Platform for producing glycoproteins, identifying glycosylation pathways
AssigneeNorthwestern University
InventorsMichael C. Jewett, Weston K. Kightlinger
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Abstract
Disclosed are components, systems, and methods for glycoprotein protein synthesis in vitro and in vivo. In particular, the disclosed components, systems, and methods relate to modular platforms for producing glycoproteins. The components, systems, and methods disclosed herein may be used in synthesizing glycoproteins and recombinant glycoproteins in cell-free protein synthesis (CFPS) and in modified cells.
Claims21
A cell-free system for glycosylating a peptide or polypeptide sequence in vitro, the peptide or polypeptide sequence comprising an asparagine residue and the system comprising as components:
- (i) a glycosyltransferase which is an N-glycosyltransferase (NGT) that catalyzes transfer to an amino group of the asparagine residue a monosaccharide to provide an N-linked glycan, or an expression vector that expresses the NGT in a cell-free protein synthesis (CFPS) reaction mixture;
- (ii) a glycosylation mixture comprising a monosaccharide donor, optionally a monosaccharide;
- wherein the peptide or polypeptide sequence is glycosylated in the glycosylation mixture in vitro to provide a peptide or polypeptide sequence comprising the N-linked glycan.
-26. (canceled)
A peptide or polypeptide sequence comprising an N-linked glycan, the N-linked glycan comprising a moiety selected from the group consisting of sialylated forms of lactose, fucosylated forms of lactose, sialylated forms of LacNAc (lactose-(poly)LacNAc), fucosylated forms of LacNAc (lactose-(poly)LacNAc), pyruvylated lactose, pyruvylated LacNAc (lactose-(poly)LacNAc), glucose, polyα1,6-linked glucose, glucose modified with β1,3 GalNAc, lactose, lactose modified with (poly)LacNAc (lactose-(poly)LacNAc), lactose modified with α1,4 galactose, lactose modified with oligo-sialic acid and an αGal epitope.
A method for preparing a glycosylated peptide or polypeptide sequence in vitro, the method comprising reacting a peptide or polypeptide sequence comprising an asparagine residue in a glycosylation mixture comprising a monosaccharide donor with a glycosyltransferase which is a N-glycosyltransferase (NGT) that catalyzes transfer of the monosaccharide from the monosaccharide donor to an amino group of the asparagine residue to provide an N-linked glycan, wherein the peptide or polypeptide sequence is glycosylated in the glycosylation mixture in vitro to provide a peptide or polypeptide sequence comprising the N-linked glycan.
A system for preparing a glycosylated peptide or polypeptide sequence, the peptide or polypeptide sequence comprising an asparagine residue and the system comprising as components:
- (i) a modified bacterial cell, optionally wherein the bacterial cell is modified to express an exogenous glycosyltransferase which is an N-glycosyltransferase (NGT) that catalyzes transfer to an amino group of the asparagine residue a monosaccharide to provide an N-linked glycan, or an expression vector that expresses the NGT in a cell-free protein synthesis (CFPS) reaction mixture;
- (ii) a glycosylation mixture comprising a non-natural sugar donor, optionally added to media for growing the modified bacterial cell;
- wherein the peptide or polypeptide sequence is glycosylated in the modified bacterial cell to provide the peptide or polypeptide sequence comprising the non-natural sugar.
Dependencies are inferred from the claim text.