Novel polysaccharide and uses thereof

A recombinant prokaryotic host cell for producing an N-glycosylated carrier protein, comprising:

• a. a nucleotide sequence encoding:
• i. dTDP-Glucose 4,6-dehydratase;
• ii. dTDP-6-Deoxy-D-glucose 3,5-epimerase;
• iii. Glucose-1-phosphate thymidylyltransferase;
• iv. dTDP-4-dehydrorhamnose 3,5-epimerase;
• v. O antigen flippase;
• vi. dTDP-Rha:Glc-Rha(Ac)-GlcNAc-UPP α-1,3-rhamnosyltransferase;
• vii. UDP-Glc:Glc-Rha(Ac)-GlcNAc-UPP α-1,6-glucosyltransferase;
• viii. O antigen polymerase;
• ix. O-acetyl transferase;
• x. UDP-Glc:Rha-GIcNAc-UPP α-1,3-glucosyltransferase;
• and
• xi. dTDP-Rha:
• GlcNAc-UPP α-1,3-rhamnosyltransferase;
• b. a nucleotide sequence encoding an oligosaccharyl transferase;
• and
• c. a nucleotide sequence encoding a carrier protein comprising a consensus sequence Asp(Glu)-X-Asn-Z-Ser(Thr), wherein X and Z are independently selected from any natural amino acid except Pro (SEQ ID NO:15).

A method of making an N-glycosylated carrier protein that comprises an E. coli O25B antigen, the method comprising:

• a. culturing a recombinant prokaryotic host cell under conditions for producing the N-glycosylated carrier protein, wherein the prokaryotic host cell comprises:
• i. a nucleotide sequence comprising a rfb gene cluster encoding the E. coli O25B antigen;
• ii. a nucleotide sequence encoding an oligosaccharyl transferase;
• and
• iii. a nucleotide sequence encoding a carrier protein comprising a consensus sequence Asn-X-Ser(Thr), wherein X is any amino acid except Pro (SEQ ID NO:14);
• and
• b. purifying the N-glycosylated carrier protein that comprises the E. coli O25B antigen.