In vitro cell-free protein synthesis system and kit containing exogenous magnesium ions and applications thereof

Abstract

The application provides an in-vitro cell-free protein synthesis system and kit containing exogenous magnesium ions and application thereof, including a D2P system (DNA-to-Protein system) and an mR2P system (mRNA-to-Protein system), and belongs to the technical field of protein synthesis. The in-vitro cell-free protein synthesis system containing exogenous magnesium ions adopts magnesium aspartate as a novel magnesium ion source, and especially in a eukaryotic cell-free system, compared with a traditional magnesium ion source, can significantly improve the protein synthesis efficiency and protein expression amount. A more efficient and higher-throughput in-vitro protein synthesis kit and a synthesis method of exogenous proteins are also provided, and the kit has the advantages of simplicity, convenience and low cost.

Description

Technical Field

[0001] This invention relates to the field of protein synthesis technology, and particularly to the field of in vitro cell-free protein synthesis technology. Specifically, it relates to an in vitro cell-free protein synthesis system containing exogenous magnesium ions, its kit, and its applications. More specifically, it relates to an in vitro cell-free protein synthesis system containing exogenous magnesium ions, including a D2P system (DNA-to-Protein system) and an mR2P system (mRNA-to-Protein system), an in vitro protein synthesis kit, and its applications. Background Technology

[0002] Proteins are essential molecules in cells, participating in almost all cellular functions. Protein synthesis mainly includes traditional intracellular synthesis techniques and next-generation in vitro synthesis techniques. Traditional protein expression systems refer to molecular biology techniques that express exogenous genes using model organisms such as bacteria, fungi, plant cells, or animal cells. In vitro protein synthesis systems, also known as cell-free expression systems, emerged in the 1960s. They use exogenous mRNA or DNA as templates for protein synthesis and achieve the synthesis of exogenous proteins by artificially controlling the addition of substrates, energy, and transcription and / or translation-related protein factors required for protein synthesis. In vitro protein synthesis systems generally refer to the rapid and efficient translation of exogenous proteins by adding nucleic acid templates (mRNA or DNA templates), RNA polymerase, amino acids, ATP, and other components to a lysed system of bacterial, fungal, plant, or animal cells. In vitro protein synthesis systems eliminate the need for plasmid construction, transformation, cell culture, cell collection, and disruption, offering a relatively rapid, time-saving, and convenient method for protein expression. This is a crucial tool in the field of protein research (“Garcia RA, Riley MR. Applied biochemistry and biotechnology. Humana Press. 1981, 263-264”; “Fromm HJ, Hargrove M. Essentials of Biochemistry. 2012”; CN109988801A; “Assenberg R, Wan PT, Geisse S, Mayr LM. Advances in recombinant protein expression for use in pharmaceutical research. Current Opinion in Structural Biology. 2013, 23(3):393-402”; “Anne Zemella, Lena Thoring, Christian Hoffmeister and Stefan Kubick. Cell-free protein synthesis: pros and cons of prokaryotic and eukaryotic…”). Systems. Chembiochem. 2015, 16:2420-2431”). In vitro protein synthesis systems can also express cellularly toxic or non-natural amino acids (such as…). DSpecialized proteins (containing amino acids) can be synthesized simultaneously and in parallel, facilitating high-throughput drug screening and proteomics research (Spirin AS, Swartz JR. Chapter 1. Cell-Free Protein Synthesis Systems: Historical Landmarks, Classification, and General Methods. Wiley-VCH Verlag GmbH & Co. KGA, 2008: 1-34.). Protein products produced using in vitro synthesis systems can be widely used in pharmaceuticals, food, nutritional products, dietary supplements, cosmetics, and other fields, including but not limited to the applicant's PROTEINN. TM Proton TM Princeton TM Protein products from brands such as [brand name missing].

[0003] Protein synthesis capacity is one of the key indicators determining whether an in vitro protein synthesis system can be industrialized, mainly including synthesis efficiency and protein synthesis amount (protein expression level). To improve protein synthesis yield, many optimizations and modifications are made to the system in terms of cell extracts, energy systems, genetic templates (nucleic acid templates), reactors, and operational methods (Zhang Xu. Key Technologies and Industrial Applications of High-Efficiency Synthesis of Complex Membrane Proteins in Cell-Free Systems [D]. Zhejiang University, 2014), and various additives are also explored. Among these, components that have beneficial effects on intracellular protein synthesis are often the first choice for research. However, due to the significant differences between intracellular synthetic biological systems and in vitro synthetic microenvironments, the effects cannot be simply predicted and require extensive experimental screening and verification. When the cells used to prepare cell extracts are derived from different bacterial species, the differences between the species can sometimes lead to unpredictable technical effects, especially when the cell source for preparing cell extracts changes between prokaryotic and eukaryotic systems, and the in vitro protein synthesis mechanism strongly depends on the inherent properties of the original bacterial species (such as unique organelles, unique biological factor components, unique metabolic mechanisms, etc.). Significant unpredictability is well known to those skilled in the art.

[0004] Inorganic salt ions are commonly used additives in in vitro protein synthesis systems, including magnesium and potassium ions. Magnesium ions play a crucial role in protein translation, promoting ribosome assembly and improving RNA stability. Furthermore, magnesium ions also promote polymerase binding. Common compounds used as magnesium ion sources include magnesium acetate, magnesium chloride, and magnesium glutamate. References include WO2016005982A1, US20060211083A1, and "L Kai, V RKaldenhoff and F Bernhard.Artificial environments for the co-translationalstabilization of cell-free expressed proteins[J].PloS one,2013,8(2):e56637" etc.

[0005] With the successful interpretation of a large amount of biological genetic information, how to achieve more efficient and higher throughput protein synthesis in in vitro systems is an urgent need in the field of protein synthesis. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention discloses a simple, convenient, more efficient, and higher-throughput in vitro cell-free protein synthesis system containing exogenous magnesium ions. This system is preferably based on eukaryotic cell extracts and uses magnesium aspartate as a novel magnesium ion source. Compared with traditional magnesium ion sources, it can improve reaction efficiency, significantly enhance protein synthesis capacity, and increase protein expression levels by more than 220% (more than three times).

[0007] 1. The first aspect of the present invention provides an in vitro cell-free protein synthesis system containing exogenous magnesium ions, wherein the "in vitro cell-free protein synthesis system containing exogenous magnesium ions" is also abbreviated as "CFPS(Mg+) system" in the present invention. The CFPS(Mg+) system comprises the following components: eukaryotic cell extract and exogenous magnesium ions; wherein the exogenous magnesium ions are derived from one or more donor sources, including at least magnesium aspartate. The CFPS(Mg+) system is capable of performing an in vitro protein synthesis reaction with a nucleic acid template encoding an exogenous protein to synthesize the exogenous protein.

[0008] The magnesium aspartate can be selected from: L-aspartate magnesium, D-aspartate magnesium, and combinations thereof; the magnesium aspartate is preferably L-aspartate magnesium.

[0009] Preferably, in a magnesium aspartate molecule, the ratio of magnesium atoms to aspartic acid residues is 1:1 or 1:2.

[0010] The exogenous magnesium ions may also optionally be derived from the group consisting of: magnesium gluconate, magnesium acetate, magnesium glutamate (L-magnesium glutamate, D-magnesium glutamate or a combination thereof), magnesium chloride, magnesium phosphate, magnesium sulfate, magnesium citrate, magnesium hydrogen phosphate, magnesium iodide, magnesium lactate, magnesium nitrate, magnesium oxalate, and combinations thereof.

[0011] In one preferred embodiment, the source of the exogenous magnesium ions includes magnesium aspartate (preferably L-aspartate magnesium), and also includes any one of magnesium gluconate, magnesium glutamate (preferably L-glutamate magnesium), and magnesium acetate.

[0012] In one preferred embodiment, the source of the exogenous magnesium ions includes magnesium aspartate (preferably L-aspartate magnesium), and also includes any two of magnesium gluconate, magnesium glutamate (preferably L-glutamate magnesium), and magnesium acetate, or further includes all three magnesium sources.

[0013] Preferably, the source of the exogenous magnesium ions is a combination of magnesium gluconate, magnesium aspartate, and magnesium glutamate. More preferably, the source of the exogenous magnesium ions is a combination of magnesium gluconate, magnesium L-aspartate, and magnesium L-glutamate.

[0014] In one preferred embodiment, the molar percentage of exogenous magnesium ions provided by the aspartic acid relative to the total exogenous magnesium ions is selected from any one of the following percentage values, or a range between any two of the following percentage values: 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, 100%; the range includes both endpoints. More preferably, the aspartic acid is L-aspartic acid magnesium.

[0015] In one preferred manner, at least 25 mol% of the exogenous magnesium ions are derived from magnesium aspartate.

[0016] In one preferred manner, at least 30 mol% of the exogenous magnesium ions are derived from magnesium aspartate.

[0017] In one preferred manner, at least 40 mol% of the exogenous magnesium ions are derived from magnesium aspartate.

[0018] In one preferred manner, at least 50 mol% of the exogenous magnesium ions are derived from magnesium aspartate.

[0019] In one preferred manner, at least 80 mol% of the exogenous magnesium ions are derived from magnesium aspartate.

[0020] In one preferred manner, at least 90 mol% of the exogenous magnesium ions are derived from magnesium aspartate.

[0021] In one preferred manner, 100 mol% of the exogenous magnesium ions are derived from magnesium aspartate.

[0022] In one preferred manner, 100 mol% of the exogenous magnesium ions are derived from L-aspartic magnesium.

[0023] The "mol% of exogenous magnesium ions" ignores the difference between free and chelated states and is calculated only based on the molar amount of exogenously added magnesium. For example, if 1 mol of magnesium chloride (completely ionized, with all magnesium ions in the free state) and 1 mol of L-aspartic magnesium (partially ionized, with some magnesium ions in the free state and some in the chelated state) are added exogenously, the exogenous magnesium ions provided by L-aspartic magnesium are calculated as 50 mol%.

[0024] In some preferred embodiments, the concentration of exogenous magnesium ions provided by the magnesium aspartate is selected from any of the following concentrations, or a concentration range between any two of the following concentration values ​​(the concentration range includes both endpoints): 0.1 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 5.5 mM, 6 mM, 6.5 mM, 7 mM M, 7.5mM, 8mM, 8.5mM, 9mM, 9.5mM, 10mM, 11mM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM, 18m M, 19mM, 20mM, 22mM, 24mM, 25mM, 28mM, 30mM, 35mM, 40mM, 45mM, 50mM, 60mM, 70mM, 80mM.

[0025] In one preferred embodiment, the concentration of exogenous magnesium ions provided by the magnesium aspartate is selected from 0.1 to 50 mM; another preferred embodiment is 0.5 to 20 mM; yet another preferred embodiment is 1 to 10 mM.

[0026] The amount of magnesium aspartate that can increase the synthesis of exogenous proteins is selected from Y. PRT (C Asp The range of magnesium aspartate dosage when the expression level of exogenous protein is greater than Y0 in the curve. The above optional dosage range can be continuous or discontinuous.

[0027] In this invention,

[0028] Q Asp This refers to the amount of magnesium aspartate used in this invention to increase the synthesis of exogenous proteins.

[0029] Unless otherwise specified, dosage generally refers to the amount of raw material added, which can be characterized by concentration, mass, amount of substance (or molar amount), etc.

[0030] C Asp The amount of magnesium aspartate used is indicated by concentration. In this invention, the amount of magnesium aspartate is preferably characterized by concentration.

[0031] Y PRT , refers to the expression level of exogenous proteins.

[0032] YPRT (C Asp The curve refers to the curve with the amount of magnesium aspartate as the independent variable, the expression level of the exogenous protein as the dependent variable, and other reaction parameters all fixed. In this invention, it is also denoted as Y. PRT ~C Asp Curve. The "other reaction parameters" include, but are not limited to: other system components, the method of adding reactants, the reaction temperature program, the reaction time, the properties of the reaction vessel, the volume of the reaction system, etc. A single formulation of the CFPS(Mg+) system can correspond to multiple Y... PRT ~C Asp Curves. A set of CFPS(Mg+) systems can react at different temperature programs and for different durations, thereby producing several different Y... PRT ~C Asp curve.

[0033] Y max , refers to Y PRT (C Asp The highest expression level of exogenous protein in the curve.

[0034] C max , refers to Y PRT (C Asp The amount of magnesium aspartate used when the exogenous protein reached its highest expression level in the curve.

[0035] Y min , refers to Y PRT (C Asp In the concentration curve, Y PRT The lowest expression level of exogenous protein within the range of >Y0.

[0036] Y0 refers to the C Asp The expression level of exogenous protein when it is 0.

[0037] Y Δ , for Y max The difference between Y0 and Y0, numerically, Y Δ =Y max -Y0.

[0038] Preferably, the Q Asp Selected from exogenous proteins with an expression level of at least Y0 + 50% Y Δ The dosage range of magnesium aspartate at that time;

[0039] More preferably, the Q Asp Selected from exogenous proteins with expression levels of at least Y0 + 60% Y Δ The dosage range of magnesium aspartate at that time;

[0040] More preferably, the Q AspSelected from exogenous proteins with an expression level of at least Y0 + 70% Y Δ The dosage range of magnesium aspartate at that time;

[0041] More preferably, the Q Asp Selected from exogenous proteins with an expression level of at least Y0 + 80% Y Δ The dosage range of magnesium aspartate at that time;

[0042] More preferably, the Q Asp Selected from exogenous proteins with an expression level of at least Y0 + 90% Y Δ The dosage range of magnesium aspartate at that time;

[0043] More preferably, the Q Asp Selected from exogenous proteins with an expression level of at least Y0 + 95% Y Δ The dosage range of magnesium aspartate at that time;

[0044] More preferably, the Q Asp The amount of magnesium aspartate used when the exogenous protein is at its highest expression level (C) max .

[0045] One preferred method is to use a concentration curve with magnesium aspartate concentration as the independent variable, where the magnesium aspartate concentration is selected from exogenous protein expression levels greater than or equal to Y. max The concentration range of Y at 50% max The highest expression level of the exogenous protein in the concentration curve; more preferably, the expression level of the exogenous protein is greater than or equal to Y. max The concentration range at 60% of the target value; more preferably, the expression level of the exogenous protein is greater than or equal to Y. max The concentration range at 70% of the target value; more preferably, the expression level of the exogenous protein is greater than or equal to Y. max The concentration range at 80% of the target value; more preferably, the expression level of the exogenous protein is greater than or equal to Y. max The concentration range. One preferred method is to determine the expression level of the exogenous protein as Y. max The concentration value at that time.

[0046] The CFPS(Mg+) system, together with the nucleic acid template encoding the foreign protein, can provide the translation-related elements required for the synthesis of the foreign protein, thereby enabling the expression of the foreign protein through an in vitro protein synthesis reaction. Preferably, the CFPS(Mg+) system contains system components capable of recognizing promoter elements in the nucleic acid template, allowing the CFPS(Mg+) system to recognize the promoter elements in the nucleic acid template encoding the foreign protein; that is, the nucleic acid template encoding the foreign protein contains promoter elements that can be recognized by the CFPS(Mg+) system. For example, the CFPS(Mg+) system contains an RNA polymerase corresponding to the promoter elements in the nucleic acid template.

[0047] System components capable of recognizing promoter elements in nucleic acid templates (such as the corresponding RNA polymerase) can be provided by eukaryotic cell extracts, other exogenous components, or a combination of both.

[0048] The CFPS(Mg+) system can synthesize foreign proteins through in vitro protein synthesis reactions by reacting with any one of the following nucleic acid templates: DNA template encoding foreign proteins, mRNA template encoding foreign proteins, or a combination thereof.

[0049] The nucleic acid template encoding the exogenous protein is preferably a DNA template.

[0050] Preferably, the transcription process of the exogenous protein is initiated by the T7 promoter on the nucleic acid template.

[0051] In one preferred embodiment, the eukaryotic cell extract of the present invention is from any of the following sources: yeast cells, mammalian cells, plant cells, insect cells, or a combination thereof.

[0052] The yeast cells are preferably Kluyveromyces, Saccharomyces cerevisiae, Pichia pastoris, or a combination thereof.

[0053] The Kluyveromyces is further preferably Kluyveromyces lactis, Kluyveromyces marx, Kluyveromyces dob, Kluyveromyces salina, Kluyveromyces wickheim, Kluyveromyces brittlewall, Kluyveromyces hubeiensis, Kluyveromyces multisporum, Kluyveromyces siamensis, Kluyveromyces yarbromentosus, or a combination thereof.

[0054] The eukaryotic cell extract is more preferably derived from any of the following sources: Kluyveromyces lactis, wheat germ cells, Spodoptera frugiperda cells (fall armyworm cells, an insect cell), rabbit reticulocytes, Chinese hamster ovary cells (CHO cells), African green monkey kidney COS cells, African green monkey kidney VERO cells, young hamster kidney cells (BHK cells), human fibrosarcoma HT1080 cells, or any combination thereof.

[0055] Preferably, the CFPS(Mg+) system includes RNA polymerase. The sources of the RNA polymerase include, but are not limited to: cell extracts containing endogenously expressed RNA polymerase, exogenous RNA polymerase, translation products of exogenous nucleic acid templates encoding RNA polymerase, and combinations thereof. Each of the above technical solutions is independently preferred, wherein the RNA polymerase is T7 RNA polymerase. The exogenous nucleic acid template encoding the RNA polymerase can be translated into RNA polymerase through an in vitro protein synthesis reaction with the CFPS(Mg+) system.

[0056] In one preferred embodiment, the eukaryotic cell extract contains endogenously expressed RNA polymerase; more preferably, the eukaryotic cell extract contains endogenously expressed T7 RNA polymerase.

[0057] In one preferred embodiment, the CFPS(Mg+) system includes a DNA polymerase. The source of the DNA polymerase includes, but is not limited to: cell extracts containing endogenously expressed DNA polymerase, exogenous DNA polymerase, translation products of exogenous nucleic acid templates encoding DNA polymerase, and combinations thereof. Each of the above technical solutions is independently preferred, wherein the DNA polymerase is phi29 DNA polymerase. The exogenous nucleic acid template encoding the DNA polymerase can be translated into DNA polymerase through an in vitro protein synthesis reaction with the CFPS(Mg+) system.

[0058] The RNA polymerase and DNA polymerase can be added directly from an exogenous source, or provided as reaction products or intermediates (e.g., by adding an exogenous nucleic acid template encoding the RNA polymerase or / and the DNA polymerase).

[0059] Typically, the transcription of foreign protein genes is initiated by a promoter on the nucleic acid template. One preferred approach is that the transcription of foreign protein genes is initiated by the T7 promoter on the nucleic acid template.

[0060] In one preferred embodiment, the CFPS(Mg+) system includes a eukaryotic cell extract containing an endogenously expressed RNA polymerase capable of recognizing a promoter in a nucleic acid template that initiates a gene transcription program for a foreign protein.

[0061] To obtain eukaryotic cell extracts containing endogenously expressed RNA polymerase, one preferred method is to genetically modify the source strain of the eukaryotic cell extract through the following methods (endogenous strain modification): inserting the coding sequence / coding gene of RNA polymerase into an intracellular free plasmid, integrating the coding gene of RNA polymerase into the cell genome, or a combination of the above two methods. It should be noted that, in performing the above-mentioned endogenous strain modification, in addition to integrating the aforementioned coding sequence / coding gene, other nucleotide sequences are also allowed to be inserted, such as non-coding sequences, enhancer sequences, Kozak sequences, leader sequences or leader peptide sequences, signal peptide sequences, tag sequences, codon sequences, etc. Through the above-mentioned endogenous strain modification, the modified strain can endogenously express RNA polymerase. The RNA polymerase is preferably T7 RNA polymerase.

[0062] In one preferred embodiment, the CFPS(Mg+) system includes exogenously added T7 RNA polymerase.

