US 12,188,072 B2
Compositions and methods for rapid in vitro synthesis of bioconjugate vaccines in vitro via production and N-glycosylation of protein carriers in detoxified 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)
Filed by Northwestern University, Evanston, IL (US); and Cornell University, Ithaca, NY (US)
Filed on Dec. 19, 2022, as Appl. No. 18/068,435.
Application 18/068,435 is a continuation of application No. 16/357,820, filed on Mar. 19, 2019, granted, now 11,530,432.
Claims priority of provisional application 62/791,425, filed on Jan. 11, 2019.
Claims priority of provisional application 62/644,811, filed on Mar. 19, 2018.
Prior Publication US 2023/0212633 A1, Jul. 6, 2023
This patent is subject to a terminal disclaimer.
Int. Cl. C12P 21/00 (2006.01); A61K 39/00 (2006.01); A61K 39/02 (2006.01); A61K 39/108 (2006.01); A61K 39/39 (2006.01); C12N 9/10 (2006.01)
CPC C12P 21/005 (2013.01) [A61K 39/00 (2013.01); A61K 39/0208 (2013.01); A61K 39/0258 (2013.01); A61K 39/39 (2013.01); C12N 9/1081 (2013.01); A61K 2039/52 (2013.01); A61K 2039/6037 (2013.01); A61K 2039/6068 (2013.01); A61K 2039/6087 (2013.01); C12Y 204/99 (2013.01)] 15 Claims
 
1. A composition comprising:
a. one or more Escherichia coli (E. coli) cell lysates from engineered E. coli strains, wherein the engineered E. coli strains comprises:
(i) a nucleic acid encoding an orthogonal or heterologous oligosaccharyltransferase (OST) which is expressed in the engineered E. coli strains, wherein the OST is a naturally occurring archaeal OST or wherein the OST is a naturally occurring single-subunit eukaryotic OST;
(ii) one or both of:
(a) a mutation of the endogenous lpxM gene wherein the mutation results in the reduced expression and/or activity of the encoded myristoyltransferase;
(b) a nucleic acid comprising an orthogonal or heterologous LpxE gene and encoding a lipidA 1-phosphatase which is expressed in the engineered E. coli strains;
(iii) a mutation in the endogenous waal gene, wherein the mutation results in the reduced expression and/or activity of the encoded O-antigen ligase;
(iv) prokaryotic transcription and translation machinery; and
b. a lipid-linked oligosaccharide (LLO) comprising a bacterial O-antigen.