Characterization and expression of proprotein convertases in CHO cells: efficient proteolytic maturation of human bone morphogenetic protein-7

Abstract

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 PC5/6ΔC, 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 PC5/6ΔC‐overexpressing cells (clone #97). Furthermore, the in vitro biological activity of the mature BMP‐7 from PC5/6ΔC‐overexpressing cells was comparable to that from CHO‐BMP‐7 cells. Taken together, the present results indicate that overexpression of PC5/6ΔC in CHO‐BMP‐7 cells is an efficient means of increasing the yield of BMP‐7. Biotechnol. Bioeng. 2015;112: 560–568. © 2014 Wiley Periodicals, Inc.