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1.
Biotechnol Appl Biochem ; 68(2): 230-238, 2021 Apr.
Article in English | MEDLINE | ID: mdl-32249976

ABSTRACT

In the pharmaceutical industry, the need for high levels of protein expression in mammalian cells has prompted the search for new strategies, including technologies to obtain cells with improved mechanisms that enhance its transcriptional activity, folding, or protein secretion. Chinese Hamster Ovary (CHO) cells are by far the most used host cell for therapeutic protein expression. However, these cells produce specific glycans that are not present in human cells and therefore potentially immunogenic. As a result, there is an increased interest in the use of human-derived cells for therapeutic protein production. For many decades, human embryonic kidney (HEK) cells were exclusively used for research. However, two products for therapeutic indication were recently approved in the United States. It was previously shown that tethered Magoh, an Exon-junction complex core component, to specific mRNA sequences, have had significant positive effects on mRNA translational efficiency. In this study, a HEK Magoh-overexpressing cell line and clones, designated here as HEK-MAGO, were developed for the first time. These cells exhibited improved characteristics in protein expression, reaching -two- to threefold increases in rhEPO protein production in comparison with the wild-type cells. Moreover, this effect was promoter independent highlighting the versatility of this expression platform.


Subject(s)
DNA-Binding Proteins/biosynthesis , Erythropoietin/biosynthesis , Gene Expression , Animals , CHO Cells , Cricetulus , DNA-Binding Proteins/genetics , Erythropoietin/genetics , HEK293 Cells , Humans , Recombinant Proteins/biosynthesis , Recombinant Proteins/genetics
2.
Appl Biochem Biotechnol ; 189(2): 661-679, 2019 Oct.
Article in English | MEDLINE | ID: mdl-31093907

ABSTRACT

Gemini-based amphiphiles are candidates for biomedical applications. In fact, most of the gemini compounds described in the literature have been prepared to be used as new synthetic vectors in gene transfection. Our group carried out an activity-structure study starting from the structure of the gemini [AG2-C18/]2, which is an effective in vitro transfection reagent. We synthesized a series of novel amphiphilic amino acid derivatives of low molecular weight, named AGn-Cm (N), in which the same apolar region (m) of oleic or palmitic acid was maintained and the peptide region was modified by amino acid insertions, deletions, and substitutions. We also determined the transfection efficiency, critical micelle concentration, particle size, and ζ-potential for these derivatives. Amphiphiles AG10-C16 and AG10-C18 were more active at a lower N/P ratio than AG2-C18. These amphiphiles showed no activity when lysine was replaced by ornithine, and the activity of all derivatives increased when there were more ornithine residues and a W/O = 1 ratio in the peptide region. It can be said that for AG10-C16, these two structural requirements on the amino acid portion predominated over the type of aliphatic chain used.


Subject(s)
Micelles , Peptides , Transfection , Animals , CHO Cells , Cricetulus , HEK293 Cells , Humans , Peptides/chemistry , Peptides/pharmacology
3.
J Biotechnol ; 286: 56-67, 2018 Nov 20.
Article in English | MEDLINE | ID: mdl-30243609

ABSTRACT

Chinese hamster ovary (CHO) derived cell lines are the preferred host system for the production of therapeutic proteins. The aim of this work was to explore the regulation of suspension-adapted CHO-K1 host cell line bioprocesses, especially under a temperature gradient from 37 °C to 31 °C. We analyzed cell cycle behavior through flow cytometry of propidium iodide stained cells and high throughput transcriptome dynamics by RNA sequencing. We found a cell culture state characterized by G0/G1 synchronization, mainly during the late exponential growth phase and towards the last days of the stationary phase. We successfully identified key genes and pathways connected with the particular culture states, such as response to low temperature, modulation of the cell cycle, regulation of DNA replication and repair, apoptosis, among others. The most important gene expression changes occurred throughout the stationary phase when gene up-regulation markedly prevailed. Our RNA-seq data analysis enabled the identification of target genes for mechanism-based cell line engineering and bioprocess modification, an essential step to translate gene expression data from CHO-K1 host cells into bioprocess-related knowledge. Further efforts aim at increasing desirable phenotypes of CHO cells, and promoting efficient production of high quality therapeutic proteins can highly benefit from this type of studies.