[0063] In one preferred embodiment, the CFPS(Mg+) system comprises at least one of the following components: exogenous RNA polymerase, exogenous nucleic acid template encoding RNA polymerase, exogenous DNA polymerase, and exogenous nucleic acid template encoding DNA polymerase.

[0064] In one preferred embodiment, the CFPS(Mg+) system includes exogenous RNA polymerase and exogenous DNA polymerase.

[0065] In one preferred embodiment, the CFPS(Mg+) system includes exogenous T7 RNA polymerase and exogenous phi29 DNA polymerase.

[0066] In one preferred embodiment, the CFPS(Mg+) system includes an energy system; the energy system is preferably selected from the following: a sugar (e.g., monosaccharide, disaccharide, oligosaccharide, polysaccharide) and phosphate energy system; a sugar and phosphocreatine energy system; a phosphocreatine and phosphocreatine enzyme system; a phosphocreatine and phosphocreatine kinase system; a glycolysis pathway and its intermediate product energy system (e.g., a monosaccharide and its glycolysis intermediate product energy system, glycogen and its glycolysis intermediate product energy system); and combinations thereof.

[0067] In one preferred embodiment, the CFPS(Mg+) system includes a substrate for synthesizing proteins; the substrate for synthesizing proteins is preferably a mixture of amino acids, including at least the mixture of amino acids required for synthesizing exogenous proteins. Preferably, the amino acid mixture is a mixture of natural amino acids.

[0068] In one preferred embodiment, the CFPS(Mg+) system includes a substrate for synthesizing RNA; the substrate for synthesizing RNA is preferably a mixture of nucleotides, more preferably selected from: nucleoside monophosphates, nucleoside triphosphates, or combinations thereof; more preferably, the substrate for synthesizing RNA is a mixture of nucleoside triphosphates.

[0069] In one preferred embodiment, the CFPS(Mg+) system includes a substrate for synthesizing DNA; the substrate for synthesizing DNA is preferably a mixture of deoxynucleotides, more preferably a mixture of deoxynucleotide triphosphates.

[0070] In one preferred embodiment, the CFPS(Mg+) system further includes at least one of the following exogenous additives: other soluble amino acid salts, translation-related elements, DNA amplification-related elements, RNA amplification-related elements, RNase inhibitors, crowding agents, potassium ions, antioxidants or reducing agents, antifreeze agents, trehalose, reaction promoters, defoamers, alkanes, buffers, and aqueous solvents.

[0071] The other soluble amino acid salts exclude magnesium aspartate, which is already included. The cations in the other soluble amino acid salts may include, but are not limited to, magnesium ions, potassium ions, sodium ions, zinc ions, calcium ions, ammonium ions, etc., and the amino acid residues may be selected from any amino acid (natural, non-natural, derivatives, etc.) and combinations thereof described in this invention.

[0072] The translation-related elements are preferably selected from: tRNA, ribosomes, other translation-related enzymes, initiation factors, elongation factors, termination factors, and combinations thereof. The translation-related elements are preferably purified translation-related elements.

[0073] The crowding agent is preferably selected from: polyethylene glycol, polyvinyl alcohol, polystyrene, dextran, sucrose polymers (including Ficoll sucrose polymers, such as...). The reagent is a nonionic synthetic sucrose polymer; it also includes polysucrose, polyvinylpyrrolidone (PVP), albumin, etc., and combinations thereof.

[0074] The potassium ion source is preferably selected from: potassium acetate, potassium glutamate, potassium chloride, potassium phosphate, potassium sulfate, potassium citrate, potassium hydrogen phosphate, potassium iodide, potassium lactate, potassium nitrate, potassium oxalate, or combinations thereof.

[0075] The antioxidant or reducing agent is preferably selected from: dithiothreitol, 2-mercaptoethanesulfonic acid, 2-mercaptoethanol, reduced glutathione, tricarboxymethyl phosphate, 3-mercapto-1,2-propanediol, and combinations thereof.

[0076] The antifreeze agent may include, but is not limited to, trehalose.

[0077] Trehalose can be used as an antifreeze agent and as a component of energy systems.

[0078] The reaction promoter is preferably an aluminum salt, an aluminum oxide (such as aluminum oxide), an iron salt, an iron oxide, a calcium salt, or a combination thereof.

[0079] The alkane is preferably C6-C6. 44 A pure or mixed alkane; the alkane is further preferably cyclohexane, isooctane, decane, tetradecane, pentadecylcyclohexane, squalane, tetradecane, petrolatum, or a combination thereof.

[0080] The buffer is preferably selected from: Tris-HCl, Tris base, HEPES, and combinations thereof.

[0081] The aqueous solvent is preferably a buffer.

[0082] Any component of the CFPS(Mg+) system involved in this invention is allowed to perform two or more functions.

[0083] The above-mentioned preferred methods can be combined in any suitable manner.

[0084] 2. A second aspect of the present invention provides an in vitro protein synthesis kit, the kit comprising:

[0085] (i) The in vitro cell-free protein synthesis system containing exogenous magnesium ions described in the first aspect (CFPS(Mg+) system);

[0086] (ii) Optionally includes a nucleic acid template encoding a foreign protein;

[0087] (iii) Labels or instructions.

[0088] The CFPS(Mg+) system, together with the nucleic acid template encoding the foreign protein, can provide the translation-related elements required for the synthesis of the foreign protein.

[0089] Preferably, the components of the CFPS(Mg+) system are placed in one or more containers in the form of solids, semi-solids (such as pastes), liquids, emulsions (also known as emulsions), suspensions, or combinations thereof.

[0090] Preferably, (i) contains individually packaged cell extracts.

[0091] The aforementioned in vitro protein synthesis kit can be used to perform in vitro protein synthesis reactions and synthesize exogenous proteins.

[0092] 3. A third aspect of the present invention provides a method for synthesizing exogenous proteins, the method comprising the following steps:

[0093] (i) Provide the in vitro cell-free protein synthesis system (CFPS(Mg+) system) containing exogenous magnesium ions as described in the first aspect of the present invention;

[0094] (ii) Add a nucleic acid template encoding the foreign protein, perform an incubation reaction, and synthesize the foreign protein;

[0095] The CFPS(Mg+) system can work with the nucleic acid template encoding the foreign protein to provide the translation-related elements required for the synthesis of the foreign protein;

[0096] Optionally, step (iii) may also be included: isolating and / or detecting the exogenous protein.

[0097] In the second and third aspects, each independently includes, but is not limited to, the following preferred methods:

[0098] (1) In one preferred manner, the nucleic acid template encoding the exogenous protein contains a promoter element that can be recognized by the CFPS(Mg+) system.

[0099] (2) In one preferred embodiment, the CFPS(Mg+) system includes a eukaryotic cell extract, and the nucleic acid template encoding the exogenous protein contains a promoter element that the eukaryotic cell extract can recognize. For example, the eukaryotic cell extract contains an endogenously expressed RNA polymerase corresponding to the promoter element on the nucleic acid template.

[0100] (3) In one preferred embodiment, the nucleic acid template encoding the exogenous protein contains a T7 promoter and the CFPS(Mg+) system contains a T7 RNA polymerase.

[0101] (4) In one preferred embodiment, the nucleic acid template encoding the exogenous protein contains a T7 promoter, the CFPS(Mg+) system includes a eukaryotic cell extract, and the eukaryotic cell extract contains an endogenously expressed T7 RNA polymerase.

[0102] (5) Preferably, the nucleic acid template encoding the foreign protein contains a T7 promoter that can initiate the gene transcription program of the foreign protein, that is, the gene transcription process of the foreign protein is initiated by the T7 promoter on the nucleic acid template.

[0103] (6) In one preferred embodiment, the nucleic acid template encoding the exogenous protein contains a T7 promoter capable of initiating the gene transcription program of the exogenous protein, and the CFPS(Mg+) system includes T7 RNA polymerase.

[0104] (7) In one preferred embodiment, the nucleic acid template encoding the exogenous protein contains a T7 promoter capable of initiating the gene transcription program of the exogenous protein (the T7 promoter is located upstream of the coding sequence of the exogenous protein in the nucleic acid template, and the gene transcription program of the exogenous protein is initiated by the T7 promoter), and the CFPS(Mg+) system includes a eukaryotic cell extract, which contains an endogenously expressed T7 RNA polymerase.

[0105] In the second and third aspects, the nucleic acid template encoding the foreign protein is a DNA template, an mRNA template, or a combination thereof; the nucleic acid template encoding the foreign protein is preferably a DNA template.

[0106] 4. The fourth aspect of this invention provides the application of the in vitro cell-free protein synthesis system (CFPS(Mg+) system) containing exogenous magnesium ions described in the first aspect, applied to protein synthesis. This application to protein synthesis includes, but is not limited to, applications in protein manufacturing or protein synthesis-based detection.

[0107] 5. The fifth aspect of the present invention provides the use of magnesium aspartate in the in vitro cell-free protein synthesis system containing exogenous magnesium ions as described in the first aspect, or in the in vitro protein synthesis kit as described in the second aspect, or in the method for synthesizing exogenous proteins as described in the third aspect.

[0108] Beneficial effects:

[0109] This invention optimizes in vitro cell-free protein synthesis systems, particularly eukaryotic-based systems. It provides a novel in vitro cell-free protein synthesis system containing exogenous magnesium ions, employing magnesium aspartate as a novel magnesium ion source. Compared to traditional magnesium ion sources (especially commonly used magnesium acetate and magnesium glutamate), this significantly improves protein synthesis efficiency and yield. Furthermore, it provides a more efficient and higher-throughput in vitro cell-free protein synthesis kit (especially the D2P kit) and a method for synthesizing exogenous proteins, while also offering the advantages of simplicity, convenience, and low cost. In addition, this invention can utilize eukaryotic cell extracts, providing superior post-translational modification mechanisms for synthesizing complex functional proteins containing post-translational modifications (such as disulfide bonds and glycosylation), thus having broad application potential.

[0110] In this invention, magnesium aspartate is used as a novel magnesium ion source. Compared with magnesium glutamate, it significantly improves the protein synthesis efficiency and protein expression level of in vitro cell-free protein synthesis systems based on eukaryotic systems (especially yeast systems, and more specifically Kluyveromyces system), even by more than three times. According to existing reports, no in vitro protein synthesis system uses magnesium aspartate as an exogenous magnesium ion source. In the in vitro protein synthesis system of this invention, it was unexpectedly found that using magnesium aspartate as an exogenous magnesium ion source better improves the protein synthesis capacity of the in vitro synthesis system compared to magnesium acetate and magnesium glutamate. We speculate that this may be because during the reaction, there is a competitive binding interaction between nucleotides and the exogenous magnesium ion source on metallic magnesium ions (i.e., the interaction between nucleotides and metallic magnesium ions is competitive with the interaction between exogenous magnesium ions and magnesium ions). With the dynamic changes in the concentration of nucleotides and nucleic acids, the magnesium ion concentration in the system is in an unstable state, thus affecting the protein synthesis efficiency and protein expression level. Compared with traditional magnesium sources (magnesium acetate, magnesium glutamate), magnesium aspartate has a strong binding force on magnesium ions (SAA Sajadi. Metal ion-binding properties of L-glutamic acid and L-aspartic acid, a comparative investigation[J]. Natural Science, 2010, 2(2): 85-90), which can provide a more stable magnesium ion concentration for the system, thereby increasing the protein synthesis capacity of the system.

[0111] When in vitro protein synthesis is primarily regulated by common properties among different bacterial species, the corresponding technical methods can achieve effects unaffected by species differences and are universally applicable across these species. The novel magnesium ion source provided in this invention is not only applicable to the optimization of in vitro protein synthesis systems using Kluyveromyces lactis cell extracts, but also to the optimization of in vitro protein synthesis systems for other yeast systems and other eukaryotic systems. Attached Figure Description

[0112] Figure 1A schematic diagram of the plasmid DNA encoding the exogenous protein mEGFP, totaling 6056 bp, denoted as plasmid D2P-mEGFP (abbreviated as pD2P-mEGFP). The mEGFP is a mutant of enhanced green fluorescent protein. This plasmid DNA includes the following elements: a T7 promoter (recognized by T7 RNA polymerase), a 5' untranslated region, a leader sequence (not shown), a purification tag (optional element, not shown), the coding sequence for the exogenous protein mEGFP, a 3' untranslated region, a LAC4 terminator, a replication origin site (f1ori), an AmpR promoter, an ampicillin resistance gene (AmpR gene), a high copy number replication origin site (ori), a gene controlling plasmid copy number (rop gene, located downstream of ori, not shown in the diagram), the coding gene for the lac repressor (lacI), and the lacI promoter.

[0113] Figure 2 The effects of different magnesium ion sources on the in vitro cell-free protein synthesis system were tested (reaction time: 3 h). Exogenous magnesium ions were provided by 0–9 mM magnesium L-aspartate (Mg-Asp), 0–9 mM magnesium L-glutamate (Mg-Glu), and 0–9 mM magnesium acetate (Mg-Ac). 0 mM corresponds to the blank control, with no exogenous magnesium ions added. Potassium acetate (80 mM) and potassium phosphate (24 mM) were used.

[0114] Figure 3 The effects of different magnesium ion sources on the in vitro cell-free protein synthesis system were tested (reaction time: 23 h). Exogenous magnesium ions were provided by 0–9 mM magnesium L-aspartate (Mg-Asp), 0–9 mM magnesium L-glutamate (Mg-Glu), and 0–9 mM magnesium acetate (Mg-Ac). 0 mM corresponds to the blank control, with no exogenous magnesium ions added. Potassium acetate (80 mM) and potassium phosphate (24 mM) were used.

[0115] Figure 4 The effects of different magnesium ion sources on the in vitro cell-free protein synthesis system were tested (reaction time: 3 h). Exogenous magnesium ions were provided by 0–9 mM magnesium L-aspartate (Mg-Asp), 0–9 mM magnesium L-glutamate (Mg-Glu), and 0–9 mM magnesium acetate (Mg-Ac). 0 mM corresponds to the blank control, with no exogenous magnesium ions added. Potassium acetate (20 mM) and potassium phosphate (24 mM) were also used.

[0116] Figure 5The effects of different magnesium ion sources on the in vitro cell-free protein synthesis system were tested (reaction time: 21 h). Exogenous magnesium ions were provided by 0–9 mM magnesium L-aspartate (Mg-Asp), 0–9 mM magnesium L-glutamate (Mg-Glu), and 0–9 mM magnesium acetate (Mg-Ac). 0 mM corresponds to the blank control, with no exogenous magnesium ions added. Potassium acetate (20 mM) and potassium phosphate (24 mM) were also used.

[0117] Figure 6 The effects of different magnesium ion sources on the in vitro cell-free protein synthesis system were tested (reaction time: 3 h). Exogenous magnesium ions were provided by 0–9 mM magnesium L-aspartate (Mg-Asp), 0–10 mM magnesium L-glutamate (Mg-Glu), and 0–13 mM magnesium acetate (Mg-Ac). 0 mM represented the blank control, with no exogenous magnesium ions added. Potassium acetate (30 mM) and potassium phosphate (18 mM) were also used.

[0118] Figure 7 The effects of different magnesium ion sources on the in vitro cell-free protein synthesis system were tested (reaction time: 22 h). Exogenous magnesium ions were provided by 0–9 mM L-aspartate magnesium (Mg-Asp), 0–10 mM L-glutamate magnesium (Mg-Glu), and 0–13 mM magnesium acetate (Mg-Ac). 0 mM corresponds to the blank control, with no exogenous magnesium ions added. Potassium acetate (30 mM) and potassium phosphate (18 mM) were also used.

[0119] Figure 8 The effects of magnesium aspartate and magnesium gluconate concentrations on the protein synthesis capacity of an in vitro cell-free protein synthesis system were investigated. The amount of exogenous protein mEGFP synthesized was indicated by RFU values. The composition of exogenous magnesium ions is shown in Table 3 of Example 4.

[0120] Figure 9 The effects of magnesium aspartate and magnesium gluconate concentrations on the protein synthesis capacity of an in vitro cell-free protein synthesis system. The amount of exogenous protein mEGFP synthesized is indicated by RFU values. The horizontal axis is labeled as "total magnesium ion concentration (aspartate magnesium percentage)".

[0121] Figure 10 The effects of magnesium aspartate, magnesium gluconate, and magnesium acetate concentrations on the protein synthesis capacity of an in vitro cell-free protein synthesis system. The amount of exogenous protein mEGFP synthesized is indicated by RFU values. The horizontal axis is labeled as "total magnesium ion concentration (aspartate magnesium percentage)".

[0122] Nucleotide and / or amino acid sequence listing

[0123] SEQ ID No.:1, the gene sequence of the exogenous protein mEGFP, is 714 bases in length.

[0124] SEQ ID No.:2, the amino acid sequence of the exogenous protein mEGFP, a total of 238 amino acids. Detailed Implementation

[0125] The meanings of the terms, nouns, and phrases in this invention.

[0126] The meanings and interpretations in this section apply to the entire text of this invention, both below and above. When references are made in this invention, the definitions of relevant terms, nouns, and phrases in the references are also cited. However, in case of conflict with the definitions in this invention, the definitions in this invention shall prevail. The conflict between the definitions in the references and the definitions in this invention does not affect the application of the referenced components, substances, compositions, materials, systems, formulations, types, methods, equipment, etc., which shall be determined in the references.

[0127] In vitro protein synthesis refers to the synthesis of proteins in a cell-free in vitro synthesis system, including at least the translation process. This includes, but is not limited to, IVT (in vitro translation), IVTT (in vitro transcription-translation), and IVDTT (in vitro replication-transcription-translation). In this invention, the IVTT reaction is preferred. The IVTT reaction, corresponding to the IVTT system, is the process of transcribing and translating DNA into protein in vitro. Therefore, we also refer to this type of in vitro protein synthesis system as a D2P system, D-to-P system, or DNA-to-Protein system; and the corresponding in vitro protein synthesis methods are also referred to as D2P methods, D-to-P methods, or DNA-to-Protein methods.

[0128] 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.

[0129] mR2P, or mRNA-to-Protein, refers to the process of converting an mRNA template into a protein product. This includes technologies, systems, methods, and kits related to mR2P.

[0130] IVTT, in vitro transcription translation.

[0131] IVDTT stands for in vitro duplication transcription translation.

[0132] CFPS system: cell-free protein synthesis system.

[0133] The CFPS(Mg+) system is a simplified way of referring to the "in vitro cell-free protein synthesis system containing exogenous magnesium ions" of this invention.

[0134] The CFPS(Mg-) system refers to the system composed of components other than degenerate magnesium ions in the CFPS(Mg+) system.

[0135] The CFPS(MgAsp-) system refers to the system composed of components other than magnesium aspartate in the CFPS(Mg+) system.

[0136] "Cell-free system" refers to a method of in vitro protein synthesis that does not involve secretion and expression by intact cells. It should be noted that in the in vitro cell-free protein synthesis system of this invention, the addition of cellular components to promote the reaction is also permitted, but the added cells are not primarily intended for the secretion and expression of exogenous target proteins. Furthermore, in the cell-free CFPS system constructed under the guidance of this invention, the intentional addition of a small number of intact cells (e.g., whose protein content does not exceed 30 wt% compared to the protein content provided by cell extracts) is also included within the scope of protection of this invention.

[0137] 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. Depending on the reaction mechanism, it may include an in vitro translation system (which can be abbreviated as IVT system, a type of mR2P system), an in vitro transcription-translation system (which can be abbreviated as IVTT system, a type of D2P system), an in vitro replication-transcription-translation system (which can be abbreviated as IVDTT system, a type of D2P system), etc. In this invention, the IVTT system is preferred. We also refer to the in vitro protein synthesis system as a "protein factory" ("Protein Factory" or "protein factory"). The in vitro protein synthesis system provided by this invention uses an open-ended description of its components.

