Method for rapid in vitro synthesis of glycoproteins via recombinant production of N-glycosylated proteins in prokaryotic cell lysates

A method for synthesizing an N-glycosylated carrier protein for a bacterial O-antigen via coordinated transcription, translation, and N-glycosylation in vitro in a single reaction vessel comprising a reaction mixture prepared by adding an aqueous solution to one or more freeze-dried Escherichia coli cell lysates,

• wherein the one or more freeze-dried Escherichia coli cell lysates comprise:
• (a) an exogenous bacterial oligosaccharyltransferase (OST);
• and
• (b) an exogenous lipid-linked oligosaccharide (LLO) comprising the bacterial O-antigen;
• the method comprising:
• (i) transcribing and translating a carrier protein in a cell-free protein synthesis reaction in the single reaction vessel, the carrier protein comprising an inserted consensus sequence, N-X-S/T, wherein X may be any natural or unnatural amino acid except proline;
• and
• (ii) N-glycosylating the carrier protein at the inserted consensus sequence with the bacterial O-antigen using the exogenous bacterial OST in a cell-free glycosylation reaction in the single reaction vessel.

A method for synthesizing an N-glycosylated carrier protein for a bacterial O-antigen via coordinated transcription, translation, and N-glycosylation in vitro in a single reaction vessel comprising one or more Escherichia coli cell lysates,

• wherein the one or more Escherichia coli cell lysates comprise:
• (a) an exogenous bacterial oligosaccharyltransferase (OST) that is a naturally occurring bacterial homolog of C. jejuni PglB or an engineered recombinant protein of C. jejuni PglB;
• and
• (b) an exogenous lipid-linked oligosaccharide (LLO) comprising the bacterial O-antigen;
• the method comprising:
• (i) transcribing and translating a carrier protein in a cell-free protein synthesis reaction in the single reaction vessel, the carrier protein comprising an inserted consensus sequence, N-X-S/T, wherein X may be any natural or unnatural amino acid except proline;
• and
• (ii) N-glycosylating the carrier protein at the inserted consensus sequence with the bacterial O-antigen using the exogenous bacterial OST in a cell-free glycosylation reaction in the single reaction vessel.