• Development and Reproduction
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• v.25 no.4
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• pp.199-211
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• 2021

Equine chorionic gonadotropin (eCG), produced by the endometrial cups of the placenta after the first trimester, is a specific glycoprotein that displays dual luteinizing hormone (LH)-like and follicle-stimulating hormone (FSH)-like effects in non-equid species. However, in equidaes, eCG exhibits only LH-like activity. To identify the specific biological functions of glycosylated sites in eCG, we constructed the following site mutants of N- and O-linked glycosylation: eCGβ/αΔ56, substitution of α-subunit⁵⁶ N-linked glycosylation site; eCGβ-D/α, deletion of the O-linked glycosylation sites at the β-subunit, and eCGβ-D/αΔ56, double mutant. We produced recombinant eCG (rec-eCG) proteins in Chinese hamster ovary suspension (CHO-S) cells. We examined the biological activity of rec-eCG proteins in CHO-K1 cells expressing the eLH/CG receptor and found that signal transduction activities of deglycosylated mutants remarkably decreased. The EC₅₀ levels of eCGβ/αΔ56, eCGβ-D/α, and eCGβ-D/αΔ56 mutants decreased by 2.1-, 5.6-, and 3.4-fold, respectively, compared to that of wild-type eCG. The Rmax values of the mutants were 56%-80% those of wild-type eCG (141.9 nmol/10⁴ cells). Our results indicate that the biological activity of eCG is greatly affected by the removal of N- and O-linked glycosylation sites in cells expressing eLH/CGR. These results provide important information on rec-eCG in the regulation of specific glycosylation sites and improve our understanding of the specific biological activity of rec-eCG glycosylation sites in equidaes.

• KSBB Journal
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• v.31 no.1
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• pp.66-72
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• 2016

Chinese hamster ovary (CHO) cells have been widely used for production of various recombinant proteins such as cytokines and monoclonal antibodies. The cell aggregation and cell death in CHO cell culture directly affect cell viability, and productivity and quality of products. In this study, we investigated preventing effects of storage-protein 2 (SP2) derived from silkworm hemolymph on cell aggregation and cell death in CHO cell culture producing albuminerythropoietin (Alb-EPO). The viable cell density in the culture supplemented with 2 mg/mL SP2 was 1.71-fold higher than that in control culture. Increased titer of Alb-EPO was also found in the culture with SP2. Morphology of CHO cells in SP2 supplemented cultures did not differ from that of control. In addition, the cell aggregation rate of the SP2 cultures was reduced 20% compared to the control. Finally, we confirmed that the apoptosis was strongly suppressed by addition of SP2 in the cultures. These results clearly demonstrate that SP2 can be served as an effective supplement for enhancing titer of Alb-EPO via reducing cell aggregation and cell death.

• 한국생물공학회:학술대회논문집
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• 2005.04a
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• pp.210-213
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• 2005

The suppression of apoptosis during the cell culture might increase recombinant protein production. In the present study, the effects of anti-apoptotic agents on the apoptosis of recombinant Chinese Hamster Ovary cells and the production of Iduronate 2-sulphatase(IDS) were investigated Cell density slightly increased when $2{\mu}M$ of EGCG and $10{\mu}g/mL$ of STR-G were added to culture medium after two days. It was observed that the percentage of apoptotic cells was decreased in the culture with STR-G, and Bcl-2 expression level was enhanced in both culture with STR-G and EGCG. These results suggest that G418 and EGCG are effective anti-apoptotic agents for increasing the productivity of IDS with recombinant CHO-DG44.

• KSBB Journal
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• v.31 no.4
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• pp.270-276
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• 2016

In glycoprotein, Terminal sialic acid residues of N-linked glycan are imperative things because they prevent the recognition from asialoglycoprotein-receptor that affect the half-life of glycoproteins. So establishment of culture process for enhancing sialic acid is important to maximize sialic acid contents of glycoprotein. In this study, we investigated effects of biphasic culture of Chinese hamster ovary (CHO) cells producing albumin-erythropoietin to increase sialylation. Biphasic cultures were performed with shift of $CO_2$ concentrations and temperatures at day 5 when viable cell density was decreased and sialidase was started to be released by cell lysis. The examined temperature set points were 33, 35 and $37^{\circ}C$ respectively and the $CO_2$ concentration was 1, 5, 10 and 15%. We confirmed that sialidase activity was the lowest in biphasic culture that was shifted from normal culture condition to 1% of $CO_2$ and $33^{\circ}C$ on day 5. However, the temperature and concentration of $CO_2$ have little effect on activity of ${\alpha}2,3$-sialyltransferase. Also, sialic acid contents were enhanced 1.13-fold higher than that in control culture. In conclusion, Biphasic cultivation in CHO cells led to inhibition of sialidase activity and increases of sialylated glycan.

• Reproductive and Developmental Biology
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• v.36 no.2
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• pp.109-114
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• 2012

We prepared the polyclonal antibody anti-$20{\alpha}$-hydroxysteroid dehydrogenase (anti-$20{\alpha}$-HSD) against the recombinant full-length protein bovine $20{\alpha}$-HSD in Escherichia coli. The specificity of anti-$20{\alpha}$-HSD was demonstrated using Chinese hamster ovary (CHO) cells transfected with recombinant bovine $20{\alpha}$-HSD and bovine placental tissues. According to western blot analysis, anti-$20{\alpha}$-HSD specifically recognizes the 37-kDa protein bovine $20{\alpha}$-HSD. The protein is not present in untransfected CHO cells. Anti-$20{\alpha}$-HSD also recognizes a specific protein in the ovaries and placenta of other animals. Immunostaining was used to detect expression of bovine $20{\alpha}$-HSD protein in the cultured luteal cells during the estrous cycle later.

