Expression and Purification of Biologically Active Human Bone Morphogenetic Protein-4 in Recombinant Chinese Hamster Ovary Cells

• Published in: Journal of microbiology and biotechnology Vol. 27; no. 7; pp. 1281 - 1287
• Main Authors: Cha, Minyub, Han, Nara, Pi, Jia, Jeong, Yongsu, Baek, Kwanghee, Yoon, Jaeseung
• Format: Journal Article
• Language: English
• Published: Korea (South) 한국미생물·생명공학회 28-07-2017
• Subjects: Animals; Batch Cell Culture Techniques; Bioreactors; Bone Morphogenetic Protein 4 - genetics; Bone Morphogenetic Protein 4 - isolation & purification; Bone Morphogenetic Protein 4 - metabolism; Bone Morphogenetic Protein 4 - pharmacology; CHO Cells; Cricetinae; Cricetulus; Gene Expression; Humans; Recombinant Proteins - metabolism; Recombinant Proteins - pharmacology; 생물학; bioassay; recombinant protein expression; stable cell line; Chinese hamster ovary (CHO) cells; bioreactor process; Bone morphogenetic protein-4 (BMP-4)
• ISSN: 1017-7825; 1738-8872
• DOI: 10.4014/jmb.1702.02011

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
-linked glycosylation of the purified rhBMP-4 were confirmed by N-terminal sequencing and de-
-glycosylation analysis, respectively. The mature purified rhBMP-4 has been proved to be functionally active, with an effective dose concentration of EC
of 2.93 ng/ml.