Optimization of expression yield in a stable cell line expressing a novel mutated chimeric tissue plasminogen activator (mt-PA)
Autor(a) principal: | |
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Data de Publicação: | 2022 |
Outros Autores: | , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Brazilian Journal of Pharmaceutical Sciences |
Texto Completo: | https://www.revistas.usp.br/bjps/article/view/204612 |
Resumo: | The development of stable cell lines producing recombinant proteins is very time-consuming and laborious. One of the practical approaches successfully performed is Fluorescence-Activated Cell Sorting (FACS). A mutated chimeric tissue plasminogen activator (mt-PA) was developed by removing the first three domains of t-PA, insertion of GHRP sequence and mutation toward resistance to plasminogen activator inhibitor-1 (PAI-1). In the current study, a new stable CHO-DG44 cell line producing mt-PA was developed by two sequential clonal selections: FACS and clonal-selection by limiting dilution. Furthermore, the expression was more evaluated using two different expression media. Finally, the high-producing clones were selected based on the dot blot and amidolytic activity test. The transfection efficiency of CHO-DG44 cells was 38% as measured by flow cytometry on green fluorescent protein (GFP). After performing FACS on stable cell pools, the expression yield was increased to fifty-fold. In terms of growth profile, CD-DG44 showed higher viability and cell density results than ProCHO5 medium. The expression of mt-PA was significantly higher in CD-DG44 than in ProCHO5, 765 and 280 IU/mL, respectively. Our data indicated that selection of an appropriate expression medium played a critical role in the development of potent producing stable cells by FACS. |
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Brazilian Journal of Pharmaceutical Sciences |
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Optimization of expression yield in a stable cell line expressing a novel mutated chimeric tissue plasminogen activator (mt-PA)CHO-DG44 Fluorescence-Activated Cell Sortingmt-PAStable cell lineTissue plasminogen activatorThe development of stable cell lines producing recombinant proteins is very time-consuming and laborious. One of the practical approaches successfully performed is Fluorescence-Activated Cell Sorting (FACS). A mutated chimeric tissue plasminogen activator (mt-PA) was developed by removing the first three domains of t-PA, insertion of GHRP sequence and mutation toward resistance to plasminogen activator inhibitor-1 (PAI-1). In the current study, a new stable CHO-DG44 cell line producing mt-PA was developed by two sequential clonal selections: FACS and clonal-selection by limiting dilution. Furthermore, the expression was more evaluated using two different expression media. Finally, the high-producing clones were selected based on the dot blot and amidolytic activity test. The transfection efficiency of CHO-DG44 cells was 38% as measured by flow cytometry on green fluorescent protein (GFP). After performing FACS on stable cell pools, the expression yield was increased to fifty-fold. In terms of growth profile, CD-DG44 showed higher viability and cell density results than ProCHO5 medium. The expression of mt-PA was significantly higher in CD-DG44 than in ProCHO5, 765 and 280 IU/mL, respectively. Our data indicated that selection of an appropriate expression medium played a critical role in the development of potent producing stable cells by FACS.Universidade de São Paulo. Faculdade de Ciências Farmacêuticas2022-11-23info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionapplication/pdfhttps://www.revistas.usp.br/bjps/article/view/20461210.1590/s2175-97902022e19692 Brazilian Journal of Pharmaceutical Sciences; Vol. 58 (2022)Brazilian Journal of Pharmaceutical Sciences; v. 58 (2022)Brazilian Journal of Pharmaceutical Sciences; Vol. 58 (2022)2175-97901984-8250reponame:Brazilian Journal of Pharmaceutical Sciencesinstname:Universidade de São Paulo (USP)instacron:USPenghttps://www.revistas.usp.br/bjps/article/view/204612/194547Copyright (c) 2022 Brazilian Journal of Pharmaceutical Scienceshttps://creativecommons.org/licenses/by/4.0info:eu-repo/semantics/openAccessRaigani, Mozhgan Barkhordari, FarzanehMoazzami, RezaDavami, Fatemeh2023-05-29T14:29:55Zoai:revistas.usp.br:article/204612Revistahttps://www.revistas.usp.br/bjps/indexPUBhttps://old.scielo.br/oai/scielo-oai.phpbjps@usp.br||elizabeth.igne@gmail.com2175-97901984-8250opendoar:2023-05-29T14:29:55Brazilian Journal of Pharmaceutical Sciences - Universidade de São Paulo (USP)false |
dc.title.none.fl_str_mv |
Optimization of expression yield in a stable cell line expressing a novel mutated chimeric tissue plasminogen activator (mt-PA) |
