title: Enhancing recombinant glycoprotein sialylation through CMP-sialic acid transporter over expression in Chinese hamster ovary cells.
abstract: Glycosylation engineering strategies that are currently used to improve quality of recombinant glycoproteins involve the manipulation of glycosyltransferase and/or glycosidase expression. We explored the possibility that over expressing nucleotide sugar transporters, particularly the CMP-sialic acid transporter (CMP-SAT) would improve the sialylation process in Chinese hamster ovary cells (CHO). Our hypothesis was that increasing CMP-SAT in the cells through recombinant means would increase the transport of CMP-sialic acid into the Golgi, resulting in an increased CMP-sialic acid intra-lumenal pool and increased sialylation of the proteins produced. We report the construction of the CMP-SAT expression vector (pcDNA-SAT) using hamster CMP-SAT (GenBank accession number Y12074) and demonstrated its functionality using Lec2 CHO mutant cells. Transfection of pcDNA-SAT into CHO IFN-gamma, a CHO cell line producing recombinant human interferon-gamma (IFN-gamma) resulted in single clones that had 2-20 fold increase in total CMP-SAT expression at the transcript level and 1.8-2.8 fold increase in CMP-SAT at the protein level when compared to untransfected parent CHO IFN-gamma. This resulted in 4%-16% increase in site sialylation of IFN-gamma. There was also a higher proportion of the more sialylated IFN-gamma glycans produced by the clones. We have thus established a novel strategy for sialylation improvement in recombinant protein production that can be considered singly or along with existing glycosylation improvement strategies, including glycosyltransferase over expression and nucleotide sugar feeding. These multiprong approaches can possibly bring us closer toward the goal of maximum and consistent sialylation in glycoprotein production using mammalian cells.
authors.affiliation: Singapore-MIT Alliance, National University of Singapore, 4 Engineering Drive 3, Singapore, Singapore.
authors.forename: Niki S C
authors.initials: NS
authors.lastname: Wong
authors.forename: Miranda G S
authors.initials: MG
authors.lastname: Yap
authors.forename: Daniel I C
authors.initials: DI
authors.lastname: Wang
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000378:metabolism*; D005434:Flow Cytometry; D015870:Gene Expression / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D007371:Interferon-gamma / Q000737:chemistry / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000032:analysis; D019158:N-Acetylneuraminic Acid / Q000032:analysis; D033704:Nucleotide Transport Proteins / Q000235:genetics / Q000378:metabolism*; D019887:Peanut Agglutinin / Q000378:metabolism; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection; D014909:Wheat Germ Agglutinins / Q000378:metabolism
chemical_list: D008562:Membrane Glycoproteins; D033704:Nucleotide Transport Proteins; D019887:Peanut Agglutinin; D011994:Recombinant Proteins; D014909:Wheat Germ Agglutinins; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D007371:Interferon-gamma; D019158:N-Acetylneuraminic Acid