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RCA-I-resistant CHO mutant cells have dysfunctional GnT I and expression of normal GnT I in these mutants enhances sialylation of recombinant erythropoietin

RCA-I-resistant CHO mutant cells have dysfunctional GnT I and expression of normal GnT I in these mutants enhances sialylation of recombinant erythropoietin

Metabolic Engineering 12(4): 360-368

A large number of CHO glycosylation mutants were isolated by Ricinus communis agglutinin-I (RCA-I). Complementation tests revealed that all these mutant lines possessed a dysfunctional N-acetylglucosaminyltransferase I (GnT I) gene. Sequencing analyses on the GnT I cDNAs isolated from 16 mutant lines led to the identification of nine different single base pair mutations. Some mutations result in a premature stop codon whereas others cause a single amino acid substitution in the GnT I protein. Interestingly, expression of the normal GnT I cDNA in mutant cells resulted in enhanced sialylation of N-glycans. The sialylation of recombinant erythropoietin (EPO) produced in mutant cells that were co-transfected with GnT I was enhanced compared to that of EPO produced in wild type CHO cells. The enhanced sialylation of EPO produced by JW152 cells in the presence of GnT I over CHO-K1 cells is a result of increased sialylated glycan structures with higher antennary branching. These findings represent a new strategy that may be utilized by the biotechnology industry to produce highly sialylated therapeutic glycoproteins.

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Accession: 055333717

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PMID: 20346410

DOI: 10.1016/j.ymben.2010.03.002

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