IP Library Granted Patent US 12,624,375
Granted Patent B2
US 12,624,375 · App. 18/343,633 · Granted May 12, 2026

Methods for co-activating in vitro non-standard amino acid (nsAA) incorporation and glycosylation in crude cell lysates

Inventors: Michael Christopher Jewett (Evanston, IL); Jessica Carol Stark (Evanston, IL); Jasmine Hershewe (Evanston, IL)
Assignee: Northwestern University
C12P21/005C12N1/20C12N9/0051C12N9/1051C12N9/16C12N9/88C12N9/93C12Y108/01007C12Y301/21001C12Y402/01047
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Quick Facts
Patent No.
US 12,624,375
App. No.
18/343,633
Granted
May 12, 2026
Kind
B2
Abstract

Disclosed are methods, systems, components, and compositions for cell-free synthesis of proteins and glycoproteins. The methods, systems, components, and compositions may be utilized for incorporating non-standard amino acids (nsAAs) into cell-free synthesized proteins and glycosylating or otherwise modifying the cell-free synthesized proteins in vitro. The nsAAs of the cell-free synthesized protein may be modified via glycosylation or other modification.

Claims (43)

1 . A cell-free glycoprotein synthesis (CFGpS) platform, wherein components of the platform are combined in a single reaction vessel, the platform comprising:

(1) a cell lysate from a genomically recoded strain of Escherichia coli ( E. coli ) comprising:

(a) a mutation in an endogenous peptide chain release factor RF1 (prfA) gene resulting in a deficiency of the encoded release factor 1 protein;

(b) a mutation in an endogenous DNA-specific endonuclease I (endA) gene encoding a DNA-specific endonuclease I protein resulting in a deficiency of the endonuclease I protein;

(c) an orthogonal oligosaccharide transferase (OST), an orthogonal ligase for synthesizing lipid-linked oligosaccharides (LLO), or both of an orthogonal oligosaccharide transferase (OST) and an orthogonal ligase for synthesizing lipid-linked oligosaccharides (LLO);

(2) a DNA template for expressing a sequence-defined amino acid polymer, a DNA-dependent RNA polymerase for transcribing an mRNA encoding the sequence-defined amino acid polymer, nucleotide triphosphates, amino acids, and an energy source; and

(3) a non-standard amino acid (nsAA), an orthogonal amino-acyl tRNA synthetase (aaRS), or both of an nsAA and an aaRS, wherein the non-standard amino acid (nsAA) comprises a moiety that reacts with a corresponding moiety on a saccharide to conjugate the nsAA to the saccharide; and

(4) one or more components for performing a strain-promoted alkyne-azide cycloaddition (SPAAC) reaction.

2 . The platform of claim 1 , wherein the strain is derived from Escherichia coli strain rEc.C321.

3 . The platform of claim 1 , wherein the strain further comprises a mutation in a glutathione reductase (gor) gene encoding a glutathione reductase protein, resulting in a knock-out of the encoded glutathione reductase protein.

4 . The platform of claim 1 , wherein the strain comprises an episomal or genomic vector for expressing an orthogonal oligosaccharide transferase (OST), an orthogonal ligase for synthesizing lipid-linked oligosaccharides (LLO), or both of an orthogonal oligosaccharide transferase (OST) and an orthogonal ligase for synthesizing lipid-linked oligosaccharides (LLO).

5 . The platform of claim 1 , wherein the nsAA is selected from para-azidophenylalanine (pAzF) and para-proparglyoxy-phenylalanine (pAcF).

6 . The platform of claim 5 , wherein the one or more components for performing the strain-promoted alkyne-azide cycloaddition (SPAAC) reaction comprise a dibenzocyclooctyne (DBCO) moiety.

7 . A method for preparing a sequence defined amino acid polymer comprising:

reacting the components of the platform of claim 1 to prepare the sequence defined amino acid polymer.

8 . A cell-free glycoprotein synthesis (CFGpS) platform, wherein the components of the platform are combined in a single reaction vessel, the platform comprising:

(1) a cell lysate from a genomically recoded strain of Escherichia coli ( E. coli ) comprising:

(a) a mutation in an endogenous peptide chain release factor RF1 (prfA) gene resulting in a deficiency of the encoded release factor 1 protein;

(b) a mutation in an endogenous DNA-specific endonuclease I (endA) gene encoding a DNA-specific endonuclease I protein resulting in a deficiency of the endonuclease I protein;

(c) a mutation in an endogenous guanosine diphosphate (GDP)-mannose 4,6-dehydratase (gmd) gene encoding a GDP-mannose 4,6-dehydratase protein resulting in reduced expression and/or activity of GDP-mannose 4,6-dehydratase as compared to wild-type E. coli ; and

