• Journal of Microbiology and Biotechnology
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• 제17권12호
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• pp.1949-1954
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• 2007

A simple way to prevent protein hyperglycosylation in Hansenula polymorpha was found. When glucose oxidase from Aspergillus niger and carboxymethyl cellulase from Bacillus subtilis were expressed under the control of an inducible methanol oxidase (MOX) promoter using methanol as a carbon source, hyperglycosylated forms occurred. In contrast, MOX-repressing carbon sources (e.g., glucose, sorbitol, and glycerol) greatly reduced the extent of hyperglycosylation. Carbon source starvation of the cells also reduced the level of glycosylation, which was reversed to hyperglycosylation by the resumption of cell growth. It was concluded that the proteins expressed under actively growing conditions are produced as hyperglycosylated forms, whereas those under slow or nongrowing conditions are as short-glycosylated forms. The prevention of hyperglycosylation in the Hansenula polymorpha expression system constitutes an additional advantage over the traditional Saccharomyces cerevisiae system in recombinant production of glycosylated proteins.

• Molecules and Cells
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• 제24권3호
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• pp.416-423
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• 2007

Alterations in the glycan chains of cell surface glycoconjugates are frequently involved biological processes such as cell-cell interaction, cell migration, differentiation and development. Cultured embryonic (E18) rat cortical neurons underwent apoptosis in response to camptothecin, and lectin histochemistry showed that binding to apoptotic neurons of FITC-conjugated Maackia amurensis agglutinin (MAA), which is specific for terminal ${\alpha}2,3$-sialic acid residues, increased progressively with increasing concentrations of camptothecin. Analysis of the total proteins of apoptotic neurons by SDS-PAGE, and lectin blotting using HRP-labeled MAA, revealed that the expression of terminal ${\alpha}2,3$-sialic acid residues on an unknown protein with an apparent molecular mass of 25.6 kDa also increased in apoptotic neurons. NP-HPLC analysis of the total cellular N-glycans of normal and apoptotic neurons demonstrated that the expression of structurally simpler biantennary types of N-glycans fell by 49% during apoptosis whereas the more branched triantennary types of N-glycans with terminal sialic acid residues increased by up to 59%. These results suggest that increased surface expression of ${\alpha}2,3$-sialic acid residues and hyperglycosylation of N-glycans is a common feature of cellular responses to changes in cell physiology such as tumorigenesis and apoptosis.

• Journal of the Korean Wood Science and Technology
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• 제33권4호통권132호
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• pp.45-52
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• 2005

백색부후균 Phanerochaete chrysosporium으로부터 유래한 Manganese peroxidase (mnp5)를 methylotrophic yeast인 Pichia pastoris에서 이종 발현을 하였다. 이종발현으로부터 얻어진 단백질은 클로닝으로부터 예상되어지는 분자량보다 높은 분자량인 45 kDa으로 나타났다. 이것은 mnp5가 가지고 있는 glycosylation site에 의한 것이며, N-linked hyperglycosylation이 효소 활성에 영향을 미치는지를 site direct mutation에 의해 확인하였다. Sodium dodesyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)와 Coomassie Brilliant Blue (CBB) 염색에 의해 분자량을 확인한 결과 약 37 kDa으로 나타났으며, 효소활성을 측정한 결과 glycosylation이 효소 활성에 영향을 미치지 않는 것으로 나타났다. 따라서 본 연구로부터 P. pastoris에서 mnp5의 이종발현이 성공적으로 이루어졌으며 이러한 결과로부터 heme을 포함하고 있는 단백질의 이종발현 생산의 가능성을 보여주었다.

• Reproductive and Developmental Biology
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• 제33권2호
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• pp.77-83
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• 2009

Erythropoietin (EPO), a glycoprotein hormone produced from primarily cells of the peritubular capillary endothelium of the kidney, is responsible for the regulation of red blood cell production. We have been investigating the roles of glycosylation site added in the biosynthesis and function of recombinant protein. We constructed three EPO mutants ($\Delta$69, $\Delta$105 and $\Delta$69,105), containing an additional oligosaccharide chains. EPOWT and EPO$\Delta$69 were effectively expressed in transient and stably transfected CHO-K1 cell lines. But, it wasn't detected any protein in the culture medium of EPO$\Delta$105 and EPO$\Delta$69,105 mutants. The growth and differentiation of EPO-dependent human leukemic cell line (F36E) were used to measure the cytokine dependency and in vitro bioactivity of rec-hEPO. MTT assay values were increased by survival of F36E cells at 24h. To analysis biological activity in vivo, two groups of ICR-mice (7 weeks old) were injected subcutaneously with 10 IU per mice of rec-hEPO molecules on days 0 and 2. Red blood cell and hematocrit values were measured on 6 days after the first injection. The hematocrit values were remarkably increased in all treatment groups. The pharmacokinetic analysis was also affected in the mice injected with rec-hEPO molecules 2.5 IU by tail intravenous. Protein samples were detected by Western blotting. An EPO$\Delta$69 protein migrated as a broad band with an average apparent molecular and detected slightly high band. Enzymatic N-deglycosylation resulted in narrow band and was the same molecular size. The biological activity of EPO$\Delta$69 was enhanced to compare with wt-hEPO. The half-life was longer than wt-hEPO. The results suggest that hyperglycosyalted recombinant human erythropoietin (EPO$\Delta$69) may have important biological and therapeutic good points.

• Reproductive and Developmental Biology
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• 제34권4호
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• pp.289-297
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• 2010

Erythropoietin (EPO) is a glycoprotein hormone secreted from primarily cells of the peritubular capillary endothelium of the kidney, and is responsible for the regulation of red blood cell production. We constructed and expressed dimeric cDNAs in Chinease hamster ovary (CHO) cells encoding a fusion protein consisting of 2 complete human EPO domains linked by a 2-amino acid linker (Ile-Asp). We described the activity of dimeric hyperglycosylated EPO (dHGEPO) mutants containing additional oligosaccharide chains and characterized the function of glycosylation. No dimeric proteins with mutation at the $105^{th}$ amino acid were found in the cell medium. Growth and differentiation of the human EPO-dependent leukemiae cell line (F36E) were used to measure cytokine dependency and in vitro bioactivity of dHGEPO proteins. MIT assay at 24 h increased due to the survival of F36E cells. The dHGEPO protein migrated as a broad band with an average molecular mass of 75 kDa. The mutant, dHGEPO, was slightly higher than the wild-type (WT) dimeri-EPO band. Enzymatic N-deglycosylation resulted in the formation of a narrow band with a molecular mass twice of that of of monomeric EPO digested with an N-glycosylation enzyme. Hematocrit values were remarkably increased in all treatment groups. Pharmacokinetic analysis was also affected when 2.5 IU of dHGEPO were intravenously injected into the tails of the mice. The biological activity and half-life of dHGEPO mutants were enhanced as compared to the corresponding items associated the WT dimeric EPO. These results suggest that recombinant dHGEPO may be attractive biological and therapeutic targets.

• 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.