Biosynthetic system that produces immunogenic polysaccharides in prokaryotic cells
The invention is directed to bioconjugate vaccines comprising N-glycosylated proteins. Further, the present invention is directed to a recombinant prokaryotic biosynthetic system comprising nucleic acids encoding an epimerase that synthesizes an oligo- or polysaccharide having N-acetylgalactosamine at the reducing terminus. The invention is further directed to N-glycosylated proteins containing an oligo- or polysaccharide having N-acetylgalactosamine at the reducing terminus and an expression system and methods for producing such N-glycosylated proteins.
1. A recombinant prokaryotic biosynthetic system comprising:
(i) a nucleic acid encoding an epimerase that synthesizes N-acetylgalactosamine on undecaprenyl pyrophosphate, wherein said epimerase comprises the amino acid sequence of SEQ ID NO. 2;
(ii) a nucleic acid encoding a glycosyltransferase that participates in the assembly of an oligo- or polysaccharide on a lipid carrier;
(iii) a nucleic acid encoding an oligosaccharyl transferase; and
(iv) a nucleic acid encoding a protein comprising one or more of an introduced consensus sequence, D/E-X-N-Z-S/T (SEQ ID NO:31), wherein X and Z can be any natural amino acid except proline.
2. The recombinant prokaryotic biosynthetic system of claim 1 , wherein said oligosaccharyl transferase is from Campylobacter jejuni.
3. The recombinant prokaryotic biosynthetic system of claim 1 , wherein said protein is P. aeruginosa exoprotein.
4. The recombinant prokaryotic biosynthetic system of claim 1 , wherein said oligo- or polysaccharide is from a Gram-negative bacterium.
5. The recombinant prokaryotic biosynthetic system of claim 4 , wherein said oligo- or polysaccharide is from Shigella flexneri.
6. The recombinant prokaryotic biosynthetic system of claim 5 , wherein said oligo- or polysaccharide is from Shigella flexneri 6.
7. The recombinant prokaryotic biosynthetic system of claim 1 , wherein said oligo- or polysaccharide comprises a structure:
8. The recombinant prokaryotic biosynthetic system of claim 4 , wherein said oligo- or polysaccharide is from E. coli.
9. The recombinant prokaryotic biosynthetic system of claim 8 , wherein said oligo- or polysaccharide is from E. coli 0157.
10. The recombinant prokaryotic biosynthetic system of claim 1 , wherein said oligo- or polysaccharide comprises a structure, α-D-PerNAc-α-L-Fuc-β-D-Glc-α-D-GalNAc.
11. A method of producing an N-linked glycosylated protein, comprising:
a.) introducing into a host organism:
i.) a nucleic acid encoding an epimerase that synthesizes N-acetylgalactosamine on undecaprenyl pyrophosphate, wherein said epimerase comprises the amino acid sequence of SEQ ID NO. 2;
ii.) a nucleic acid encoding a glycosyltransferase that participates in the assembly of an oligo- or polysaccharide on a lipid carrier;
iii.) a nucleic acid encoding an oligosaccharyl transferase; and
iv.) a nucleic acid encoding a protein comprising one or more of an introduced consensus sequence, D/E-X-N-Z-S/T (SEQ ID NO:31), wherein X and Z can be any natural amino acid except proline; and
b.) culturing said host organism until at least one N-glycosylated protein is produced.
12. The method of claim 11 , wherein said oligosaccharyl transferase is from Campylobacter jejuni.
13. The method of claim 11 , wherein said protein is P. aeruginosa exoprotein.
14. The method of claim 11 , wherein said oligo- or polysaccharide is from a Gram-negative bacterium.
15. The method of claim 14 , wherein said oligo- or polysaccharide is from Shigella flexneri.
16. The method of claim 15 , wherein said oligo- or polysaccharide is from Shigella flexneri 6.
17. The method of claim 11 , wherein said oligo- or polysaccharide comprises a structure:
18. The method of claim 14 , wherein said oligo- or polysaccharide is from E. coli.
19. The method of claim 18 , wherein said oligo- or polysaccharide is from E. coli 0157.
20. The method of claim 11 , wherein said oligo- or polysaccharide comprises a structure, α-D-PerNAc-α-L-Fuc-β-D-Glc-α-D-GalNAc.