title |
Optimization of expression yield in a stable cell line expressing a novel mutated chimeric tissue plasminogen activator (mt-PA) |
spellingShingle |
Optimization of expression yield in a stable cell line expressing a novel mutated chimeric tissue plasminogen activator (mt-PA) Raigani, Mozhgan CHO-DG44 Fluorescence-Activated Cell Sorting mt-PA Stable cell line Tissue plasminogen activator |
title_short |
Optimization of expression yield in a stable cell line expressing a novel mutated chimeric tissue plasminogen activator (mt-PA) |
title_full |
Optimization of expression yield in a stable cell line expressing a novel mutated chimeric tissue plasminogen activator (mt-PA) |
title_fullStr |
Optimization of expression yield in a stable cell line expressing a novel mutated chimeric tissue plasminogen activator (mt-PA) |
title_full_unstemmed |
Optimization of expression yield in a stable cell line expressing a novel mutated chimeric tissue plasminogen activator (mt-PA) |
title_sort |
Optimization of expression yield in a stable cell line expressing a novel mutated chimeric tissue plasminogen activator (mt-PA) |
author |
Raigani, Mozhgan |
author_facet |
Raigani, Mozhgan Barkhordari, Farzaneh Moazzami, Reza Davami, Fatemeh |
author_role |
author |
author2 |
Barkhordari, Farzaneh Moazzami, Reza Davami, Fatemeh |
author2_role |
author author author |
dc.contributor.author.fl_str_mv |
Raigani, Mozhgan Barkhordari, Farzaneh Moazzami, Reza Davami, Fatemeh |
dc.subject.por.fl_str_mv |
CHO-DG44 Fluorescence-Activated Cell Sorting mt-PA Stable cell line Tissue plasminogen activator |
topic |
CHO-DG44 Fluorescence-Activated Cell Sorting mt-PA Stable cell line Tissue plasminogen activator |
description |
The development of stable cell lines producing recombinant proteins is very time-consuming and laborious. One of the practical approaches successfully performed is Fluorescence-Activated Cell Sorting (FACS). A mutated chimeric tissue plasminogen activator (mt-PA) was developed by removing the first three domains of t-PA, insertion of GHRP sequence and mutation toward resistance to plasminogen activator inhibitor-1 (PAI-1). In the current study, a new stable CHO-DG44 cell line producing mt-PA was developed by two sequential clonal selections: FACS and clonal-selection by limiting dilution. Furthermore, the expression was more evaluated using two different expression media. Finally, the high-producing clones were selected based on the dot blot and amidolytic activity test. The transfection efficiency of CHO-DG44 cells was 38% as measured by flow cytometry on green fluorescent protein (GFP). After performing FACS on stable cell pools, the expression yield was increased to fifty-fold. In terms of growth profile, CD-DG44 showed higher viability and cell density results than ProCHO5 medium. The expression of mt-PA was significantly higher in CD-DG44 than in ProCHO5, 765 and 280 IU/mL, respectively. Our data indicated that selection of an appropriate expression medium played a critical role in the development of potent producing stable cells by FACS. |
publishDate |
2022 |
dc.date.none.fl_str_mv |
2022-11-23 |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
https://www.revistas.usp.br/bjps/article/view/204612 10.1590/s2175-97902022e19692 |
url |
https://www.revistas.usp.br/bjps/article/view/204612 |
identifier_str_mv |
10.1590/s2175-97902022e19692 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
https://www.revistas.usp.br/bjps/article/view/204612/194547 |
dc.rights.driver.fl_str_mv |
Copyright (c) 2022 Brazilian Journal of Pharmaceutical Sciences https://creativecommons.org/licenses/by/4.0 info:eu-repo/semantics/openAccess |
rights_invalid_str_mv |
Copyright (c) 2022 Brazilian Journal of Pharmaceutical Sciences https://creativecommons.org/licenses/by/4.0 |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
Universidade de São Paulo. Faculdade de Ciências Farmacêuticas |
publisher.none.fl_str_mv |
Universidade de São Paulo. Faculdade de Ciências Farmacêuticas |
dc.source.none.fl_str_mv |
Brazilian Journal of Pharmaceutical Sciences; Vol. 58 (2022) Brazilian Journal of Pharmaceutical Sciences; v. 58 (2022) Brazilian Journal of Pharmaceutical Sciences; Vol. 58 (2022) 2175-9790 1984-8250 reponame:Brazilian Journal of Pharmaceutical Sciences instname:Universidade de São Paulo (USP) instacron:USP |
instname_str |
Universidade de São Paulo (USP) |
instacron_str |
USP |
institution |
USP |
reponame_str |
Brazilian Journal of Pharmaceutical Sciences |
collection |
Brazilian Journal of Pharmaceutical Sciences |
repository.name.fl_str_mv |
Brazilian Journal of Pharmaceutical Sciences - Universidade de São Paulo (USP) |
repository.mail.fl_str_mv |
bjps@usp.br||elizabeth.igne@gmail.com |
_version_ |
1800222916116217856 |