Optimization of expression yield in a stable cell line expressing a novel mutated chimeric tissue plasminogen activator (mt-PA)
Braz. J. Pharm. Sci. (Online)
; 58: e19692, 2022. graf
Article
de En
| LILACS
| ID: biblio-1384014
Bibliothèque responsable:
BR40.1
Localisation: BR40.1
ABSTRACT
Abstract 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.
Mots clés
Texte intégral:
1
Indice:
LILACS
Sujet Principal:
Activateur tissulaire du plasminogène
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Optimisation du Processus
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Cytométrie en flux
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Fluorescence
langue:
En
Texte intégral:
Braz. J. Pharm. Sci. (Online)
Thème du journal:
Farmacologia
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Teraputica
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Toxicologia
Année:
2022
Type:
Article