• Biotechnology and Bioprocess Engineering:BBE
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• v.6 no.2
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• pp.117-127
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• 2001

The high-level expression of a human single chain urokinase-type plasminogen activator (scu-PA) was achieved by employing a methotrexate (MTX)-dependent gene amplification system in Chinese hamster ovary (CHO) cells. By cotransfecting and coamplifying a scu-PA expression plasmid and dihydrofolate reductase (DHFR) minigene, several scu-PA expressing CHO cell lines were selected and gene-amplified. These recombinant cell lines, NGpUKs, secreted a completely processed scu-PA of 54 kD and up to 60mg/L was accumulated in the culture medium when they were adapted to an optimal MTX concentration. Over 95% of the scu-PA expressed was secreted in the culture medium and identified having the proper function of a plasminogen activator when activated by plasmin. Based on a genomic Southern analysis, a representative subclone, MGpUK-5, exhibited MTX-dependent scu-PA gene amplification, plus the initial single-copy gene of scu-PA eventually turned into about 150 copies of the amplified gene of scu-PA after gradual adaptation to 2.0$\mu$M of MTX. Meanwhile, the transcripts kof the scu-PA gene increased, although -early saturation of transcription was identified at 0.1$\mu$M of MTX. The scu-PA production by the MGpUK-5 subclone also increased relative to the gene amplification and increased transcripts, however, the relationship was not linearly proportional. Accordingly, since the MGpUK cell lines expressed elevated levels of enzymatically active scu-PA, these cell lines could be applied to the largescale production of scu-PA.

• Microbiology and Biotechnology Letters
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• v.16 no.4
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• pp.282-286
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• 1988

Growth kinetic parameters for mass cultivation of Chinese Hamster Ovary (CHO) cells are estimated by measuring oxygen uptake rates. It Is found that there is strong correlation between cell growth and oxygen consumption, showing that correlation factor is 0.83. Derived linear model predicts actual cell density very well. It tells that oxygen uptake rate can play important role in indirectly measuring cell density when conventional method of estimating cell density is no longer meaningful due to heavy cell clumpings. Cell yield per oxygen consumption, $Y_{\chi}o$ and mass transfer coefficient for oxygen, Ka are also estimated as 1.26$\times$10$^4$cells/mmole $O_2$ consumed and 1.01/h, respectively. Average specific growth rate over all runs is 2.891/day for CHO cells with producting 2 grams of tPA per day under continuous perfusion operations.

• Biotechnology and Bioprocess Engineering:BBE
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• v.6 no.5
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• pp.332-336
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• 2001

When parental Chinese hamster ovary (CHO) cell clones that are capable of producing thrombopoietin (TPO) were subjected to high methotrexate (MTX) concentrations, clonal variations in cell growth were apparent. In the clones that had no significant enhancement in specific TPO productivity (q$\_$Tpo/)when a higher level of MTX was administered, their growth was not depressed significantly nor their cell size changed significantly. On the other hand, those clones that showed a significant-enhancement in q$\_$Tpo/ at higher a MTX dosage, cell growth was depressed initially but recovered during successive sub-cultures. Furthermore, their cell size increased, which suggested that changes in cell size may be indicative of an enhanced q$\_$Tpo/. When the enhancement of the q$\_$Tpo/ of 9 clones after a high MTX dosage was plotted against the extent of the increase of their size, there was a linear correlation (γ$^2$=0.80, p<0.001, ANOVA), which suggested that an enhancement of q$\_$Tpo/ after high MTX administration can be measured by the increase in their cell size. Taken together, our data demonstrate that the selection of amplified CHO cell clones with enhanced q$\_$Tpo/ can be done upon their increased size and growth pattern. This facilitates the development of highly productive recombinant CHO cell lines.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.457-458
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• 2001

To determine the effect of ERp57 expression on thrombopoiein (TPO) productivity in recombinant Chinese hamster ovary (rCHO) cells, TPO producing rCHO cell line with doxycycline-regulated ERp57 expression \Vas developed. The Erp57 expression level could be regulated by addition of different concentrations of doxycycline to culture medium. The doxycycline concentration of I ${\mu}g/mL$ was high enough to suppress the ERp57 expression. Up to 5 ${\mu}g/mL$ doxycycline concentration used in culture medium, no observable cytotoxic effect of doxycycline was detected during culture. Overexpression of ERp57 、 vas found to increase the specific TPO productivity ($q_{Tpo}$) without growth inhibition, probably due to the chaperone-like activity of ERp57 in CHO cells.

• Journal of Biomedical Engineering Research
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• v.10 no.2
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• pp.195-202
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• 1989

Chinese Hamster Ovary( CHO) cells were cultured on the surface-modified polymers described in the previous study( "Polymer Surfaces for Cell Adhesion. 1. Surface Modification of Polymers and ESCA Analysis, " J. of KOSOMBE, Vol. 10, No. 1, 43-51, 1989). Among the physicochemical treatment methods. the chloric acid treatment was found to be the best method of rendering the polymer surfaces adhesive for CHO cells probably due to the high density of hydroxyl groups on the surface. Among the biological methods, the fibronectin treatment was best for CHO cell-compatibility probably due to specific active sites existed on the tell-binding domains of the fibronectin structure. When we compare the cell-compatibility of the chloric acid - and the fibronectin -treated PET surfaces, the number of cells attached on the surfaces were increased by 460.5 % and 559.0 % and, respectively, after 32 hr CHO cell culture, compared to that of untreated PET.eated PET.