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Optimization of expression yield in a stable cell line expressing a novel mutated chimeric tissue plasminogen activator (mt-PA)
Raigani, Mozhgan; Barkhordari, Farzaneh; Moazzami, Reza; Davami, Fatemeh; Mahboudi, Fereidoun.
  • Raigani, Mozhgan; Pasteur Institute of Iran. Biotechnology Research Center. Tehran. IR
  • Barkhordari, Farzaneh; Pasteur Institute of Iran. Biotechnology Research Center. Tehran. IR
  • Moazzami, Reza; Pasteur Institute of Iran. Biotechnology Research Center. Tehran. IR
  • Davami, Fatemeh; Pasteur Institute of Iran. Biotechnology Research Center. Tehran. IR
  • Mahboudi, Fereidoun; Pasteur Institute of Iran. Biotechnology Research Center. Tehran. IR
Braz. J. Pharm. Sci. (Online) ; 58: e19692, 2022. graf
Article in English | LILACS | ID: biblio-1384014
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.
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Full text: Available Index: LILACS (Americas) Main subject: Tissue Plasminogen Activator / Process Optimization / Flow Cytometry / Fluorescence Language: English Journal: Braz. J. Pharm. Sci. (Online) Journal subject: Farmacologia / Terapˆutica / Toxicologia Year: 2022 Type: Article Affiliation country: Iran Institution/Affiliation country: Pasteur Institute of Iran/IR

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Full text: Available Index: LILACS (Americas) Main subject: Tissue Plasminogen Activator / Process Optimization / Flow Cytometry / Fluorescence Language: English Journal: Braz. J. Pharm. Sci. (Online) Journal subject: Farmacologia / Terapˆutica / Toxicologia Year: 2022 Type: Article Affiliation country: Iran Institution/Affiliation country: Pasteur Institute of Iran/IR