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Sialylation of recombinant therapeutic glycoproteins modulates their pharmacokinetic properties by affecting their in vivo half-life. N-glycan branching on glycoproteins increases the number of potential attachment sites for sialic acid. Here, we introduce a new approach for increasing the sialylation of recombinant human erythropoietin (rhEPO) produced in CHO cells by modulating poly-N-acetyllactosamine (poly-LacNAc) biosynthesis. We did not observe an increase in rhEPO sialylation, however, until the feedback inhibition by intracellular cytidine monophosphate-N-acetylneuraminic acid (CMP-Neu5Ac), which is a limiting factor for sialylation, was released. Thus, we found that a combined approach inhibiting poly-LacNAc biosynthesis and releasing CMP-Neu5Ac feedback inhibition produces the most significant increase in rhEPO sialylation in metabolically engineered CHO cells. Furthermore, a detailed analysis of the resulting N-glycan structures using LC/MS revealed increased tri- and tetra- sialylated N-glycan structures accompanied by a reduction of di-sialylated N-glycan structures. These results validate our new approach for glycosylation engineering, and we expect this approach will be useful in future efforts to enhance the efficacy of other therapeutic glycoproteins.
Pubmed ID: 29740059
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NIH genetic sequence database that provides annotated collection of all publicly available DNA sequences for almost 280 000 formally described species (Jan 2014) .These sequences are obtained primarily through submissions from individual laboratories and batch submissions from large-scale sequencing projects, including whole-genome shotgun (WGS) and environmental sampling projects. Most submissions are made using web-based BankIt or standalone Sequin programs, and GenBank staff assigns accession numbers upon data receipt. It is part of International Nucleotide Sequence Database Collaboration and daily data exchange with European Nucleotide Archive (ENA) and DNA Data Bank of Japan (DDBJ) ensures worldwide coverage. GenBank is accessible through NCBI Entrez retrieval system, which integrates data from major DNA and protein sequence databases along with taxonomy, genome, mapping, protein structure and domain information, and biomedical journal literature via PubMed. BLAST provides sequence similarity searches of GenBank and other sequence databases. Complete bimonthly releases and daily updates of GenBank database are available by FTP.
View all literature mentionsOpen source Java based image processing software program designed for scientific multidimensional images. ImageJ has been transformed to ImageJ2 application to improve data engine to be sufficient to analyze modern datasets.
View all literature mentionsCommercial instrument and chemical vendor. Developer and manufacturer of specialized technological products for life science research and clinical diagnostics markets.
View all literature mentionsA commercial organization which provides assay technologies to isolate DNA, RNA, and proteins from any biological sample. Assay technologies are then used to make specific target biomolecules, such as the DNA of a specific virus, visible for subsequent analysis.
View all literature mentionsCompany provides laboratories worldwide with analytical instruments and supplies, clinical and diagnostic testing services, consumables, applications and expertise in life sciences and applied chemical markets.
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