[0138] The in vitro protein synthesis reaction mixture, also described as a reaction mixture, reaction mixture system, or in vitro protein synthesis reaction mixture system, refers to a mixture system including the in vitro protein synthesis system and the nucleic acid template encoding the exogenous protein; it can be homogeneous or heterogeneous, and is allowed to be a liquid system such as a solution, emulsion, or suspension.

[0139] Magnesium source: The magnesium source of exogenous magnesium ions in this invention is classified according to its negative valence portion, which includes, but is not limited to, negative valence groups and negative valence ions. Unless otherwise specified, there is no particular limitation on the ratio between magnesium atoms and the negative valence portion. For example, in this invention, "magnesium aspartate" specifies that the magnesium source is provided by the negative valence portion of aspartic acid residues (specifically, carboxyl groups), and there is no particular limitation on the chelation ratio between it and magnesium. For example, one magnesium atom can chelate two aspartic acid molecules, or one magnesium atom can chelate one aspartic acid molecule through intramolecular chelation. Moreover, there is no particular limitation on the site of complexation / binding / chelation with magnesium ions; it can be an α-carboxyl group, an ε-carboxyl group, or a combination of both. As another example, magnesium diglutamate is specifically defined as one magnesium atom chelating two glutamate molecules. Furthermore, there is no limitation on whether the magnesium source contains bound water. The quantitative determination of exogenous magnesium ions in this invention is achieved by controlling the content of magnesium atoms.

[0140] RFU stands for Relative Fluorescence Unit.

[0141] Mg-Asp: Magnesium Aspartate

[0142] Mg-Ac: Magnesium acetate

[0143] Mg-Glu: Magnesium glutamate

[0144] Mg-Glc: Magnesium gluconate

[0145] eGFP: Enhanced green fluorescence protein

[0146] meGFP or mEGFP: an A206K mutant of eGFP.

[0147] mol%: Mole percentage, which represents the percentage of a substance in terms of amount.

[0148] wt% or %(wt): These are units of mass concentration, both representing a mass percentage.

[0149] (v / v)% or %(v / v): both represent volume percentage.

[0150] % (w / v): a unit of mass-volume concentration, corresponding to g / 100mL.

[0151] In this invention, "protein" and "protein protein" have the same meaning and are both translated as protein, and can be used interchangeably.

[0152] In this invention, "system" and "structure" are both translated as "system" and can be used interchangeably.

[0153] In this invention, "protein synthesis amount", "protein expression amount" and "protein expression yield" have the same meaning and can be used interchangeably.

[0154] In this invention, cell extract, cell extract solution, cell lysate, cell fragments, and cell lysate have the same meaning and can be used interchangeably. In English, they can be described as cell extract, cell lysate, etc.

[0155] In this invention, the terms "energy system," "energy system," and "energy supply system" have equivalent meanings and can be used interchangeably. Similarly, "energy regeneration system" and "energy regeneration system" have equivalent meanings and can be used interchangeably. An energy regeneration system is a preferred embodiment or component of an energy system.

[0156] Post-translational modification (PTM), also known as post-translational processing, plays a crucial role in the proper folding, activity, and stability of proteins.

[0157] In this invention, "translation-related elements" refers to the relevant functional elements required in the synthesis process from a nucleic acid template to a protein product, and is not limited to functional elements required for the translation process; when the nucleic acid template is DNA, it also broadly includes functional elements required for the transcription process. These translation-related elements can be provided through cell extracts (various endogenous factors), other exogenous additives to the in vitro protein synthesis system (such as exogenous RNA polymerase, tRNA, ribosomes, other translation-related enzymes, initiation factors, elongation factors, termination factors, etc., or combinations thereof), and functional elements on the nucleic acid template (such as functional elements controlling exogenous protein transcription / translation, resistance gene translation systems, lac repressor translation systems, plasmid copy number control translation systems, etc.). Examples of functional elements controlling exogenous protein transcription / translation include promoters, terminators, enhancers, IRES elements, Kozak sequences, other elements regulating translation levels, signal sequences, leader sequences, and functional tags (such as selection marker tags, tags enhancing translation levels).

[0158] DNA amplification-related elements include at least DNA polymerase. Depending on the different amplification mechanisms, other factors may also be included, such as helicase (HDA amplification), recombinase, and single-stranded DNA-binding protein (RPA amplification).

[0159] Genes include coding and non-coding regions.

[0160] Nucleotide sequence: A sequence composed of nucleotide units.

[0161] Nucleic acid sequence: The sequence of nucleic acid material, including DNA sequence and RNA sequence.

[0162] Coding sequence (CDS): A nucleotide sequence that perfectly corresponds to the codons of a protein, containing no other sequences that do not correspond to that protein (sequence changes during mRNA processing are not considered).

[0163] Encoding gene: A valid gene segment that encodes a protein; it can be continuous or discontinuous. A coding gene must contain a coding sequence.

[0164] Nucleic acid template: also known as a genetic template, refers to a nucleic acid sequence that serves as a template for protein synthesis, including DNA templates, mRNA templates, and combinations thereof. In any embodiment of the present invention, the nucleic acid template can be independently a DNA template, an mRNA template, or a combination thereof. In any embodiment of the present invention, the nucleic acid template is preferably an independent DNA template. In the present invention, unless otherwise specified, the nucleic acid template encoding a foreign protein is preferably a DNA template.

[0165] "A nucleic acid template encoding protein X" refers to a nucleic acid template containing the coding sequence of protein X. Protein X can be synthesized based on this template through at least a translation process (or, for example, transcription-translation). The template may contain non-coding regions and may also contain coding sequences for other polypeptides or proteins besides protein X. For example, "a nucleic acid template encoding RNA polymerase" includes at least the coding sequence of RNA polymerase, and may also include non-coding regions, fusion tags, and other nucleic acid sequences. Correspondingly, the expression product must contain at least an RNA polymerase structure, which can be an RNA polymerase molecule or its fusion protein, or a mixture including an RNA polymerase molecule and / or its fusion protein molecule.

[0166] Enhancer element: Unless otherwise specified, in this invention, it refers to a sequence in a nucleic acid sequence located between the promoter and the coding sequence of the target protein, which plays a role in promoting transcription and / or translation. Examples include Ω sequences, kozak sequences, and IRES sequences. This includes transcriptional enhancers and translational enhancers.

[0167] Endogenous / Endogenous: Dependent on the metabolic activity of living cells. Endogenously expressed proteins are secreted endogenously during cell growth and can be present in the cell extracts of this invention after processing.

[0168] Exogenous / Exogenous: Independent of active cellular metabolic activity. Exogenous components are added directly to the in vitro protein synthesis system, rather than through the addition of cells or cell extracts. For example, exogenous RNA polymerase can be added to the reaction system via the addition of precursors (e.g., inactive precursors that can be activated by enzymatic digestion or other methods to generate RNA polymerase), nucleic acid templates (which can be translated into proteins by the system), fusion proteins, pure substances, or mixtures. Similarly, exogenous DNA polymerase can also be added to the reaction system via the aforementioned exogenous methods.

[0169] Exogenous protein: The target expression product of the in vitro protein synthesis system of this invention is not synthesized by the secretion of host cells. It can be a protein, a fusion protein, or a mixture containing protein molecules or fusion protein molecules; it also broadly includes polypeptides. The product obtained after in vitro protein synthesis based on a nucleic acid template encoding an exogenous protein can be a single substance or a combination of two or more substances.

[0170] Exogenous RNA polymerase: has the same meaning as exogenous RNA polymerase.

[0171] Exogenous DNA polymerase: has the same meaning as exogenous DNA polymerase.

[0172] "A nucleic acid template encoding RNA polymerase (or a nucleic acid template encoding DNA polymerase)" must include at least the coding sequence of RNA polymerase (or DNA polymerase), and may also include other nucleic acid sequences such as non-coding regions and fusion tags; correspondingly, the expression product must contain at least an RNA polymerase structure (or a DNA polymerase structure). Taking RNA polymerase as an example, it can be an RNA polymerase molecule or its fusion protein, or a mixture of RNA polymerase molecules and / or their fusion protein molecules.

[0173] A peptide is a compound formed by two or more amino acids linked together by peptide bonds. In this invention, peptide and peptide segment have the same meaning and can be used interchangeably.

[0174] Polypeptides are peptides composed of 10 to 50 amino acids.

[0175] Proteins are peptides composed of 50 or more amino acids. Fusion proteins are also a type of protein.

[0176] Derivatives of peptides and proteins: Unless otherwise specified (e.g., a specific sequence is designated), the present invention also includes derivatives of any peptide or protein. These peptide and protein derivatives include at least those containing a C-terminal tag, an N-terminal tag, or both C-terminal and N-terminal tags. The C-terminus refers to the COOH terminus, and the N-terminus refers to the NH2 terminus; their meanings are understood by those skilled in the art. The tags can be peptide tags or protein tags. Examples of tags include, but are not limited to, 6-histidine (6×-His, HHHHHH), Glu-Glu, and c-myc epitopes (EQKLISEEDL). Octapeptide (DYKDDDDK), Protein C (EDQVDPRLIDGK), Tag-100 (EETARFQPGYRS), V5 epitope (GKPIPNPLLGLDST), VSV-G (YTDIEMNRLGK), Xpress (DLYDDDDK), hemagglutinin (YPYDVPDYA), β-galactosidase, thioredoxin, histidine-patch thioredoxin, IgG-binding domain, intein-chitin binding domain, T7 gene 10, glutathione-S-transferase (GST), green fluorescent protein (GFP), maltose-binding protein (MBP), etc.

[0177] Homology, unless otherwise specified, refers to at least 50% homology; preferably at least 60% homology, more preferably at least 70% homology, more preferably at least 75% homology, more preferably at least 80% homology, more preferably at least 85% homology, more preferably at least 90% homology; and also, for example, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% homology. Examples of objects described include homologous sequences of the Ω sequence mentioned in this invention.

[0178] A "variant" refers to a substance that has a different structure (including but not limited to minor variations) but still retains or substantially retains its original function or performance. Variants include, but are not limited to, nucleic acid variants, peptide variants, and protein variants. Methods for obtaining variants include, but are not limited to, recombination, deletion or omission, insertion, translocation, and substitution of structural units. Variants include, but are not limited to, modified products, genetically modified products, and fusion products. Methods for genetic modification to obtain genetically modified products include, but are not limited to, gene recombination (corresponding to gene recombination products), gene deletion or omission, insertion, frameshift, and base substitution. Gene mutation products, also known as gene mutants, are a type of genetically modified product.

[0179] Modified products include, but are not limited to, chemically modified products, amino acid modifiers, peptide modifiers, and protein modifiers. Chemically modified products refer to products modified using chemical synthesis methods such as organic chemistry, inorganic chemistry, and polymer chemistry. Examples of modification methods include ionization, salting, desalination, complexation, decomplexation, chelation, dechelation, addition reactions, substitution reactions, elimination reactions, insertion reactions, oxidation reactions, reduction reactions, and post-translational modifications. Specific examples include oxidation, reduction, methylation, demethylation, amination, carboxylation, and sulfidation.

[0180] In this invention, unless otherwise specified, a "mutant" refers to a mutated product that retains or substantially retains its original function or performance, and there is no particular limitation on the number of mutation sites. Mutants include, but are not limited to, gene mutants, polypeptide mutants, and protein mutants. A mutant is a type of variant. Methods for obtaining relevant mutants include, but are not limited to, recombination, deletion or omission, insertion, shift, and substitution of structural units. The structural unit of a gene is a base, while the structural units of polypeptides and proteins are amino acids. Types of gene mutations include, but are not limited to, gene deletion or omission, insertion, frameshift, and base substitution.

[0181] An amino acid mixture refers to a mixture containing at least two amino acids.

[0182] In this invention, unless otherwise specified, the amino acids can be natural amino acids or non-natural amino acids. L -amino acids, D - Amino acids or combinations thereof, and may also be radioisotope-labeled amino acids, modified amino acids, etc. Modified amino acids refer to amino acids linked with chemically modified groups, and their structure is not particularly limited, including but not limited to modifications via amino acid side groups. The above definition of amino acids covers any substance comprising an amino acid unit in this invention, including but not limited to: polypeptides and their derivatives, proteins and their derivatives, polypeptide tags, protein tags, polypeptide sequences, protein sequences, amino acid modifications, polypeptide modifications, protein modifications, partial domains of any of the aforementioned, subunits or fragments of any of the aforementioned (including domains of any of the aforementioned), and variants of any of the aforementioned (including variants of domains, subunits, and fragments of any of the aforementioned). The "variants of any of the aforementioned" include, but are not limited to, "mutants of any of the aforementioned." In this invention, for the chiral type " L -", " D The '-' symbol has the same meaning as the non-subscript form.

[0183] Crowding agents are reagents used to simulate the crowded macromolecular environment within cells in vitro. References include “X Ge, D Luo and J Xu. Cell-free protein expression under macromolecular crowding conditions[J]. PLoS One, 2011, 6(12):e28707” and its cited references.

[0184] Sucrose polymers: polymers containing at least two sucrose units. This includes, but is not limited to, polysucrose.

[0185] Ficoll sucrose polymer: unless otherwise specified, refers to... The reagent, a nonionic synthetic sucrose polymer, is a highly branched polymer copolymerized from sucrose and epichlorohydrin, and can be selected from commercially available products. Examples include Ficoll-400 (polysucrose 400, CAS: 26873-85-8) and Ficoll-70 (polysucrose 70, CAS: 72146-89-5). PM 400 (Sigma Aldrich) is a highly branched polymer copolymerized from sucrose and epichlorohydrin, with an average molecular weight of 400 kg / mol; Ficoll PM 70 (Sigma Aldrich) has an average molecular weight of 70 kg / mol.

[0186] Phosphoric acid compounds include both organic and inorganic compounds.

[0187] Unless otherwise specified, phosphates refer to inorganic phosphates.

[0188] In this invention, "room temperature" is preferably room temperature to 37°C, specifically, preferably 20-37°C, and more preferably 25-37°C.

[0189] In this invention, preferred embodiments, better embodiments, more preferred embodiments, and most preferred embodiments are not intended to limit the implementation of this invention, but are only used to provide examples of embodiments with better technical effects.

[0190] In the description of this invention, the terms "preferred method," "preferred embodiment," "preferred example," "preferred example," "in a preferred embodiment," "preferred," "preferred," "more preferred," "more preferably," "further preferred," "most preferred," etc., as well as the illustrative enumeration terms such as "one embodiment," "one method," "example," "specific example," "for instance," "as an example," "for example," "like," "as," etc., all describe specific features included in at least one specific embodiment of this invention. In this invention, the specific features described by each method can be combined in any suitable manner in one or more specific embodiments. In this invention, the technical solutions corresponding to each preferred method can also be combined in any suitable manner; for example, exogenous RNA polymerase and exogenous DNA polymerase can be added simultaneously, see patent document CN108642076A.

[0191] In this invention, "optionally" means that it may or may not be present. The selection is based on whether it is suitable for the corresponding system.

[0192] In this invention, "any combination thereof" means "greater than 1" in quantity and "a group consisting of the following situations in terms of scope: "any one of them, or a group consisting of at least two of them".

[0193] In this invention, the descriptions of "one or more", "one or more", "one or more or all", etc., have the same meaning as "at least one", "at least one", "or a combination thereof", "or any combination thereof", "or any combination thereof", etc., and can be used interchangeably to indicate a quantity equal to "1" or "greater than 1".

[0194] In this invention, the terms "or / and" and "and / or" are used to indicate "either one or a combination thereof," or at least one of them. For example, "including substrates for synthesizing RNA and / or substrates for synthesizing proteins" means that it may include only substrates for synthesizing RNA, only substrates for synthesizing proteins, or both substrates for synthesizing RNA and substrates for synthesizing proteins.

[0195] The prior art methods described in this invention using terms such as "usually," "conventionally," "generally," "frequently," and "often" are also cited as references to the content of this invention, and unless otherwise specified, can be regarded as one of the preferred embodiments of this invention.

[0196] All documents mentioned in this invention, and those directly or indirectly cited by such documents, are incorporated herein by reference as if each document were cited individually.

[0197] 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 the embodiments) can be combined with each other to form new or preferred technical solutions, as long as they can synthesize exogenous proteins in vitro or preferably synthesize exogenous proteins efficiently. Due to space limitations, they will not be described in detail here.

[0198] 1. A first aspect of the present invention provides an in vitro cell-free protein synthesis system containing exogenous magnesium ions, wherein the "in vitro cell-free protein synthesis system containing exogenous magnesium ions" is also referred to in the present invention as the "CFPS(Mg+) system", comprising the following components: eukaryotic cell extract and exogenous magnesium ions; wherein the exogenous magnesium ions are derived from one or more donor sources, including at least magnesium aspartate; wherein the CFPS(Mg+) system is capable of reacting with a nucleic acid template encoding an exogenous protein, thereby expressing the exogenous protein.

[0199] The CFPS(Mg+) system can work with the nucleic acid template encoding the foreign protein to provide the translation-related elements required for the synthesis of the foreign protein.

[0200] Preferably, the CFPS(Mg+) system contains a component (e.g., an RNA polymerase corresponding to a promoter element) capable of recognizing promoter elements in a nucleic acid template, such that the CFPS(Mg+) system can recognize promoter elements of nucleic acid templates encoding exogenous proteins.

[0201] By limiting the technical function to "expressing exogenous proteins," this invention only covers combinations of technical features that can achieve the aforementioned function; combinations of technical features that cannot achieve the aforementioned function are naturally excluded from the scope of this invention. That is, the CFPS(Mg+) system must first be a working system, a system capable of expressing exogenous proteins.

[0202] 1.1. In vitro cell-free protein synthesis system containing exogenous magnesium ions (CFPS(Mg+) system)

[0203] The in vitro protein synthesis reaction of the present invention is carried out in an in vitro cell-free protein synthesis system containing exogenous magnesium ions.

[0204] The CFPS(Mg+) system can provide various factors required for in vitro protein synthesis. It can be provided integratedly via cell extracts or added separately (e.g., the PURE system from Japan, such as the PURExpress kit).

[0205] The types and amounts of the components in the CFPS(Mg+) system are not particularly limited, as long as the system can react with the nucleic acid template encoding the foreign protein to synthesize the foreign protein. Preferably, combinations that can efficiently express the foreign protein are preferred. Combinations that cannot express the foreign protein due to excessively low or high concentrations of certain components are naturally excluded from the scope of this invention.

[0206] There are no particular restrictions on the order in which the components of the CFPS(Mg+) system are added.

[0207] Unless otherwise specified, the concentrations of each component in the CFPS(Mg+) system refer to the initial concentrations in the in vitro protein synthesis reaction mixture.

[0208] Preferably, the CFPS(Mg+) system contains system components capable of recognizing promoter elements on nucleic acid templates, such as RNA polymerases corresponding to promoter elements.

[0209] The system components capable of recognizing promoter elements in nucleic acid templates, such as the corresponding RNA polymerase, can be provided by cell extracts in the system, by exogenous addition, or by a combination of the two methods.

[0210] In one preferred embodiment, the CFPS(Mg+) system includes at least a cell extract. The cell extract is intended to provide structural or biological factors for protein transcription and translation. The selection criteria for the cell extract are: it enables the synthesis of a foreign protein using a nucleic acid template encoding a foreign protein as a template via an in vitro protein synthesis reaction. The cell extract of this invention can be derived from wild-type or non-wild-type cells. Modification methods for non-wild-type cells include, but are not limited to, genetic modification. The cell extract of this invention is preferably derived from eukaryotic cells.

[0211] In one preferred embodiment, the CFPS(Mg+) system includes a cell extract containing an endogenously expressed RNA polymerase corresponding to a promoter element on a nucleic acid template. Specifically, for example, Kluyveromyces lactis cell extract contains an endogenously expressed T7 RNA polymerase capable of recognizing the T7 promoter on a nucleic acid template.

[0212] In one embodiment, the CFPS(Mg+) system includes eukaryotic cell extracts and exogenous magnesium ions (including at least magnesium aspartate).

