Methods for co-activating in vitro non-standard amino acid (nsAA) incorporation and glycosylation in crude cell lysates
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.
1. A cell-free glycoprotein synthesis (CFGpS) reaction mixture in a single reaction vessel comprising:
(1) a cell lysate from a genomically recoded strain of Escherichia coli ( E. coli ) comprising:
(a) a mutation in an endogenous (prfA) gene resulting in a deficiency 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) a mutation in an endogenous 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
(e) 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 cell-free glycoprotein synthesis (CFGpS) reaction mixture of claim 1 , wherein the strain is derived from Escherichia coli strain rEc.C321.
3. The cell-free glycoprotein synthesis (CFGpS) reaction mixture 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 cell-free glycoprotein synthesis (CFGpS) reaction mixture of claim 1 , wherein the strain comprises a 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 cell-free glycoprotein synthesis (CFGpS) reaction mixture of claim 1 comprising a non-standard amino acid (nsAA), wherein the nsAA is selected from para-azidophenylalanine (pAzF) and para-propargyloxy-phenylalanine (pAcF).
6. The cell-free glycoprotein synthesis (CFGpS) reaction mixture of claim 5 , wherein the one or more components for performing the SPAAC reaction comprise a dibenzocyclooctyne (DBCO) moiety.
7. A method for preparing a sequence defined amino acid polymer comprising: reacting the cell-free glycoprotein synthesis (CFGpS) reaction mixture of claim 1 to prepare the sequence defined amino acid polymer.
8. The method of claim 7 , wherein the CFGpS reaction mixture comprises the non-standard amino acid (nsAA) selected from para-azido-phenylalanine (pAzF) and para-propargyloxy-phenylalanine (pAcF).
9. The method of claim 8 , wherein the one or more components for performing the strain-promoted alkyne-azide cycloaddition (SPAAC) reaction comprise a dibenzocyclooctyne (DBCO) moiety for performing the SPAAC reaction with the nsAA.