Subject(s)
CHO Cells/cytology , Cell Culture Techniques/methods , Gene Expression Profiling/methods , Sequence Analysis, RNA/methods , Animals , Cell Cycle , Cricetulus , Gene Expression Regulation , High-Throughput Nucleotide Sequencing/methods , Temperature
4.
J Biosci Bioeng ; 124(5): 591-598, 2017 Nov.
Article in English | MEDLINE | ID: mdl-28688754

ABSTRACT

If cultured in appropriate conditions, such as supplementing culture media with costly cytokines and growth factors, hematopoietic stem/progenitor cells (HSPCs) from different origins have shown to be an adequate source of erythroid cells. This requirement turns erythroid cells production into a complicated process to be scaled-up for future applications. The aim of our work was to genetically modify HSPCs with human erythropoietin (hEPO) sequence by lentiviral transgenesis in order for cells to secrete the hormone into the culture medium. Initially, we evaluated erythroid differentiation in colony forming units (CFU) assays and further analyzed cell expansion and erythroid differentiation throughout time in suspension cultures by flow cytometry and May-Grünwald-Giemsa staining. Additionally, we studied hEPO production and its isoforms profile. The different assessment approaches demonstrated erythroid differentiation, which was attributed to the hEPO secreted by the HSPCs. Our data demonstrate that it is possible to develop culture systems in which recombinant HSPCs are self-suppliers of hEPO. This feature makes our strategy attractive to be applied in biotechnological production processes of erythroid cells that are currently under development.


Subject(s)
Cell Culture Techniques/methods , Cell Differentiation/genetics , Erythroid Cells/cytology , Erythropoietin/genetics , Erythropoietin/metabolism , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Lentivirus/genetics , Biotechnology/methods , Cells, Cultured , Colony-Forming Units Assay , Erythroid Cells/metabolism , Erythropoietin/biosynthesis , Erythropoietin/chemistry , Humans , Lentivirus/metabolism
5.
Oncogene ; 23(50): 8196-205, 2004 Oct 28.
Article in English | MEDLINE | ID: mdl-15378003

ABSTRACT

Kruppel-like transcription factors (KLFs) represent one of the most diverse set of regulators in vertebrate organisms. KLF family members are involved in cell proliferation and differentiation control in normal as well as in pathological situations. Here, we demonstrate that KLF6 behaves as a functional antagonist of the c-Jun proto-oncoprotein. Thus, KLF6 overexpression downregulated c-Jun-dependent transcription and a physical interaction between c-Jun and KLF6 was detected. Moreover, cell proliferation induced by c-Jun was significantly decreased by KLF6. The inhibition of c-Jun functions correlates directly with c-Jun protein degradation induced by KLF6. We also show that all KLF6 effects on c-Jun were largely dependent on phorbol ester (TPA/ionomycin) extracellular stimulation, which enhanced KLF6 nuclear translocation and transcriptional activity and modified its phosphorylation status. Our data are consistent with a novel mechanism of KLF6's role as an inhibitor of cell proliferation by counteracting the function of the c-Jun proto-oncoprotein involving enhanced c-Jun degradation by the proteasome-dependent pathway, and further reinforces KLF6 as a potential tumor suppressor gene product.


Subject(s)
Proto-Oncogene Proteins c-jun/antagonists & inhibitors , Proto-Oncogene Proteins/physiology , Trans-Activators/physiology , Animals , COS Cells , Cell Division/physiology , Cell Nucleus/metabolism , Humans , Hydrolysis , Jurkat Cells , Kruppel-Like Factor 6 , Kruppel-Like Transcription Factors , Phosphorylation , Protein Transport , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-jun/metabolism , Proto-Oncogene Proteins c-jun/physiology , Trans-Activators/metabolism , Transcription, Genetic/physiology
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