[0213] The in vitro cell-free protein synthesis system of the present invention includes exogenous magnesium ions, and the CFPS(Mg+) system includes at least magnesium aspartate; preferably at least 25 mol%, more preferably at least 30 mol%, more preferably at least 40 mol%, and more preferably at least 50 mol%, wherein the exogenous magnesium ions are provided by magnesium aspartate.

[0214] The process of synthesizing proteins in vitro includes at least translation and optionally transcription.

[0215] The transcription process, which converts DNA into mRNA, is inseparable from RNA polymerase. Therefore, the corresponding CFPS(Mg+) system preferably also includes RNA polymerase, which can be sourced from: endogenously expressed RNA polymerase (provided via cell extracts), exogenously added RNA polymerase, translation products of exogenous nucleic acid templates encoding RNA polymerase, and combinations thereof.

[0216] The endogenously expressed RNA polymerase is not added independently, but is present in the cell extract.

[0217] To ensure that the cell extract contains endogenously expressed RNA polymerase, the coding sequence / gene of RNA polymerase is preferably integrated into the host cell for which the cell extract is prepared. Specifically, this is preferably achieved through the following methods: inserting the coding sequence / gene of RNA polymerase into a cell-free plasmid, integrating the coding gene of RNA polymerase into the cell genome, or a combination of the above two methods to modify the bacterial strain, followed by the preparation of the cell extract. The methods for integrating the coding sequence / gene of RNA polymerase into the cell genome include, but are not limited to: insertion into the cell genome, in situ replacement of a portion of the genome, and combinations thereof.

[0218] The exogenously added or endogenously expressed RNA polymerase is preferably T7 RNA polymerase.

[0219] In one preferred embodiment, the CFPS(Mg+) system includes a DNA polymerase, the source of which may be selected from: endogenously expressed DNA polymerase (provided via cell extract), exogenously added DNA polymerase, translation products of exogenous nucleic acid templates encoding DNA polymerase, and combinations thereof.

[0220] The CFPS(Mg+) system may optionally include an exogenous RNA polymerase and / or a nucleic acid template encoding the RNA polymerase.

[0221] The CFPS(Mg+) system may optionally include an exogenous DNA polymerase and / or a nucleic acid template encoding the DNA polymerase.

[0222] In one preferred embodiment, the CFPS(Mg+) system includes exogenous RNA polymerase and exogenous DNA polymerase. (Reference: CN108642076A)

[0223] In one preferred embodiment, the CFPS(Mg+) system includes an energy system.

[0224] In one preferred embodiment, the CFPS(Mg+) system includes a substrate for synthesizing RNA.

[0225] In one preferred embodiment, the CFPS(Mg+) system includes a substrate for synthesizing proteins.

[0226] In one preferred embodiment, the CFPS(Mg+) system includes DNA polymerase and a substrate for DNA synthesis.

[0227] In one preferred embodiment, the CFPS(Mg+) system includes eukaryotic cell extracts, exogenous magnesium ions (including at least magnesium aspartate; preferably magnesium aspartate), an energy system, a substrate for RNA synthesis, and a substrate for protein synthesis.

[0228] In one preferred embodiment, the CFPS(Mg+) system includes eukaryotic cell extract, exogenous magnesium ions (including at least magnesium aspartate; preferably, magnesium aspartate), an energy system, a substrate for protein synthesis, RNA polymerase (included in the cell extract or added exogenously independently), and a substrate for RNA synthesis.

[0229] In one preferred embodiment, the CFPS(Mg+) system includes eukaryotic cell extract, magnesium aspartate, an energy system, a substrate for protein synthesis, RNA polymerase (included in the cell extract or added exogenously), a substrate for RNA synthesis, DNA polymerase (included in the cell extract or added exogenously), and a substrate for DNA synthesis.

[0230] In one preferred embodiment, the CFPS(Mg+) system comprises Kluyveromyces lactis cell extract (containing endogenously expressed T7 RNA polymerase), magnesium aspartate, optional magnesium gluconate, an energy system, a substrate for RNA synthesis, and a substrate for protein synthesis.

[0231] In one preferred embodiment, the CFPS(Mg+) system comprises Kluyveromyces lactis cell extract (encoding gene for host cell non-endogenously integrated RNA polymerase), magnesium aspartate, optional magnesium gluconate, an energy system, exogenous RNA polymerase, substrate for RNA synthesis, and substrate for protein synthesis.

[0232] The CFPS(Mg+) system may also optionally include at least one of the following exogenous additives: other soluble amino acid salts, translation-related elements, DNA amplification-related elements, RNA amplification-related elements, RNase inhibitors, crowding agents, potassium ions, antioxidants or reducing agents, antifreeze agents, trehalose, reaction promoters, defoamers, alkanes, buffers, and aqueous solvents.

[0233] 1.1.1. Exogenous magnesium ions

[0234] The in vitro cell-free protein synthesis system of the present invention includes exogenous magnesium ions, constituting the CFPS(Mg+) system.

[0235] The source of the exogenous magnesium ions includes at least magnesium aspartate.

[0236] In any CFPS(Mg+) system of the present invention, the magnesium aspartate can be L-aspartate magnesium, D-aspartate magnesium, or a combination thereof, preferably L-aspartate magnesium.

[0237] The source of the exogenous magnesium ions may optionally be from the group consisting of: magnesium gluconate, magnesium acetate, magnesium glutamate (MgO), ... L- type, D- Type or combination thereof, preferably L- Magnesium chloride, magnesium phosphate, magnesium sulfate, magnesium citrate, magnesium hydrogen phosphate, magnesium iodide, magnesium lactate, magnesium nitrate, magnesium oxalate, and combinations thereof.

[0238] In this invention, unless otherwise specified, "magnesium ion source" specifically refers to "exogenous magnesium ion supply".

[0239] One preferred method is that the magnesium ion source includes magnesium aspartate (preferably L-aspartate magnesium), and also includes any one of magnesium gluconate, magnesium glutamate (preferably L-glutamate magnesium), and magnesium acetate.

[0240] One preferred method is that the magnesium ion source is a combination of magnesium aspartate (preferably L-aspartate magnesium) and magnesium gluconate.

[0241] One preferred method is that the magnesium ion source is a combination of magnesium aspartate (preferably L-aspartate magnesium) and magnesium glutamate (preferably L-glutamate magnesium).

[0242] One preferred method is that the magnesium ion source is a combination of magnesium aspartate (preferably L-aspartate magnesium) and magnesium acetate.

[0243] One preferred method is that the magnesium ion source includes magnesium aspartate (preferably L-aspartate magnesium), and also includes any two of magnesium gluconate, magnesium glutamate (preferably L-glutamate magnesium), and magnesium acetate.

[0244] One preferred embodiment is that the magnesium ion source is a combination of magnesium aspartate (preferably L-aspartate magnesium), magnesium gluconate, and magnesium glutamate (preferably L-glutamate magnesium). A more preferred embodiment is that the magnesium ion source is a combination of magnesium gluconate, magnesium L-aspartate, and magnesium L-glutamate.

[0245] In one preferred embodiment, the source of the exogenous magnesium ions includes four types: magnesium aspartate (preferably L-aspartate magnesium), magnesium gluconate, magnesium glutamate (preferably L-glutamate magnesium), and magnesium acetate.

[0246] The amount of magnesium aspartate (Q) that can increase the synthesis of exogenous proteins Asp According to Y PRT (C Asp The expression levels of exogenous proteins in the curve were determined and selected from Y. PRT (C Asp The dosage range in the curve where the expression level of the exogenous protein is greater than Y0. The Y... PRT (C Asp The curve can be obtained through preliminary experiments.

[0247] The Q Asp Preferably selected from exogenous proteins with an expression level of at least Y0 + 50% Y Δ The dosage range of magnesium aspartate at that time;

[0248] The Q Asp More preferably, the protein selected is an exogenous protein with an expression level of at least Y0 + 60% Y. Δ The dosage range of magnesium aspartate at that time;

[0249] The Q Asp More preferably, the protein selected is an exogenous protein with an expression level of at least Y0 + 70% Y. Δ The dosage range of magnesium aspartate at that time;

[0250] The Q Asp More preferably, the protein selected is an exogenous protein with an expression level of at least Y0 + 80% Y. Δ The dosage range of magnesium aspartate at that time;

[0251] The Q Asp More preferably, the protein selected is an exogenous protein with an expression level of at least Y0 + 90% Y. Δ The dosage range of magnesium aspartate at that time;

[0252] The Q Asp More preferably, the protein selected is an exogenous protein with an expression level of at least Y0 + 95% Y. Δ The dosage range of magnesium aspartate at that time;

[0253] More preferably, the Q AspThe amount of magnesium aspartate used when the exogenous protein is at its highest expression level (C) max .

[0254] Preferably, the Q Asp C Asp C max The same characterization method is used, such as using concentration, mass, or molarity for all. For example, the amount of magnesium aspartate (C) is characterized by concentration. Asp Different amounts of magnesium aspartate were added to CFPS(MgAsp-) systems to obtain a series of CFPS(Mg+) systems with different magnesium aspartate concentrations; in vitro protein synthesis reactions were performed, and the expression levels of exogenous proteins (Y) were measured. PRT ), which can obtain the expression level of exogenous proteins (Y). PRT ) and the concentration of magnesium aspartate (C Asp The relationship curve between (Y) PRT (C Asp ( ) curve); determine the highest expression level of the exogenous protein (Y) from the curve. max The corresponding concentration of magnesium aspartate added at (C) max Furthermore, the amount of magnesium aspartate (Q) protected by this invention, which can increase the expression level of exogenous proteins, was determined by concentration characterization. Asp The concentration range of the dosage method.

[0255] In this invention, the amount of magnesium aspartate is preferably measured and controlled by the amount of exogenous reagent added.

[0256] For the expression level of exogenous proteins (Y) PRT Appropriate assay methods and characterization techniques can be selected at will. Methods for determining protein expression levels include, but are not limited to, ultraviolet absorption assay, biuret assay, BCA method, Lowry method, Coomassie brilliant blue assay, and Kjeldahl method. Different characterization methods can be used to indicate the concentration, mass, or amount of protein, such as, but not limited to, absorbance values ​​(OD values) and relative fluorescence unit values ​​(RFU values) of fluorescent proteins.

[0257] In this invention, the amount of magnesium aspartate is preferably characterized by concentration.

[0258] One implementation method uses a mutant mEGFP or its fusion protein with enhanced green fluorescent protein as the exogenous protein (PRT), wherein the mEGFP portion is fluorescent. After the in vitro protein synthesis reaction, the RFU value of the solution sample is tested using ultraviolet absorption under conditions of excitation wavelength 488 nm and emission wavelength 507 nm. The expression level (γ) of the exogenous protein product containing the mEGFP structure is also measured.PRT The result can be obtained using the following formula: Where RFU is the relative fluorescence unit value reading, C PRT M represents the concentration of the exogenous protein product (in μg / mL). mEGFP M represents the relative molecular weight of the mEGFP fluorescent protein structure. PRT This represents the relative molecular weight of the exogenous protein product. Within the standard testing range, C... PRT The relationship between M and RFU is essentially linear. When mEGFP is used directly as the exogenous protein, M... PRT With M mEGFP The values ​​are equal, as in Examples 1-6. Combined with the solution volume of the exogenous protein product, the mass and molar amount of the exogenous protein product can be calculated.

[0259] One preferred method is to determine the amount of magnesium aspartate as follows: when the types and contents of the components in the CFPS(Mg+) system are determined, the amount of magnesium aspartate is adjusted within a relatively wide concentration range. Under specified reaction conditions (reaction temperature, reaction time, etc.), the amount of magnesium aspartate C at which the expression level of the exogenous protein is highest is determined. max This is the optimal amount of magnesium aspartate under this technical solution.

[0260] The Y PRT (C Asp ) curve, Q Asp C Asp Y PRT Y max C max Y0, Y Δ The definition is the same as above.

[0261] One preferred method is to provide at least 25 mol% of the exogenous magnesium ions from magnesium aspartate.

[0262] One preferred method is to provide at least 30 mol% of the exogenous magnesium ions from magnesium aspartate.

[0263] One preferred method is to provide at least 40 mol% of the exogenous magnesium ions from magnesium aspartate.

[0264] One preferred method is to provide at least 50 mol% of the exogenous magnesium ions from magnesium aspartate.

[0265] One preferred method is to provide at least 80 mol% of the exogenous magnesium ions from magnesium aspartate.

[0266] One preferred method is to provide at least 90 mol% of the exogenous magnesium ions from magnesium aspartate.

[0267] One preferred method is to provide 100 mol% of the exogenous magnesium ions from magnesium aspartate.

[0268] One preferred method is to provide 100 mol% of the exogenous magnesium ions from L-aspartic magnesium.

[0269] The concentration of exogenous magnesium ions provided by the magnesium aspartate is, for example, any one of the following concentrations, or a concentration range between any two of the following concentration values ​​(the concentration range includes both endpoints): 0.1 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 5.5 mM, 6 mM, 6.5 mM, 7 mM, 7 .5mM, 8mM, 8.5mM, 9mM, 9.5mM, 10mM, 11mM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM, 18mM, 19mM, 20mM, 22mM, 24mM, 25mM, 28mM, 30mM, 35mM, 40mM, 45mM, 50mM, 60mM, 70mM, 80mM.

[0270] In one preferred embodiment, the concentration of exogenous magnesium ions is 0.1–50 mM.

[0271] In another preferred embodiment, the concentration of exogenous magnesium ions is 0.5–20 mM.

[0272] In another preferred embodiment, the concentration of exogenous magnesium ions is 1–10 mM.

[0273] In another preferred embodiment, the concentration of exogenous magnesium aspartate is 0.1–50 mM, more preferably 0.5–20 mM, and even more preferably 1–10 mM.

[0274] In another preferred embodiment, the concentration of exogenous L-aspartic magnesium is 0.1–50 mM, more preferably 0.5–20 mM, and even more preferably 1–10 mM.

[0275] In one preferred embodiment, the selectable concentration range of the exogenous magnesium aspartate is determined based on the concentration value at which the protein expression level is highest in a concentration curve with the concentration of magnesium aspartate as the independent variable. The concentration of the exogenous magnesium aspartate is preferably selected from a concentration range where the protein expression level is at least 50% of the highest expression level (e.g., see attached...). Figure 3 The concentration range of Mg-Asp in the medium was 2–4 mM; (Attached) Figure 5 The concentration range of Mg-Asp in the medium was 2–6 mM; (Attached) Figure 7The concentration range of Mg-Asp is 3 to 8 mM, more preferably at least 60% of the highest expression level, more preferably at least 70% of the highest expression level, more preferably at least 80% of the highest expression level, more preferably at least 90% of the highest expression level, and more preferably at least 95% of the highest expression level.

[0276] One preferred method is to determine the concentration of magnesium aspartate as follows: When the types and contents of other components in the CFPS(Mg+) system are determined, the concentration of magnesium aspartate is adjusted within a relatively wide range, such as 0–50 mM. Under specified reaction conditions (reaction temperature, synthesis time, etc.), the concentration at which the expression level of the exogenous protein is highest is the optimal concentration (most preferred concentration) of magnesium aspartate under this technical solution. For example, see Appendix Figure 3 In the corresponding technical solution, the optimal Mg-Asp concentration is 3 mM; Appendix Figure 5 In the corresponding technical solution, the optimal Mg-Asp concentration is 2 mM; Appendix Figure 7 In the corresponding technical solution, the optimal Mg-Asp concentration is 4 mM.

[0277] 1.1.2. Cell extract (preferably, eukaryotic cell extract)

[0278] The cell extract should be able to express a nucleic acid template encoding a foreign protein, that is, be able to synthesize the foreign protein encoded by the nucleic acid template encoding the foreign protein.

[0279] The cell extract is intended to provide structural and / or biological factors for protein expression (such as transcription and translation).

[0280] Cell extracts can provide many of the key translation-related elements needed to synthesize exogenous proteins; this is an endogenous way of providing them.

[0281] The cell extracts, including yeast cell extracts, are typically used to provide substances such as ribosomes, transfer RNA (tRNA), aminoacyl-tRNA synthetase, initiation and elongation factors and termination release factors required for protein synthesis, and can also endogenously provide other enzymes such as polymerases (RNA polymerase and / or DNA polymerase) after strain modification.

[0282] The cell extracts described herein do not contain intact cells in principle, because the preparation methods for cell extracts involve a cell disruption step (also known as cell lysis or lysis). Compared to traditional methods of protein synthesis using intact cells for secretion and expression, the in vitro protein synthesis system constructed in this way is called a cell-free system.

[0283] Cell extracts may also contain other proteins derived from the cytoplasm of cells, especially soluble proteins.

[0284] Preferably, the cell extract contains various factors required for protein synthesis.

[0285] The various protein factors provided by the aforementioned cell extracts, and the corresponding encoding genes, can be naturally present in the cell genome, or the relevant encoding genes can be integrated into the cell genome (integrated into chromosomes), or the relevant genes, gene fragments, or coding sequences can be inserted into intracellular free plasmids. RNA polymerase and DNA polymerase are used as examples. In one preferred embodiment, the cell extract contains endogenously expressed RNA polymerase and / or DNA polymerase.

[0286] The CFPS(Mg+) system optionally includes purified translation-related elements (CRLEs). When cell extracts are insufficient to provide all the CRLEs required for the synthesis of exogenous proteins (insufficient variety and / or insufficient quantity), the missing CRLEs can be added exogenously. This is particularly relevant when the endogenous secretion products of the source strain lack a certain component for the expression of a foreign protein, which can be supplemented exogenously. For example, the purified CRLEs include, but are not limited to, any one or a combination thereof selected from the following group: tRNA, ribosomes, other translation-related enzymes, initiation factors, elongation factors, and termination factors. The translation-related enzymes include, but are not limited to, various aminoacyl-tRNA synthetases and peptidyl transferases.

[0287] By endogenously integrating the coding sequence or coding gene of a heterologous protein into the source cells of the cell extract, the modified strain can endogenously express the heterologous protein. The heterologous protein may include, but is not limited to, RNA polymerase, DNA polymerase, etc. The methods for endogenously integrating the coding sequence or coding gene of the heterologous protein can refer to the methods provided in existing literature and its cited literature, including but not limited to CN109423496A, CN10697843A, CN2018116198190, "Molecular and Cellular Biology, 1990, 10(1): 353-360", etc. Specifically, these methods include, but are not limited to, inserting the coding sequence into a free intracellular plasmid, inserting the coding gene into the cell genome, replacing part of the gene in the cell genome with the coding gene in situ, and combinations thereof.

[0288] In one preferred embodiment, the cell extract originates from cells that intrinsically integrate the encoding gene of RNA polymerase, enabling endogenous expression of RNA polymerase. This allows for cell-free protein synthesis in vitro without the addition of exogenous RNA polymerase, replacing the exogenous addition method, simplifying the formulation, improving operational convenience, and saving costs. The methods for achieving endogenous integration of RNA polymerase include, but are not limited to: inserting the RNA polymerase encoding gene into a cell plasmid or into the cell genome; replacing a portion of a gene or sequence in the cell genome with the RNA polymerase encoding gene in situ (i.e., including the step of knocking out a portion of the original gene or sequence); knocking out a portion of the original gene and inserting the RNA polymerase encoding gene; and combinations thereof. More preferably, the cell extract originates from yeast. Even more preferably, the cell extract originates from Kluyveromyces lactis. For example, in Examples 1-6, the coding gene for T7 RNA polymerase was integrated into the genome of *Kluyveromyces lactis*, which endogenously expresses T7 RNA polymerase. The resulting cell extract contains endogenously expressed T7 RNA polymerase, and no additional RNA polymerase is added to the in vitro cell-free protein synthesis system. This allows for in vitro cell-free protein synthesis without the addition of exogenous RNA polymerase. In other specific embodiments, the coding gene for RNA polymerase is inserted into a cell plasmid, such as a *Kluyveromyces lactis* cell plasmid, to prepare the cell extract. Refer to the preparation method in CN109423496A for details.

