Introduction: Mammalian cell recombinant bone morphogenetic protein (rBMP) synthesis is reported to be poor. The BMP pro-domain may be involved in folding, stability and secretion. Objectives: Investigate the effect of inhibition of pro-domain cleavage on rhBMP-2 production. Methods: CHO cells transfected with human BMP-2 (hBMP-2) were cultured in the presence of the proprotein convertase inhibitor 9DR in short (multi-well) and long-term (bioreactor) cultures. Mature and proBMP secretion was measured by ELISA and characterized by Western blot. BMP activity was determined by C2C12 bioassay. Results: 9DR significantly enhanced the yields of both pro- and mature hBMP-2 in short and long-term cultures, without any negative effects on cell growth or viability. The rhBMP-2 was biologically active. ProBMP-2 could be converted by exogenous furin treatment into mature BMP-2 as shown by Western blot. Conclusions: 9DR increases rhBMP-2 production and is a simple, but effective way to enhance the yield of active, mature BMP-2 in CHO cells.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/11175 |
| Date | 30 July 2008 |
| Creators | Zhou, Jing-Jing Aileen |
| Contributors | Cameron, Clokie, Sean, Peel |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
| Format | 8503668 bytes, application/pdf |
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