Jee Yon Kim
KAIST
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Featured researches published by Jee Yon Kim.
Biotechnology and Bioengineering | 2010
Young Kue Han; Yeon-Gu Kim; Jee Yon Kim; Gyun Min Lee
During recombinant Chinese hamster ovary (rCHO) cell culture, various events, such as feeding with concentrated nutrient solutions or the addition of base to maintain an optimal pH, increase the osmolality of the medium. To determine the effect of hyperosmotic stress on two types of programmed cell death (PCD), apoptosis and autophagy, of rCHO cells, two rCHO cell lines, producing antibody and erythropoietin, were subjected to hyperosmotic stress resulting from NaCl addition (310-610 mOsm/kg). For both rCHO cell lines, hyperosmolality up to 610 mOsm/kg increased cleaved forms of PARP, caspase-3, caspase-7, and fragmentation of chromosomal DNA, confirming the previous observation that apoptosis was induced by hyperosmotic stress. Concurrently, hyperosmolality increased the level of accumulation of LC3-II, a widely used autophagic marker, which was determined by Western blot analysis and confocal microscopy. When glucose and glutamine concentrations were measured during the cultures, glucose and glutamine concentrations in the culture medium at various osmolalities (310-610 mOsm/kg) showed no significant differences. This result suggests that induction of PCD by hyperosmotic stress occurred independently of nutrient depletion. Taken together, autophagy as well as apoptosis was observed in rCHO cells subjected to hyperosmolality.
Biotechnology and Bioengineering | 2009
Yeon-Gu Kim; Jee Yon Kim; Chaya Mohan; Gyun Min Lee
Upon nutrient deprivation during culture, recombinant Chinese hamster ovary (rCHO) cells are subjected to two types of programmed cell death (PCD), apoptosis and autophagy. To investigate the effect of Bcl‐xL overexpression on apoptosis and autophagy in rCHO cells, an erythropoietin (EPO)‐producing rCHO cell line with regulated Bcl‐xL overexpression (EPO‐off‐Bcl‐xL) was established using the Tet‐off system. The expression level of Bcl‐xL in EPO‐off‐Bcl‐xL cells was tightly regulated by doxycycline in a dose‐dependent manner. Bcl‐xL overexpression enhanced cell viability and extended culture longevity in batch culture. Upon nutrient depletion in the later stage of batch culture, Bcl‐xL overexpression suppressed apoptosis by inhibiting the activation of caspase‐3 and ‐7. Simultaneously, Bcl‐xL overexpression also delayed autophagy, characterized by LC3‐II accumulation. Immunoprecipitation analysis with a Flag‐tagged Bcl‐xL revealed that Bcl‐xL interacts with Bax and Bak, essential mediators of caspase‐dependent apoptosis, as well as with Beclin‐1, an essential mediator of autophagy, and may inhibit their pro‐cell death function. Taken together, it was found that Bcl‐xL overexpression inhibits both apoptosis and autophagy in rCHO cell culture. Biotechnol. Bioeng. 2009;103: 757–766.
Applied Microbiology and Biotechnology | 2011
Jee Yon Kim; Yeon-Gu Kim; Young Kue Han; Hyun Soo Choi; Young Hwan Kim; Gyun Min Lee
In order to understand the intracellular responses in recombinant CHO (rCHO) cells producing antibody in serum-free medium (SFM) supplemented with optimized hydrolysates mixtures, yielding the highest specific growth rate (μ, SFM#S1) or the highest specific antibody productivity (qAb, SFM#S2), differentially expressed proteins in rCHO cells are measured by two-dimensional gel electrophoresis combined with nano-LC-ESI-Q-TOF tandem MS. The comparative proteomic analysis with basal SFM without hydrolysates revealed that the addition of hydrolysate mixtures significantly altered the profiles of CHO proteome. In SFM#S1, the expression of metabolism-related proteins, cytoskeleton-associated proteins, and proliferation-related proteins was up-regulated. On the other hand, the expression of anti-proliferative proteins and pro-apoptotic protein was down-regulated. In SFM#S2, the expression of various chaperone proteins and proliferation-linked proteins was altered. 2D-Western blot analysis of differentially expressed proteins confirmed the proteomic results. Taken together, identification of differentially expressed proteins in CHO cells by a proteomic approach can provide insights into understanding the effect of hydrolysates on intracellular events and clues to find candidate genes for cell engineering to maximize the protein production in rCHO cells.
