|
KMID : 0545120170270071281
|
|
Journal of Microbiology and Biotechnology
2017 Volume.27 No. 7 p.1281 ~ p.1287
|
|
|
Expression and Purification of Biologically Active Human Bone Morphogenetic Protein-4 in Recombinant Chinese Hamster Ovary Cells
|
|
Cha Min-Yub
Han Na-Ra
Pi Jia
Jeong Yong-Su
Baek Kwang-Hee
Yoon Jae-Seung
|
|
|
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.
|
|
|
KEYWORD
|
|
|
Bone morphogenetic protein-4 (BMP-4), Chinese hamster ovary (CHO) cells, recombinant protein expression, stable cell line, bioreactor process, bioassay
|
|
|
FullTexts / Linksout information
|
|
|
|
|
|
Listed journal information
|
|
  
|