(d) an orthogonal oligosaccharide transferase (OST), an orthogonal ligase for synthesizing lipid-linked oligosaccharides (LLO), or both of an orthogonal oligosaccharide transferase (OST) and an orthogonal ligase for synthesizing lipid-linked oligosaccharides (LLO);

(2) a DNA template for expressing a sequence-defined amino acid polymer, a DNA-dependent RNA polymerase for transcribing an mRNA encoding the sequence-defined amino acid polymer, nucleotide triphosphates, amino acids, and an energy source; and

(3) a non-standard amino acid (nsAA), an orthogonal amino-acyl tRNA synthetase (aaRS), or both of an nsAA and an aaRS, wherein the non-standard amino acid (nsAA) comprises a moiety that reacts with a corresponding moiety on a saccharide to conjugate the nsAA to the saccharide; and

(4) one or more components for performing a strain-promoted alkyne-azide cycloaddition (SPAAC) reaction.

9 . The platform of claim 8 , wherein the strain is derived from Escherichia coli strain rEc.C321.

10 . The platform of claim 8 , wherein the strain further comprises a mutation in a glutathione reductase (gor) gene encoding a glutathione reductase protein, resulting in a knock-out of the encoded glutathione reductase protein.

11 . The platform of claim 8 , wherein the strain comprises an episomal or genomic vector for expressing an orthogonal oligosaccharide transferase (OST), an orthogonal ligase for synthesizing lipid-linked oligosaccharides (LLO), or both of an orthogonal oligosaccharide transferase (OST) and an orthogonal ligase for synthesizing lipid-linked oligosaccharides (LLO).

12 . The platform of claim 8 , wherein the nsAA is selected from para-azidophenylalanine (pAzF) and para-proparglyoxy-phenylalanine (pAcF).

13 . The platform of claim 12 , wherein the one or more components for performing the strain-promoted alkyne-azide cycloaddition (SPAAC) reaction comprise a dibenzocyclooctyne (DBCO) moiety.

14 . A method for preparing a sequence defined amino acid polymer comprising:

reacting the components of the platform of claim 8 to prepare the sequence defined amino acid polymer.

15 . A cell-free glycoprotein synthesis (CFGpS) platform, wherein the components of the platform are combined in a single reaction vessel, the platform comprising:

(1) a cell lysate from a genomically recoded strain of Escherichia coli ( E. coli ) comprising:

(a) a mutation in an endogenous peptide chain release factor RF1 (prfA) gene resulting in a deficiency of the encoded release factor 1 protein;

(b) a mutation in an endogenous O-antigen ligase (waaL) gene encoding an O-antigen ligase protein resulting in a deficiency of the O-antigen ligase protein;

(c) a mutation in an endogenous DNA-specific endonuclease I (endA) gene encoding a DNA-specific endonuclease I protein resulting in a deficiency of the endonuclease I protein;

(d) an orthogonal oligosaccharide transferase (OST), an orthogonal ligase for synthesizing lipid-linked oligosaccharides (LLO), or both of an orthogonal oligosaccharide transferase (OST) and an orthogonal ligase for synthesizing lipid-linked oligosaccharides (LLO).

16 . The platform of claim 15 , wherein the strain further comprises a mutation in a glutathione reductase (gor) gene encoding a glutathione reductase protein, resulting in a knock-out of the encoded glutathione reductase protein.

17 . The platform of claim 15 , wherein the strain comprises an episomal or genomic vector for expressing an orthogonal oligosaccharide transferase (OST), an orthogonal ligase for synthesizing lipid-linked oligosaccharides (LLO), or both of an orthogonal oligosaccharide transferase (OST) and an orthogonal ligase for synthesizing lipid-linked oligosaccharides (LLO).

18 . The platform of claim 15 , wherein the nsAA is selected from para-azidophenylalanine (pAzF) and para-proparglyoxy-phenylalanine (pAcF).

19 . The platform of claim 18 , wherein the one or more components for performing the strain-promoted alkyne-azide cycloaddition (SPAAC) reaction comprise a dibenzocyclooctyne (DBCO) moiety.

20 . A method for preparing a sequence defined amino acid polymer comprising:

reacting the components of the platform of claim 15 to prepare the sequence defined amino acid polymer.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jun 30, 2023
From: JEWETT, MICHAEL CHRISTOPHER; STARK, JESSICA CAROL; HERSHEWE, JASMINE M
To: NORTHWESTERN UNIVERSITY
Reel/Frame 064126/0824 →
Continuity (3)
Continuation 17048331
Provisional Application 62658181 · Apr 16, 2018
Related Publication 20240026411A1 · Jan 25, 2024
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