A method for inserting non-natural amino acids and uses thereof

Abstract

The application provides a method for inserting unnatural amino acids and application thereof, the method introduces unnatural amino acids into proteins by using pyrrolysine aminoacyl-tRNA synthetase mutants as orthogonal translation elements to obtain proteins containing unnatural amino acids, and further provides an in vitro cell-free protein synthesis system for inserting unnatural amino acids, the system can efficiently introduce unnatural amino acids, especially lysine analog unnatural amino acids. The technical bottleneck of low recognition efficiency of natural translation system for unnatural amino acids is solved, and an efficient and controllable technical tool is provided for protein function research and biological medicine development.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a method for inserting non-natural amino acids and its application. Background Technology

[0002] Gene code expansion refers to the use of orthogonal tRNA synthetase and its paired tRNA (O-aaRs / O-tRNA) to insert non-natural amino acids (ncaa) into protein sequences at specific codon positions. With the continuous development of orthogonal translation elements, more and more non-natural amino acids with novel chemical properties can be introduced into protein molecules, greatly promoting the research and development of proteins and biological processes.

[0003] Site-directed incorporation of non-natural amino acids is a key tool in protein engineering. Wang et al. reported a modified pyrrolysyl-tRNA synthetase variant, named mpylRs(302I / 346T / 348I / 384L / 417K). )[1] This orthogonal translation system can efficiently express proteins containing fluorosulfonyl-L-tyrosine (FSY) in prokaryotic and eukaryotic cells. However, the FSY side chain groups are small and lack flexibility; its sulfonyl fluoride group can only be covalently linked to nucleophilic amino acid side chains within a radius of approximately 9 Å, limiting its application range.

[0004] To expand the application range of sulfonyl fluoroamino acids, and to screen for orthogonal translation systems targeting the non-natural amino acid fluorosulfonyloxybenzoyl-L-lysine (FSK), which has a longer and more flexible side chain, the Wang laboratory selected *Methanomethylophilus alvus* PylRS (maPylRs) derived from archaea. Compared to mpylRs, maPylRs lack the poorly water-soluble N-terminal domain, thus facilitating heterologous expression. The orthogonal translation system of mapylRs (Y126G / M129A / V168F / H227T / Y228P / L229I) demonstrated that it can achieve efficient site-specific delivery of FSK in both prokaryotic and eukaryotic cells, utilizing proximity reactions to generate intramolecular or intermolecular covalent bonds. Due to the longer side chain of FSK, it can covalently cross-link with the nucleophilic side chains of surrounding amino acids within a radius of 13.8 Å, expanding the targetable range and avoiding the impact of FSK introduction on protein-protein interactions. [2]However, in Wang's experiment, all sulfonyl fluoride amino acids were expressed intracellularly. Since FSY or FSK have poor water solubility, even if the concentration of FSK or FSY used in the culture medium is 1 mM, the actual concentration of non-natural amino acids entering the cell membrane will be lower. This will affect the expression level of the target protein, making it difficult to scale up production for proteins with clinical therapeutic value.

[0005] Cell-free synthesis systems have made significant progress in the insertion of non-natural amino acids in recent years, but still face several challenges. For example, endogenous aminoacyl-tRNA synthetases may still exhibit non-specific recognition of structurally similar UAAs. Some non-natural amino acids, such as those with long side chains, may interfere with ribosome function, leading to protein misfolding. Furthermore, different cell-free systems have varying compatibility with non-natural amino acids. These challenges limit the efficient insertion and application of non-natural amino acids in cell-free systems. Therefore, there is an urgent need in this field to develop a method for efficient, site-specific insertion of non-natural amino acids and a corresponding cell-free synthesis system.

[0006] References:

[0007] [1]Wang, N., et al., Genetically Encoding Fluorosulfate-l-tyrosine ToReact with Lysine, Histidine, and Tyrosine via SuFEx in Proteins in Vivo . J Am Chem Soc. , 2018. 140(15): p. 4995-4999.

[0008] [2]Liu, J., et al., A Genetically Encoded Fluorosulfonyloxybenzoyl-l-lysine for Expansive Covalent Bonding of Proteins via SuFEx Chemistry. J Am Chem Soc. , 2021. 143(27): p. 10341-10351. Summary of the Invention

[0009] The purpose of this invention is to address the shortcomings of existing technologies by providing a method for inserting non-natural amino acids and its application. This involves a method for inserting non-natural amino acids into proteins using a pyrrolidone lysine aminoacyl-tRNA synthetase mutant and a cell-free protein synthesis system containing the mutant. This method can be used for efficient, site-specific insertion of non-natural amino acids, particularly lysine analogs.

[0010] On one hand, the present invention provides a method for inserting non-natural amino acids, using a pyrrolidone aminoacyl-tRNA synthetase mutant as an orthogonal translation element to introduce non-natural amino acids into a protein, thereby obtaining a protein containing non-natural amino acids. The pyrrolidone aminoacyl-tRNA synthetase mutant is an enzyme mutant obtained by mutating the amino acid at position 126 of the sequence shown in SEQ ID NO. 14, wherein the mutation is as follows:

[0011] The tyrosine at position 126 is replaced with any one of alanine, glycine, or serine, and the non-natural amino acid is a lysine analogue, as shown in Table 1.

[0012] More preferably, the pyrrolidone aminoacyl-tRNA synthetase mutant is obtained by protein synthesis and expression of the nucleic acid molecule encoding it.

[0013] More preferably, the pyrrolidone aminoacyl-tRNA synthetase mutant is expressed in a host cell via an expression vector containing the above-mentioned nucleic acid molecule, or is obtained by cell-free in vitro protein synthesis using a nucleic acid construct containing the above-mentioned nucleic acid molecule as a template.

[0014] More preferably, the host cell contains at least one of the above-described expression vectors or at least one site in the genome of the host cell that integrates the above-described nucleic acid molecules, and the host cell is a prokaryotic cell or a eukaryotic cell.

[0015] More preferably, the method uses a nucleic acid construct containing the coding sequence of a protein into which a non-natural amino acid is to be inserted as a template, wherein the coding codon of at least one target amino acid in the coding sequence is replaced with a stop codon, and under suitable conditions, the non-natural amino acid is introduced into the protein in cell-free in vitro protein synthesis using the pyrrolidone aminoacyl-tRNA synthetase mutant to obtain a protein containing the non-natural amino acid.

