Bacterial system for producing human o-glycoproteins
AssigneeCornell University
InventorsMatthew P. DELISA, Aravind NATARAJAN
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Abstract
The present application relates to recombinant prokaryotic host cells expressing one or more 4-epimerases, one or more glycosyl-1-phosphate transferases, one or more O-oligosaccharyltransferases, and, optionally, one or more ß1,3-galactosyltransferase enzymes capable of transferring galactose to undecaprenyl pyrophosphate-linked N-Acetylgalactosamine. Also disclosed are methods for producing an O-glycosylated protein.
Claims70
A recombinant prokaryotic host cell expressing one or more 4-epimerases, one or more glycosyl-1-phosphate transferases, and one or more O-oligosaccharyltransferases.
A recombinant prokaryotic host cell expressing one or more 4-epimerases, one or more glycosyl-1-phosphate transferases, one or more O-oligosaccharyltransferases, and one or more ß1,3-galactosyltransferase enzymes capable of transferring galactose (Gal) to undecaprenyl pyrophosphate (Und-PP)-linked N-Acetylgalactosamine (GalNAc).
The recombinant prokaryotic host cell of any one of claims 2-9, wherein the one or more ß1,3-galactosyltransferase enzymes capable of transferring galactose (Gal) to undecaprenyl pyrophosphate (Und-PP)-linked N-Acetylgalactosamine (GalNAc) is the ß-1,3;
- galactosyltransferase derived from the O-antigen biosynthesis pathway of enterohemorrhagic Escherichia coli O104 (EcWbwC).
A method for producing an O-glycosylated protein comprising:
- providing a recombinant host cell expressing one more 4-epimerases, one or more glycosyl-1-phosphate transferases, one or more O-oligosaccharyltransferases, and a glycoprotein target comprising one or more serine and/or threonine residues;
- and
- culturing said host cell under conditions effective to:
- (i) produce N-acetylgalactosamine (GalNAc) linked to undecaprenyl pyrophosphate (Und-PP);
- and
- (ii) transfer the N-acetylgalactosamine (GalNAc) linked to undecaprenyl pyrophosphate (Und-PP) en bloc to a serine or threonine amino acid of the glycoprotein target.
A method for producing an O-glycosylated protein comprising:
- providing a recombinant host cell expressing one more 4-epimerases, one or more glycosyl-1-phosphate transferases, one or more O-oligosaccharyltransferases, one or more ß1,3-galactosyltransferase enzymes capable of transferring galactose (Gal) to undecaprenyl pyrophosphate (Und-PP)-linked N-Acetylgalactosamine (GalNAc), and a glycoprotein target comprising one or more serine and/or threonine residues;
- and
- culturing said host cell under conditions effective to:
- (i) produce N-acetylgalactosamine (GalNAc) linked to undecaprenyl pyrophosphate (Und-PP);
- (ii) extend Und-PP-GalNAc by a single galactose (Gal) monosaccharide to yield lipid-linked Gal-ß1,3-GalNAc;
- and
- (iii) transfer the lipid-linked Gal-ß1,3-GalNAc en bloc to a serine or threonine amino acid of the glycoprotein target.
An in vitro method for producing an O-glycosylated protein comprising:
- providing glycosylation reagents comprising one more 4-epimerases, one or more glycosyl-1-phosphate transferases, and one or more O-oligosaccharyltransferases,
- providing a glycoprotein target comprising one or more serine and/or threonine residues, and
- incubating said glycosylation reagents and said glycoprotein target under conditions effective to:
- (i) yield N-acetylgalactosamine (GalNAc) linked to undecaprenyl pyrophosphate (Und-PP), and
- (ii) transfer the N-acetylgalactosamine (GalNAc) linked to undecaprenyl pyrophosphate (Und-PP) en bloc to a serine or threonine amino acid of the glycoprotein target.
