidade Estadual Paulista (UNESP) |
instacron_str |
UNESP |
institution |
UNESP |
reponame_str |
Repositório Institucional da UNESP |
collection |
Repositório Institucional da UNESP |
repository.name.fl_str_mv |
Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP) |
repository.mail.fl_str_mv |
|
_version_ |
1808129084994093056 |