[0016] Secondly, this invention provides an in vitro cell-free protein synthesis system.

[0017] The reaction system includes:

[0018] (1) Cell extracts;

[0019] (2) Non-natural amino acids;

[0020] (3) Using a nucleic acid construct containing the coding sequence of a protein into which a non-natural amino acid is to be inserted as a template, wherein the coding codon of at least one target amino acid in the coding sequence is replaced with a stop codon.

[0021] (4) An orthogonal tRNA capable of recognizing the stop codon and the corresponding exogenous orthogonal aminoacyl tRNA synthetase, wherein the exogenous orthogonal aminoacyl tRNA synthetase is any of the pyrrolidone aminoacyl-tRNA synthetase mutants described above.

[0022] The exogenous orthogonal aminoacyl-tRNA synthetase is a pyrrolysine aminoacyl-tRNA synthetase mutant, which is obtained by mutating the 126th amino acid in the sequence shown in SEQ ID NO.14. The mutation is as follows:

[0023] Replace the tyrosine at position 126 with any one of alanine, glycine, or serine;

[0024] The non-natural amino acid in question is a lysine analogue.

[0025] Preferably, the cells in the cell extract are selected from one or any combination of bacteria, mammalian cells, human cells, plant cells, yeast cells, and insect cells.

[0026] More preferably, the cells are selected from yeast cells.

[0027] More preferably, the yeast cells are selected from Pichia pastoris (Pichia pastoris). Shepherd's pie Pichia pastoris (Finnish Pichia pastoris) Finnish Pichia ), Pichia pastoris (trehalose) Pichia trehalophila ), Pichia pastoris ( ), Pichia koclamae ), Pichia pastoris () Pichia membranaefaciens ), Pichia pastoris ( Pichiaminata Methanol-induced yeast Ogataea minuta ), Pichia pastoris ( Pichia lindneri Pichia pastoris (cactus yeast) Prickly pear ), heat-resistant Pichia pastoris ( Pichia thermotolerans ), Pichia pastoris ( Willowherb Pichia pastoris () Pichia guercuumum ), Pichia pastoris ( Peach piper Pichia pastoris (Tree trunk yeast) Prickly pear ), Pichia pastoris () Pichia methanolica Pichia pastoris () Peach sp.), brewer's yeast ( Saccharomyces cerevisiae ), brewer's yeast ( Saccharomyces cerevisiae Hansen ), yeast ( Saccharomyces sp.), Hansenula polymorpha ( Hansenula polymorpha ), Candida utilis ( Useful Candida Kluyveromycin ( Kluyveromyces One or a combination thereof.

[0028] More preferably, the cells are selected from Escherichia coli.

[0029] More preferably, the in vitro cell-free protein synthesis system further includes one or more components selected from the following: a substrate for RNA synthesis, a substrate for protein synthesis, polyethylene glycol, magnesium ions, potassium ions, a buffer, RNA polymerase, an energy regeneration system, dithiothreitol (DTT), and optionally water or an aqueous solvent.

[0030] More preferably, its features also include:

[0031] (1) The cell extract has a (v / v) ratio of 20-80% relative to the reaction system;

[0032] (2) It also includes polyethylene glycol, wherein the polyethylene glycol accounts for 0.1-8%, 0.5%-4%, or 1%-2% (w / v) of the reaction system;

[0033] (3) The concentration range of the exogenous orthogonal tRNA synthetase relative to the reaction system is 0.001~1 mmol / L, 0.002~0.1 mmol / L or 0.002~0.01 mmol / L;

[0034] (4) The concentration range of the orthogonal tRNA relative to the reaction system is 0.1~10 mmol / L, 0.5~0.1 mmol / L or 0.5~1 mmol / L;

[0035] (5) The concentration range of the non-natural amino acids relative to the reaction system is 0.1~10 mmol / L, 0.5~5 mmol / L or 0.5~1 mmol / L;

[0036] (6) The mass concentration range of the template of the coding sequence relative to the reaction system is 1~500ng / µl, 10~300ng / µl or 10~50ng / µl.

[0037] The advantages of this invention are:

[0038] 1. This invention provides a method for inserting non-natural amino acids. A pyrrolidone-lysine aminoacyl-tRNA synthetase mutant is used as an orthogonal translation element to introduce non-natural amino acids into proteins, resulting in proteins containing these amino acids. Specifically, the mutant mutates the tyrosine residue at position 126 of the pyrrolidone-lysine-tRNA synthetase (maPylRs) to alanine, glycine, or serine. Experimental verification shows that these mutants significantly improve the recognition efficiency of lysine analogs of non-natural amino acids. In a cell-free system, these mutants exhibit a significantly higher RFP / EGFP fluorescence ratio than the control group, enabling efficient insertion of lysine analogs into non-natural amino acids.

[0039] 2. The present invention also provides an in vitro cell-free protein synthesis system containing a pyrrolidine lysine aminoacyl-tRNA synthetase mutant. This synthesis system can be used to efficiently synthesize proteins containing non-natural amino acids with lysine analogues, thus expanding the application range of non-natural amino acids. Attached Figure Description

[0040] Figure 1 Here is the chemical structure diagram of FSK;

[0041] Figure 2 The 3D structure of maPylRs is shown (with key amino acid residues of the active pocket labeled).

[0042] Figure 3 This is a schematic diagram of random mutations using the megaprimer PCR method.

[0043] Figure 4~11 The sequencing results are for the mutations in Example 1;

[0044] Figure 12 The purification results are for the 12 mutant proteins screened in Example 1;

[0045] Figure 13 This is a comparison of the EGFP fluorescence expression results measured in Example 2;

[0046] Figure 14 This is a comparison of the RFP fluorescence expression results of FSKRS measured in Example 2;

[0047] Figure 15 This is a graph showing the comparison of the absolute fluorescence values ​​of the two fluorescent proteins after overnight in vitro in vitro systems containing FSKRS, as measured in Example 2.