Journal of Biotechnology | 2009
Yeon-Gu Kim; Jee Yon Kim; Gyun Min Lee
Previously, overexpression of X-linked inhibitor of apoptosis (XIAP), which is known to inhibit activities of caspase-3, -7, and -9 in CHO-K1 cells offered protection against Sindbis virus-induced apoptosis. In this study, the potential role of XIAP overexpression in recombinant CHO (rCHO) cells treated with sodium butyrate (NaBu), which can increase the specific productivity, was investigated by establishing erythropoietin (EPO)-producing rCHO cells with regulated XIAP overexpression (EPO-off-XIAP). The XIAP overexpression in EPO-off-XIAP was tightly regulated by doxycycline. The XIAP overexpression could simultaneously reduce the activation of caspase-3, -7, and -9 induced by NaBu addition. However, XIAP overexpression could not inhibit NaBu-induced apoptosis, as evidenced by DNA fragmentation. In addition, it also did not help the maintenance of the mitochondrial membrane potential in the presence of NaBu, suggesting that the release of mitochondrial proteins might induce caspase-independent apoptosis. As a result, XIAP overexpression did not affect cell growth and EPO production significantly. Taken together, XIAP overexpression, which was reported to inhibit Sindbis virus-induced apoptosis, could not inhibit the NaBu-induced apoptosis in rCHO cells.
Biotechnology and Bioengineering | 2012
Jee Yon Kim; Yeon-Gu Kim; Gyun Min Lee
The use of glycine betaine combined with hyperosmolality is known to be an efficient means for achieving high protein production in recombinant Chinese hamster ovary (rCHO) cells. In order to understand the intracellular events and identify the key factors in rCHO cells cultivated with glycine betaine under hyperosmotic conditions, two‐dimensional differential in‐gel electrophoresis (2D‐DIGE) followed by mass spectrometric analysis was applied. Differentially expressed 19 protein spots were selected and 16 different kinds of proteins were successfully identified. The identified proteins were associated with cellular metabolism (PEPCK, GAPDH, and PK), cellular architecture (β‐tubulin and β‐actin), protein folding (GRP78 and OSP94), mRNA processing (Rbm34, ACF, and IPMK), and protein secretion (γ‐COP). 2D‐Western blot analysis of β‐tubulin, GAPDH, Peroxidoxin‐1, and GRP78 confirmed the proteomic findings. The proteins identified from this study, which are related to cell growth and antibody production, can be applied to cell engineering for maximizing the efficacy of the use of glycine betaine combined with hyperosmolality in rCHO cells. Biotechnol. Bioeng. 2012; 109:1395–1403.
Biotechnology Progress | 2009
Jee Yon Kim; Yeon-Gu Kim; Jong Youn Baik; Eun Jin Joo; Young Hwan Kim; Gyun Min Lee
Identification of the cellular proteins interacting with incompletely folded and unfolded forms of erythropoietin (EPO) in recombinant CHO (rCHO) cells leads to better insight into the possible genetic manipulation approaches for increasing EPO production. To do so, a pull‐down assay was performed with dual‐tagged (N‐terminal GST‐ and C‐terminal hexahistidine‐tagged) EPO expressed in E. coli as bait proteins and cell lysates of rCHO cells (DG44) as prey proteins. Cellular proteins interacting with dual‐tagged EPO were then resolved by two‐dimensional gel electrophoresis (2DE) and identified by MALDI‐TOF MS/MS. A total of 27 protein spots including glucose‐regulated protein 78 (GRP78) were successfully identified. Western blot analysis of GRP78 confirmed the results of the MS analyses. Taken together, a pull‐down assay followed by a proteomic approach is found to be an efficient means to identify cellular proteins interacting with foreign protein in rCHO cells.
