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- 저자 : Chaya MOHAN (검색결과 7 건)
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Mohan, Chaya,Park, Soon Hye,Chung, Joo Young,Lee, Gyun Min John Wiley & Sons 2007 Biotechnology and bioengineering Vol.98 No.3
<P>Protein disulfide isomerase (PDI), one of the ER-resident molecular chaperones, forms and isomerizes disulfide bonds. This study attempts to investigate the effect of PDI expression level on specific productivity (q) of recombinant Chinese hamster ovary (rCHO) cells producing thrombopoietin (TPO) and antibody (Ab). To regulate the PDI expression level, the Tet-Off system was introduced in TPO and Ab producing CHO cells, and stable Tet-Off cells (TPO-Tet-Off and Ab-Tet-Off) were screened using the luciferase assay. The doxycycline-regulated PDI expression system in Tet-Off rCHO cells (Tet-TPO-PDI and Tet-Ab-PDI) was established by the cotransfection of pTRE-PDI and pTK-Hyg expression vector into TPO-Tet-Off and Ab-Tet-Off cells, respectively. Subsequent screening was done by Western blot analysis of PDI and an enzyme-linked immunosorbent assay of the secreted TPO and antibody. We cultured two Tet-TPO-PDI and two Tet-Ab-PDI clones, and all these clones showed an average of 2.5-fold increase in PDI expression when compared to the basal level. In both these cell lines the PDI expression was tightly controlled by various concentrations of doxycycline. The q of TPO (q<SUB>TPO</SUB>) was unaffected but that of antibody producing cells was increased by 15–27% due to the PDI expression level. Biotechnol. Bioeng. 2007;98:611–615. © 2007 Wiley Periodicals, Inc.</P>
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Mohan, Chaya,Lee, Gyun Min Wiley Subscription Services, Inc., A Wiley Company 2010 Biotechnology and bioengineering Vol.107 No.2
<P>To enhance specific antibody (Ab) productivity (q<SUB>Ab</SUB>) of recombinant Chinese hamster ovary (rCHO) cells, post-translational limitations in the endoplasmic reticulum during antibody production should be relieved. Previously, we reported that overexpression of protein disulfide isomerase (PDI), which catalyzes disulfide bond exchanges and assists in protein folding of newly synthesized proteins, enhanced q<SUB>Ab</SUB> of rCHO cells by about 27% (Mohan et al., 2007, Biotechnol Bioeng 98:611–615) . Since the rate limiting step in disulfide bond formation is found to be the regeneration of oxidized PDI, the oxidation state of PDI, as well as the amount of PDI, might be important. Endoplasmic reticulum oxidoreductase (ERO1L) maintains PDI in an oxidized state so that disulfide bond formation occurs. Here, PDI and its helper protein, ERO1L were overexpressed in rCHO cells producing an Ab in an attempt to ease the bottleneck in disulfide bond formation, and hence, Ab folding and secretion. Transient expression of ERO1L alone and with PDI resulted in enhanced q<SUB>Ab</SUB> by 37% and 55%, respectively. In contrast, under stable inducible co-overexpression of PDI and ERO1L, the q<SUB>Ab</SUB> was unaffected or negatively affected by varying degrees, depending on the individual expression levels of these genes. In stable clones with altered oxidation state of PDI due to co-overexpression of PDI and ERO1L, secretion of Ab was hindered and PDI-associated retention of Ab was seen in the cells. Under transient gene expression, secretion of Ab was not compromised. The data presented here suggests a possible mechanism of PDI/ERO1L interaction with the target Ab and shows how the expression levels of these proteins could affect the q<SUB>Ab</SUB> of this Ab-producing rCHO cell line. Biotechnol. Bioeng. 2010;107: 337–346. © 2010 Wiley Periodicals, Inc.</P>
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A Role of GADD153 in ER Stress-induced Apoptosis in Recombinant Chinese Hamster Ovary Cells
Chaya Mohan,Madhavi Sathyamurthy,이균민 한국생물공학회 2012 Biotechnology and Bioprocess Engineering Vol.17 No.3
