Granted Patent
B2
US 12,410,456 · App. 17/811,051 · Granted Sep 9, 2025
Cell-free glycoprotein synthesis (CFGPS) in prokaryotic cell lysates enriched with components for glycosylation
Inventors:
Michael Christopher Jewett (Evanston, IL); Jessica Carol Stark (Evanston, IL); Matthew P. DeLisa (Ithaca, NY); Thapakorn Jaroentomeechai (Ithaca, NY)
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Abstract
Disclosed are components and systems for cell-free glycoprotein synthesis (CFGpS). In particular, the components and systems include and utilize prokaryotic cell lysates from engineered prokaryotic cell strains that have been engineered to enable cell-free synthesis of glycoproteins.
Claims (18)
1. A cell-free platform for performing glycoprotein synthesis of a target protein, the platform comprising a cell-free lysate from a genetically modified strain of Escherichia coli ( E. coli ) bacteria comprising:
a heterologous oligosaccharyltransferase (OST) gene which is expressed in the modified strain of E. coli bacteria and which encodes an OST enzyme, wherein the OST enzyme is a component of the cell-free lysate; and wherein the lysate does not comprise the target protein.
2. The cell-free platform of claim 1 , wherein the heterologous OST gene is from Campylobacter jejuni, Campylobacter coli, Campylobacter Zari, Desulfovibrio desulfricans, Desulfovibrio gigas , or Desulfovibrio vulgaris.
3. The cell-free platform of claim 1 , wherein the heterologous OST gene is from Campylobacter jejuni ( C. jejuni ).
4. The cell-free platform of claim 3 , wherein the heterologous OST gene comprises pglB.
5. The cell-free platform of claim 1 , wherein the genetically modified strain of bacteria comprises one or both of (a) an inactivated or deleted gene encoding an enzyme in the glycosyltransferase pathway; (b) an inactivated or deleted gene encoding an enzyme that metabolizes a monosaccharide or polysaccharide that is a substrate in glycosylation.
6. The cell-free platform of claim 5 , wherein the inactivated or deleted gene encoding an enzyme in the glycosyltransferase pathway comprises a mutation in an endogenous waaL gene that inactivates the waaL gene.
7. The cell-free platform of claim 5 , wherein the inactivated or deleted gene encoding an enzyme that metabolizes a monosaccharide or polysaccharide that is a substrate in glycosylation comprises a mutation in an endogenous gmd gene that inactivates the gmd gene.
8. The cell-free platform of claim 5 , wherein the genetically modified strain of bacteria comprises a mutation in an endogenous waaL gene that inactivates the waaL gene and a mutation in an endogenous gmd gene that inactivates the gmd gene.
9. The cell-free platform of claim 1 , wherein the lysate comprises crude cell lysates.
10. The cell-free platform of claim 1 , wherein the cell-free lysate is enriched in lipid-linked oligosaccharides (LLO).
11. The cell-free platform of claim 10 , wherein the cell-free lysate is enriched in (a) Man 3 GlcNAc 2 LLO; or (b) Man 3 GlcNAc 4 Gal 2 Neu 5 Ac 2 LLO.
12. The cell-free platform of claim 1 , wherein the cell-free lysate is enriched in (a) Man 3 GlcNAc 2 ; or (b) Man 3 GlcNAc 4 Gal 2 Neu 5 Ac 2 .
13. The cell-free platform of claim 1 , wherein the cell-free lysate is enriched in glycosyltransferases (GTs).
14. The cell-free platform of claim 1 , comprising bacterial transcription and translation machinery.
15. The cell-free platform of claim 1 , wherein the cell-free lysate has been preserved by freeze-drying or lyophilization.
16. The cell-free platform of claim 10 , wherein the LLOs comprise a truncated form of Man 3 GlcNAc 4 Gal 2 Neu 5 Ac 2 .
17. The cell-free platform of claim 10 , wherein the LLOs comprising GlcNAc as the reducing end sugar.
Assignments (2)
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Jun 22, 2023
From: JEWETT, MICHAEL CHRISTOPHER; STARK, JESSICA CAROL
To: NORTHWESTERN UNIVERSITY
Reel/Frame 064028/0228 →
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Jun 22, 2023
From: DELISA, MATTHEW P.; JAROENTOMEECHAI, THAPAKORN
To: CORNELL UNIVERSITY
Reel/Frame 064028/0443 →
Continuity (4)
Continuation In Part
PCTUS2016069512
· Dec 30, 2016
Provisional Application
62273124
· Dec 30, 2015
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