Characterization and Expression of Proprotein Convertases in CHO Cells: Efficient Proteolytic Maturation of Human Bone Morphogenetic Protein-7

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Bone morphogenetic protein-7 (BMP-7) is synthesized as a precursor that requires proteolytic cleavage of the propeptide by proprotein convertases (PCs) for its functional activity. A high-level expression of BMP-7 in CHO cells (CHO-BMP-7) resulted in secretion of a mixture of inactive precursor and active BMP-7. In an effort to achieve efficient processing of BMP-7 in CHO cells, PCs responsible for cleavage of the precursors in CHO cells were characterized. Analysis of the mRNA expression levels of four PCs (furin, PACE4, PC5/6, and PC7) revealed that only furin and PC7 genes are expressed in CHO-BMP-7 cells. Specific inhibition of the PCs by hexa-D-arginine (D6R) or decanoyl-RVKR-chloromethyl ketone (RVKR-CMK) further revealed that furin is mainly responsible for the proteolytic processing of BMP-7. To identify a more efficient PC for BMP-7 processing, the four PC genes were transiently expressed in CHO-BMP-7 cells, respectively. Among these, PC5/6 was found to be the most efficient in BMP-7 processing. Stable overexpression of PC5/6C, a secreted form of PC5/6, significantly improved mature BMP-7 production in CHO-BMP-7 cells. When the maximum BMP-7 concentration was obtained in the culture of CHO-BMP-7 cells, approximately 88% of BMP-7 was unprocessed. In contrast, no precursor was found in the culture of PC5/6C-overexpressing cells (clone #97). Furthermore, the in vitro biological activity of the mature BMP-7 from PC5/6C-overexpressing cells was comparable to that from CHO-BMP-7 cells. Taken together, the present results indicate that overexpression of PC5/6C in CHO-BMP-7 cells is an efficient means of increasing the yield of BMP-7. Biotechnol. Bioeng. 2015;112: 560-568.
Publisher
WILEY-BLACKWELL
Issue Date
2015-03
Language
English
Article Type
Article
Citation

BIOTECHNOLOGY AND BIOENGINEERING, v.112, no.3, pp.560 - 568

ISSN
0006-3592
DOI
10.1002/bit.25458
URI
http://hdl.handle.net/10203/195589
Appears in Collection
BS-Journal Papers(저널논문)
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