[0048] Figure 16 The ratio of RFP and EGFP fluorescence measured in Example 2 (*: P < 0.05, significant difference; **: P <0.01, highly significant difference; ***:P < 0.001, extremely significant difference; ns: P > 0.05, no significant difference);

[0049] Figure 17 Here is the chemical structure diagram of Prock;

[0050] Figure 18 This is a comparison of EGFP fluorescence expression results of the mutants in Example 3;

[0051] Figure 19 The image shows the comparison results of RFP fluorescence expression measured in Example 3 (*: P < 0.05, significant difference; **: P <0.01, highly significant difference; ***: P < 0.001, extremely significant difference; ns: P > 0.05, no significant difference);

[0052] Figure 20 The graph shows the comparison results of the RFP / EGFP fluorescence ratio measured in Example 3 (*). P < 0.05, significant difference;** P < 0.01, highly significant difference; *** P < 0.001, extremely significant difference; ns: P > 0.05, no significant difference). Detailed Implementation

[0053] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the description of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0054] Unless otherwise specified, the methods used in the embodiments are conventional methods, and the materials and reagents used are commercially available.

[0055] The definitions of terms used herein are intended to incorporate the generally accepted prior art definitions of each term in the field of biotechnology. Examples are provided where appropriate. Unless otherwise limited, either individually or as part of a larger group, these definitions apply to the terminology used throughout this specification.

[0056] In this invention, preferred embodiments such as “preferred,” “better,” “more preferred,” “even better,” “most preferred,” and “further preferred” do not constitute any limitation on the scope of the invention or its protection scope, and are not intended to limit the scope and implementation of the invention, but are only used to provide some embodiments as examples.

[0057] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (including but not limited to embodiments) can be combined with each other to form new or preferred technical solutions, as long as they can be used to implement this invention. Due to space limitations, they will not be described in detail here.

[0058] In vitro protein synthesis refers to the reaction that synthesizes proteins in an in vitro cell-free synthesis system, including at least the translation process. This includes, but is not limited to, IVT (in vitro translation reaction), IVTT (in vitro transcription-translation reaction), and IVDTT (in vitro replication-transcription-translation reaction). In this invention, the IVTT reaction is preferred.

[0059] The IVTT reaction, corresponding to the IVTT system, is a process of transcribing and translating DNA into protein in vitro. Therefore, we also refer to this type of in vitro protein synthesis system as the D2P system, D-to-P system, D_to_P system, or DNA-to-Protein system; the corresponding in vitro protein synthesis methods are also called the D2P method, D-to-P method, D_to_P method, or DNA-to-Protein method.

[0060] D2P, DNA-to-Protein, refers to the process of converting a DNA template into a protein product. Examples include D2P technology, D2P systems, D2P methods, and D2P kits.

[0061] "Cell-free" refers to a method of in vitro protein synthesis that does not rely on the secretion and expression of intact cells. The in vitro protein synthesis system provided by this invention uses an open-ended description of its components. The cell-free protein synthesis system of this invention uses exogenous DNA, mRNA, or a combination thereof as the nucleic acid template for protein synthesis. By artificially controlling the addition of substrates and transcription / translation-related protein factors required for protein synthesis, the in vitro synthesis of the target protein is achieved. It should be noted that the in vitro cell-free protein synthesis system of this invention also allows the addition of cellular components to promote the reaction.

[0062] The terms "expression system of the present invention", "in vitro expression system of the present invention", "in vitro cell-free expression system", and "in vitro cell-free expression system" are used interchangeably and all refer to the in vitro protein expression system of the present invention. Other descriptive methods may also be used, such as: in vitro protein synthesis system, in vitro protein synthesis system, cell-free system, cell-free protein synthesis system, cell-free in vitro protein synthesis system, in vitro cell-free protein synthesis system, in vitro cell-free synthesis system, CFS system (cell-free system), CFPS system (cell-free protein synthesis system), etc.

[0063] In this invention, the in vitro protein synthesis system is also referred to as a protein synthesis factory. The components of the in vitro protein synthesis system provided by this invention are described in an open-ended manner.

[0064] In this invention, "protein" and "protein protein" have the same meaning and are both translated as protein, and can be used interchangeably.

[0065] This invention provides an in vitro cell-free protein synthesis system, which includes at least a cell extract. In this application, "cell extract" refers to a mixture containing cell contents obtained by disrupting cells using physical, chemical, or enzymatic methods. It typically contains metabolic enzymes and cofactors that support gene transcription, protein translation, and energy provision, and may also contain some soluble proteins from the cytoplasm. The terms "cell extract," "cell lysate," "cell lysate," "cell fragments," and "cell lysate" used herein, along with "cell extract," all refer to substances containing the aforementioned components and can be used interchangeably in the in vitro cell-free protein synthesis system. In English, terms such as "cell extract" and "cell lysate" can be used.

[0066] Furthermore, the synthetic system further includes one or more components selected from the group consisting of: a substrate for protein synthesis, a substrate for RNA synthesis, RNA polymerase, magnesium ions, potassium ions, a buffer, an energy regeneration system, polyethylene glycol (PEG) or an analogue thereof, dithiothreitol (DTT), and optionally a solvent, said solvent being water or an aqueous solvent.

[0067] Furthermore, the cell is a eukaryotic cell. The eukaryotic cell is one of mammalian cells, plant cells, yeast cells, insect cells, or any combination thereof. Specifically, the yeast cell is selected from Pichia pastoris and Pichia finnica. Finnish Pichia ), Pichia pastoris (trehalose) Pichia trehalophila), Pichia pastoris ( ), Pichia koclamae ), Pichia pastoris () Pichia membranaefaciens ), Pichia pastoris ( Pichiaminata Methanol-induced yeast Ogataea minuta ), Pichia pastoris ( Pichia lindneri Pichia pastoris (cactus yeast) Prickly pear ), heat-resistant Pichia pastoris ( Pichia thermotolerans ), Pichia pastoris ( Willowherb Pichia pastoris () Pichia guercuumum ), Pichia pastoris ( Peach piper Pichia pastoris (Tree trunk yeast) Pichiasis ), Pichia pastoris () Pichia methanolica Pichia pastoris () Peach sp.), brewer's yeast ( Saccharomyces cerevisiae ), brewer's yeast ( Saccharomyces cerevisiae Hansen ), yeast ( Saccharomyces sp.), Hansenula polymorpha (sp.), Hansenula polymorpha ), Candida utilis ( Useful Candida Kluyveromycin ( Kluyveromyces One or a combination thereof.

