Granted Patent
B2
US 12,365,930 · App. 18/065,689 · Granted Jul 22, 2025
Method for rapid in vitro synthesis of glycoproteins via recombinant production of N-glycosylated proteins in prokaryotic cell lysates
Inventors:
Michael Christopher Jewett (Evanston, IL); Jessica Carol Stark (Evanston, IL); Matthew P. DeLisa (Ithaca, NY); Thapakorn Jaroentomeechai (Ithaca, NY)
C12P21/005A61K38/04A61K38/16A61K39/0208A61K39/0258C07K7/06C07K14/47C12N1/205C12N9/10C12N9/1081C12P21/02C12Y204/99019A61K2039/6037A61K2039/6068C07K9/00C07K14/00C07K2319/91C12N1/00C12P19/00C12P21/00C12R2001/00C12R2001/145C12R2001/19Y02A50/30
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Abstract
Disclosed are methods, systems, components, and compositions for cell-free synthesis of glycosylated proteins. The glycosylated proteins may be utilized in vaccines, including anti-bacterial vaccines. The glycosylated proteins may include a bacterial polysaccharide conjugated to a carrier, which may be utilized to generate an immune response in an immunized host against the polysaccharide conjugated to the carrier. The glycosylated proteins may be synthesized in cell-free glycoprotein synthesis (CFGpS) systems using prokaryote cell lysates that are enriched in components for glycoprotein synthesis such as oligosaccharyltransferases (OSTs) and lipid-linked oligosaccharides (LLOs) including OSTs and LLOs associated with synthesis of bacterial O antigens.
Claims (37)
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:
A) providing, in a single reaction vessel, a cell-free transcription, translation, and glycosylation reaction mixture, the cell-free reaction mixture comprising:
(i) a transcription template encoding the carrier protein, 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) an exogenous lipid-linked oligosaccharide (LLO) comprising a bacterial O-antigen;
(iii) an exogenous oligosaccharide transferase (OST) that facilitates transfer of the O-antigen to the consensus sequence on the carrier protein;
(iv) lysate enriched in the LLO of (ii) and the OST of (iii), and comprising endogenous transcription and translation components, wherein the lysate is derived from one or more engineered Escherichia coli ( E. coli ) strains, wherein at least one engineered E. coli strain produces the exogenous OST of (iii), and at least one engineered E. coli strain produces the exogenous LLO of (ii);
B) transcribing and translating the carrier protein in the cell-free reaction mixture;
C) glycosylating the carrier protein in the cell-free reaction mixture with the bacterial O-antigen.
2. The method of claim 1 , wherein the bacterial O-antigen is from Escherichia coli ( E. coli ).
3. The method of claim 1 , wherein the bacterial O-antigen is from Franciscella tularensis.
4. The method of claim 1 , further comprising formulating the N-glycosylated carrier protein as an antigenic composition comprising the N-glycosylated carrier protein.
5. The method of claim 1 , further comprising formulating the N-glycosylated carrier protein a vaccine composition comprising the N-glycosylated carrier protein.
6. The method of claim 1 , wherein the carrier protein is an engineered variant of E. coli maltose binding protein (MBP), wherein the engineered variant comprises the inserted consensus sequence.
7. The method of claim 1 , wherein the carrier protein is selected from a detoxified variant of the toxin from Clostridium tetani , a detoxified variant of the toxin from Corynebacterium diptheriae, Haemophilus influenzae protein D (PD), and a variant of Neisseria meningitidis porin protein (PorA), wherein the variants comprise the inserted consensus sequence.
8. The method of claim 1 , wherein the glycosylating step utilizes an oligosaccharyltransferase (OST) which is a naturally occurring bacterial homolog of Campylobacter jejuni ( C. jejuni ) PglB.
9. The method of claim 1 , wherein the glycosylating step utilizes an OST that is an engineered variant of C. jejuni PglB.
10. The method of claim 1 , wherein the glycosylating step utilizes an OST that is a naturally occurring archaeal OST.
11. The method of claim 1 , wherein the glycosylating step utilizes an OST which is a naturally occurring single-subunit eukaryotic OST.
12. The method of claim 1 , wherein the one or more engineered E. coli strains comprises an inactivating modification or deletion in an endogenous waal gene.
13. The method of claim 1 , wherein the cell lysate has an endotoxin unit (EU) concentration of less than about 180,000 EU/ml.
14. A kit for synthesizing a N-glycosylated carrier protein in vitro, the kit comprising as components:
(i) one or more cell lysates, wherein the one or more cell lysates are enriched in an orthogonal oligosaccharyltransferase (OST) and an O-antigen;
(ii) a transcription template encoding a carrier protein, the carrier protein comprising an inserted consensus sequence, N-X-S/T, wherein X may be any natural or unnatural amino acid except proline.
15. The kit of claim 14 comprising one cell lysate prepared from a single source strain, wherein the orthogonal OST and the O-antigen are present in the one cell lysate.
16. The kit of claim 14 comprising two cell lysates prepared from two source strains, wherein the orthogonal OST is present in one cell lysate of the two cell lysates and the O-antigen is present in the other cell lysate of the two cell lysates.
17. The kit of claim 14 , wherein OST is produced from a source strain that overexpresses a gene encoding the orthogonal OST.
18. The kit of claim 14 , wherein the O-antigen is produced from a source strain that overexpresses a synthetic glycosyltransferase pathway for producing the O-antigen.
19. A cell-free reaction mixture comprising an N-glycosylated carrier protein, wherein the N-glycosylated carrier protein is synthesized via a method comprising coordinated transcription, translation, and N-glycosylation in vitro, the method comprising:
A) providing, in a single reaction vessel, a cell-free transcription, translation, and glycosylation reaction mixture, the cell-free reaction mixture comprising:
(i) a transcription template encoding the carrier protein, 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) an exogenous lipid-linked oligosaccharide (LLO) comprising a bacterial O-antigen;
(iii) an exogenous oligosaccharide transferase (OST) that facilitates transfer of the O-antigen to the consensus sequence on the carrier protein;
(iv) lysate enriched in the LLO of (ii) and the OST of (iii), and comprising endogenous transcription and translation components, wherein the lysate is derived from one or more engineered Escherichia coli ( E. coli ) strains, wherein at least one engineered E. coli strain produces the exogenous LLO of (ii), and at least one engineered E. coli strain produces the exogenous OST of (iii), wherein the one or more engineered E. coli strains comprise an inactivating modification or deletion in an endogenous waaL gene;
B) transcribing and translating the carrier protein in the cell-free reaction mixture;
C) glycosylating the carrier protein in the cell-free reaction mixture the bacterial O-antigen.
20. A method of vaccinating a subject in need thereof against a bacterial O-antigen, the method comprising administering to the subject the cell-free reaction mixture of claim 19 and/or a purified N-glycosylated carrier protein therefrom.
21. The method of claim 5 , wherein the vaccine composition further comprises an adjuvant.
Assignments (2)
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Nov 28, 2023
From: DELISA, MATTHEW; JAROENTOMEECHAI, THAPAKORN
To: CORNELL UNIVERSITY
Reel/Frame 065687/0825 →
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Nov 28, 2023
From: JEWETT, MICHAEL CHRISTOPHER; STARK, JESSICA CAROL
To: NORTHWESTERN UNIVERSITY
Reel/Frame 065696/0724 →
Continuity (4)
Provisional Application
62362327
· Jul 14, 2016
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