A novel peptide to enhance recombinant BMP-2 production in mammalian cell cultures
| Autor: | Aileen J Zhou, Cameron M L Clokie, Sean A. F. Peel |
|---|---|
| Jazyk: | angličtina |
| Předmět: |
Gene knockdown
animal structures Cell growth Cell HEK 293 cells lcsh:R lcsh:Medicine Biological activity General Medicine Transfection biochemical phenomena metabolism and nutrition Biology Bioinformatics Molecular biology General Biochemistry Genetics and Molecular Biology medicine.anatomical_structure Cell culture Meeting Abstract embryonic structures biology.protein medicine lcsh:Q lcsh:Science Furin |
| Zdroj: | BMC Proceedings, Vol 5, Iss Suppl 8, p P96 (2011) BMC Proceedings |
| ISSN: | 1753-6561 |
| DOI: | 10.1186/1753-6561-5-s8-p96 |
| Popis: | Materials and methods Two stable cell lines expressing the hBMP2 gene, CHOBMP2 and HEK-BMP2, were cultured in the presence of IND-1 in short-term (24 h, multi-well) and long-term (two-month, perfusion flasks) cultures. The rhBMP-2 produced was characterized by Western blot and its activity assessed using the C2C12 cell-based assay. The amount of proBMP-2 and mature BMP-2 produced was quantified by ELISA. The mRNA level of BMP-2 and furin in cells treated with or without IND-1 was compared by real-time RT-PCR. Cellular uptake of IND-1 was estimated by measuring the fluorescence of cell lysates following incubation with FITC labeled IND-1. Cellular PC activity post IND-1 incubation was measured using the Boc-RVRR-AMC substrate. Furin-specific siRNA was used to knock down the furin expression in CHO-BMP2 cells and its effect on the rhBMP-2 production was determined. Results Stably transfected CHO-BMP2 cells secreted 36 kDa rhBMP-2 dimers that were biologically active. In 24 h cell cultures, IND-1 treated cells produced significantly greater amounts of proBMP-2 (≥ 10-fold, P |
| Databáze: | OpenAIRE |
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