[0068] Furthermore, the Kluyveromyces species mentioned include: Kluyveromyces lactis (… Kluyveromyces, K.lactis ), Max Kluyveromycin ( Kluyveromyces marxianus Dobkluyces yeast ( Kluyveromyces dobzhanskii ), Kluyveromycin from sea mud ( Kluyveromyces aestuarii ), non-fermented Kluyveromycin ( Kluyveromyces nonfermentans ), Kluyveromyces wilfordii ( Kluyveromyceswickerhamii ), heat-resistant Kluyveromycin ( Kluyveromyces thermotolerans Kluyveromyces brittle-walled Kluyveromyces fragilis Kluyveromycin (Hubei) Kluyveromyces hubeiensis Kluyveromyces multisporum ( Kluyveromyces polysporus Kluyveromycin (Siamese yeast) Kluyveromyces siamensis ), Kluyveromyces yoloviae ( Kluyveromyces yarrowii One or a combination thereof; preferably, the yeast cells are Kluyveromyces lactis cells.

[0069] More preferably, the cell extract is an aqueous extract of yeast cells.

[0070] More preferably, the cell extract does not contain yeast endogenous long-chain nucleic acid molecules.

[0071] More preferably, the substrate for the synthesized RNA includes one or a combination of nucleoside monophosphate, nucleoside triphosphate, or a combination thereof.

[0072] More preferably, the substrate for the synthesized protein includes 20 natural amino acids and non-natural amino acids.

[0073] More preferably, the magnesium ions are derived from a magnesium ion source selected from the group consisting of magnesium acetate, magnesium glutamate, or a combination thereof.

[0074] More preferably, the potassium ions are derived from a potassium ion source, which is selected from the group consisting of potassium acetate, potassium glutamate, or a combination thereof.

[0075] More preferably, the energy regeneration system is selected from the group consisting of: creatine phosphate / creatine phosphate enzyme system, energy system of intermediate products in glycolysis pathway, sucrose, or a combination thereof.

[0076] More preferably, the buffer is selected from the group consisting of 4-hydroxyethylpiperazine ethanesulfonic acid, tris(hydroxymethyl)aminomethane, or a combination thereof.

[0077] More preferably, the protein synthesis system contains polyethylene glycol (PEG) or an analogue thereof. The concentration of PEG or an analogue thereof is not particularly limited, but typically, the concentration (w / v) of PEG or an analogue thereof is 0.1% to 8%, more preferably 0.5% to 4%, and even more preferably 1% to 2%, based on the total weight of the protein synthesis system. Representative PEGs are selected from the group consisting of PEG3000, PEG3350, PEG6000, PEG8000, or combinations thereof.

[0078] More preferably, the polyethylene glycol includes polyethylene glycol with a molecular weight (Da) of 200-10000, such as PEG200, 400, 1500, 2000, 4000, 6000, 8000, 10000, etc., and more preferably, polyethylene glycol with a molecular weight of 3000-10000.

[0079] More preferably, the optional scheme is that the in vitro cell-free protein synthesis system provided by the present invention includes one, more or all of the following components: yeast cell extract, 4-hydroxyethylpiperazine ethanesulfonic acid, potassium acetate, magnesium acetate, adenine triphosphate (ATP), guanine triphosphate (GTP), cytosine triphosphate (CTP), thymidine triphosphate (TTP), amino acid mixture, creatine phosphate, dithiothreitol (DTT), creatine phosphate kinase, RNA polymerase, polyethylene glycol, and sucrose.

[0080] In this invention, the cell extract does not contain intact cells. Typical cell extracts include ribosomes for protein translation, transfer RNA, aminoacyl-tRNA synthetase, initiation and elongation factors required for protein synthesis, and termination release factors. Furthermore, the cell extract also contains other proteins derived from the cytoplasm of cells, especially soluble proteins.

[0081] The nucleoside triphosphate mixture in the in vitro cell-free protein synthesis system of this invention comprises adenine nucleoside triphosphate, guanine nucleoside triphosphate, cytosine nucleoside triphosphate, and uracil nucleoside triphosphate. In this invention, the concentration of each mononucleotide is not particularly limited; typically, the concentration of each mononucleotide is 0.5-5 mM, preferably 1.0-2.0 mM.

[0082] The amino acid mixture in the in vitro cell-free protein synthesis system of this invention may include natural or non-natural amino acids, and may include D-type or L-type amino acids. Representative amino acids include (but are not limited to) 20 natural amino acids: glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine. The concentration of each amino acid is typically 0.01–0.5 mM, preferably 0.02–0.2 mM, such as 0.05, 0.06, 0.07, or 0.08 mM.

[0083] In a preferred embodiment, the in vitro cell-free protein synthesis system further contains sucrose, the concentration of which is 0.03wt% to 40wt%, more preferably 0.08wt% to 10wt%, and even more preferably 0.1wt% to 5wt% based on the total weight of the protein synthesis system.

[0084] In the in vitro cell-free synthesis method of the present invention, the technical elements such as the in vitro protein synthesis system, template, plasmid, target protein, in vitro protein synthesis reaction (incubation reaction), various preparation methods, and various detection methods can each be independently selected from the following documents for suitable implementation methods or methods, including but not limited to CN111484998A, CN106978349A, CN108690139A, and CN108949801A. References include CN108642076A, CN109022478A, CN109423496A, CN109988801A, CN110093284A, CN110408635A, CN110408636A, CN110551745A, CN110551700A, CN110551785A, CN110819647A, CN110845622A, etc. Unless they conflict with the purpose of this invention, these documents and their cited references are cited in their entirety and for all purposes.

[0085] In this invention, cell extract and cell lysate have the same meaning; both refer to substances obtained after cell lysis. In this embodiment, the cell extract is derived from Kluyveromyces lactis, specifically Kluyveromyces lactis (Lactobacillus lactis). Kluyveromyces lactis , K. lactis However, the same design, analysis, and experimental methods can also be applied to extracts from other cell sources.

[0086] The purpose of this invention is to screen mutant proteins with non-natural amino acid insertion activity, i.e., orthogonal translation elements capable of efficiently inserting non-natural amino acids, using a cell-free expression system and a dual-fluorescent reporter gene expression system. The core technical approach is to integrate a biological orthogonal translation system (OTS) into a cell-free expression system and combine it with a dual-fluorescent reporter gene expression system for screening. This OTS consists of two key functional components: one is a non-canonical amino acid (ncaa) tRNA synthetase mutant (O-aaRs) using non-canonical amino acids (ncaaRSs) as substrates; the other is an orthogonal tRNA (O-tRNA) that specifically recognizes this mutant (ncaaRSs).