[0289] Other gene modification methods can also be used to modify the source cells to improve the activity of cell extracts and better promote in vitro protein synthesis, such as the gene knockout methods in CN2018116083534, CN2019107298813, and CN108949801A, and the gene modification methods in CN2018112862093.

[0290] The preparation method of the cell extract can employ reported techniques. In brief, it typically includes the following steps: providing a sufficient quantity of cells, flash-freezing the cells with liquid nitrogen, lysing the cells, centrifuging to collect the supernatant, thus obtaining the cell extract. Refer to documents CN106978349A, CN108535489A, CN108642076A, CN109593656A, CN109971783A, etc. Alternatively, seed cells can be fermented, centrifuged, the culture medium removed, and a sufficient quantity of cells collected to prepare the cell extract.

[0291] The cell extract prepared by the method provided in this invention enables in vitro protein synthesis reactions to proceed normally and contains essential components required for protein synthesis, such as tRNA with amino acid transport function and aminoacyl-tRNA synthetase. In some embodiments, the cell extract is a yeast cell extract, prepared by a method comprising the following steps: (i) providing source cells; (ii) washing the yeast cells to obtain washed yeast cells; (iii) performing cell disruption on the washed yeast cells to obtain a crude yeast extract; and (iv) performing solid-liquid separation on the crude yeast extract, with the collected supernatant being the cell extract. Preferably, the yeast cell extract is a Kluyveromyces lactis cell extract.

[0292] In this invention, one preferred method for determining the protein content in the cell extract is 20–100 mg / mL. Another preferred method is 20–50 mg / mL. Yet another preferred method is 50–100 mg / mL. Another preferred method is any one of the following concentrations: 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, and 90 mg / mL, or a concentration range (including both endpoints) between any two concentrations. Methods for determining the protein content include, but are not limited to, ultraviolet absorption method, biuret method, BCA method, Lowry method, Coomassie brilliant blue method, and Kjeldahl method. One preferred method is the Coomassie brilliant blue assay.

[0293] There is no particular limitation on the concentration of the cell extract in the in vitro protein synthesis reaction mixture. It can be a volume ratio or a weight ratio; unless otherwise specified, the final volume ratio refers to the total volume ratio. In one preferred embodiment, the concentration of the cell extract is 20%–80% (v / v); in another preferred embodiment, the concentration is 20%–70% (v / v); in yet another preferred embodiment, the concentration is 30%–60% (v / v); in yet another preferred embodiment, the concentration is 40%–50% (v / v); and in yet another preferred embodiment, the concentration is 80% (v / v); all based on the total volume of the in vitro cell-free protein synthesis system. Examples of cell extract concentrations include, but are not limited to, any one of the following volume percentages, or a range between any two of the following volume percentages (the range may or may not include two endpoints): 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%.

[0294] The cell extracts of this invention are preferably derived from eukaryotic cells. Prokaryotic and eukaryotic cells differ fundamentally in their translation initiation and regulation mechanisms, and prokaryotic expression systems lack many post-translational protein processing mechanisms. Typically, cell-free systems based on *E. coli* cells lack the translation or post-translational modification capabilities that only eukaryotic cell-free systems can perform, making many eukaryotic proteins unsuitable for expression in *E. coli* systems; the synthesized proteins contain incomplete novel polypeptides. The synthetic mechanisms of cell-free protein synthesis systems based on prokaryotic systems differ significantly from those based on eukaryotic systems, particularly regarding the various factor levels involved in the cell extracts. References include, but are not limited to, the following literature: “Nicole E. Gregorio, Max Z. Levine and Javin P. Oza. Methods Protoc. 2019, 2, 24”, “Edited by Alexander S. Spirin and James R. Swartz. Cell-free protein synthesis: methods and protocols [M]. 2008, p. 5”, “Zhang Xu. Key technologies and industrial applications of efficient synthesis of complex membrane proteins in cell-free systems [D]. Zhejiang University, 2014”, etc. The in vitro cell-free protein synthesis system of this invention preferably uses eukaryotic cell extracts.

[0295] The cell source of the cell extract may be selected from one or more types of eukaryotic cells from the group consisting of, but not limited to, mammalian cells (such as rabbit reticulocytes, HF9, HeLa, CHO, K562, HEK293), plant cells (such as wheat germ cells, tobacco BY-2 cells), yeast cells, insect cells, and combinations thereof. The mammalian cell sources include, but are not limited to, mouse, rabbit, monkey, human, sheep, pig, and bovine sources.

[0296] The cell source and preparation method of the cell extract can also refer to existing literature, including but not limited to the cell sources reported in the following literature, which are all incorporated into this invention: "Nicole E. Gregorio, Max Z. Levine and Javin P. Oza. A User's Guide to Cell-Free Protein Synthesis[J]. Methods Protoc. 2019, 2, 24", "Y Lu. Advances in cell-free biosynthetic technology[J]. Current Developments in Biotechnology and Bioengineering, 2019, Chapter 2, 23-45", and other literature directly or indirectly cited. For example, eukaryotic cell sources include, but are not limited to, Saccharomyces cerevisiae, Streptomyces lividans, wheat germ, Tobacco BY-2 cell, Spodopterafrugiperda cell (sf cell, an insect cell), Trichoplusiani cell (an insect cell), rabbit reticulocyte, CHO cell (Chinese hamsterovary cell), human K562 cell, HEK293 cell, HeLa cell, mouse fibroblast, and Leishmania tarentolae cell (protozoan, a single-celled organism).

[0297] One preferred embodiment of the yeast cells is *Saccharomyces cerevisiae*, *Pichia pastoris*, *Kluyveromyces*, or a combination thereof; the *Kluyveromyces* is further preferably *Kluyveromyces lactis* (K. lactis), *Kluyveromyces lactis var. drosophilarum*, *Kluyveromyces lactis var. lactis*, *Kluyveromyces marxianus*, *Kluyveromyces marxianus var. lactis*, *Kluyveromyces marxianus var. marxianus*, *Kluyveromyces marxianus var. vanudenii*, *Kluyveromyces dobzhanskii*, *Kluyveromyces aestuarii*, *Kluyveromyces nonfermentans*, or *Kluyveromyces* Wickheim. Kluyveromyces wickerhamii), Kluyveromyces thermomotolerans, Kluyveromyces fragilis, Kluyveromyces hubeiensis, Kluyveromyces polysporus, Kluyveromyces siamensis, Kluyveromyces yarrowii, etc., or combinations thereof; References include, but are not limited to, the following: EP1197560A1, "Marc-André Lachance. The Yeasts (Fifth Edition), Chapter 35, Kluyveromyces van der Walt (1971). 2011, Pages 471-481", "JL Souciet, B Dujon, C Gaillardin, M Johnston et al. Comparative genomics of protoploid Saccharomycetaceae[J]. Genome Res. 2009, 19: 1696-1709.

[0298] Kluyveromyces is an ascospore-forming yeast, among which Kluyveromyces marxianus and Kluyveromyces lactis are widely used industrially. Compared with other yeasts, Kluyveromyces lactis has many advantages, such as superior secretion capacity, better large-scale fermentation characteristics, higher food safety levels, and the ability to perform post-translational protein modifications. The genome of wild-type Kluyveromyces lactis does not contain the gene encoding T7 RNA polymerase.

[0299] In one preferred embodiment, the cell extract is derived from Kluyveromyces lactis and endogenously integrates any one or a combination of the following gene sequences: the gene encoding RNA polymerase, or the gene encoding DNA polymerase. Preferably, the endogenous integration occurs through integration into a free intracellular plasmid or into the cellular genome.

[0300] In one preferred embodiment, the cell extract is derived from Kluyveromyces lactis and endogenously integrates any one or a combination of the following gene sequences: the encoding gene for T7 RNA polymerase and the encoding gene for phi29 DNA polymerase. Preferably, the endogenous integration occurs either by integration into a free intracellular plasmid or by integration into the cellular genome.

[0301] In one preferred embodiment of the cell extract of the present invention, the cell extract may be selected from any of the following sources: yeast cells, mammalian cells, plant cells, insect cells, and combinations thereof. The yeast cells are more preferably Kluyveromyces, Saccharomyces cerevisiae, Pichia pastoris, or combinations thereof; the Kluyveromyces is further preferably Kluyveromyces lactis var. drosophilarum, Kluyveromyces lactis var. lactis, Kluyveromyces marxianus var. lactis, Kluyveromyces marxianus var. marxianus, Kluyveromyces marxianus var. vanudenii, Kluyveromyces dobbit, non-fermenting Kluyveromyces, Kluyveromyces var. salina, Kluyveromyces wickheim, Kluyveromyces thermostable, Kluyveromyces brittlewall, Kluyveromyces hubeiensis, Kluyveromyces polysporus, Kluyveromyces siamensis, Kluyveromyces yarrowensis, or combinations thereof.

[0302] In another preferred embodiment, the cell extract is a yeast cell extract, more preferably a Kluyveromyces cell extract, even more preferably a Kluyveromyces marxosa cell extract or a Kluyveromyces lactis cell extract.

[0303] In another preferred embodiment, the cell extract may be selected from any of the following sources: Kluyveromyces lactis, wheat germ cells, Spodoptera frugiperda cells (sf cells, an insect cell), rabbit reticulocytes, Chinese hamster ovary cells (CHO cells), African green monkey kidney COS cells, African green monkey kidney VERO cells, young hamster kidney cells (BHK cells), human fibrosarcoma HT1080 cells, and combinations thereof.

[0304] 1.1.3. Exogenous RNA polymerase, exogenous DNA polymerase

[0305] When the genome of the cells from which the cell extract is derived does not contain the gene for RNA polymerase, nor does it have the coding sequence / gene for endogenous integrative RNA polymerase, exogenous RNA polymerase is usually required to promote the reaction. For example, when using cell extracts from wild-type Kluyveromyces lactis, the cell extract prepared from the wild-type Kluyveromyces lactis strain cannot recognize the T7 promoter.

[0306] Adding exogenous RNA polymerase to in vitro protein synthesis systems is a traditional technique. Existing in vitro protein synthesis systems with added exogenous RNA polymerases reported in the art are all included in this invention as optional methods for the CFPS(MgAsP-) system of this invention. The CFPS(MgAsP-) system refers to the system composed of components other than magnesium aspartate in the CFPS(Mg+) system. For example, the Kluyveromyces lactis in vitro protein synthesis system with added exogenous RNA polymerase (such as T7 RNA polymerase) described in CN108535489A is also included in this invention as an optional method for CFPS(MgAsP-).

[0307] The CFPS(Mg+) system may also include at least one of the following components: exogenous RNA polymerase, exogenous nucleic acid template encoding RNA polymerase, exogenous DNA polymerase, and exogenous nucleic acid template encoding DNA polymerase.

[0308] In one preferred embodiment, the CFPS(Mg+) system includes exogenous RNA polymerase and exogenous DNA polymerase.

[0309] In one preferred embodiment, the CFPS(Mg+) system includes exogenous T7 RNA polymerase and exogenous phi29 DNA polymerase.

[0310] You can add exogenous RNA polymerase directly, or you can add an exogenous nucleic acid template encoding RNA polymerase, or a combination thereof. The gene encoding RNA polymerase can be constructed together with the nucleic acid template encoding the exogenous protein, or it can be constructed separately in a separate exogenous nucleic acid template.

[0311] Similarly, DNA polymerase can be added directly, or by adding a foreign nucleic acid template containing its encoding gene, or by adding a combination thereof. It can be a nucleic acid template encoding a foreign protein, or a standalone foreign nucleic acid template.

[0312] When the nucleic acid template encoding the exogenous protein is a DNA template, the DNA amplification process may or may not be included. If the in vitro protein synthesis reaction also includes a DNA amplification process, especially when the amount of DNA template is insufficient, the system needs to contain endogenously expressed and / or exogenously added DNA polymerase, such as the exogenous phi 29 DNA polymerase added in CN108642076A. In Examples 1-6 of the present invention, after the DNA encoding the exogenous protein mEGFP is amplified in vitro, the amplification product is added to the reaction system as an exogenous DNA template, and the in vitro protein synthesis reaction no longer needs to include a DNA amplification process. When DNA polymerase is added to the system, that is, when the in vitro reaction process includes a DNA amplification process, a substrate for DNA synthesis usually also needs to be added.

[0313] DNA polymerases can be polymerases derived from eukaryotes or prokaryotes. Examples of eukaryotic polymerases include any one or any combination thereof: pol-α, pol-β, pol-δ, pol-ε, etc., any of the aforementioned fragments, and variants of any of the aforementioned fragments (including variants of any of the aforementioned fragments). Examples of prokaryotic polymerases include any one or any combination thereof: E. coli DNA polymerase I (e.g., the Klenow fragment), E. coli DNA polymerase II, E. coli DNA polymerase III, E. coli DNA polymerase IV, E. coli DNA polymerase V, bacteriophage T4 DNA polymerase, Bacillus stearothermophilus polymerase I, Phi29 DNA polymerase, T7 DNA polymerase, Bacillus subtilis Pol I, Staphylococcus aureus Pol I, etc., any domain of any of the foregoing, any subunit or fragment of any of the foregoing, and any variant of any of the foregoing (including variants of any of the foregoing fragments). The variants include, but are not limited to, mutants.

[0314] The polymerase (exogenous RNA polymerase, exogenous DNA polymerase) is preferably a polymerase capable of room-temperature amplification, wherein room temperature is preferably room temperature to 37°C, specifically, preferably 20–37°C, more preferably 25–37°C. The room-temperature amplification polymerase can be selected based on the exogenous nucleic acid template; room-temperature amplification polymerases suitable for use in cell-free in vitro systems are included in the scope of this invention, including but not limited to phi29 DNA polymerase, T4 DNA polymerase, T7 DNA polymerase, exo-klenow DNA polymerase, Bsu DNA polymerase, Pol III DNA polymerase, T7 RNA polymerase, T3 RNA polymerase, T4 RNA polymerase, T5 RNA polymerase, etc., any partial domain of any of the aforementioned polymerases, any subunit or fragment of any of the aforementioned polymerases, any variant of any of the aforementioned polymerases, and any combination of the aforementioned polymerases and their partial domains, subunits, fragments, variants (including but not limited to mutants). This invention may also employ other DNA polymerases such as Taq DNA polymerase, Pfu DNA polymerase, Pol I DNA polymerase, and Pol II DNA polymerase.

[0315] In some preferred embodiments, the DNA polymerase has strand displacement function.

[0316] In some preferred embodiments, the DNA polymerase lacks 3'-5' exonuclease activity.

[0317] The amplification techniques that can be used in this invention, especially room temperature amplification methods, are not particularly limited. Room temperature amplification techniques that can be used in cell-free in vitro systems are all included in the scope of this invention for reference.

[0318] 1.1.4. Energy System / Energy Regeneration System

[0319] Energy systems / energy regeneration systems are used to provide the energy required for protein synthesis.

[0320] The energy systems / energy regeneration systems previously reported for in vitro cell-free protein synthesis systems can all provide energy for the in vitro protein synthesis of this invention. Including but not limited to literature: CN109988801A, CN2018116198186, CN2018116198190, US20130316397A, US20150376673A, "MJ Anderson, JC Stark, CE Hodgman and MC Jewett. Energizing eukaryotic cell-free protein synthesis with glucose metabolism[J].FEBS Letters,2015,589(15):1723-1727","Y Lu.Advances inCell-Free Biosynthetic Technology[J].Current Developments in Biotechnologyand Bioengineering,2019,Chapter 2,23-45","P Shrestha,MT Smith and BCBundy.Cell-free unnatural amino acid incorporation with alternative energysystems and linear expression templates[J].New Energy systems / energy regeneration systems reported in literature such as Biotechnology, 2014, 31(1):28-34, and other directly or indirectly cited literature are all incorporated herein by reference.

[0321] In one preferred embodiment, the energy system is a sugar (e.g., monosaccharide, disaccharide, oligosaccharide, or polysaccharide) and phosphate energy system, a sugar and phosphocreatine energy system, a phosphocreatine and phosphocreatine kinase system, a glycolysis pathway and its intermediate product energy system (monosaccharide and its glycolysis intermediate product energy system, glycogen and its glycolysis intermediate product energy system), or a combination thereof. Specifically, the phosphate refers to inorganic phosphate, preferably orthophosphate, dihydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, or a combination thereof. The polysaccharide can be selected from, but is not limited to, starch, glycogen, dextrin (e.g., maltodextrin, corn dextrin, cyclodextrin), etc. The disaccharide is, for example, sucrose, maltose, etc. The monosaccharide can be a hexose or a pentose. Examples of monosaccharides are glucose, mannose, lactose, etc. The glycolysis pathway and its intermediate product energy system includes, but is not limited to, a glucose-based energy system.

[0322] In one preferred embodiment, the energy system is a sugar and phosphate energy system. Depending on the strain of the cell extract used, the sugar may be selected from, but is not limited to, glucose, fucose, mannose, galactose, lactose, xylose, arabinose, sucrose, maltose, starch, glycogen, dextrin (such as maltodextrin, corn dextrin, cyclodextrin, and any combination thereof).

[0323] There are no particular restrictions on the concentration of each component in the energy system, including but not limited to existing reported technical solutions and their equivalents. Examples 1-6 use a composite energy system composed of glucose, maltodextrin or corn dextrin, and potassium phosphate.

[0324] 1.1.5. Substrates for RNA synthesis

[0325] The substrate for synthesizing RNA refers to a raw material capable of providing structural units of RNA. The substrate for synthesizing RNA is preferably a mixture of nucleotides. In one embodiment, the substrate for synthesizing RNA is a nucleoside monophosphate, a nucleoside triphosphate, or a combination thereof. The substrate for synthesizing RNA is preferably a mixture of nucleoside triphosphates (NTPs). The nucleoside triphosphate mixture is preferably a mixture of adenine nucleoside triphosphate, guanine nucleoside triphosphate, cytosine nucleoside triphosphate, and / or uracil nucleoside triphosphate; more preferably a mixture of the aforementioned four nucleoside triphosphates. In this invention, the concentration of each mononucleotide is not particularly limited, and is measured according to the nucleotides required for protein synthesis. In one preferred embodiment, the concentration of each mononucleotide is 0.5–5 mM; in another preferred embodiment, it is 1.0–2.0 mM. Each mononucleotide concentration is independently exemplified by any of the following concentrations, or a concentration range between any two of the following concentration values ​​(the concentration range includes both endpoints): 0.5 mM, 1.0 mM, 1.5 mM, 2.0 mM, 2.5 mM, 3.0 mM, 3.5 mM, 4.0 mM, 4.5 mM, 5.0 mM, 5.5 mM, 6.0 mM. The above concentrations refer to the initial concentrations in the in vitro protein synthesis reaction mixture.

[0326] 1.1.6 Substrates for DNA Synthesis

[0327] When DNA amplification or in vitro protein synthesis reactions involve DNA replication, a substrate for DNA synthesis is usually added. The substrate for DNA synthesis refers to a raw material capable of providing the structural units of DNA. Preferably, the substrate for DNA synthesis is a mixture of deoxynucleotides, and more preferably, a mixture of deoxynucleoside triphosphates (dNTPs).

[0328] When the CFPS(Mg+) system contains DNA polymerase, it preferably also contains a substrate for DNA synthesis.

[0329] 1.1.7. Substrates for protein synthesis

[0330] The substrate for synthesizing the protein refers to the raw materials that can provide the amino acid units that constitute the protein. The substrate for synthesizing the protein is preferably a mixture of amino acids. Measurements are taken based on the amino acids required for protein synthesis.

[0331] The substrate raw materials for the synthesized protein are supplied with different kinds of amino acids, wherein the amounts of any two amino acids can be independently the same or different from each other.

