Methods for co-activating in vitro non-standard amino acid (nsAA) incorporation and glycosylation in crude cell lysates
AssigneeNorthwestern University
InventorsMichael Christopher Jewett, Jessica Carol Stark, Jasmine Hershewe
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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.
Claims20
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.
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.
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).
Dependencies are inferred from the claim text.