[0087] It is noteworthy that O-tRNA possesses strict bioorthogonal properties; it does not bind to naturally occurring aminoacyl-tRNA synthetases within cells, thus preventing it from participating in the natural aminoacylation process as a substrate. In stark contrast, O-aaRs can highly specifically catalyze the aminoacylation reaction between O-tRNA and ncaa. Typically, the anticodon of O-tRNA is precisely modified to achieve complementary binding with the amber stop codon (TAG). This characteristic allows the mRNA signal, which would normally terminate during natural translation, to be re-recognized, leading to the insertion of ncaa into the peptide chain to complete translational elongation. Furthermore, dual-fluorescent reporter gene expression systems can visually reflect the activity of introducing non-natural amino acids into mutant proteins through changes in the reporter gene's fluorescence signal, aiding in the screening of highly efficient orthogonal translation elements. Moreover, these introduced non-natural amino acids carry unique chemically active groups that can form stable covalent bonds with probe molecules or other target molecules through click chemistry, ultimately achieving specific modification of the target protein.

[0088] The non-natural amino acid of this invention is a lysine analogue, which is shown in Table 1 below:

[0089] Table 1

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101] In the examples, fluorosulfonyloxybenzoyl-L-lysine (i.e., FSK, structure shown in Appendix) was selected. Figure 1 ( ) as non-natural amino acids to screen orthogonal translation elements with non-natural amino acid introduction activity.

[0102] The concentrations of each component in the "Protein factory" in this embodiment of the invention are as follows: 22 mM 4-hydroxyethylpiperazine ethanesulfonic acid at pH 7.4, 30-150 mM potassium acetate, 1.0-5.0 mM magnesium acetate, 1.5-4 mM nucleoside triphosphate mixture (adenine nucleoside triphosphate, guanine nucleoside triphosphate, cytosine nucleoside triphosphate, and uracil nucleoside triphosphate), 0.08-0.24 mM amino acid mixture (glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine), 25 mM creatine phosphate, 1.7 mM dithiothreitol, 0.027-0.054 mg / mL T7 RNA polymerase, 0.27 mg / mL creatine phosphate kinase, 1% - 4% polyethylene glycol, 0.5% - 2% sucrose, and finally 50% by volume of Kluyveromyces lactis cell extract.

[0103] The preparation process of the Kluyveromyces lactis cell extract adopted conventional technical methods, referring to the method described in CN109593656A. In summary, the preparation steps include: providing an appropriate amount of fermented Kluyveromyces lactis cells as raw material; flash-freezing the cells with liquid nitrogen; breaking the cells; centrifuging and collecting the supernatant to obtain the cell extract; and the protein concentration in the obtained Kluyveromyces lactis cell extract is 20–40 mg / mL.

[0104] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0105] Example 1: Purification of Mapylrs mutant protein

[0106] 1. Construction of Mapylrs protein mutant library

[0107] Based on the crystal structure characteristics of maPylRs, key sites in the substrate binding pocket were selected for mutation. The amino acid sequence of maPylRs is shown in SEQ ID NO.14 of Sequence Listing 3, and the nucleotide sequence is shown in SEQ ID NO.15.

[0108] Random mutations of the target amino acids were performed using the "megaprimer PCR" method (refer to Tyagi, R., R. Lai, and RG Duggleby, Anew approach to 'megaprimer' polymerase chain reaction mutagenesis without an intermediate gel purification step. BMC Biotechnol, 2004. 4: p. 2.). The primers used are shown in Table 2 below.

[0109] Table 2

[0110]

[0111] Sequencing results for random mutations are shown in Figures 4-11 34 mutations were generated after specific saturation mutations.

[0112] 2. Megaprimer PCR

[0113] First round PCR reaction conditions

[0114] The total volume was 50 μL, including 25 μL of 2X phanta max buffer (Novizan), 50-100 ng of pET28-mapyl RsDNA, 0.2 mM dNTPs, 0.5 U phanta max super-fidelity DNA polymerase, 1.0 pmol of each mutant primer, and 0.05 pmol of reverse-flow primer. PCR reaction conditions were set as follows: denaturation at 95°C for 1 min, annealing at 58°C for 15 sec, and extension at 72°C for 1 min. Five cycles were performed, followed by a final extension at 72°C for 35 min.

[0115] Second round PCR reaction conditions

[0116] Add 1 pmol of forward primer to the same sample tube. PCR reaction conditions: denaturation at 95°C for 1 min, followed by 25 cycles: 95°C for 15 sec; 58°C for 15 sec; 72°C for 2 min. Finally, extend at 72°C for 10 min.

[0117] The vector was amplified using primers vecF / VecR under the same conditions as the second round of PCR described above.

[0118] The amplified products were recovered via gel electrophoresis, and the concentration of the recovered PCR products was determined using nanodrop. The target gene fragment and vector fragment were mixed at equimolar concentrations, and 1 Unit of DpnI (NEB) was added for digestion at 37°C for 3 hours. The ligation products were transformed into Ecoli DH5α chemocompetent cells (Video Biotechnology), and 12 clones were randomly selected for gene sequencing (Sangon Biotech). These plasmids with known sequences were then transformed into BL21 (DE3) strain for expression of mutant proteins. The remaining clones were merged for in vivo bacterial activity screening.

[0119] 3. Expression and purification of mutant proteins

[0120] (1) Select BL21(DE3) bacterial single clones transformed from the above 12 mutant plasmids, inoculate with 100 ml LB (containing 100 mg / L kanamycin), and incubate overnight at 37°C;

[0121] (2) On the second day, the inoculum was inoculated into fresh LB medium (containing 100 mg / L kanamycin) at a ratio of 1:100, and cultured at 37°C until OD600≈0.6. IPTG was added to a final concentration of 0.1 mM, and expression was induced at 16°C for 20 h.

[0122] (3) Collect bacterial cells by centrifugation at 5000 rpm for 20 min at 4℃. Resuspend the bacterial cells in 100 ml of lysis buffer (25 mM Tris-HCl, 500 mM NaCl, 25 mM imidazole, 5 mM β-mercaptoethanol, 1 mM PMSF, 0.1% Triton X-100) using a high-pressure homogenizer.