The imbalance between the folding capacity and the folding demand imposed on the endoplasmic reticulum (ER) of therapeutic protein-producing host cells results in a stressed ER. This initiates a series of cellular signaling events termed the unfolded protein response (UPR) aimed at restoring homeostasis. In order to alleviate ER stress and ER stress-induced apoptosis in recombinant Chinese hamster ovary (rCHO) cells, silencing of the growth arrest and DNA damage 153 gene (GADD153), the main pro-apoptotic factor of UPR, was attempted. The rCHO cells were cultured under four ER stress inducing conditions, including thapsigargin, brefeldin A, glucose deprivation, glucose and glutamine deprivation. In these conditions, the functions of stably GADD153-silenced clones were investigated. It was found that under exclusive ER stress-inducing conditions of thapsigargin and brefeldin A treatments, the GADD153-silenced clones showed a less incidence of apoptosis (about 38%) and less cell viability (about 58% non-viable cells) than the control cells. However, under nutrient deprivation, the beneficial effect of GADD153 silencing was not pronounced because nutrient deprivation led to a cascade of various events including GADD153-induced cell death. GADD153-overexpressing pool cells also substantiated the findings of GADD153 downregulation. Investigation of the underlying mechanism revealed that increased GADD153 expression results in an exaggerated production of reactive oxygen species (ROS) and that GADD153 silencing promotes translational attenuation facilitating cell recovery from stress. Taken together, this study suggests that GADD153 sensitizes cells to ER stress through mechanisms that involve enhanced oxidative injury and by manipulating the ER client protein load in rCHO cells. The imbalance between the folding capacity and the folding demand imposed on the endoplasmic reticulum (ER) of therapeutic protein-producing host cells results in a stressed ER. This initiates a series of cellular signaling events termed the unfolded protein response (UPR) aimed at restoring homeostasis. In order to alleviate ER stress and ER stress-induced apoptosis in recombinant Chinese hamster ovary (rCHO) cells, silencing of the growth arrest and DNA damage 153 gene (GADD153), the main pro-apoptotic factor of UPR, was attempted. The rCHO cells were cultured under four ER stress inducing conditions, including thapsigargin, brefeldin A, glucose deprivation, glucose and glutamine deprivation. In these conditions, the functions of stably GADD153-silenced clones were investigated. It was found that under exclusive ER stress-inducing conditions of thapsigargin and brefeldin A treatments, the GADD153-silenced clones showed a less incidence of apoptosis (about 38%) and less cell viability (about 58% non-viable cells) than the control cells. However, under nutrient deprivation, the beneficial effect of GADD153 silencing was not pronounced because nutrient deprivation led to a cascade of various events including GADD153-induced cell death. GADD153-overexpressing pool cells also substantiated the findings of GADD153 downregulation. Investigation of the underlying mechanism revealed that increased GADD153 expression results in an exaggerated production of reactive oxygen species (ROS) and that GADD153 silencing promotes translational attenuation facilitating cell recovery from stress. Taken together, this study suggests that GADD153 sensitizes cells to ER stress through mechanisms that involve enhanced oxidative injury and by manipulating the ER client protein load in rCHO cells.
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Kim, Yeon-Gu,Kim, Jee Yon,Mohan, Chaya,Lee, Gyun Min Wiley Subscription Services, Inc., A Wiley Company 2009 Biotechnology and bioengineering Vol.103 No.4
<P>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-x<SUB>L</SUB> overexpression on apoptosis and autophagy in rCHO cells, an erythropoietin (EPO)-producing rCHO cell line with regulated Bcl-x<SUB>L</SUB> overexpression (EPO-off-Bcl-x<SUB>L</SUB>) was established using the Tet-off system. The expression level of Bcl-x<SUB>L</SUB> in EPO-off-Bcl-x<SUB>L</SUB> cells was tightly regulated by doxycycline in a dose-dependent manner. Bcl-x<SUB>L</SUB> overexpression enhanced cell viability and extended culture longevity in batch culture. Upon nutrient depletion in the later stage of batch culture, Bcl-x<SUB>L</SUB> overexpression suppressed apoptosis by inhibiting the activation of caspase-3 and -7. Simultaneously, Bcl-x<SUB>L</SUB> overexpression also delayed autophagy, characterized by LC3-II accumulation. Immunoprecipitation analysis with a Flag-tagged Bcl-x<SUB>L</SUB> revealed that Bcl-x<SUB>L</SUB> 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-x<SUB>L</SUB> overexpression inhibits both apoptosis and autophagy in rCHO cell culture. Biotechnol. Bioeng. 2009;103: 757–766. © 2009 Wiley Periodicals, Inc.</P>
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