Overexpression of PACEsol improves BMP-7 processing in recombinant CHO cells

J Biotechnol. 2012 Dec 15;164(2):336-9. doi: 10.1016/j.jbiotec.2013.01.012. Epub 2013 Jan 31.

Abstract

Bone morphogenetic protein-7 (BMP-7) is a member of the TGF-β 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biotechnology
  • Blotting, Western
  • Bone Morphogenetic Protein 7 / analysis
  • Bone Morphogenetic Protein 7 / genetics
  • Bone Morphogenetic Protein 7 / metabolism*
  • CHO Cells
  • Cell Growth Processes / physiology
  • Cell Survival / physiology
  • Cloning, Molecular
  • Cricetinae
  • Cricetulus
  • Furin / genetics
  • Furin / metabolism*
  • Humans
  • Recombinant Proteins / analysis
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism*

Substances

  • BMP7 protein, human
  • Bone Morphogenetic Protein 7
  • Recombinant Proteins
  • Furin