[0123] (4) Centrifuge twice at 20,000 rpm for 20 min at 4℃, repeating to remove bacterial debris. Use the supernatant to separate and purify the target protein using a HisTrap affinity chromatography column. Buffer A: 25 mM TrisHCl pH 7.8, 500 mM NaCl, 25 mM imidazole; Buffer B: 25 mM TrisHCl pH 7.8, 150 mM NaCl, 500 mM imidazole;

[0124] (5) After the cell lysis supernatant is passed through the affinity chromatography column, the affinity column is repeatedly washed with 10 cv buffer A, and then the target protein is eluted from 0% to 100% gradient with 10 cv buffer B.

[0125] (6) Collect and combine the components containing the target protein, and concentrate the sample using an ultrafiltration centrifuge tube;

[0126] (7) The concentrated sample was dialyzed thoroughly with dialysis buffer (50% glycerol, 25mM Hepes, pH 7.5). After dialysis, the protein concentration was measured to be 20~30mg / L. The sample was then stored at -80℃.

[0127] Each sample in step (7) was treated in the same way as in step (6), concentrated using ultrafiltration centrifuge tubes, and then dialyzed in storage buffer (25 mM Heps pH 7.5, 15 mM MgCl2). The concentration of the 12 mutant proteins was quantified using a Nanodrop micro spectrophotometer, and the final quantified concentration of the mutant proteins was 20~30 mg / L.

[0128] To verify the purity and integrity of the purified products, the 12 selected mutant proteins were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The results are as follows: Figure 12 As shown, all 12 mutant proteins exhibited a single, clear band without any obvious contaminating protein bands, indicating that the purity of the target protein met the experimental requirements and could provide standardized protein samples for subsequent functional experiments.

[0129] Example 2: The cell-free expression system and dual-fluorescent reporter gene expression system were used to verify the delivery activity of non-natural amino acids.

[0130] 1.1 Establishment of an expression system containing non-natural amino acids and dual-fluorescent reporters:

[0131] Protein factory: 100 μl;

[0132] FSK (non-natural amino acids) 500mM: 1 μl (final concentration 1mM);

[0133] matRNA CUA pyl In vitro transcription product (unpurified): 10 μl (final concentration 8 μM);

[0134] EGFP-TAG-RFP dual fluorescent reporter gene PCR product: 3 μl (final concentration 30ng / ul);

[0135] The final concentration of the Mapylrs mutant protein was 5 μM.

[0136] matRNA CUA pyl The in vitro transcription process is as follows:

[0137] 80 Mm Heps pH7.5 + 10 mM DTT + 15 mM GMP + 3.75 mM rNTP + 2 U / ml RNase inhibitor + 0.2% Triton-X + 0.2 mM Speridin + 32 μg / ml T7 RNAP + 275 μg / ml Ppase + 30 mM MgCl2 + 1.2 ng / μl template DNA. The reaction was carried out at 37℃ for 12 h. This in vitro transcription product was not purified.

[0138] The EGFP-TAG-RFP dual fluorescent reporter gene was synthesized by Sangon Biotech. Its protein sequence is shown in SEQ ID NO. 13 of Sequence Listing 3. This protein sequence is the protein into which non-natural amino acids are to be inserted, and TAG is the "recognition site" and "insertion signal" of the non-natural amino acid.

[0139] 1.2 Fluorescence Intensity Detection Method

[0140] Detection was performed using a fluorescent microplate reader:

[0141] Detection of EGFP fluorescence: The excitation wavelength (Ex) was set to 485 nm and the emission wavelength (Em) to 535 nm, and the fluorescence intensity value of each well was measured.

[0142] Detection of RFP fluorescence: Set the excitation wavelength (Ex) to 535 nm and the emission wavelength (Em) to 595 nm, and measure the fluorescence intensity values ​​of the same wells.

[0143] EGFP is an enhanced green fluorescent protein, and RFP is a red fluorescent protein. The fluorescence intensity of RFP and the RFP / EGFP ratio can be used to determine the introduction activity and efficiency of non-natural amino acids. It should be noted that in fluorescence detection, the RFP / EGFP ratio is the ratio of the fluorescence value of RFP to the fluorescence value of EGFP in the same expression system. The fluorescence values ​​measured in the experiment are all relative fluorescence units (RFU).

[0144] 1.3 Confirmation that FSKRS possesses FSK-specific delivery activity

[0145] The expression system containing non-natural amino acids and dual fluorescence reported in 1.1 was used to detect the FSK-specific delivery activity of FSKRS reported in reference [2]. The FSKRS sequence in that reference is mapylRs (containing the Y126G / M129A / V168F / H227T / Y228P / L229I mutation), and the sequence details are shown in SEQ ID NO.16 of sequence listing 3.

[0146] During the detection process, the fluorescence intensity of the system was measured, including the fluorescence intensity after overnight incubation. The fluorescence intensity of EGFP and RFP was detected, and the fluorescence intensity ratio of RFP to EGFP was calculated as the basis for determining FSK delivery activity.

[0147] To ensure the reliability and repeatability of the test results, six replicate samples were set up for each test group. A control group was also set up, which differed from the expression system described in 1.1 in that it did not contain FSK, while all other conditions in the expression system were the same.

[0148] 1.4 Results

[0149] The results are as follows Figure 13-16 As shown, "FSK" indicates an expression system containing FSK, and "non" indicates an expression system that does not contain FSK.

[0150] Figure 13 The results showed that in the cell-free system containing FSKRS, EGFP expression increased continuously over time regardless of the addition of FSK. This indicates that EGFP expression, located upstream of the TAG stop codon in the dual-fluorescent reporter gene, was not affected by non-natural amino acids, demonstrating the stability of the cell-free system.

[0151] Figure 14 The display shows that the expression of RFP after the stop codon only increases with time after the addition of FSK, indicating that it is not dependent on natural amino acids, which also verifies the functional effectiveness of the system.

[0152] Figure 15 The results showed that after overnight incubation, both EGFP and RFP were expressed after the addition of FSK. However, the fluorescence value of EGFP decreased due to the fluorescence resonance energy transfer effect triggered by the expression of RFP.

[0153] Figure 16 The results showed that in the system containing FSK, the fluorescence ratio of RFP to EGFP was significantly higher than that in the system without FSK. P <0.001). This indicates that FSKRS can effectively suppress the termination effect of the TAG stop codon in the presence of FSK, thereby restoring RFP expression, indicating that the FSKRS reported in the literature has the activity of specifically introducing FSK.

