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Suigen, South Korea

Paredes V.,Kyung Hee University | Park J.S.,PanGen Biotech Inc. | Jeong Y.,Kyung Hee University | Yoon J.,Kyung Hee University | Baek K.,Kyung Hee University
Biotechnology Letters | Year: 2013

The gradual loss of recombinant protein expression in CHO cell lines during prolonged subculture is a common issue, referred to as instability, which seriously affects the industrial production processes of therapeutic proteins. Loss of recombinant gene copies, due to the genetic instability of CHO cells, and epigenetic silencing of transgene sequences, are the main reported causes of production instability. To increase our understanding on the molecular mechanisms inherent to CHO cells involved in production instability, we explored the molecular features of stable and unstable antibody producing cell lines obtained without gene amplification, to exclude the genetic instability induced by the gene amplification process. The instability of recombinant antibody production during long-term culture was caused by a 48-53 % decrease in recombinant mRNA levels without significant loss of recombinant gene copies, but accompanied by a ~45 % decrease in histone H3 acetylation (H3ac). Thus, our results suggest a critical role of H3ac in the stability of recombinant protein production. © 2013 Springer Science+Business Media Dordrecht. Source


Yoon S.,Kyung Hee University | Lee Y.,Kyung Hee University | Pi J.,Kyung Hee University | Jeong Y.,Kyung Hee University | And 3 more authors.
Protein Expression and Purification | Year: 2016

Bone morphogenetic protein-7 is a multifunctional growth factor involved in various cellular processes such as osteogenesis, kidney and eye development, brown adipogenesis, and bone metastasis, and thus has been considered to have therapeutic potential for treating various diseases. In this study, we established a Chinese hamster ovary (CHO) cell line stably overexpressing recombinant human BMP-7 (rhBMP-7). Over the course of a 14-day fed-batch culture process in a 7.5-l bioreactor (5-l working volume) using chemically defined medium, the established cells could produce over 188 mg/l of rhBMP-7 protein. The rhBMP-7 was purified to homogeneity from the culture supernatant using a two-step chromatographic procedure that resulted in a recovery rate of approximately 55%, with protein purity greater than 95%. The purified rhBMP-7 was further demonstrated to be functionally active by measuring the proliferation of MC3T3-E1 cells, revealing a half-maximal effective concentration of 28.31 ng/ml. © 2015 Elsevier Inc. All rights reserved. Source


Patent
Pangen Biotech Inc. | Date: 2012-03-30

There is provided an expression vector for animal cell having an increased gene expression efficiency, and particularly, an expression vector for animal cells including a MAR element and a SAR element, which are gene expression increasing factors, at a 5 end of a promoter, a 3 end of a transcription termination site, or at both of the 5 end of the promoter and the 3 end of the transcription termination site. The expression vector for animal cells according to the present invention exhibits remarkably increased gene expression efficiency as compared to conventional expression vectors for animal cells, such that protein expression of foreign genes may be significantly increased using this expression vector for animal cells. Particularly, the expression vector for animal cells according to the present invention may be useful in that a high-expression cell line may be secured even without MTX amplification.


Song K.,Seoul National University | Song K.,Research South, Inc. | Yoon I.-S.,Mokpo National University | Kim N.A.,Dongguk University | And 11 more authors.
PLoS ONE | Year: 2014

The purpose of this study was to develop a biobetter version of recombinant human interferon-β 1a (rhIFN-β 1a) to improve its biophysical properties, such as aggregation, production and stability, and pharmacokinetic properties without jeopardizing its activity. To achieve this, we introduced additional glycosylation into rhIFN-β 1a via site-directed mutagenesis. Glycoengineering of rhIFN-β 1a resulted in a new molecular entity, termed R27T, which was defined as a rhIFN-β mutein with two N-glycosylation sites at 80th (original site) and at an additional 25th amino acid due to a mutation of Thr for Arg at position 27th of rhIFN-β 1a. Glycoengineering had no effect on rhIFN-β ligand-receptor binding, as no loss of specific activity was observed. R27T showed improved stability and had a reduced propensity for aggregation and an increased half-life. Therefore, hyperglycosylated rhIFN-β could be a biobetter version of rhIFN-β1a with a potential for use as a drug against multiple sclerosis. © 2014 Song et al. Source


Yu S.,Kyung Hee University | Park J.S.,PanGen Biotech Inc. | Paredes V.,Kyung Hee University | Song M.-C.,Kyung Hee University | And 6 more authors.
Journal of Biotechnology | Year: 2010

To develop a cell-based assay to screen for human dopamine D1 receptor agonists or antagonists from medicinal plant extracts, a stable Chinese hamster ovary (CHO) cell line (CHO-D1R) expressing the human dopamine D1 receptor was established using an expression vector containing a scaffold attachment region (SAR) element. CHO-D1R cells showed specific binding to [3H]-SCH23390 with high affinity (Kd = 1.47 ± 0.17 nM) and dose-dependent responses for the dopamine-mediated stimulation of cAMP concentrations (EC50 = 20.6 ± 1.44 nM). The screening of medicinal plant extracts using cell-based cAMP assays revealed that an extract of Gleditsia sinensis Lam., which is known to be rich in saponin, had strong antagonist activity for the D1 receptor. From the activity-guided fractionation and chemical structural analysis of the G. sinensis extract, a compound called gleditsioside F was isolated and was identified to have antagonist activity for the D1 receptor. Gleditsioside F showed very effective D1 antagonist activity by inhibiting ligand binding to the D1 receptor as well as by inhibiting dopamine-mediated increases in cAMP concentration. © 2009 Elsevier B.V. All rights reserved. Source

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