Human hepatoma cell line HuH-7 is an effective cellular system to produce recombinant factor IX with improved post-translational modifications
| Autor: | Michel Becchi, Nathalie Enjolras, Florine Guillaume, Claude Negrier, Eloïse Perot, Yesim Dargaud |
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| Rok vydání: | 2012 |
| Předmět: |
endocrine system
Glycosylation CHO Cells Biology Transfection law.invention Factor IX chemistry.chemical_compound Sulfation law Cell Line Tumor Cricetinae medicine Animals Humans Secretion Chinese hamster ovary cell Liver Neoplasms Hematology Recombinant Proteins chemistry Biochemistry Recombinant DNA Phosphorylation Protein Processing Post-Translational medicine.drug |
| Zdroj: | Thrombosis research. 130(5) |
| ISSN: | 1879-2472 |
| Popis: | Recombinant factor IX (rFIX) is increasingly used to treat patients with hemophilia B. CHO (Chinese Hamster Ovary) cells are commonly used for the production of rFIX but they have a limited capacity for introducing post-translational modifications (PTM) leading to incomplete γ-carboxylation, low phosphorylation and sulfation profiles as compared with plasma-derived preparations. Imperfect PTM might have an impact on the activity of Factor IX molecule. Several studies in animal models as well as clinical trials have previously reported a lower recovery of rFIX compared to plasma-derived FIX concentrates. In the present study, we aimed to produce a rFIX having a profile of PTM similar to plasma-derived FIX, using human hepatoma cell line HuH-7. We showed that rFIX produced by HuH-7 cells followed the classical intracellular pathway before secretion. In addition, improved PTM were associated with fully active molecule compared to plasma-derived and recombinant control FIX molecules. Secreted rFIX presented as a single band at the correct molecular weight. HuH-7 cellular clones were obtained and they secreted a biologically active human FIX. FIX was then purified for a detailed evaluation of PTM. Glycosylation and sialylation profiles were similar to plasma-derived and rFIX and mass spectrometry analysis demonstrated the presence of phosphorylated and sulfated forms of rFIX. These data strongly support that HuH-7 cells may represent an effective cellular system for production of rFIX exhibiting PTM similar to plasma-derived FIX. |
| Databáze: | OpenAIRE |
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