[0332] The concentration of each amino acid, independently and typically, is 0.01–5 mM in one preferred embodiment and 0.1–1 mM in another preferred embodiment. Examples of the concentrations of each amino acid independently include any of the following concentrations, or concentration ranges between any two of the following concentration values ​​(the concentration range includes both endpoints): 0.1 mM, 0.2 mM, 0.4 mM, 0.5 mM, 1.0 mM, 1.2 mM, 1.5 mM, 1.8 mM, 2.0 mM, 2.5 mM, 3.0 mM, 3.5 mM, 4.0 mM, 4.5 mM, 5.0 mM, 5.5 mM, and 6.0 mM. The above concentrations refer to the initial concentrations in the in vitro protein synthesis reaction mixture.

[0333] The amino acid mixture includes at least the amino acid mixture required for the synthesis of the exogenous protein, selected from, but not limited to: glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, histidine, and combinations thereof. One preferred embodiment is a mixture of the aforementioned twenty amino acids. The amino acid mixture required for the synthesis of the exogenous protein includes not only the amino acids constituting the primary sequence of the exogenous protein but may also include other amino acids involved in the synthesis process.

[0334] The amino acid mixture may include natural amino acids and non-natural amino acids.

[0335] The amino acid mixture may include L -amino acids, D - Amino acids and their combinations.

[0336] In addition to natural amino acids, the amino acid mixture may also include non-natural amino acids. D- Components including amino acids, radioisotope-labeled amino acids, and modified amino acids. The non-natural amino acids are not particularly limited and can be selected from, but are not limited to, those reported or cited in the following literature: “Y Lu. Cell-free synthetic biology: Engineering in an open world[J]. Synthetic and Systems Biotechnology, 2017, 2, 23-27”, “W Gao, E Cho, Y Liu and Y Lu. Advances and challenges in cell-free incorporation of unnatural amino acids into proteins[J]. Frontiers in pharmacology, 2019, 10: 611”, and other directly or indirectly cited literature. The radioisotope-labeled amino acids are not particularly limited and include, but are not limited to, isotope labeling used in the field of protein synthesis as reported. The modified amino acids are not particularly limited and include, but are not limited to, modifications via amino acid side groups.

[0337] Preferably, the amino acid mixture is a mixture of natural amino acids.

[0338] In one preferred embodiment, the amino acid mixture is a mixture of twenty natural amino acids.

[0339] 1.1.8. Other additive components

[0340] The CFPS(Mg+) system may further include at least one of the following exogenous additives: other soluble amino acid salts, translation-related elements, DNA amplification-related elements, RNA amplification-related elements, RNase inhibitors, crowding agents (preferably polyethylene glycol and / or its analogues), potassium ions, antioxidants or reducing agents, antifreeze agents, trehalose, reaction promoters, defoamers, alkanes, buffers, and aqueous solvents. See references WO2016005982A1, US20060211083A1, and "LKai, V". R Kaldenhoff and F Bernhard. Artificial environments for the co-translational stabilization of cell-free expressed proteins[J]. PloS one,2013,8(2):e56637”, US20030119091A1, US20180245087A1, US5665563, WO2019033095A1, US9410170B2, US9528137B2 and other literature and their direct or indirect citations.

[0341] The other soluble amino acid salts mentioned do not include magnesium aspartate. The cations may include, but are not limited to, magnesium ions, potassium ions, sodium ions, zinc ions, calcium ions, ammonium ions, etc., and the amino acid residues may be selected from any amino acid (natural, non-natural, derivatives, etc.) and combinations thereof described in this invention. Other soluble amino acid salts include potassium aspartate, sodium aspartate, monosodium glutamate, etc.

[0342] The necessary components for the translation of exogenous proteins can also be provided or supplemented by adding exogenous translation-related elements. These translation-related elements are preferably selected from: tRNA, ribosomes, other translation-related enzymes, initiation factors, elongation factors, termination factors, and combinations thereof. The translation-related elements are preferably purified translation-related elements.

[0343] When protein synthesis involves DNA amplification, in addition to endogenous provision, DNA amplification-related elements can also be added exogenously. These DNA amplification-related elements, besides DNA polymerases, may also include other factors such as helicases (HDA amplification), recombinases, and single-stranded DNA-binding proteins (RPA amplification), depending on the specific amplification mechanism.

[0344] When protein synthesis involves RNA amplification, in addition to endogenous provision, RNA amplification-related elements can also be added exogenously.

[0345] The RNA inhibitor can stabilize RNA.

[0346] In some preferred embodiments, the CFPS(Mg+) system also contains crowding agents to mimic the crowded macromolecular environment within cells. The structure of the crowding agents is not particularly limited and can be linear or nonlinear. Nonlinear structures include, but are not limited to, branched, multi-armed, cyclic, comb-like, dendritic, and star-shaped structures. In some preferred embodiments, the crowding agents can be selected from the group consisting of: polyethylene glycol, polyvinyl alcohol (PVA), polystyrene, dextran, sucrose polymers (such as Ficoll sucrose polymers, such as polysucrose, such as Ficoll-400), poly(vinylpyrrolidone) (PVP), albumin, etc., and any combination thereof. The albumin sources include, but are not limited to: human serum albumin, bovine serum albumin, porcine serum albumin, and combinations thereof; preferably, the albumin is human serum albumin. The crowding agent may also refer to the crowding agents disclosed in the following literature: "X Ge, D Luo and J Xu. Cell-free protein expression under macromolecular crowding conditions[J]. PLoS One, 2011, 6(12):e28707" and its cited references. In some preferred embodiments, the concentration of the crowding agent in the in vitro protein synthesis reaction mixture is sufficient to increase the amount of protein synthesized.

[0347] In some preferred embodiments, the molecular weight of the crowding agent does not exceed 400 kDa. In some preferred embodiments, the molecular weight of the crowding agent does not exceed 200 kDa. Typically, the molecular weight specification preferably has a molecular weight distribution of ±10% or narrower. In one preferred manner, the amount of the crowding agent, expressed as a weight percentage (wt%), volume percentage (%(v / v)), or mass-volume concentration (%(w / v)) of the crowding agent in the in vitro protein synthesis reaction mixture, is selected from 0.5% to 15%, more preferably from 1% to 12%; for example, any of the following concentration values, or a concentration range between any two of the following concentration values ​​(the concentration range includes both endpoints): 1.0 %, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0%, 10.5%, 11.0%, 11.5%, 12.0%, 12.5%, 13.0%, 13.5%, 14.0%, 14.5%, 15.0%.

[0348] In one preferred embodiment, the CFPS(Mg+) system further contains polyethylene glycol and / or its analogues as a congesting agent. Polyethylene glycol, for example, can also adjust the viscosity of the system. Polyethylene glycol, having repeating CH2CH2O units (EO units), is commonly abbreviated as PEG (polyethylene glycol), PEO (poly(ethylene oxide)), or POE (polyoxyethylene). The polyethylene glycol analogues include, but are not limited to, copolymers rich in EO units, polyethylene glycol derivatives, other polyoxyolefins that can act as congesting agents (e.g., polyoxypropylene, POP), and derivatives of these other polyoxyolefins; the derivatives, taking polyethylene glycol derivatives as an example, include, but are not limited to, chemical modifiers (e.g., methoxy polyethylene glycol, amino modifiers, carboxyl modifiers, etc.), amino acid modifiers, peptide modifiers, protein modifiers, block polymers containing polyethylene glycol blocks, and polymers containing polyethylene glycol side chains. There is no particular limitation on the concentration of polyethylene glycol or its analogues. Typically, the concentration of polyethylene glycol or its analogues is 0.1% to 10%, preferably 0.1% to 8%, more preferably 0.5% to 4%, and even more preferably 1% to 2%, expressed as a mass-volume concentration (% (w / v)) in the in vitro protein synthesis reaction mixture or as a total weight (wt%). Unless otherwise specified, all values ​​in this invention refer to mass-volume concentration, expressed in % (w / v), such as 2%, which means 2% (w / v), corresponding to 2 g / 100 mL or 20 mg / mL. In some preferred embodiments, the molecular weight of polyethylene glycol and / or its analogues does not exceed 40,000 Da. Representative molecular weights include any one of the following molecular weights or a range between any two of the following molecular weights (including both endpoints): 200, 400, 500, 600, 800, 1000, 1200, 1400, 1450, 1500, 1600, 1800, 2000, 2500, 3000, 3350, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 12000, 14000, 15000, 16000, 18000, 20000, 25000, 30000, 35000, 40000; unit Da; each of the above numbers is numerically equal to the weight-average molecular weight or the number-average molecular weight. Typically, the molecular weight specification preferably has a molecular weight distribution of ±10% or narrower. The polyethylene glycol and / or its analogues preferably have a molecular weight of 200 Da to 10000 Da, more preferably 3000 Da to 10000 Da. Another preferred option is 200 Da to 8000 Da. Another preferred method is 3000Da to 8000Da.In this invention, unless otherwise specified, the molecular weight of polyethylene glycol or its analogues refers to the weight-average molecular weight M. w Representative PEGs are selected from the following groups: PEG200, PEG400, PEG1000, PEG1500, PEG2000, PEG3000, PEG3350, PEG5000, PEG6000, PEG8000, PEG10000, etc., and combinations thereof; among them, the numbers such as 3350 are numerically equal to the weight-average molecular weight.

[0349] The potassium ions are derived from a potassium ion source, which is not particularly limited. The potassium ion source may be selected from the group consisting of: potassium acetate, potassium glutamate (preferably L-glutamate), potassium chloride, potassium phosphate, potassium sulfate, potassium citrate, potassium hydrogen phosphate, potassium iodide, potassium lactate, potassium nitrate, potassium oxalate, or combinations thereof. One preferred embodiment has a concentration range of 0–500 mM. Another preferred embodiment has a concentration range of 1–250 mM. Another preferred embodiment has a concentration range of 5–200 mM. Another preferred embodiment has a concentration range of 10–100 mM. In one preferred embodiment, the potassium ion source is selected from any one, two, or all of potassium aspartate, potassium glutamate, and potassium acetate.

[0350] The optimization effects and preferred methods of polyethylene glycol, magnesium ions, and potassium ions reported in patent document WO2016005982A1 are incorporated into this invention for reference.

[0351] The antioxidant, also known as a reducing agent, may include, but is not limited to, dithiothreitol (DTT), 2-mercaptoethanesulfonic acid, 2-mercaptoethanol, reduced glutathione (GSH), tricarboxymethyl phosphate (TCEP), 3-mercapto-1,2-propanediol (MPD), etc. One preferred embodiment is dithiothreitol. DTT can be used at its conventional concentration, such as 0.5–10 mM in one embodiment; another embodiment uses a concentration of 0–1.7 mM.

[0352] The cryoprotectant may be selected from, but is not limited to, cryoprotectants for solid dosage forms as described in patent document WO2018138195A1 and its cited references. For example, trehalose. The purpose of adding the cryoprotectant is primarily to enable low-temperature storage of the entire system or a portion of its dispensed components, especially when stored in kit form. The added cryoprotectant may also have the function of regulating in vitro protein synthesis reactions.

[0353] Some antifreeze agents, including but not limited to trehalose, can also serve as components of energy systems.

[0354] The reaction promoter includes, but is not limited to, the reaction promoter provided in CN109971783A (such as aluminum salt). In one preferred embodiment, the reaction promoter is an aluminum salt, aluminum oxide (such as alumina), iron salt, iron oxide, calcium salt, or a combination thereof.

[0355] The defoamer, for example, is the defoamer provided in CN1934276A and its cited documents. Specific examples include, but are not limited to, alkyl polyoxyalkylene glycol ethers, esters, siloxanes, polysiloxanes, sulfites, sulfonates, fatty acids and their derivatives.

[0356] The alkane can serve to provide a hydrophobic interface or simulate a hydrophobic environment. The relevant content of patent application CN202010179689.4 is incorporated herein by reference. For example, C 6～44 Pure or mixed alkanes, further examples include cyclohexane, isooctane, decane, tetradecane, pentadecylcyclohexane, squalane, tetradecane, petrolatum, etc.

[0357] The buffer is primarily used to maintain the pH environment of the system. One preferred embodiment is selected from any one or a combination thereof: Tris-HCl, Tris base, or HEPES (4-hydroxyethylpiperazine ethanesulfonic acid system).

[0358] The aqueous solvent is preferably a buffer.

[0359] It should be noted that any component of the CFPS(Mg+) system involved in this invention may be added to the system for other functions or purposes besides those mentioned above.

[0360] Any component of the CFPS(Mg+) system involved in this invention is allowed to perform two or more functions. For example, some sugar components can function as components of an energy system, and are also allowed to function as functional components such as crowding agents and antifreeze agents.

[0361] 1.1.9. Example of specific implementation of the in vitro protein synthesis system containing exogenous magnesium ions (CFPS(Mg+) system)

[0362] In the following specific embodiments, the concentrations of each component are final concentrations (relative to the mother liquor), corresponding to the initial concentrations in the in vitro protein synthesis reaction mixture.

[0363] One preferred embodiment is that the CFPS(Mg+) system contains eukaryotic cell extract, magnesium aspartate, endogenously expressed RNA polymerase (included in the aforementioned eukaryotic cell extract) or exogenously added RNA polymerase, an energy system, a substrate for RNA synthesis, a substrate for protein synthesis, a congestant, potassium ions, and a buffer solution. Optionally, it may also include any of the following exogenous components: other exogenous magnesium ions, an exogenous nucleic acid template encoding RNA polymerase (independently preferably a DNA template), endogenously expressed DNA polymerase or exogenously added DNA polymerase, an exogenous nucleic acid template encoding DNA polymerase (independently preferably a DNA template), other DNA amplification-related elements, a substrate for DNA synthesis, other soluble amino acid salts, translation-related elements, RNA amplification-related elements, RNase inhibitors, antioxidants or reducing agents, antifreeze agents, trehalose, stress promoters, defoamers, alkanes, and aqueous solvents. The eukaryotic cell extract is preferably a yeast cell extract, more preferably a Kluyveromyces cerevisiae cell extract, and one of the more preferred methods is a Kluyveromyces lactis cell extract.

[0364] One preferred embodiment is that the CFPS(Mg+) system contains eukaryotic cell extract (cell source modified by strains, with the RNA polymerase encoding gene integrated into the cell genome or inserted into an intracellular free plasmid), magnesium aspartate (L-aspartate magnesium, D-aspartate magnesium, or a combination thereof), and one or more exogenous components selected from the group consisting of: potassium 4-hydroxyethylpiperazine ethanesulfonate (HEPES-K) or tris(hydroxymethyl)aminomethane (Tris), potassium acetate, and potassium glutamate (preferably L-glutamate). Potassium), potassium chloride, magnesium acetate, magnesium glutamate (preferably L-glutamate magnesium), a mixture of nucleoside triphosphates (NTPs), a mixture of amino acids, creatine phosphate, creatine phosphokinase, creatine phosphokinase, glucose, L-arabinose, sucrose, maltose, starch, glycogen, dextrin, corn dextrin, maltodextrin, cyclodextrin, phosphates (such as potassium phosphate), DNA amplification-related elements, a mixture of deoxynucleoside triphosphates, RNA amplification-related elements, RNase inhibitors, polyethylene glycol, dextran, sucrose polymers, and dithiothreitol (DTT). The eukaryotic cell extract is preferably a yeast cell extract, more preferably a Kluyveromyces cell extract, and one of the more preferred methods is a Kluyveromyces lactis cell extract.

[0365] One preferred embodiment is that the CFPS(Mg+) system contains eukaryotic cell extract, magnesium aspartate, and one or more exogenous components selected from the group consisting of: potassium 4-hydroxyethylpiperazine ethanesulfonate (HEPES-K) or tris(hydroxymethyl)aminomethane (Tris), potassium acetate, potassium glutamate (preferably L-glutamate), potassium chloride, magnesium acetate, magnesium glutamate (preferably L-glutamate), a mixture of nucleoside triphosphates (NTPs), a mixture of amino acids, creatine phosphate, creatine phosphokinase, creatine phosphokinase, glucose, L-arabinose, sucrose, maltose, starch, glycogen, dextrin, corn dextrin, maltodextrin, cyclodextrin, phosphates (such as potassium phosphate), RNase inhibitors, polyethylene glycol, dextran, sucrose polymers, dithiothreitol, exogenous T7 RNA polymerase, exogenous phi29 DNA polymerase, other DNA amplification-related elements, a mixture of deoxynucleoside triphosphates, and RNA amplification-related elements. The eukaryotic cell extract is preferably a yeast cell extract, more preferably a Kluyveromyces oryzae cell extract, and one of the more preferred methods is a Kluyveromyces lactis cell extract.

[0366] In one preferred embodiment, the CFPS(Mg+) system contains eukaryotic cell extracts (the source cells may optionally be modified by strains, optionally integrating the RNA polymerase encoding gene into the cell genome or inserting it into an intracellular free plasmid), magnesium aspartate, and one or more exogenous components selected from the group consisting of: potassium acetate, potassium glutamate (preferably L-glutamate), potassium chloride, magnesium acetate, magnesium glutamate (preferably L-glutamate), HEPES-K, Tris-HCl, a mixture of nucleoside triphosphates (NTPs), a mixture of amino acids, creatine phosphate, creatine phosphokinase, creatine phosphokinase, glucose, L-arabinose, sucrose, maltose, starch, glycogen, dextrin, corn dextrin, maltodextrin, cyclodextrin, potassium phosphate, RNase inhibitors, polyethylene glycol, dextran, sucrose polymers, dithiothreitol, trehalose, alumina accelerators, defoamers, alkanes, exogenous T7 RNA polymerase, exogenous phi29 DNA polymerase, and T7-encoding DNA polymerase. The DNA template for RNA polymerase, the DNA template encoding phi29 DNA polymerase, other DNA amplification-related elements, a mixture of deoxynucleoside triphosphates, and RNA amplification-related elements. The eukaryotic cell extract is preferably a yeast cell extract. The yeast cell extract is preferably Kluyveromyces oryzae cell extract, and more preferably Kluyveromyces lactis cell extract.

[0367] Another preferred embodiment is that the CFPS(Mg+) system contains eukaryotic cell extract, L-aspartate magnesium, and one or more exogenous components selected from the group consisting of: D-aspartate magnesium, Tris-HCl (pH 8.0), potassium acetate, potassium glutamate (preferably L-glutamate potassium), potassium chloride, magnesium acetate, magnesium glutamate (preferably L-glutamate magnesium), glucose, L-arabinose, sucrose, maltose, maltodextrin, corn dextrin, a mixture of nucleoside triphosphates (a mixture of four nucleoside triphosphates, wherein the concentration of each nucleoside triphosphate is the same), a mixture of amino acids (glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and / or histidine; preferably a mixture of twenty amino acids; wherein the concentration of each amino acid can be the same), potassium phosphate, exogenous T7 RNA polymerase, and exogenous phi29. DNA polymerase, other DNA amplification-related elements, a mixture of deoxyribonucleoside triphosphates, RNA amplification-related elements, RNase inhibitors, polyethylene glycol, dextran, sucrose polymers, and dithiothreitol. The eukaryotic cell extract is preferably a yeast cell extract. The yeast cell extract is preferably Kluyveromyces yeast extract, and more preferably Kluyveromyces lactis cell extract.

