• Journal of Microbiology and Biotechnology
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• 제18권12호
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• pp.1945-1952
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• 2008

Sialylation, the attachment of sialic acid residues to a protein, can affect the biological activity and in vivo circulatory half-life of glycoproteins. Human ${\alpha}2$,3-sialyltransferase (${\alpha}2$,3-ST) and ${\beta}1$,4-galactosyltransferase (${\beta}1$,4-GT) are responsible for terminal sialylation and galactosylation, respectively. Enhanced sialylation of human erythropoietin (EPO) by the expression of ${\alpha}2$,3-ST and ${\beta}1$,4-GT was achieved using recombinant Chinese hamster ovary (CHO) cells (EC1). The sialic acid content and sialylation of N-glycans were evaluated by HPLC. When ${\alpha}2$,3-ST was expressed in CHO cells (EC1-ST2), the sialic acid content (moles of sialic acid/mole of EPO) increased from 6.7 to 7.5. In addition, the amount of trisialylated glycans increased from 17.3% to 26.1 %. When ${\alpha}2$,3-ST and ${\beta}1$,4-GT were coexpressed in CHO cells (EC1-GTST15), the degree of sialylation was greater than that in EC1-ST2 cells. In the case of EC1-GTST15 cells, the sialic acid content increased to 8.2 and the proportion of trisialylated glycans was markedly increased from 17.3% to 35.5%. Interestingly, the amount of asialoglycans decreased only in the case of GTST15 cells (21.4% to 14.2%). These results show that coexpression of ${\alpha}2$,3-ST and ${\beta}1$,4-GT is more effective than the expression of ${\alpha}2$,3-ST alone. Coexpression of ${\alpha}2$,3-ST and ${\beta}1$,4-GT did not affect CHO cell growth and metabolism or EPO production. Thus, coexpression of ${\alpha}2$,3-ST and ${\beta}1$,4-GT may be beneficial for producing therapeutic glycoproteins with enhanced sialylation in CHO cells.

• Journal of Microbiology and Biotechnology
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• 제23권5호
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• pp.699-706
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• 2013

We have previously reported that N-acetylcysteine (NAC) not only delayed apoptosis but also enhanced the production of recombinant erythropoietin (EPO) in Chinese hamster ovary (CHO) cell culture. To investigate the production enhancement mechanism, the effects of similar thiol-reducing agents were studied. Intriguingly, all mild reducing agents examined including mercaptoethanesulfonic acid (MESNA), thiolactic acid (TLA), and thioglycolate (TG) were shown to block apoptosis and increase EPO production. A pulse-chase study of EPO secretion revealed that all four thiol-reducing agents increased the EPO secretion rate; among them TLA showed the highest rate. In terms of product quality, the sialic acid content of the glycoprotein is one of the most important factors. It was reported that a number of glycoproteins produced by CHO cells often have incomplete sialylation, particularly under high-producing conditions. Human ${\alpha}2,3$-sialyltransferase (${\alpha}2,3$-ST) was introduced into EPO-producing CHO cells in order to compensate for the reduced sialylation during supplementation with NAC. When ${\alpha}2,3$-ST was expressed in the presence of NAC, reduced sialylation was restored and an even more sialylated EPO was produced. Thus, our study is significant in that it offers increased EPO production while still allowing the prevention of decreased sialylation of EPO.

• KSBB Journal
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• 제32권3호
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• pp.193-198
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• 2017

Sialylation is important in producing therapeutic proteins such as antibody, cytokine and fusion protein. Thus, enhancement of sialylation is usually performed in CHO cell cultures. ${\alpha}2,6$-Sialyltransferase (ST), which plays a key role in the attachment of ${\alpha}2,6-sialic$ acid, is present in human cells but not in Chinese hamster ovary (CHO) cells. Overexpression of ${\alpha}2,6-ST$ can be used for enhancing the degree of sialylation and achieving human-like glycosylation. In this study, we constructed CHO cells producing human cytotoxic T-lymphocyte antigen4-immunoglobulin (hCTLA4-Ig) as well as ${\alpha}2,6-ST$. Transfected CHO cells were selected using G418 and stable cell line was established. Profiles of viable cell density and hCTLA4-Ig titer in an overexpressed cell line were similar to those of a wild-type cell line. It was confirmed that the total amount of sialic acid was increased and ${\alpha}2,6-sialic$ acid was attached to the terminal residues of N-glycan of hCTLA4-Ig by ESI-LC-MS. Compared to 100% of ${\alpha}2,3-sialic$ acid in wild type cells, 70.9% of total sialylated N-glycans were composed of ${\alpha}2,6-sialic$ acid in transfected cells. In conclusion, overexpression of ${\alpha}2,6-ST$ in CHO cells led to the increase of both the amount of total sialylated N-glycan and the content of ${\alpha}2,6-sialic$ acid, which is more resemble to human-like structure of glycosylation.

