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.
Subject(s)
Bone Morphogenetic Protein 7/metabolism , Proprotein Convertases/metabolism , Recombinant Proteins/metabolism , Animals , Bone Morphogenetic Protein 7/chemistry , Bone Morphogenetic Protein 7/genetics , CHO Cells , Cricetinae , Cricetulus , Humans , Proprotein Convertases/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/geneticsABSTRACT
Bone morphogenetic protein-7 (BMP-7) is a member of the TGF-ß superfamily and plays a critical role in cartilage, bone, and kidney development. BMP-7 is synthesized as a large precursor and undergoes proteolytic cleavage by subtilisin-like proprotein convertase to secrete the functionally active mature dimer. When CHO cells producing recombinant human BMP-7 (rhBMP) (CHO-BMP-7) were cultivated in a serum-free suspension culture, a significant amount of unwanted precursor forms of rhBMP-7 (ca. 69% of total rhBMP-7), along with the mature form of rhBMP-7, was secreted into the culture medium, likely due to the insufficient amount of the proteolytic cleaving enzyme within the secretory pathway. In order to solve this problem, a soluble form of the paired basic amino acid cleaving enzyme (PACEsol), responsible for the majority of the processing events occurring in the constitutive secretory pathway in mammalian cells, was overexpressed in CHO-BMP-7 cells. Overexpression of PACEsol was effective in processing the precursor forms of BMP-7, while it did not significantly affect cell growth. As a result, the culture supernatants of CHO-BMP-7 cells overexpressing PACEsol contained almost 100% of the mature BMP-7 form. Taken together, the results show that PACEsol overexpression in CHO-BMP-7 cells is an efficient means of increasing the production of mature BMP-7 and facilitating the downstream purification steps by eliminating the need to remove the precursor forms.