[0368] Specifically, one preferred embodiment is that the CFPS(Mg+) system contains 50%–80% (v / v) cell extract, 1.5–8 mM L-aspartic acid magnesium (more preferably 1.5–6 mM), and also contains one or more components selected from the group consisting of: 0–20 mM D-aspartic acid magnesium, 9.78 mM The following solutions are used: pH 8.0 Tris-HCl, 20–80 mM potassium acetate, 2–10 mM magnesium acetate, 0.5–5 mM four nucleoside triphosphates (each nucleoside triphosphate has the same concentration, e.g., 1.8 mM), 0.1–1 mM a mixture of twenty 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, each amino acid has the same concentration, e.g., 0.5 mM), 10–40 mM glucose, and 5–110 mM... L-arabinose, 200–400 mM maltodextrin (measured as glucose monomer, e.g., 320 mM corresponds to approximately 52 mg / mL), 10–40 mM potassium phosphate, 0.5%–5% (w / v) polyethylene glycol (e.g., 2% (w / v)), and 0.4–5 mM dithiothreitol (e.g., 0.44 mM). The eukaryotic cell extract is preferably a yeast cell extract. The yeast cell extract is preferably Kluyveromyces oryzae cell extract, and more preferably Kluyveromyces lactis cell extract.

[0369] One specific implementation of the CFPS(MgAsP-) system without the addition of magnesium aspartate includes, but is not limited to, in vitro cell-free protein synthesis systems based on wheat germ cells, rabbit reticulocytes, Saccharomyces cerevisiae, Pichia pastoris, and Kluyveromyces martensii, as described in other cited literature of this invention, including but not limited to those based on wheat germ cells, rabbit reticulocytes, Saccharomyces cerevisiae, Pichia pastoris, and Kluyveromyces martensii. These systems are all included in this invention as optional implementations of the CFPS(MgAsP-) system. For example, the in vitro cell-free protein synthesis systems described in the literature “Lu, Y. Advances in Cell-Free Biosynthetic Technology. Current Developments in Biotechnology and Bioengineering, 2019, Chapter 2, 23-45”, including but not limited to those cited in the “2.1 Systems and Advantages” section on pages 27-28, are all optional implementations of the CFPS(MgAsP-) system of this invention without the addition of magnesium aspartate. For example, documents CN106978349A, CN108535489A, CN108690139A, CN108949801A, CN108642076A, CN109022478A, CN109423496A, CN109423497A, CN109837293A, CN109971783A, CN109988801A, CN110551700A, CN109971775A, CN110551745A, CN110551700A, CN2018116083534, CN2018116198186, CN201811619819 The in vitro cell-free protein synthesis systems described in CN2019102128619, CN2019102355148, CN2019107298813, CN2019112066163, CN2018108881848, CN2018109550734, CN2018111131300, CN2018111423277, CN2018112862093, CN201911418151.8, CN202010069383.3, CN202010179689.4 and their cited references are all optional embodiments of the CFPS(MgAsP-) system of the present invention.

[0370] 1.2. Exogenous Proteins

[0371] There are no particular limitations on the exogenous proteins applicable to the CFPS(Mg+) system described in this invention, as long as they can be synthesized in vitro based on cell extracts (preferably eukaryotic cell extracts, particularly yeast cell extracts, and more particularly Kluyveromyces lactis cell extracts). Exogenous proteins already disclosed in the prior art that are applicable to in vitro protein synthesis systems derived from eukaryotic cell extracts, yeast cell extracts, or Kluyveromyces lactis, or endogenous proteins that are applicable to intracellular synthesis in eukaryotic cell systems (preferably yeast cell systems, more preferably Kluyveromyces lactis systems), can also be synthesized using the system of this invention, or attempts can be made to synthesize them using the in vitro protein synthesis system provided by this invention.

[0372] The application fields of the exogenous protein include, but are not limited to, biomedicine, molecular biology, medicine, in vitro detection, medical diagnosis, regenerative medicine, bioengineering, tissue engineering, stem cell engineering, genetic engineering, polymer engineering, surface engineering, nanoengineering, cosmetics, food, food additives, nutrients, agriculture, feed, daily necessities, detergents, environment, chemical staining, and fluorescent labeling.

[0373] The exogenous protein can be a natural protein or its modified product, or it can be an artificially synthesized sequence. There are no particular restrictions on the source of the natural protein, including but not limited to: eukaryotic cells and prokaryotic cells; wherein the eukaryotic cell sources include but are not limited to: mammalian cells, plant cells, yeast cells, insect cells, nematode cells, and combinations thereof; the mammalian cell sources include but are not limited to mouse, rabbit, monkey, human, pig, sheep, and bovine sources.

[0374] The types of exogenous proteins include, but are not limited to, polypeptides (in this invention, "exogenous protein" broadly includes polypeptides), fluorescent proteins, enzymes and their corresponding zymogens, antibodies and their fragments, antigens, immunoglobulins, hormones, collagen, polyamino acids, vaccines, etc., as well as partial domains of any of the aforementioned proteins, subunits or fragments of any of the aforementioned proteins, and variants of any of the aforementioned proteins. The phrase "subunits or fragments of any of the aforementioned proteins" includes subunits or fragments of "partial domains of any of the aforementioned proteins." The phrase "variants of any of the aforementioned proteins" includes variants of "partial domains of any of the aforementioned proteins, subunits or fragments of any of the aforementioned proteins." The phrase "variants of any of the aforementioned proteins" includes, but is not limited to, mutants of any of the aforementioned proteins. In this invention, the meaning of two or more consecutive instances of "as described above" in other positions is interpreted similarly.

[0375] The structure of exogenous proteins can be a complete structure or selected from corresponding partial domains, subunits, fragments, dimers, multimers, fusion proteins, glycoproteins, etc. For example, nanobodies (heavy chain antibodies lacking light chains) are incomplete antibody structures.

[0376] For example, the exogenous proteins synthesizable by the CFPS(Mg+) system of the present invention can be selected from, but are not limited to, any one of the following proteins, fusion proteins in any combination, or mixtures in any combination: luciferase (e.g., firefly luciferase), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), yellow fluorescent protein (YFP), aminoacyl-tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase (e.g., mouse catalase), actin, variable regions of antibodies (e.g., single-chain variable regions of antibodies, scFV), single chains of antibodies and their fragments (e.g., heavy chains of antibodies, light chains of antibodies), α-amylase, enterotoxin A, hepatitis C virus E2 glycoprotein, insulin, and... The precursors, glucagon-like peptide-1 (GLP-1), interferons (including but not limited to interferon α, such as interferon αA, interferon β, interferon γ, etc.), interleukins (such as interleukin-1β, interleukin 2, interleukin 12, etc.), lysozyme, serum albumin (including but not limited to human serum albumin, bovine serum albumin), thyroxine transporter protein, tyrosinase, xylanase, β-galactosidase (LacZ, for example, Escherichia coli β-galactosidase), etc., partial domains of any of the aforementioned proteins, subunits or fragments of any of the aforementioned proteins, or variants of any of the aforementioned proteins (as defined above, the variants include mutants, for example, luciferase mutants, eGFP mutants). The aminoacyl-tRNA synthetase, for example, human lysine-tRNA synthetase, human leucine-tRNA synthetase, etc. The glyceraldehyde-3-phosphate dehydrogenase, for example, Arabidopsis thaliana glyceraldehyde-3-phosphate dehydrogenase. See also patent document CN109423496A. The mixture of any combination may include any of the aforementioned proteins, or may include fusion proteins of any of the aforementioned combinations.

[0377] In one preferred embodiment, the protein synthesis capacity of the CFPS(Mg+) system is evaluated using exogenous proteins with fluorescent properties, such as GFP, eGFP, similar substances, or mutants thereof.

[0378] 1.3. Exogenous nucleic acid templates (including nucleic acid templates encoding exogenous proteins)

[0379] Unless otherwise specified, the exogenous nucleic acid template of the present invention specifically refers to a nucleic acid template encoding an exogenous protein. Furthermore, when specified, the exogenous nucleic acid template of the present invention may also include a nucleic acid template encoding a protein factor or protease required for in vitro protein synthesis, such as an exogenous nucleic acid template encoding RNA polymerase or an exogenous nucleic acid template encoding DNA polymerase.

[0380] If there is no nucleic acid template encoding a foreign protein in the synthesis system, the in vitro synthesis reaction of the foreign protein cannot proceed.

[0381] In any embodiment of the present invention, the nucleic acid template encoding the foreign protein can be independently a DNA template, an mRNA template, or a combination thereof.

[0382] In any embodiment of the present invention, the nucleic acid template encoding the foreign protein can be independently preferred to be a DNA template.

[0383] The nucleic acid template encoding the foreign protein serves as a direct template (mRNA), an indirect template (DNA), or a combination thereof for synthesizing the foreign protein.

[0384] The nucleic acid template encoding the foreign protein may include non-coding regions. The expression product may be a polypeptide or a protein, or a fusion protein. One translation (or transcription-translation) process is completed for one nucleic acid template molecule, and the number of polypeptide or protein molecules that can be synthesized may be one, two, or more.

[0385] Protein synthesis via transcription and translation uses a DNA template as an indirect template, while protein synthesis via translation alone can use an mRNA template as a direct template.

[0386] Preferably, the CFPS(Mg+) system of the present invention is an in vitro transcription and translation system, i.e., an IVTT system, which uses a DNA template as a nucleic acid template encoding a foreign protein.

[0387] The nucleic acid template encoding the foreign protein contains translation-related elements required for the synthesis of the foreign protein.

[0388] In any embodiment of the present invention, preferably independently, the nucleic acid template encoding the exogenous protein further contains a promoter element that can be recognized by the cell extract.

[0389] In one preferred embodiment, the nucleic acid template encoding the exogenous protein contains a promoter element that the cell extract can recognize.

[0390] In one preferred embodiment, the nucleic acid template encoding the foreign protein contains a T7 promoter capable of initiating the gene transcription program of the foreign protein, that is, the gene transcription process of the foreign protein is initiated by the T7 promoter on the nucleic acid template.

[0391] In one preferred embodiment, the nucleic acid template encoding the exogenous protein contains a T7 promoter capable of initiating the gene transcription program of the exogenous protein (at this time, the T7 promoter is located upstream of the coding sequence of the exogenous protein in the nucleic acid template, and the gene transcription program of the exogenous protein is initiated by the T7 promoter), and the cell extract in the CFPS(Mg+) system contains endogenously expressed T7 RNA polymerase.

[0392] In one preferred embodiment, the nucleic acid template encoding the exogenous protein includes an exogenous protein translation system, an resistance gene translation system, and a lac repressor translation system; each of the above translation systems includes a corresponding promoter.

[0393] In one preferred embodiment, the nucleic acid template encoding the foreign protein further contains a gene that controls the copy number of the plasmid.

[0394] In one preferred embodiment, the nucleic acid template encoding the foreign protein further contains a transcriptional enhancement element, such as a kozak sequence.

[0395] In one preferred embodiment, the nucleic acid template encoding the exogenous protein further contains translation enhancement elements, such as translation enhancer elements, IRES elements, kozak sequences, etc.

[0396] 1.3.1. Exogenous DNA template (including DNA templates of exogenous proteins)

[0397] Unless otherwise specified, the exogenous DNA template of this invention refers specifically to the DNA template encoding the exogenous protein.

[0398] The exogenous DNA template of this invention can be DNA, cDNA, methylated DNA, or a combination thereof. cDNA can be obtained by reverse transcription of RNA or miRNA. miRNA (microRNA) is a class of non-coding single-stranded RNA molecules approximately 20-25 nucleotides in length encoded by endogenous genes.

[0399] The DNA template encoding the foreign protein contains the coding sequence of the foreign protein.

[0400] Preferably, the DNA template encoding the foreign protein contains the gene encoding the foreign protein.

[0401] The DNA template encoding the foreign protein is determined based on the foreign protein.

[0402] The DNA template encoding the exogenous protein may also contain other functional elements selected from promoters, terminators, enhancers (e.g., enhancer elements described in CN109423497A, CN109022478A, CN109837293A (CN201711194355.9), CN109971775A, etc., such as Ω sequences and their homologous sequences, combined enhancer elements), kozak sequences (refer to CN109022478A, CN109837293A, CN109971775A, etc., and their cited references), IRES elements (internal ribosome entry sequences, refer to CN109022478A, CN109423497A, etc., and their cited references), multiple cloning sites (MCS), genes controlling plasmid copy number, etc. It may also contain coding sequences / genes for other amino acid chains, such as signal sequences, leader sequences, functional tags (e.g., purification tags, solubilization tags), and linker peptides. It may also contain 5' untranslated sequences and 3' untranslated sequences. Solubilization tags directly or indirectly disclosed in patent application CN201911204796.1 and its cited references are also incorporated herein by reference.

[0403] The DNA template encoding the exogenous protein preferably contains a promoter element. The promoter element is required to be recognizable by the cell extract used or by other components of the CFPS(Mg+) system; it can be a promoter recognizable by the wild-type cell extract, or the source strain of the cell extract can be modified to recognize the promoter. The promoter in the DNA template of this invention can be selected from the following group: AOD1, MOX, AUG1, AOX1, GAP, FLD1, PEX8, YPT1, LAC4, PGK, ADH4, AMY1, GAM1, XYL1, XPR2, TEF, RPS7, T7, or combinations thereof. References include, but are not limited to, the following literature and its cited references: “Cereghino G. Applications of yeast in biotechnology: protein production and genetic analysis. Current Opinion in Biotechnology, 1999, 10(5), 422-427.”

[0404] In Examples 1-6, the transcription process of the exogenous protein was initiated by the T7 promoter using the exogenous DNA template; the T7 promoter is a strong promoter that can specifically respond to T7 RNA polymerase.

[0405] Preferably, the exogenous DNA template contains a T7 promoter capable of initiating gene transcription of the exogenous protein.

[0406] The concentration of the exogenous DNA template is selected based on the amount of exogenous protein to be expressed in the experimental protocol. One preferred embodiment is a concentration of 1–400 ng / μL for the exogenous DNA template. Another preferred embodiment is a concentration of 1–80 ng / μL for the exogenous DNA template. Yet another preferred embodiment is a concentration of 5–50 ng / μL for the exogenous DNA template. In this invention, unless otherwise specified, the concentration of the added DNA template is the final concentration, referring to the initial concentration in the in vitro protein synthesis reaction mixture.

[0407] The exogenous DNA template can be circular DNA or linear DNA. It can be single-stranded or double-stranded. The gene encoding the exogenous protein can be selected from, but is not limited to, genomic sequences, cDNA sequences, and combinations thereof. The exogenous DNA template may also contain a promoter sequence, a 5' untranslated sequence, and a 3' untranslated sequence.

[0408] In one preferred embodiment, the exogenous DNA template further includes any one or a combination of elements selected from the group consisting of: promoters, terminators, poly(A) elements, transport elements, gene targeting elements, selection marker genes, enhancers, IRES elements, Kozak sequences, resistance genes, transposase-encoding genes, signal sequences, leading sequences (e.g., the leading sequences described and cited in CN109022478A), genes controlling plasmid copy number (rop genes), tags that enhance translation (e.g., polypeptide tags described in CN2019112066163), and other functional tags (e.g., purification tags, fluorescent tags, solubilization tags, etc.). See also US20060211083A1, etc.

[0409] The exogenous DNA template can also be constructed in an expression vector. Those skilled in the art can construct expression vectors containing genes encoding exogenous proteins using well-known methods. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology.

[0410] For example, a nucleic acid construct with a "Z1-Z2" structure can be inserted into the cloning site of a plasmid vector to serve as plasmid DNA; where Z1 is the promoter, "-" represents a covalent bond or nucleotide fragment, and Z2 is the coding sequence of the foreign protein. One preferred method for Z1 is the T7 promoter.

[0411] In one preferred embodiment, the exogenous DNA template is circular DNA, more preferably plasmid DNA. The plasmid DNA is not particularly limited, as long as it can react with the cell extract of the system to synthesize exogenous proteins. Typically, plasmids contain functional elements such as promoters, terminators, and untranslated regions (UTRs). In one preferred embodiment, the plasmid contains a promoter that can be recognized by the in vitro protein synthesis system; specifically, in one preferred embodiment, the plasmid contains a promoter that can be recognized by the cell extract. For example, plasmids containing the T7 promoter can generally be used as expression vectors for the exogenous DNA templates used in Examples 1-6. For example, pET series plasmids and pGEM series plasmids of *E. coli* can be used to replace the plasmid vectors of *Kluyveromyces lactis* extract in Examples 1-6 to implement the present invention. In another preferred embodiment, the plasmid contains a promoter that can be recognized by the exogenously added component.

[0412] Taking the transcription program of exogenous protein initiated by the T7 promoter using an exogenous DNA template as an example, the T7 promoter can be recognized and initiated by the T7 RNA polymerase expressed endogenously in cell extracts, or by the T7 RNA polymerase added exogenously.

[0413] Linear DNA can be obtained through in vitro nucleic acid amplification. There are no particular restrictions on the amplification techniques that can be used, including but not limited to PCR amplification, isothermal amplification, room temperature amplification, and room temperature amplification. Among these, isothermal amplification is preferred.

[0414] One preferred embodiment is that the exogenous DNA template is linear DNA, and specifically a PCR linear fragment. The PCR linear fragment can be obtained using reported PCR techniques.

[0415] Another preferred embodiment is that the exogenous DNA template is linear DNA, specifically double-stranded linear DNA obtained through an amplification system. The amplification system is not particularly limited and can be selected from, but is not limited to, existing commercial kits and amplification systems reported in the literature, as long as they can amplify the DNA template encoding the exogenous protein of this invention. Examples include, but are not limited to, commercial DNA amplification systems provided by companies such as Biocompare, Neta Scientific Inc., ABM, Thermo Fisher Scientific, Expedeon, and Vivantis.

[0416] Another preferred embodiment uses double-stranded DNA as the exogenous DNA template and constructs it in a circular plasmid vector. The plasmid vector typically contains functional elements such as a T7 promoter, a T7 or LAC4 terminator, a 5'UTR, and a 3'UTR.

[0417] As one of the preferred embodiments, in Examples 1-6, double-stranded DNA was used as the exogenous DNA template and constructed in a circular plasmid vector. These plasmids contain the T7 promoter, which serves as a promoter for initiating the transcription and translation of exogenous proteins. In Examples 1-6, the modified Kluyveromyces lactis can endogenously express T7 RNA polymerase. Cell extracts were prepared from the modified strain, and an in vitro cell-free protein synthesis system was constructed. The T7 promoter in this system is suitable for the in vitro cell-free expression of various proteins. The plasmid also contains functional elements such as the LAC4 terminator and UTR.

[0418] In some specific embodiments, the plasmid DNA includes the following functional elements: promoter, 5' untranslated region, coding sequence of foreign protein, 3' untranslated region, terminator, replication initiation site (f1 ori), AmpR promoter, ampicillin resistance gene (AmpR gene), high copy number replication initiation site (ori), gene controlling plasmid copy number (rop gene), lacI promoter, and coding sequence of lacI.

[0419] In one specific implementation, the plasmid DNA includes at least the structural elements shown in Table 1.

[0420] Table 1. Description of the main structural elements of a plasmid DNA.

[0421]

[0422] In other specific embodiments, in addition to the functional elements in Table 1, there is also a purification tag, such as a polyhistidine tag (His-tag), between the coding sequence of the 5'UTR and mEGFP.

[0423] In other specific implementations, in addition to the functional elements in Table 1, there is a kozak sequence downstream of the 5'UTR to improve the translation level.

[0424] In other specific embodiments, in addition to the functional elements in Table 1 of the specific embodiments, there is also a coding sequence for a signal peptide (signal sequence) between the coding sequence of the 5'UTR and the mEGFP, downstream of the 5'UTR.

[0425] In other specific embodiments, the plasmid DNA includes the following functional elements: promoter, 5' untranslated region, leader sequence, coding sequence of foreign protein, 3' untranslated region, terminator, replication initiation site (f1 ori), AmpR promoter, AmpR gene, high copy number replication initiation site (ori), gene controlling plasmid copy number (rop gene), lacI promoter, and coding sequence of lacI.

