Prokaryote-based cell-free system for the synthesis of glycoproteins

Abstract

The present invention is directed to a cell-free system for producing a glycosylated protein. This system comprises an isolated oligosaccharyltransferase capable of transferring a glycan from a lipid carrier molecule to a glycoprotein target, one or more isolated glycans, where each glycan is linked to a lipid carrier molecule, and a glycoprotein target comprising one or more glycan acceptor amino acid residues or a nucleic acid molecule encoding said glycoprotein target. The present invention further relates to kits and methods for producing a glycosylated protein in this cell-free system.

What is claimed is: 
     
         1 . A cell-free system for producing a glycosylated protein comprising:
 an isolated oligosaccharyltransferase capable of transferring a glycan from a lipid carrier molecule to a glycoprotein target;   one or more isolated glycans, wherein each glycan is linked to a lipid carrier molecule; and   a glycoprotein target comprising one or more glycan acceptor amino acid residue, or a nucleic acid molecule encoding said glycoprotein target.   
     
     
         2 . The system of  claim 1 , wherein the oligosaccharyltransferase is a prokaryotic oligosaccharyltransferase. 
     
     
         3 . The system of  claim 2 , wherein the prokaryotic oligosaccharyltransferase is derived from  Campylobacter.    
     
     
         4 . The system of  claim 1 , wherein the oligosaccharyltransferase is an archaea oligosaccharyltransferase. 
     
     
         5 . The system of  claim 1 , wherein the oligosaccharyltransferase is a eukaryotic oligosaccharyltransferase. 
     
     
         6 . The system of  claim 1 , wherein the lipid carrier molecule comprises undecaprenyl-phosphate. 
     
     
         7 . The system of  claim 1 , wherein the one or more isolated glycans comprise a prokaryotic glycan. 
     
     
         8 . The system of  claim 1 , wherein the prokaryotic glycan comprises GlcGalNAc 5 Bac. 
     
     
         9 . The system of  claim 1 , wherein the one or more isolated glycans comprise a eukaryotic glycan. 
     
     
         10 . The system of  claim 9 , wherein the eukaryotic glycan comprises GlcNAc 2 . 
     
     
         11 . The system of  claim 10 , wherein the eukaryotic glycan further comprises at least one mannose residue. 
     
     
         12 . The system of  claim 9 , wherein the eukaryotic glycan comprises a composition selected from Man 1 GlcNAc 2 , Man 2 GlcNAc 2 , and Man 3 GlcNAc 2 . 
     
     
         13 . The system of  claim 1 , wherein the one or more glycan acceptor amino acid residues of the glycoprotein target is an asparagine residue. 
     
     
         14 . The system of  claim 13 , wherein glycoprotein target further comprising an N-X 1 -S/T or a D/E-X 1 -N-X 2 -S/T (SEQ ID NO: 1) glycan acceptor amino acid sequence motif wherein D is aspartic acid, X 1  and X 2  are any amino acid other than proline, N is asparagine, and T is threonine. 
     
     
         15 . The system of  claim 1  further comprising:
 reagents suitable for synthesizing the glycoprotein target from said nucleic acid molecule. 
 
     
     
         16 . The system of  claim 1 , wherein the glycoprotein target comprises an antibody. 
     
     
         17 . A kit comprising:
 an isolated oligosaccharyltransferase capable of transferring a glycan from a lipid carrier molecule to a glycoprotein target and   one or more isolated glycans, wherein each glycan is linked to a lipid carrier molecule.   
     
     
         18 . The kit of  claim 17  further comprising:
 reagents suitable for synthesizing a glycoprotein target from a nucleic acid molecule encoding said glycoprotein target. 
 
     
     
         19 . A method for producing a glycosylated protein in a cell-free system comprising:
 providing an isolated oligosaccharyltransferase capable of transferring a glycan from a lipid carrier molecule to a glycoprotein target;   providing one or more isolated glycans, wherein each glycan is linked to a lipid carrier molecule;   providing a glycoprotein target comprising one or more glycan acceptor amino acid residues;   combining the oligosaccharyltransferase, one or more isolated glycans and glycoprotein target to form a cell-free glycosylation reaction mixture; and   subjecting the cell-free glycosylation reaction mixture to conditions effective for the oligosaccharyltransferase to transfer the glycan from the lipid carrier molecule to the one or more glycan acceptor residues of the glycoprotein target to produce a glycosylated protein.   
     
     
         20 . The method of  claim 19 , wherein the oligosaccharyltransferase is a prokaryotic oligosaccharyltransferase. 
     
     
         21 . The method of  claim 20 , wherein the prokaryotic oligosaccharyltransferase is derived from  Campylobacter.    
     
     
         22 . The method of  claim 19 , wherein the oligosaccharyltransferase is an archaea oligosaccharyltransferase. 
     
     
         23 . The method of  claim 19 , wherein the oligosaccharyltransferase is a eukaryotic oligosaccharyltransferase. 
     
     
         24 . The method of  claim 19 , wherein the lipid carrier molecule comprises undecaprenyl phosphate. 
     
     
         25 . The method of  claim 19 , wherein the one or more isolated glycans comprise a prokaryotic glycan. 
     
     
         26 . The method of  claim 25 , wherein the one or more prokaryotic glycans comprise GlcGalNAc 5 Bac. 
     
     
         27 . The method of  claim 19 , wherein one or more isolated glycans comprise a eukaryotic glycan. 
     
     
         28 . The method of  claim 27 , wherein the one or more eukaryotic glycans comprise GlcNAc 2 . 
     
     
         29 . The method of  claim 28 , wherein the one or more eukaryotic glycans further comprise at least one mannose residue. 
     
     
         30 . The method of  claim 28 , wherein the one or more eukaryotic glycans comprise a composition selected from Man 1 GlcNAc 2 , Man 2 GlcNAc 2 , and Man 3 GlcNAc 2 . 
     
     
         31 . The method of  claim 19 , wherein said providing a glycoprotein target comprises providing a nucleic acid molecule encoding the glycoprotein, said method further comprising:
 providing reagents suitable for synthesizing a glycoprotein target from said nucleic acid molecule and   blending the reagents with the glycosylation reaction under conditions effective to synthesize the glycoprotein target from the nucleic acid molecule prior to, or concurrent with, said subjecting.   
     
     
         32 . The method of  claim 19 , wherein the one or more glycan acceptor amino acid residues of the glycoprotein target is an asparagine residue. 
     
     
         33 . The method of  claim 32 , wherein the glycoprotein target further comprising an N-X 1 -S/T or a D/E-X 1 -N-X 2 -S/T (SEQ ID NO: 1) glycan acceptor amino acid sequence motif wherein D is aspartic acid, X 1  and X 2  are any amino acid other than proline, N is asparagine, and T is threonine. 
     
     
         34 . The method of  claim 19 , wherein the protein comprises an antibody.