Yeast strain for the production of proteins with terminal alpha-1,3-linked galactose
View Patent ↗Lower eukaryotic host cells have been engineered to produce glycoprotein having at least one terminal α-galactosyl epitope. The glycoproteins are useful for the production of highly antigenic glycoprotein compositions with advantages for the production of vaccines.
1. A recombinant Pichia host cell producing human-like glycoproteins, comprising a nucleic acid encoding a fusion protein that comprises an α-1, 3 galactosyltransferase catalytic domain fused to a yeast golgi localization peptide which is capable of transferring an α-galactose residue onto a terminal β-galactose residue of an N-linked oligosaccharide branch of an N-glycan having a trimannose core of a glycoprotein produced by the host cell, wherein the N-linked oligosaccharide branch has a structure selected from the group consisting of:
Galβ1, 4-GlcNAcβ1, 2-Manα1, 3;
Galβ1, 4-GlcNAcβ1, 4-Manα1, 3;
Galβ1, 4-GlcNAcβ1, 2-Manα1, 6;
Galβ1, 4-GlcNAcβ1, 4-Manα1, 6; and
Galβ1, 4-GlcNacβ1, 6-Manα1, 6.
2. The recombinant Pichia host cell of claim 1 which is Pichia pastoris.
3. The recombinant Pichia host cell of claim 1 wherein the yeast golgi localization peptide is a Saccharomyces cerevisiae Mnn2p transmembrane Golgi targeting localization domain.
4. A recombinant Pichia host cell producing human-like glycoproteins, comprising a nucleic acid encoding a fusion protein that comprises a Mus Musculus α-1, 3 -galactosyl transferase that lacks a putative transmembrane Golgi localization domain, which is fused to a Saccharomyces cerevisiae Mnn2p transmembrane Golgi targeting localization domain, which is capable of transferring an α-galactose residue onto a terminal β-galactose residue of an N-linked oligosaccharide branch of an N-glycan having a trimannose core of a glycoprotein produced by the host cell, wherein the N-linked oligosaccharide branch has a structure selected from the group consisting of:
Galβ1, 4-GlcNAcβ1, 2-Manα1, 3;
Galβ1, 4-GlcNAcβ1, 4-Manα1, 3;
Galβ1, 4-GlcNAcβ1, 2-Manα1, 6;
Galβ1, 4-GlcNAcβ1, 4-Manα1, 6; and
Galβ1, 4-GlcNacβ1, 6-Manα1, 6.
5. The recombinant Pichia host cell of claim 4 which lacks sialyltransferase.
6. The recombinant Pichia host cell of claim 4 which is Pichia pastoris.