[0154] Example 3: Screening for maPylRs mutants with FSK introduction activity

[0155] The fluorescence intensity of the 12 mutant proteins purified in Example 1 (sequences detailed in Sequence Listing 3, SEQ ID NO. 17-28) was detected using the expression system containing non-natural amino acids and dual fluorescence reporting as described in Example 2, 1.1. The detection method was the same as in Example 1, 1.2, to screen mutants with FSK introduction activity. Each mutant was a group, and each group was tested in 6 replicates. The negative control was an expression system without FSK, and two positive controls were set up. One of them was the non-natural amino acid Prock (i.e., N-E-propyneoxycarbonyl-L-lysine hydrochloride, structure shown in Appendix) which has been verified to be successfully and efficiently inserted into this system. Figure 17 The control group differed from the system in 1.1 in that the inserted non-natural amino acid was Prock, and Mapylrs was a wild-type enzyme (the negative control did not contain Prock). Another control group used FSKRS, which had been validated in the aforementioned literature, as a control for orthogonal translation elements. The inserted non-natural amino acid was FSK, and all other conditions were the same as the experimental group.

[0156] It should be noted that, to verify the reliability of the experiment, two groups were set up for the mutant Y126G, with 6 replicates in each group.

[0157] The results are as follows Figure 18-20 As shown, for ease of comparison, the control group and the experimental group are placed in the same results figure, where Prock represents the expression system containing prock and FSKRS represents the expression system containing FSKRS. Figure 18-20 In the experimental group, "non-natural amino acids" refers to the presence of FSK; in the prock expression system, it refers to the presence of Prock; in the FSKRS expression system, it refers to the presence of FSK; and "non" means that it does not contain non-natural amino acids.

[0158] Figure 18 The results show that EGFP can be expressed in all expression systems regardless of whether the system contains non-natural amino acids, which is the same as the results in 1.3 above. EGFP expression is not affected by non-natural amino acids.

[0159] Figure 19 The results showed that in the experimental groups, the RFP expression levels of each expression system containing Y126I, Y126K, L125GW239G, L125GN166GV168S, A223SW239Q, N166AV168A, N166TV168K, N166GV128G, and L125G were not significantly different from those without FSK. P>0.05), indicating that none of the expression systems containing the above 9 mutants could successfully introduce FSK; however, the RFP expression levels of the expression systems containing Y126G, Y126A, and Y126S were significantly higher than those without FSK, indicating that the expression systems containing the above 3 mutants could successfully introduce FSK; the RFP expression levels of the expression systems containing Prock and FSKRS in the positive control group were significantly higher than those without non-natural amino acids, indicating that the expression system containing Prock in the positive control could successfully introduce Prock, and the expression system containing FSKRS could successfully introduce FSK, indicating that the results of the expression systems containing Y126G, Y126A, and Y126S mutants obtained in this study are reliable.

[0160] Figure 20 The results showed that in the experimental groups, the fluorescence ratios (RFP / EGFP) of the expression systems containing Y126I, Y126K, L125GW239G, L125GN166GV168S, A223SW239Q, N166AV168A, N166TV168K, N166GV128G, and L125G were not significantly different from those without FSK. P >0.05), indicating that the expression system containing the above 9 mutants does not have the activity of specifically introducing FSK;

[0161] The fluorescence ratios (RFP / EGFP) of the expression systems containing Y126G (average of two groups), Y126A, and Y126S were 2.37, 2.57, and 2.08, respectively. These ratios were 3.5 times, 3.8 times, and 3.2 times higher than those of the corresponding negative control groups (RFP / EGFP ratios were 0.68, 0.68, and 0.66, respectively). Statistical analysis showed that the values ​​in each group were significantly higher than those in the negative control group. P <0.001), indicating that the expression systems containing the above three mutants had high FSK delivery activity; at the same time, the fluorescence ratios (RFP / EGFP) of the expression systems of Prock and FSKRS in the control group were 2.75 and 2.72, respectively, which were 2.89 times and 4.77 times that of their respective negative controls (RFP / EGFP ratios were 0.95 and 0.57, respectively). Statistical analysis showed that the values ​​of each group were significantly higher than those without non-natural amino acids. P <0.001), indicating that the expression system containing Prock has high activity for Prock introduction and the expression system containing FSKRS has high activity for FSK introduction, which also shows that the introduction activity results of the various expression systems containing Y126G, Y126A and Y126S mutants obtained in this study are reliable.

[0162] The fluorescence ratios (RFP / EGFP) of the expression systems containing Y126G (average of two groups), Y126A, and Y126S mutants were 87.1%, 94.5%, and 76.5% of the RFP / EGFP of the control group FSKRS (containing multiple mutations of Y126G / M129A / V168F / H227T / Y228P / L229I), respectively; and 86.2%, 93.5%, and 75.6% of the RFP / EGFP of the control group prock, respectively. This indicates that the orthogonal translation elements with single-site mutations of the maPylRs mutants Y126G, Y126A, and Y126S of the present invention have comparable FSK introduction efficiency to the multi-site mutants of FSKRS, confirming that the above-mentioned orthogonal translation elements with single-site mutations have excellent non-natural amino acid introduction effects, and that the expression system has good reliability and stability.

[0163] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

[0164] The sequences involved in this invention are summarized in Table 3. It should be noted that there are sequences containing modified and degenerate bases in SEQ ID NO.1-SEQ ID NO.12 in Table 3, which cannot be identified in the separately submitted WIPO ST.26 sequence listing. Therefore, the modified and degenerate bases involved are deleted. The sequences used in the actual experiment are as shown in Table 3.

[0165] Table 3

[0166]

[0167]

[0168]

[0169]

Claims

1. A method for inserting non-natural amino acids, characterized in that, In an in vitro cell-free protein synthesis system, a pyrrolidone aminoacyl-tRNA synthetase mutant was used as an orthogonal translation element to introduce non-natural amino acids into a protein, resulting in a protein containing non-natural amino acids. The pyrrolidone aminoacyl-tRNA synthetase mutant was obtained by mutating the 126th amino acid in the sequence shown in SEQ ID NO.

14. The mutation was as follows: Replace the tyrosine at position 126 with alanine; The non-natural amino acid is FSK.

2. The method according to claim 1, characterized in that, The pyrrolidone aminoacyl-tRNA synthetase mutant was obtained by protein synthesis and expression of the nucleic acid molecule encoding it.

