Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Expression and Purification of Biologically Active Human Bone Morphogenetic Protein-4 in Recombinant Chinese Hamster Ovary Cells

  • Cha, Minyub (Graduate School of Biotechnology, Kyung Hee University) ;
  • Han, Nara (Graduate School of Biotechnology, Kyung Hee University) ;
  • Pi, Jia (Graduate School of Biotechnology, Kyung Hee University) ;
  • Jeong, Yongsu (Graduate School of Biotechnology, Kyung Hee University) ;
  • Baek, Kwanghee (Graduate School of Biotechnology, Kyung Hee University) ;
  • Yoon, Jaeseung (Graduate School of Biotechnology, Kyung Hee University)
  • Received : 2017.02.07
  • Accepted : 2017.05.18
  • Published : 2017.07.28
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Abstract

Bone morphogenetic protein-4 (BMP-4) is considered to have therapeutic potential for various diseases, including cancers; however, the high expression of biologically active recombinant human BMP-4 (rhBMP-4) needed for its manufacture for therapeutic purposes has yet to be established. In the current study, we established a recombinant Chinese hamster ovary (rCHO) cell line overexpressing rhBMP-4 as well as a production process using 7.5-l bioreactor (5 L working volume). The expression of the mature rhBMP-4 was significantly enhanced by recombinant furin expression. The combination of a chemically defined medium and a nutrient supplement solution for high expression of rhBMP-4 was selected and used for bioreactor cultures. The 11-day fed-batch cultures of the established rhBMP-4-expressing rCHO cells in the 7.5-L bioreactor produced approximately 32 mg/l of rhBMP-4. The mature rhBMP-4 was purified to homogeneity from the culture supernatant using a two-step chromatographic procedure, resulting in a recovery rate of approximately 55% and a protein purity greater than 95%. The N-terminal amino acid sequences and N-linked glycosylation of the purified rhBMP-4 were confirmed by N-terminal sequencing and de-N-glycosylation analysis, respectively. The mature purified rhBMP-4 has been proved to be functionally active, with an effective dose concentration of $EC_{50}$ of 2.93 ng/ml.

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