| Enhanced sialylation of recombinant human EPO in Chinese hamster ovary cells by combinatorial engineering of selected genes. | |
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MedLine Citation:
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PMID: 21436238 Owner: NLM Status: Publisher |
Abstract/OtherAbstract:
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Therapeutic glycoproteins with exposed galactose residues are cleared rapidly from the bloodstream by asialoglycoprotein receptors in hepatocytes. Various approaches have been used to increase content of sialic acid, which occupies terminal sites of N- or O-linked glycans and thereby increases half-life of therapeutic glycoproteins. We enhanced sialylation of human erythropoietin (EPO) by genetic engineering of the sialylation pathway in Chinese hamster ovary (CHO) cells. The enzyme GNE (UDP-GlcNAc 2-epimerase)/ MNK (ManNAc kinase), which play a key role in the initial two steps of sialic acid biosynthesis, is regulated by CMP-sialic acid through a feedback mechanism. Since sialuria patient cells fail in regulating sialic acid biosynthesis by feedback mechanism, various sialuria-like mutated rat GNEs were established, and subjected to in vitro activity assay. GNE/MNK-R263L-R266Q mutant showed 93.6% relative activity compared to wild-type, and did not display feedback inhibition. Genes for sialuria-mutated rat GNE/MNK, Chinese hamster CMP-sialic acid transporter, and human α2,3-sialyltransferase were transfected simultaneously into recombinant human (rh)EPO-producing CHO cells. CMP-sialic acid concentration of engineered cells was significantly (>10-fold) increased by sialuria-mutated GNE/MNK (R263L-R266Q) expression. Sialic acid content of rhEPO produced from engineered cells was 43% higher than that of control cells. Ratio of tetra-sialylated glycan of rhEPO produced from engineered cells was increased ~32%, but ratios of asialo- and mono-sialylated glycan were decreased ~50%, compared to control. These findings indicate that sialuria-mutated rat GNE/MNK effectively increases intracellular CMP-sialic acid level. The newly-constructed host CHO cell lines produced more highly-sialylated therapeutic glycoproteins through overexpression of sialuria-mutated GNE/MNK, CMP-SAT, and α2,3-ST. |
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Authors:
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Young-Dok Son; Yeon Tae Jeong; Seung-Yeol Park; Jung Hoe Kim |
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Publication Detail:
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Type: JOURNAL ARTICLE Date: 2011-3-24 |
Journal Detail:
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Title: Glycobiology Volume: - ISSN: 1460-2423 ISO Abbreviation: - Publication Date: 2011 Mar |
Date Detail:
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Created Date: 2011-3-25 Completed Date: - Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 9104124 Medline TA: Glycobiology Country: - |
Other Details:
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Languages: ENG Pagination: - Citation Subset: - |
Affiliation:
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Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 335 Gwahangno, Yuseong-gu, Daejeon 305-701, Republic of Korea. |
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From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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