• Bulletin of the Korean Chemical Society
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• 제31권3호
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• pp.720-723
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• 2010

• Biotechnology and Bioprocess Engineering:BBE
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• 제3권1호
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• pp.40-43
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• 1998

The glycosylation pattern of Erythropoietin (EPO), produced by recombinant CHO cells, was studied using the simple and rapid technique of 'Lectin-blotting'. In this experiment we used three different kinds of lectins, MAA(Maackia amurensis agglutinine), RCA(Ricinus communis agglutinine), and DSA(Datura stramonium agglutinine), which bind to the terminal sialic acid, galactose, and the N-acetyllactosamine chain respectively. The lectin-blotting technique was used to analyze the carbohydrate structure of EPO produced in the presence of two physiologically active chemical compounds, ammonium and chloroquine. The effect of the ammonium ion on the glycosylation of EPO was studied because it accumulated in the medium mainly as a by-product of glutamine matabolism. Ammonium chloride significantly inhibited the sialylation of the terminal galactose residue at concentrations of 8mM or more. Chloroquine, a potent inhibitor of glycosylation, inhibited terminal sialylation at concentrations of 100 and 200 $\mu$M, and at a concentration of 300 $\mu$M, also inhibited Nacetyllactosamine chain synthesis.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVII)
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• pp.148-149
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• 2005

• Experimental and Molecular Medicine
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• 제50권12호
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• pp.9.1-9.12
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• 2018

Endometriosis is a disease characterized by implants of endometrial tissue outside the uterine cavity and is strongly associated with infertility. Focal adhesion of endometrial tissue to the peritoneum is an indication of incipient endometriosis. In this study, we examined the effect of various cytokines that are known to be involved in the pathology of endometriosis on endometrial cell adhesion. Among the investigated cytokines, transforming growth factor-${\beta}1$ ($TGF-{\beta}1$) increased adhesion of endometrial cells to the mesothelium through induction of ${\alpha}2-6$ sialylation. The expression levels of ${\beta}$-galactoside ${\alpha}2-6$ sialyltransferase (ST6Gal) 1 and ST6Gal2 were increased through activation of $TGF-{\beta}RI/SMAD2/3$ signaling in endometrial cells. In addition, we discovered that terminal sialic acid glycan epitopes of endometrial cells engage with sialic acid-binding immunoglobulin-like lectin-9 expressed on mesothelial cell surfaces. Interestingly, in an in vivo mouse endometriosis model, inhibition of endogenous sialic acid binding by a $NeuAc{\alpha}2-6Gal{\beta}1$-4GlcNAc injection diminished $TGF-{\beta}1$-induced formation of endometriosis lesions. Based on these results, we suggest that increased sialylation of endometrial cells by $TGF-{\beta}1$ promotes the attachment of endometrium to the peritoneum, encouraging endometriosis outbreaks.

• BMB Reports
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• 제46권3호
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• pp.157-162
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• 2013

Human ${\alpha}$-galactosidase A (GLA) has been used in enzyme replacement therapy for patients with Fabry disease. We expressed recombinant GLA from Chinese hamster ovary cells with very high productivity. When compared to an approved GLA (agalsidase beta), its size and charge were found to be smaller and more neutral. These differences resulted from the lack of terminal sialic acids playing essential roles in the serum half-life and proper tissue targeting. Because a simple sialylation reaction was not enough to increase the sialic acid content, a combined reaction using galactosyltransferase, sialyltransferase, and their sugar substrates at the same time was developed and optimized to reduce the incubation time. The product generated by this reaction had nearly the same size, isoelectric points, and sialic acid content as agalsidase beta. Furthermore, it had better in vivo efficacy to degrade the accumulated globotriaosylceramide in target organs of Fabry mice compared to an unmodified version.

• 한국미생물·생명공학회지
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• 제32권2호
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• pp.142-148
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• 2004

에리스로포이에틴은 인간 적혈구분화의 조절인자로 작용한다. 유전자 재조합 인간에리스로포이에틴(rhEPO)은 동물세포에서 생산되고 있는 재조합 당단백질의 하나이며 당쇄부분이 전체 분자량의 40％를 차지한다. 시알산 함량은 체내약물 투여 지속기간과 직접적인 연관이 있어 시알산 함량은 의약용 당단백질의 중요한 성질로 여겨진다. 본 연구에서는 CHO세포 배양액에 시알산 생합성 전구물질인 N-acetylmannosamine(ManNAc)과 sialidase 저해제인 2-deoxy-2,3-hyo-N-acetylneuraminic acid(NeuAc2en)를 첨가하여 rhEPO의 sialic acid함량을 증가시킬 수 있었다. 특히, 배양액에 20 mM ManNAc/0.5 mM NeuAc2en를 첨가할 때 대조구에 비하여 약 10배의 시알산 함량이 증가하였으며 세포성장이나 배양액의 rhEPO생산량에는 영향이 없었다. rhEPO의 정제시 시알산 함량이 11∼15％인 다당쇄 rhEPO분획을 얻었으며, 배양액 내에 20 mM ManNAc와 0.5 mM NeuAc2en를 동시에 첨가함으로 대조구에 비하여 시말산함량이 높은다당쇄 rhEPO의 생산성이 50％ 증가하였다.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVII)
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• pp.415-416
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• 2005