2017 ; Vol.27-7: 1281~1287
|Author||Minyub Cha, Nara Han, Jia Pi, Yongsu Jeong, Kwanghee Baek, Jaeseung Yoon|
|Place of duty||Graduate School of Biotechnology, Kyung Hee University, Yongin 17104, Republic of Korea|
|Title||Expression and Purification of Biologically Active Human Bone Morphogenetic Protein-4 in Recombinant Chinese Hamster Ovary Cells|
J. Microbiol. Biotechnol.2017 ;
|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 EC50 of 2.93 ng/ml.|
|Key_word||Bone morphogenetic protein-4 (BMP-4), Chinese hamster ovary (CHO) cells, recombinant protein expression, stable cell line, bioreactor process, bioassay|
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