3. The method according to claim 2, characterized in that, The pyrrolidone lysine aminoacyl-tRNA synthetase mutant was subjected to The expression vector containing the nucleic acid molecule of claim 2 is expressed after being introduced into a host cell, or it is obtained by cell-free in vitro protein synthesis using a nucleic acid construct containing the nucleic acid molecule as a template.

4. The method according to claim 3, characterized in that, The host cell contains at least one expression vector as described in claim 3, or the host cell has at least one site in its genome that integrates the nucleic acid molecule as described in claim 2, and the host cell is a prokaryotic cell or a eukaryotic cell.

5. The method according to any one of claims 1 to 4, characterized in that, Using a nucleic acid construct containing the coding sequence of a protein into which a non-natural amino acid is to be inserted as a template, the coding codon of at least one target amino acid in the coding sequence is replaced with a stop codon. Under suitable conditions, the non-natural amino acid is introduced into the protein in cell-free in vitro protein synthesis using the pyrrolidone aminoacyl-tRNA synthetase mutant, resulting in a protein containing the non-natural amino acid.

6. An in vitro cell-free protein synthesis system, characterized in that, The reaction system includes: (1) Cell extracts; (2) Non-natural amino acids; (3) Using a nucleic acid construct containing the coding sequence of a protein into which a non-natural amino acid is to be inserted as a template, wherein the coding codon of at least one target amino acid in the coding sequence is replaced with a stop codon. (4) It can recognize the orthogonal tRNA of the stop codon and the corresponding exogenous orthogonal aminoacyl-tRNA synthetase. The exogenous orthogonal aminoacyl-tRNA synthetase is a pyrrolysine aminoacyl-tRNA synthetase mutant, which is a mutant obtained by mutating the 126th amino acid in the sequence shown in SEQ ID NO.

14. The mutation is as follows: Replace the tyrosine at position 126 with alanine; The non-natural amino acid is FSK.

7. The in vitro cell-free protein synthesis system according to claim 6, characterized in that, The cells used in the cell extract are selected from bacteria, mammalian cells, yeast cells, or any combination thereof.

8. The in vitro cell-free protein synthesis system according to claim 7, characterized in that, The cells were selected from yeast cells.

9. The in vitro cell-free protein synthesis system according to claim 8, characterized in that, The yeast cells mentioned are selected from Pichia pastoris (Pichia pastoris). Pichia pastoris Pichia pastoris (Finnish Pichia pastoris) Pichia finlandica ), Pichia pastoris (trehalose) Pichia trehalophila ), Pichia pastoris ( ), Pichia koclamae ), Pichia pastoris () Pichia membranaefaciens ), Pichia pastoris ( Pichiaminuta Methanol-induced yeast Ogataeaminuta ), Pichia pastoris ( Pichia lindneri Pichia pastoris (cactus yeast) Pichia opuntiae ), heat-resistant Pichia pastoris ( Pichia thermotolerans ), Pichia pastoris ( Pichiasalictaria Pichia pastoris () Pichia guercuumum ), Pichia pastoris ( Pichia pijperi Pichia pastoris (Tree trunk yeast) Pichiastiptis ), Pichia pastoris () Pichia methanolica Pichia pastoris () Pichia sp.), brewer's yeast ( Saccharomyces cerevisiae ), brewer's yeast ( Saccharomyces cerevisiae Hansen ), yeast ( Saccharomyces sp.), Hansenula polymorpha (sp.), Hansenulapolymorpha ), Candida utilis ( Candida utilis Kluyveromycin ( Kluyveromyces One or a combination thereof.

10. The in vitro cell-free protein synthesis system according to claim 7, characterized in that, The cells were selected from Escherichia coli.

11. The in vitro cell-free protein synthesis system according to any one of claims 7 to 10, characterized in that, The in vitro cell-free protein synthesis system further includes one or more components selected from the following: a substrate for RNA synthesis, a substrate for protein synthesis, polyethylene glycol, magnesium ions, potassium ions, a buffer, RNA polymerase, an energy regeneration system, dithiothreitol, and optionally an aqueous solvent.

12. The in vitro cell-free protein synthesis system according to any one of claims 7 to 10, characterized in that, (1) The cell extract has a (v / v) ratio of 20% to 80% relative to the reaction system; (2) It also includes polyethylene glycol, wherein the polyethylene glycol accounts for 0.1% to 8% (w / v) of the reaction system; (3) The concentration range of the exogenous orthogonal tRNA synthetase relative to the reaction system is 0.001~1 mmol / L; (4) The concentration range of the orthogonal tRNA relative to the reaction system is 0.001~1 mmol / L; (5) The concentration range of the non-natural amino acids relative to the reaction system is 0.1~10 mmol / L; (6) The mass concentration of the template of the coding sequence relative to the reaction system is in the range of 1~500 ng / µl.

13. The in vitro cell-free protein synthesis system according to claim 12, characterized in that, The polyethylene glycol is 0.5% to 4% (w / v) relative to the reaction system.

14. The in vitro cell-free protein synthesis system according to claim 13, characterized in that, The polyethylene glycol is 1% to 2% (w / v) relative to the reaction system.

15. In the in vitro cell-free protein synthesis system according to claim 12, the concentration of the exogenous orthogonal tRNA synthetase relative to the reaction system is in the range of 0.002~0.1 mmol / L.

16. In the in vitro cell-free protein synthesis system according to claim 15, the concentration of the exogenous orthogonal tRNA synthetase relative to the reaction system is in the range of 0.002~0.01 mmol / L.

17. The in vitro cell-free protein synthesis system according to claim 12, wherein the concentration of the orthogonal tRNA relative to the reaction system is in the range of 0.005~0.1 mmol / L.

18. The in vitro cell-free protein synthesis system according to claim 17, wherein the concentration of the orthogonal tRNA relative to the reaction system is in the range of 0.005~0.01 mmol / L.

19. The in vitro cell-free protein synthesis system according to claim 12, wherein the concentration of the non-natural amino acid relative to the reaction system is in the range of 0.5~5 mmol / L.

20. The in vitro cell-free protein synthesis system according to claim 19, wherein the concentration of the non-natural amino acid relative to the reaction system is in the range of 0.5~1 mmol / L.

21. The in vitro cell-free protein synthesis system according to claim 12, wherein the mass concentration of the template encoding the sequence relative to the reaction system is in the range of 10~300 ng / µl.

22. The in vitro cell-free protein synthesis system according to claim 21, wherein the mass concentration of the template encoding the sequence relative to the reaction system is in the range of 10~50 ng / µl.

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