Optimization of expression yield in a stable cell line expressing a novel mutated chimeric tissue plasminogen activator (mt-PA)
DOI:
https://doi.org/10.1590/s2175-97902022e19692%20Keywords:
CHO-DG44, Fluorescence-Activated Cell Sorting, mt-PA, Stable cell line, Tissue plasminogen activatorAbstract
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.
Downloads
References
Azarian B, Sajedin SM, Azimi A, Raigani M, Vaziri B, Davami F. Proteomics profiling of chimeric-truncated tissue plasminogen activator producing- chinese hamster ovary cells cultivated in a chemically defined medium supplemented with protein hydrolysates. Iran Biomed J. 2017;21(3):154-66.
Barnes LM, Bentley CM, Dickson AJ. Characterization of the stability of recombinant protein production in the GS-NS0 expression system. Biotechnol Bioeng. 2001;73(4):261-70.
Barreto AD. Intravenous thrombolytics for ischemic stroke. Neurotherapeutics. 2011;8(3):388-99.
Beckers T, Hauser HJ, Husken D, Engels JW. Expression and characterization of a des-methionine mutant interleukin-2 receptor (Tac protein) with interleukin-2 binding affinity. J Biol Chem. 1988;263(17):8359-65.
Browne SM, Al-Rubeai M. Selection methods for high-producing mammalian cell lines. Trends Biotechnol. 2007;25(9):425-32.
Butler M. Animal cell cultures: recent achievements and perspectives in the production of biopharmaceuticals. Appl Microbiol Biotechnol. 2005;68(3):283-91.
Carroll S, Al-Rubeai M. ACSD labelling and magnetic cell separation: a rapid method of separating antibody secreting cells from non-secreting cells. J Immunol Methods. 2005;296(1-2):171-8.
Choe J, Guo HH, van den Engh G. A dual-fluorescence reporter system for high-throughput clone characterization and selection by cell sorting. Nucleic Acids Res. 2005;33(5):e49.
Davami F, Barkhordari F, Alebouyeh M, Adeli A, Mahboudi F. Combined TGE-SGE expression of novel PAI-1-resistant t-PA in CHO DG44 cells using orbitally shaking disposable bioreactors. J Microbiol Biotechnol. 2011a;21(12):1299-305.
Davami F, Sardari S, Majidzadeh AK, Hemayatkar M, Barkhordari F, Enayati S, et al. A novel variant of t-PA resistant to plasminogen activator inhibitor-1; expression in CHO cells based on in silico experiments. BMB Rep. 2011b;44(1):34-9.
Davami F, Sardari S, Majidzadeh AK, Hemayatkar M, Barkhrdari F, Omidi M, et al. Expression of a novel chimeric truncated t-PA in CHO cells based on in silico experiments. J Biomed Biotechnol. 2010;2010:108159.
Dean PN. Helpful hints in flow cytometry and sorting. Cytometry. 1985;6(1):62-4.
DeMaria CT, Cairns V, Schwarz C, Zhang J, Guerin M, Zuena E, et al. Accelerated clone selection for recombinant CHO CELLS using a FACS-based high-throughput screen. Biotechnol Prog. 2007;23(2):465-72.
Kavathas P, Herzenberg LA. Amplification of a gene coding for human T-cell differentiation antigen. Nature. 1983;306(5941):385-7.
Kayser O, Warzecha H. Pharmaceutical Biotechnology: Drug Discovery and Clinical Applications. 2012.
Kim NS, Kim SJ, Lee GM. Clonal variability within dihydrofolate reductase-mediated gene amplified Chinese hamster ovary cells: stability in the absence of selective pressure. Biotechnol Bioeng . 1998a;60(6):679-88.
Kim SJ, Kim NS, Ryu CJ, Hong HJ, Lee GM. Characterization of chimeric antibody producing CHO cells in the course of dihydrofolate reductase-mediated gene amplification and their stability in the absence of selective pressure. Biotechnol Bioeng . 1998b;58(1):73-84.
Kim YG, Park B, Ahn JO, Jung JK, Lee HW, Lee EG. New cell line development for antibody-producing Chinese hamster ovary cells using split green fluorescent protein. BMC Biotechnol. 2012;12:24.
Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227(5259):680-5.
Lamond AI, Earnshaw WC. Structure and function in the nucleus. Science. 1998;280(5363):547-53.
Mancia F, Patel SD, Rajala MW, Scherer PE, Nemes A, Schieren I, et al. Optimization of protein production in mammalian cells with a coexpressed fluorescent marker. Structure. 2004;12(8):1355-60.
Martinez R, Schwaneberg U. A roadmap to directed enzyme evolution and screening systems for biotechnological applications. Biol Res. 2013;46(4):395-405.
Mattanovich D, Borth N. Applications of cell sorting in biotechnology. Microb Cell Fact. 2006;5:12.
Meng YG, Liang J, Wong WL, Chisholm V. Green fluorescent protein as a second selectable marker for selection of high producing clones from transfected CHO cells. Gene. 2000;242(1-2):201-7.
Mohan C, Kim YG, Koo J, Lee GM. Assessment of cell engineering strategies for improved therapeutic protein production in CHO cells. Biotechnol J. 2008;3(5):624-30.
Pandya BV, Gabriel JL, O'Brien J, Budzynski AZ. Polymerization site in the beta chain of fibrin: mapping of the B beta 1-55 sequence. Biochemistry. 1991;30(1):162-8.
Pendse GJ, Karkare S, Bailey JE. Effect of cloned gene dosage on cell growth and hepatitis B surface antigen synthesis and secretion in recombinant CHO cells. Biotechnol Bioeng . 1992;40(1):119-29.
Puck TT, Marcus PI. A Rapid Method for Viable Cell Titration and Clone Production with Hela Cells in Tissue Culture: The Use of X-Irradiated Cells to Supply Conditioning Factors. Proc Natl Acad Sci USA. 1955;41(7):432-7.
Raigani M, Rouini M-R, Golabchifar A-A, Mirabzadeh E, Vaziri B, Barkhordari F, et al. Scale up and pharmacokinetic study of a novel mutated chimeric tissue plasminogen activator (mt-PA) in rats. Scientific Reports. 2017;7:43028.
Sambrook J, Fritsch EF, Maniatis T. Molecular cloning : a laboratory manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory. 1989.
Sleiman RJ, Gray PP, McCall MN, Codamo J, Sunstrom NA. Accelerated cell line development using two-color fluorescence activated cell sorting to select highly expressing antibody-producing clones. Biotechnol Bioeng . 2008;99(3):578-87.
Vinzon SE, Pirpignani ML, Nowicki C, Biscoglio de Jimenez Bonino M. Molecular cloning and expression in Pichia pastoris of a hypoallergenic antigen 5. Protein Expr Purif. 2010;73(1):23-30.
Walsh G. Biopharmaceutical benchmarks 2006. Nat Biotechnol. 2006;24(7):769-76.
Welsh S, Kay SA. Reporter gene expression for monitoring gene transfer. Curr Opin Biotechnol. 1997;8(5):617-22.
Wulhfard S. Transient recombinant protein expression in mammalian cells - the role of mRNA level and stability: EPFL. 2009.
Wurm FM. Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotech. 2004a;22(11):1393-8.
Wurm FM. Production of recombinant protein therapeutics in cultivated mammalian cells. Nat Biotechnol. 2004b;22(11):1393-8.
Yoshikawa T, Nakanishi F, Ogura Y, Oi D, Omasa T, Katakura Y, et al. Flow cytometry: an improved method for the selection of highly productive gene-amplified CHO cells using flow cytometry. Biotechnol Bioeng . 2001;74(5):435-42.
Zang R, Li D, Tang I-C, Wang J, Yang S-T. Cell-Based Assays in High-Throughput Screening for Drug Discovery. Int J Biotechnol Wellness Industries. 2012;1:31-51.
Downloads
Published
Issue
Section
License
Copyright (c) 2022 Brazilian Journal of Pharmaceutical Sciences
This work is licensed under a Creative Commons Attribution 4.0 International License.
All content of the journal, except where identified, is licensed under a Creative Commons attribution-type BY.
The on line journal has open and free access.