An in vitro method for producing an O-glycosylated protein comprising:
- providing glycosylation reagents comprising one more 4-epimerase enzymes, one or more heterologous N,N′-diacetylbacilliosaminyl-1-phosphate transferase enzymes, one or more heterologous O-oligosaccharyltransferases, and one or more ß1,3-galactosyltransferase enzymes capable of transferring galactose (Gal) to undecaprenyl pyrophosphate (Und-PP)-linked N-Acetylgalactosamine (GalNAc),
- providing a glycoprotein target comprising one or more serine and/or threonine residues, and
- incubating said glycosylation reagents and said glycoprotein target under conditions effective to:
- (i) yield lipid-linked Gal-ß1,3-GalNAc, and
- (ii) transfer the lipid-linked Gal-ß1,3-GalNAc and any additional sugars en bloc to a serine or threonine amino acid of the glycoprotein target.
An in vitro method for producing an O-glycosylated protein comprising:
- providing reagents suitable for synthesizing a glycoprotein target;
- providing glycosylation reagents comprising one more 4-epimerases, one or more glycosyl-1-phosphate transferases, and one or more O-oligosaccharyltransferases;
- providing a nucleic acid molecule encoding a glycoprotein target;
- and
- incubating said reagents suitable for synthesizing a glycoprotein target, glycosylation reagents, and nucleic acid molecule encoding a glycoprotein target under conditions effective to:
- (i) synthesize the glycoprotein target encoded by the nucleic acid molecule encoding a glycoprotein target,
- (ii) yield N-acetylgalactosamine (GalNAc) linked to undecaprenyl pyrophosphate (Und-PP), and
- (iii) transfer the N-acetylgalactosamine (GalNAc) linked to undecaprenyl pyrophosphate (Und-PP) en bloc to a serine or threonine amino acid of the glycoprotein target.
An in vitro method for producing an O-glycosylated protein comprising:
- providing reagents suitable for synthesizing a glycoprotein target:
- providing glycosylation reagents comprising one more 4-epimerase enzymes, one or more heterologous N,N′-diacetylbacilliosaminyl-1-phosphate transferase enzymes, one or more heterologous O-oligosaccharyltransferases, and one or more ß1,3-galactosyltransferase enzymes capable of transferring galactose (Gal) to undecaprenyl pyrophosphate (Und-PP)-linked N-Acetylgalactosamine (GalNAc),
- providing a nucleic acid molecule encoding a glycoprotein target;
- and
- incubating said reagents suitable for synthesizing a glycoprotein target, glycosylation reagents, and nucleic acid molecule encoding a glycoprotein target under conditions effective to:
- (i) synthesize the glycoprotein target encoded by the nucleic acid molecule encoding a glycoprotein target,
- (ii) yield lipid-linked Gal-ß1,3-GalNAc, and
- (iii) transfer the lipid-linked Gal-ß1,3-GalNAc and any additional sugars en bloc to a serine or threonine amino acid of the glycoprotein target.
A prokaryotic host cell expressing an α2,6-sialyltransferases and an α2,3-sialyltransferase, wherein the α2,6-sialyltransferases is the α2,6-sialyltransferases from Photobacterium sp. JT-ISH-224 (PspST6) and where the α2,3-sialyltransferase is the α2,3-sialyltransferase from E. coli O104 (EcWbwA).
A prokaryotic host cell expressing one or more 4-epimerases, one or more glycosyl-1-phosphate transferases, one or more O-Antigen ligases, and, optionally, one or more ß1,3-galactosyltransferase enzymes capable of transferring galactose (Gal) to undecaprenyl pyrophosphate (Und-PP)-linked N-Acetylgalactosamine (GalNAc).
A method for producing a lipid linked Gal-β1,3-GalNAcα (T antigen or core 1) comprising:
- providing a recombinant host cell expressing one or more 4-epimerases, one or more glycosyl-1-phosphate transferases, one or more ß1,3-galactosyltransferase enzymes capable of transferring galactose (Gal) to undecaprenyl pyrophosphate (Und-PP)-linked N-Acetylgalactosamine (GalNAc), and one or more O-Antigen ligases (e.g., EcWaaL) and
- culturing the host cell under conditions effective to:
- (i) produce Gal-ß1,3-GalNAc linked to undecaprenyl pyrophosphate (Und-PP) and (ii) transfer Gal-ß1,3-GalNAc linked to undecaprenyl pyrophosphate (Und-PP) en bloc to a lipid target.
Dependencies are inferred from the claim text.