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US 2012/0237973 A1Published application
Production of glycoproteins with reduced o-glycosylation
AssigneeGlycoFi, Inc.
InventorsPiotr Bobrowicz, W. James Cook, Warren Kett
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Abstract
A method is described for producing protein compositions having reduced amounts of O-linked glycosylation. The method includes producing the protein in cells cultured in the presence of an inhibitor of Pmt-mediated O-linked glycosylation and/or in the presence of one or more α-1,2-mannosidases.
Claims19
A method of producing a protein having reduced O-linked glycosylation comprising:
- (a) providing Pichia pastoris or Saccharomyces cerevisiae host cells comprising a nucleic acid encoding a protein operably linked to an inducible promoter;
- (b) growing the host cells for a time sufficient to provide a multiplicity of the host cells before contacting the host cells with one or more inhibitors of Pmt-mediated O-glycosylation;
- (c) contacting the host cells with an inducer of the promoter to induce expression of the protein at a time before or at the same time as contacting the host cells with the one or more inhibitors of Pmt-mediated O-linked glycosylation;
- and
- (d) isolating the protein produced by the host cells in the presence of the one or more inhibitors to produce the protein having reduced O-linked glycosylation.
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A method of producing a protein having reduced O-linked glycosylation comprising:
- (a) providing Pichia pastoris or Saccharomyces cerevisiae host cells comprising a nucleic acid encoding a protein operably linked to an inducible promoter and a nucleic acid encoding an α-1,2-mannosidase enzyme;
- (b) growing the host cells for a time sufficient to provide a multiplicity of the host cells before contacting the host cells with one or more inhibitors of Pmt-mediated O-glycosylation;
- (c) contacting the host cells with an inducer of the promoter to induce expression of the protein at a time before or at the same time as contacting the host cells with the one or more inhibitors of Pmt-mediated O-linked glycosylation;
- and
- (d) isolating the protein produced by the host cells in the presence of the one or more inhibitors to produce the protein having reduced O-linked glycosylation.
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Dependencies are inferred from the claim text.