US 12,404,533 B2
Bioconjugate vaccines' synthesis in prokaryotic cell lysates
Michael Christopher Jewett, Evanston, IL (US); Jessica Carol Stark, Evanston, IL (US); Matthew P. DeLisa, Ithaca, NY (US); and Thapakorn Jaroentomeechai, Ithaca, NY (US)
Assigned to Northwestern University, Evanston, IL (US); and Cornell University, Ithaca, NY (US)
Appl. No. 17/310,022
Filed by Northwestern University, Evanston, IL (US); and Cornell University, Ithaca, NY (US)
PCT Filed Jan. 10, 2020, PCT No. PCT/US2020/013207
§ 371(c)(1), (2) Date Jul. 12, 2021,
PCT Pub. No. WO2020/146814, PCT Pub. Date Jul. 16, 2020.
Claims priority of provisional application 62/791,425, filed on Jan. 11, 2019.
Prior Publication US 2022/0267821 A1, Aug. 25, 2022
This patent is subject to a terminal disclaimer.
Int. Cl. C12P 21/00 (2006.01); A61K 39/00 (2006.01); A61K 39/39 (2006.01)
CPC C12P 21/005 (2013.01) [A61K 39/00 (2013.01); A61K 39/39 (2013.01)] 23 Claims
 
1. A method for synthesizing an N-glycosylated carrier protein for a polysaccharide via coordinated transcription, translation, and N-glycosylation in vitro, the method comprising:
(i) transcribing and translating a carrier protein in a cell-free protein synthesis reaction, the carrier protein comprising an inserted consensus sequence, N—X—S/T, wherein X may be any natural or unnatural amino acid except proline;
(ii) glycosylating the carrier protein in the cell-free protein synthesis reaction with at least one polysaccharide, wherein the at least one polysaccharide is at least one bacterial O-antigen;
wherein the cell-free protein synthesis reaction comprises:
(i) lysate derived from one or more genetically modified Escherichia coli (E. coli) strains, wherein the genetically modified E. coli strains (a) are deficient in lpxM gene product, (b) comprise an orthogonal or heterologous LpxE gene that is expressed in the engineered E. coli strains and is present in the lysate, and (c) comprise an inactivating modification in the waaL gene;
(ii) transcription and translation machinery;
(iii) at least one bacterial O-antigen as part of lipid-linked oligosaccharides (LLOs);
(iii) an oligosaccharide transferase which recognizes the inserted consensus sequence;
(iv) a transcription template encoding the carrier protein.