Biotechnology Progress | 2011
Yeon-Gu Kim; Young Kue Han; Jee Yon Kim; Eun Gyo Lee; Hong Weon Lee; Gyun Min Lee
Constitutively active Ras (CA‐Ras) is known to enhance cell growth through the induction of various signaling cascades including the phosphoinositide 3‐kinase (PI3K)/Akt and mitogen‐activated protein kinase (MAPK)/ERK signaling pathways, although the cellular response is highly dependent on the cell type. To evaluate the effect of CA‐Ras overexpression on cell growth in recombinant Chinese hamster ovary (rCHO) cells, an erythropoietin (EPO)‐producing rCHO cell line with regulated CA‐Ras overexpression (EPO‐off‐CA‐Ras) was established using the Tet‐off system. The CA‐Ras expression level in EPO‐off‐CA‐Ras cells was tightly regulated by doxycycline addition. Although CA‐Ras overexpression slightly increased the viable cell concentration during the late exponential phase, it did not increase the maximum viable cell concentration or specific growth rate to a significant degree. Unexpectedly, CA‐Ras overexpression in rCHO cells led only to the enhancement in the activation of the MAPK/ERK signaling pathway and not the PI3K/Akt signaling pathway. Taken together, CA‐Ras overexpression in rCHO cells did not significantly affect cell growth; it also had no critical impact on viable cell concentration or EPO production, possibly due to a failure to activate the PI3K/Akt signaling pathway.
Journal of Biotechnology | 2018
Sung-Min Lim; Sun-Hye Park; Joo-Hyoung Lee; Sun Hong Kim; Jee Yon Kim; Jeong-Ki Min; Gyun Min Lee; Yeon-Gu Kim
Sodium butyrate (NaBu) is an efficient supplement for increasing recombinant protein production in Chinese hamster ovary (CHO) cell culture. To elucidate the effects of NaBu on miRNA expression profile in recombinant CHO (rCHO) cells, differentially expressed miRNAs in NaBu-treated rCHO cells were assessed by NanoString nCounter analysis. This result showed that eight mature mouse miRNAs (let-7b, let-7d, miR-15b, miR-25, miR-27a, miR-99a, miR-125a-5p, and miR-125b-5p) were differentially expressed. Furthermore, quantitative real-time RT-PCR analysis of eight mature CHO miRNAs, annotated using a miRBase database, confirmed the transcriptomic findings. Among the potential corresponding target mRNAs for the selected mature miRNAs, seven cell growth-related target genes (e2f2, akt2, mtor, bcl-2, bim, p38α, and bmf) and five N-glycosylation-related target genes (neu1, b4galt3, gale, man1b1 and mgat4a) were selected by considering the effectiveness of NaBu on rCHO cell culture. The altered expression patterns of the 12 target mRNAs were inversely correlated with those of the selected mature miRNAs. Altogether, NanoString nCounter analysis may be useful for identifying differentially expressed miRNAs in rCHO cells.
Applied Microbiology and Biotechnology | 2018
Joo-Hyoung Lee; Jong-Ho Park; Sun-Hye Park; Sun-Hong Kim; Jee Yon Kim; Jeong-Ki Min; Gyun Min Lee; Yeon-Gu Kim
Despite the relatively low transfection efficiency and low specific foreign protein productivity (qp) of Chinese hamster ovary (CHO) cell-based transient gene expression (TGE) systems, TGE-based recombinant protein production technology predominantly employs CHO cells for pre-clinical research and development purposes. To improve TGE in CHO cells, Epstein-Barr virus nuclear antigen-1 (EBNA-1)/polyoma virus large T antigen (PyLT)-co-amplified recombinant CHO (rCHO) cells stably expressing EBNA-1 and PyLT were established using dihydrofolate reductase/methotrexate-mediated gene amplification. The level of transiently expressed Fc-fusion protein was significantly higher in the EBNA-1/PyLT-co-amplified pools compared to control cultures. Increased Fc-fusion protein production by EBNA-1/PyLT-co-amplification resulted from a higher qp attributable to EBNA-1 but not PyLT expression. The qp for TGE-based production with EBNA-1/PyLT-co-amplified rCHO cells (EP-amp-20) was approximately 22.9-fold that of the control culture with CHO-DG44 cells. Rather than improved transfection efficiency, this cell line demonstrated increased levels of mRNA expression and replicated DNA, contributing to an increased qp. Furthermore, there was no significant difference in N-glycan profiles in Fc-fusion proteins produced in the TGE system. Taken together, these results showed that the use of rCHO cells with co-amplified expression of the viral elements EBNA-1 and PyLT improves TGE-based therapeutic protein production dramatically. Therefore, EBNA-1/PyLT-co-amplified rCHO cells will likely be useful as host cells in CHO cell-based TGE systems.
Applied Microbiology and Biotechnology | 2012
Jee Yon Kim; Yeon-Gu Kim; Gyun Min Lee
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Korea Research Institute of Bioscience and Biotechnology
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