US-12624375-B2 - Methods for co-activating in vitro non-standard amino acid (nsAA) incorporation and glycosylation in crude cell lysates

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.

Inventors

• Michael Christopher Jewett
• Jessica Carol Stark
• Jasmine Hershewe

Assignees

• NORTHWESTERN UNIVERSITY

Dates

Publication Date

20260512

Application Date

20230628

Claims (20)

1. 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. 2 . The platform of claim 1 , wherein the strain is derived from Escherichia coli strain rEc.C321.
3. 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. 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. 5 . The platform of claim 1 , wherein the nsAA is selected from para-azidophenylalanine (pAzF) and para-proparglyoxy-phenylalanine (pAcF).
6. 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. 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. 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. 9 . The platform of claim 8 , wherein the strain is derived from Escherichia coli strain rEc.C321.
10. 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. 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. 12 . The platform of claim 8 , wherein the nsAA is selected from para-azidophenylalanine (pAzF) and para-proparglyoxy-phenylalanine (pAcF).
13. 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. 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. 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. 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. 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. 18 . The platform of claim 15 , wherein the nsAA is selected from para-azidophenylalanine (pAzF) and para-proparglyoxy-phenylalanine (pAcF).
19. 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. 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.

Description

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS This application is a continuation of U.S. application Ser. No. 17/048,331, filed Oct. 16, 2020, which is the U.S. national stage entry of PCT/US2019/027733 filed Apr. 16, 2019, which claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62/658,181, filed Apr. 16, 2018. The content of each of the aforementioned applications is incorporated herein by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under MCB1413563 awarded by the National Science Foundation. The government has certain rights in the invention. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING The contents of the electronic sequence listing (702581_02369.xml; Size: 19,800 bytes; and Date of Creation: Jun. 28, 2023) is herein incorporated by reference in its entirety. BACKGROUND The field of the invention relates to in vitro synthesis of proteins. In particular, the field of the invention relates to one-pot systems for incorporating non-standard amino acids and glycosylation into cell-free synthesized proteins. Cell-free protein synthesis (CFPS) using extracts from prokaryotic source strains such as E. coli has undergone a transformational shift from an exploratory platform used in the discovery of the genetic code to a present-day, high-yielding protein production platform [1]. This shift is fueled by the open nature of this system, allowing for rapid combination, supplementation, and optimization of the physiochemical environment for increasing protein yields and batch reaction duration [2, 3]. Now, cell-free systems are seen as a complement to in vivo protein expression and can be used as both a prototyping platform due to its simplicity, easiness, and modular design for protein expression [4-6] as well as a large-scale production platform for difficult to express proteins in vivo [7]. The transition from exploratory platform to high-yielding protein production platform has come about, at least in part, by complex strain engineering to stabilize biological substrates in the cell-free reaction mixtures [8, 9]. These genetic modifications targeted the deletion of proteins known to affect the stability of DNA [10], mRNA [8, 11], protein [12], energy [13], and amino acids [14, 15] in the cell-free reaction. In addition to strain engineering efforts, activation of multiple biological pathways [16], decreases in cost [17], and improved understanding of reaction contents makes CFPS an attractive platform for the production of new kinds of high-value proteins. One area of great interest for the application of cell-free systems is the production of modified proteins containing non-standard amino acids. Incorporating non-standard amino acids or unnatural amino acids (nsAAs) allows for the production of proteins with novel structures and functions that are difficult or impossible to create using the 20 canonical amino acids [18, 19]. Recently, cell-free protein synthesis (CFPS) systems have been employed to increase yields of proteins bearing nsAAs [20, 21], achieve direct protein-protein conjugation [22], explore drug discovery [23], and enhance enzyme activity [24, 25]. Typically, nsAA incorporation systems use amber suppression technology to insert nsAAs into proteins, a method by which an in-frame amber (TAG) stop codon is utilized as a sense codon for assigning nsAAs [26, 27]. Amber suppression technology, however, has limited efficiency for nsAA incorporation because of the presence of release factor 1 (RF1). RF1 naturally binds the amber stop codon (TAG) [28] and prematurely terminates protein translation. Methods to counteract this competitive termination of the TAG stop codon include increasing the addition of competing tRNA [21], tagging and purifying out RFT [29], release factor engineering [30], and genomically recoding strains to remove RF1 and reassigning all occurrences to the synonymous TAA codon [31]. High-yield protein production with multiple-site incorporation of NSAAs still remains a critical challenge. Glycosylation is possible in some CFPS systems. The development of a highly active E. coli CFPS platform has prompted recent efforts to enable glycoprotein production in E. coli lysates through the addition of orthogonal glycosylation components. In one study, Guarino and DeLisa demonstrated the ability to produce glycoproteins in E. coli CFPS by adding purified lipid-linked oligosaccharides (LLOs) and the C. jejuni OST to a CFPS reaction. Yields of between 50-100 μg/mL of AcrA, a C. jejuni glycoprotein, were achieved [64]. Despite these recent advances, bacterial cell-free glycosylation systems have been limited by their inability to co-activate efficient protein synthesis and glycosylation. We recently developed a cell-free glycoprotein synthesis (CFGpS) system that addresses this limitation by enabling modular, coordinated transcription, translation, and N-glycosy