Production of glycoproteins with reduced O-glycosylation
View Patent ↗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.
1. A method for producing a heterologous protein from a yeast host cell comprising:
(a) providing a yeast host cell having a nucleic acid encoding the heterologous protein operably linked to an inducible promoter;
(b) contacting the yeast host cell with an inducer of the promoter to induce expression of the heterologous protein at a time before or at the same time as contacting the yeast host cell with 5-[[3-(1-Phenyl-2-hydroxy)ethoxy)-4-(2-phenylethoxy)]phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineacetic Acid; and
(e) isolating the heterologous protein produced by the host cell.
2. The method of claim 1 , wherein the host cell has been genetically modified to produce glycoproteins with a predominant N-glycan glycoform.
3. The method of claim 1 , wherein the host cell is selected from the group consisting of Saccharomyces cerevisiae, K. lactis, Pichia pastoris, Pichia methanolica , and Hansenula.
4. The method of claim 1 , wherein the host cell is Pichia pastoris.
5. The method of claim 1 , wherein the host cell is Saccharomyces cerevisiae.
6. A method for producing a heterologous protein from a yeast host cell comprising:
(a) providing a yeast host cell having a nucleic acid encoding the heterologous protein operably linked to an inducible promoter and a second nucleic acid encoding a Trichoderma reesei α-1,2-mannosidase enzyme;
(b) contacting the yeast host cell with an inducer of the promoter to induce expression of the heterologous protein at a time before or at the same time as contacting the yeast host cell with 5-[[3-(1-Phenyl-2-hydroxy)ethoxy)-4-(2-phenylethoxy)]phenyl]methylene]-4-oxo-2-thioxo-3-thiazolidineacetic Acid; and
(e) isolating the heterologous protein produced by the host cell.
7. The method of claim 6 , wherein the α-1,2-mannosidase is operably linked to an inducible promoter.
8. The method of claim 6 , wherein the host cell is selected from the group consisting of Saccharomyces cerevisiae, K. lactic, Pichia pastoris, Pichia methanolica , and Hansenula.
9. The method of claim 6 , wherein the host cell is Pichia pastoris.
10. The method of claim 6 , wherein the host cell is Saccharomyces cerevisiae.