[0426] In other specific embodiments, the plasmid DNA includes the following functional elements: a promoter, a 5' untranslated region, a coding sequence for a signal peptide, a coding sequence for a foreign protein, a 3' untranslated region, a terminator, f1 ori, an AmpR promoter, an AmpR gene, ori, a rop gene, a lacI promoter, and a coding sequence for lacI. Specifically, for example, the plasmid DNA includes the following functional elements: a T7 promoter, a 5' untranslated region, a coding sequence for a signal peptide, a coding sequence for the foreign protein mEGFP, a 3' untranslated region, a T7 terminator, f1 ori, an AmpR promoter, an AmpR gene, ori, a rop gene, a lacI promoter, and a coding sequence for lacI.

[0427] In other specific embodiments, the plasmid DNA includes the following functional elements: a promoter, a 5' untranslated region, a coding sequence for a signal peptide, a coding sequence for a purification tag, a multiple cloning site (MCS), a coding sequence for a foreign protein, a 3' untranslated region, a terminator, f1 ori, an AmpR promoter, an AmpR gene, ori, a rop gene, a lacI promoter, and a coding sequence for lacI. Specifically, for example, the plasmid DNA includes the following functional elements: a T7 promoter, a 5' untranslated region, a coding sequence for a signal peptide, a coding sequence for a purification tag, an MCS, a coding sequence for the foreign protein mEGFP, a 3' untranslated region, a LAC4 terminator or a T7 terminator, f1 ori, an AmpR promoter, an AmpR gene, ori, a rop gene, a lacI promoter, and a coding sequence for lacI.

[0428] The basic structure construction of plasmids and the method of inserting the coding gene of exogenous proteins into plasmid vectors can be carried out using conventional techniques in the field, and will not be elaborated here. For example, refer to patent documents such as CN108690139A, CN107574179A, and CN108949801A. For example, the basic structure of plasmids can also be seen in the figures of Chinese patent application CN201910460987.8.

[0429] In this invention, the concentration of the DNA template encoding the non-exogenous protein can be determined based on the amount of DNA template encoding the exogenous protein used, as described above, and according to the desired expression level of the non-exogenous protein. The non-exogenous protein refers to a translation product synthesized to promote the reaction, rather than the intended expression protein.

[0430] 1.3.2. Exogenous mRNA template

[0431] The present invention can also use an exogenous mRNA template instead of an exogenous DNA template, or use a mixture of an exogenous mRNA template and an exogenous DNA template, add it to the above-mentioned CFPS(Mg+) system, and carry out an in vitro protein synthesis reaction to synthesize the exogenous protein encoded by the mRNA template.

[0432] 1.3.3. In vitro nucleic acid amplification (in vitro nucleic acid amplification technology, in vitro nucleic acid amplification methods)

[0433] "In vitro nucleic acid amplification" is the process of replicating nucleic acids in vitro.

[0434] The nucleic acid templates used in the in vitro protein synthesis system of the present invention include nucleic acid templates encoding exogenous proteins, and optionally also include nucleic acid templates encoding other proteins. All of these can be prepared using in vitro nucleic acid amplification technology, and can also include a nucleic acid amplification process during the in vitro protein synthesis reaction.

[0435] There are no particular restrictions on the in vitro nucleic acid amplification techniques that can be used; they can be non-isothermal amplification or isothermal amplification (also known as isothermal amplification). These include, but are not limited to, polymerase chain reaction (PCR) amplification, isothermal amplification, room temperature amplification, and room temperature amplification. Among these, room temperature amplification is preferred.

[0436] The isothermal amplification technique can be referenced from the following literature: "J Kim et al. Isothermal DNA amplification in bioanalysis: strategies and applications[J]. Bioanalysis, 2011, 3(2): 227–239", "Gill P et al. Nucleic Acid Isothermal Amplification Technologies—A Review[J]. Nucleosides, Nucleotides, and Nucleic Acids, 2008, 27(3): 224-243", "Yong-Joo Jeong, Kkothanahreum Park and Dong-Eun Kim. Isothermal DNA amplification in vitro: the helicase-dependent amplification system[J]. Cell. Mol. Life “Sci., 2009, 66:3325–3336”, “Lv Bei et al. Development and continuous innovation of rapid nucleic acid amplification technology in vitro [J]. China Biotechnology Journal, 2011, 31(3):91-96”, “Wang Lin et al. Research progress of nucleic acid isothermal amplification technology [J]. Biotechnology Communications, 2011, 22(2)” and other cited literature. Specifically, the nucleic acid isothermal amplification methods that can be used in the technical means of this invention include, but are not limited to: loop-mediated isothermal amplification / loop-mediated isothermal amplification (LAMP), chain substitution amplification / chain displacement amplification (SDA), nucleic acid sequence-dependent amplification (NASBA), rolling circle amplification (RCA), nicking enzyme nucleic acid isothermal amplification (nicking enzyme amplification reaction, NEAR), helicase-dependent isothermal amplification (HDA), transcription-dependent amplification, hybridization capture, transcription-mediated amplification (TMA), recombinase-mediated amplification (RAA), recombinase polymerase amplification (RPA), etc. One of the preferred methods is rolling circle amplification.

[0437] The in vitro nucleic acid amplification methods used in this invention, especially room temperature amplification methods, are not particularly limited. All room temperature amplification techniques that can be used in cell-free in vitro systems in the prior art are included in the scope of this invention, including but not limited to rolling circle amplification (RCA), recombinase polymerase amplification (RPA), chain displacement amplification (SDA), helicase dependent amplification (HDA), 3SR (self-sustained sequence replication), etc. The in vitro nucleic acid amplification methods (especially room temperature amplification methods) disclosed in the following references can all be used as technical means of the present invention, and are all incorporated into the present invention by reference. These references include, but are not limited to: “Nicole E. Gregorio, Max Z. Levine and Javin P. Oza. A User's Guide to Cell-Free Protein Synthesis[J]. Methods Protoc. 2019, 2, 24”, “Y Lu. Advances in Cell-Free Biosynthetic Technology[J]. Current Developments in Biotechnology and Bioengineering, 2019, Chapter 2, 23-45”, “Y Lu. Cell-free synthetic biology: Engineering in an open world[J]. Synthetic and Systems Biotechnology, 2017, 2, 23-27”, and other references directly or indirectly cited.

[0438] The in vitro nucleic acid amplification of the present invention can also employ amplification techniques such as SMART amplification (SMAP), single primer isothermal amplification (SPIA), exponential amplification reaction (EXPAR), thermally stable HDA (tHDA), multiple substitution amplification (MDA), and restriction-assisted RCA.

[0439] The in vitro nucleic acid amplification reaction of the present invention can be carried out continuously at a specific temperature or temperature range that is favorable for the reaction. Any of the room-temperature amplification techniques of the present invention also allows for operation under conditions with minor temperature fluctuations. The reaction conditions of any of the room-temperature amplification techniques of the present invention also allow for fluctuations within an acceptable temperature range.

[0440] 1.4. Incubation reaction (in vitro protein synthesis reaction)

[0441] A nucleic acid template (preferably a DNA template) encoding a foreign protein is added to the CFPS(Mg+) system, and the reaction is incubated for a period of time to express and synthesize the foreign protein.

[0442] The conditions for in vitro protein synthesis reactions are determined based on the specific in vitro cell-free protein synthesis system. Reported reaction conditions can be referenced, including but not limited to those described in documents such as CN106978349A, CN108535489A, and CN108642076A. The in vitro protein synthesis reaction can be carried out continuously at a specific temperature or within a temperature range favorable to the reaction. Preferably, the temperature fluctuation of the mixture in Celsius is less than 25% (e.g., less than 20%, less than 15%, less than 10%, less than 5%) and / or the temperature fluctuation of the mixture is less than 15°C (e.g., less than 10°C, less than 5°C, less than 2°C, or less than 1°C) throughout the reaction. It is preferred to conduct the in vitro protein synthesis reaction at room temperature. The room temperature is preferably from room temperature to 37°C, specifically, preferably 20–37°C. One preferred method is 25–37°C. Another preferred method is 20–30°C. All reported methods for synthesizing proteins at room temperature or isothermal conditions can be used to implement the technical solution of this invention.

[0443] The reaction time can be determined comprehensively based on factors such as the amount of raw materials used (e.g., the amount of reaction substrate, the expected protein expression level, etc.) and the efficiency of protein synthesis reaction.

[0444] In one embodiment, the reaction time is 1 to 72 hours.

[0445] In another embodiment, the reaction time is 3 to 24 hours.

[0446] In another embodiment, the reaction time is 3 to 21 hours.

[0447] In another embodiment, the reaction time is 6 to 21 hours.

[0448] The reaction time may also be selected from any of the following time lengths, or the time length between any two time lengths: 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, 24h, 36h, 48h, 72h; the time length range includes two endpoints.

[0449] The determination of exogenous protein expression levels can draw upon methods used to test protein content in cell extracts, and a suitable method can be selected based on the characteristics of the exogenous protein. For example, in Examples 1-6, ultraviolet absorption was used to determine the RFU value, thereby calculating the amount of exogenous protein synthesized.

[0450] 2. A second aspect of the present invention provides an in vitro protein synthesis kit, the kit comprising:

[0451] (i) The in vitro cell-free protein synthesis system containing exogenous magnesium ions described in the first aspect (CFPS(Mg+) system);

[0452] (ii) Optionally includes a nucleic acid template encoding a foreign protein;

[0453] (iii) Labels or instructions.

[0454] The CFPS(Mg+) system, together with the nucleic acid template encoding the foreign protein, can provide the translation-related elements required for the synthesis of the foreign protein.

[0455] The nucleic acid template encoding the exogenous protein can be designed and provided by the user, and then used in conjunction with the CFPS(Mg+) system provided in the kit (i).

[0456] In one preferred manner, the nucleic acid template encoding the exogenous protein is used as a reference control.

[0457] Preferably, the components of the kit are placed in one or more containers in the form of solids, semi-solids, liquids, emulsions, suspensions, or combinations thereof. One preferred form of the dry powder is lyophilized powder or vacuum-dried powder. The liquid includes pure substances and solutions.

[0458] In one preferred embodiment, (k1) and (k2) are packaged separately.

[0459] The solid, such as powder (or dry powder) or granules.

[0460] The semi-solid, such as a paste.

[0461] The liquid can be a pure substance or a mixture.

[0462] The emulsion refers to a mixed system of immiscible liquid phases, also known as an emulsion.

[0463] The suspension refers to a mixed system of immiscible liquid phase and solid.

[0464] Preferably, (i) contains individually packaged cell extracts.

[0465] The aforementioned in vitro protein synthesis kit can be used to perform in vitro protein synthesis reactions and synthesize exogenous proteins.

[0466] In one preferred embodiment, the components of the kit together constitute an aqueous solution. The kit includes a container for holding the aqueous solution.

[0467] In one preferred embodiment, the reagent kit comprises two parts: a dry powder (e.g., lyophilized powder, vacuum-dried powder) and a liquid reagent. The reagent kit includes two containers, one for the dry powder component and the other for the liquid reagent component. The liquid reagent includes all systems containing a liquid phase, which can be homogeneous or mixed, including but not limited to pure substances, solutions, emulsions, suspensions, and combinations thereof.

[0468] In one preferred embodiment, the kit includes dispensing containers comprising the following components: (a) cell extract; (b) energy system; (c) optionally, nucleic acid template; (d) buffer; (e) optionally, pH adjustment component; (f) optionally, several other solid components; and (h) optionally, several other liquid components. Components (a), (b), and (c) are each independently a dry powder or an aqueous solution. Components (c), (e), and (f) are independently present or absent. "Several" here means one, two, or more. Exogenous magnesium ions may be dispensed into one or more containers from (d), (e), (f), and (h), or into other containers.

[0469] When the kit contains liquid reagents, it is preferable that it includes an antifreeze component.

[0470] In one preferred embodiment, the components of the kit are dispensed as dry powder, buffer solution, or other liquid reagents, optionally including water as a solvent.

[0471] In one preferred manner, the following components may be aliquoted separately or in appropriate combinations into different containers: eukaryotic cell extract (containing endogenously expressed RNA polymerase, optionally containing endogenously expressed DNA polymerase), exogenous magnesium ions (including at least magnesium aspartate), an energy system, a substrate for RNA synthesis, a substrate for protein synthesis, a congestant, exogenous potassium ions, and a buffer solution. Optionally, the container may also include any one of the following exogenous components or appropriate combinations thereof: a nucleic acid template encoding an exogenous protein, other exogenous magnesium ions, exogenously added RNA polymerase, an exogenous DNA template encoding RNA polymerase, exogenous added DNA polymerase, an exogenous DNA template encoding RNA polymerase, other DNA amplification-related elements, a substrate for DNA synthesis, other soluble amino acid salts, translation-related elements, RNA amplification-related elements, RNase inhibitors, antioxidants or reducing agents, antifreeze, trehalose, reaction promoters, defoamers, alkanes, and aqueous solvents. The cell extract preferably contains transfer RNA (tRNA) and ribosomes. Each RNA polymerase is preferably a T7 RNA polymerase. Each DNA polymerase is preferably a phi29 DNA polymerase. When providing DNA polymerase, the substrate for DNA synthesis is usually provided also, via an endogenous, exogenous, or combination thereof. The eukaryotic cell extract is preferably a Kluyveromyces cell extract, and more preferably a Kluyveromyces lactis cell extract.

[0472] In one preferred manner, the following components may be aliquoted separately or in appropriate combinations into different containers: eukaryotic cell extract (from cells lacking the coding sequence / gene for endogenous integrated RNA polymerase and the coding sequence / gene for endogenous integrated DNA polymerase), exogenous magnesium ions (including at least magnesium aspartate), exogenously added RNA polymerase, energy system, substrate for RNA synthesis, substrate for protein synthesis, congestant, exogenous potassium ions, and buffer solution. Optionally, the container may also include any one of the following exogenous components or appropriate combinations thereof: nucleic acid template encoding exogenous protein, other exogenous magnesium ions, exogenous DNA template encoding RNA polymerase, exogenously added DNA polymerase, exogenous DNA template encoding DNA polymerase, other DNA amplification-related elements, substrate for DNA synthesis, other soluble amino acid salts, translation-related elements, RNA amplification-related elements, RNase inhibitors, antioxidants or reducing agents, antifreeze, trehalose, reaction promoters, defoamers, alkanes, and aqueous solvents. The cell extract preferably contains transfer RNA and ribosomes. Each of the RNA polymerases is more preferably T7 RNA polymerase. Each of the DNA polymerases is more preferably phi29 DNA polymerase. The eukaryotic cell extract is more preferably Kluyveromyces cell extract, and more preferably Kluyveromyces lactis cell extract.

[0473] 3. A third aspect of the present invention provides a method for synthesizing exogenous proteins, the method comprising the following steps:

[0474] (i) Provides the in vitro cell-free protein synthesis system (CFPS(Mg+) system) containing exogenous magnesium ions provided in the first aspect of the present invention;

[0475] (ii) Add a nucleic acid template encoding the foreign protein, perform an incubation reaction, and synthesize the foreign protein;

[0476] The CFPS(Mg+) system can work with the nucleic acid template encoding the foreign protein to provide the translation-related elements required for the synthesis of the foreign protein;

[0477] Optionally, step (iii) may also be included: isolating and / or detecting the exogenous protein.

[0478] The incubation reaction refers to an in vitro protein synthesis reaction, which includes at least the translation process (in which case the nucleic acid template may only include the mRNA template), and optionally includes the transcription process and the nucleic acid replication process (the replication process of DNA and / or RNA).

[0479] One preferred approach is to use a DNA template encoding a foreign protein, and the incubation reaction accordingly includes transcription and translation processes.

[0480] Separation and / or detection steps

[0481] The method for synthesizing the exogenous protein may optionally include the steps of isolating and / or detecting the exogenous protein. The isolation and / or detection methods can be implemented using conventional techniques.

[0482] Through detection and calculation, results can be obtained, including but not limited to yield, purity, molecular weight, and protein function.

[0483] 4. In the second and third aspects, each independently includes, but is not limited to, the following preferred methods:

[0484] (1) In one preferred manner, the nucleic acid template encoding the exogenous protein contains a promoter element that can be recognized by the CFPS(Mg+) system.

[0485] (2) In one preferred embodiment, the nucleic acid template encoding the exogenous protein contains a promoter element that can be recognized by the cell extract of the CFPS(Mg+) system. For example, the cell extract contains an endogenously expressed RNA polymerase corresponding to the promoter element on the nucleic acid template.

[0486] (3) In one preferred embodiment, the nucleic acid template encoding the exogenous protein contains a T7 promoter, and the CFPS(Mg+) system includes a T7 RNA polymerase.

[0487] (4) In one preferred embodiment, the nucleic acid template encoding the exogenous protein contains a T7 promoter, and the cell extract of the CFPS(Mg+) system contains endogenously expressed T7 RNA polymerase.

[0488] (5) Preferably, the nucleic acid template encoding the foreign protein contains a T7 promoter that can initiate the gene transcription program of the foreign protein, that is, the gene transcription process of the foreign protein is initiated by the T7 promoter on the nucleic acid template.

[0489] (6) In one preferred embodiment, the nucleic acid template encoding the exogenous protein contains a T7 promoter capable of initiating the gene transcription program of the exogenous protein, and the CFPS(Mg+) system includes T7 RNA polymerase.

[0490] (7) In one preferred manner, the T7 promoter is located upstream of the coding sequence of the exogenous protein in the nucleic acid template and initiates the transcription program of the exogenous protein. The cell extract of the CFPS(Mg+) system contains endogenously expressed T7 RNA polymerase.

[0491] In the second and third aspects, the nucleic acid templates encoding the foreign protein are each independently a DNA template, an mRNA template, or a combination thereof; the nucleic acid templates encoding the foreign protein are each preferably DNA templates.

[0492] 5. A fourth aspect of the present invention provides the application of the in vitro cell-free protein synthesis system (CFPS(Mg+) system) containing exogenous magnesium ions described in the first aspect, applied to protein synthesis. The application to protein synthesis includes, but is not limited to, its application in protein manufacturing or in protein synthesis-based detection.

[0493] The application fields of the CFPS(Mg+) system include, but are not limited to, biomedicine, molecular biology, medicine, in vitro detection, medical diagnostics, regenerative medicine, bioengineering, tissue engineering, stem cell engineering, genetic engineering, polymer engineering, surface engineering, nanoengineering, cosmetics, food, food additives, nutrients, agriculture, feed, daily necessities, detergents, environment, chemical staining, and fluorescent labeling.

[0494] 6. The fifth aspect of the present invention provides the use of magnesium aspartate in the in vitro cell-free protein synthesis system containing exogenous magnesium ions as described in the first aspect, or in the in vitro protein synthesis kit as described in the second aspect, or in the method for synthesizing exogenous proteins as described in the third aspect.

[0495] 7. The present invention will be further described below with reference to specific embodiments and accompanying drawings. 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 without specific conditions are preferably performed according to the conditions indicated in the specific embodiments described above, and then generally according to conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), Cell-free Protein Synthesis: Methods and Protocols [M]. 2008, or according to the manufacturer's recommendations. Unless otherwise stated, percentages and parts mentioned in this invention are weight percentages and parts by weight.

[0496] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available products.

[0497] This invention uses Kluyveromyces lactis (K. lactis or kl) as the source of cell extracts in the examples, and all cell extracts are prepared using modified strains of Kluyveromyces lactis. It should be noted that the same design, analysis, and experimental methods are also applicable to other cell extract sources described herein, such as other yeast cells, animal cells, and other eukaryotic cells (mammalian cells, plant cells, insect cells).

[0498] The plasmid expression vectors used in the following embodiments of the present invention are only used to illustrate the implementation of the present invention and are not intended to limit the scope of the present invention; other plasmid vectors that can be used to implement the present invention include, but are not limited to, common plasmid vectors that are available through existing commercial channels, such as: pET series plasmids, pGEM series plasmids, etc.

[0499] The concentrations of each component and the concentration of the exogenous DNA template in the in vitro cell-free protein synthesis system of the following embodiments of the present invention are all final concentrations unless otherwise specified; the final concentration refers to the initial concentration in the in vitro protein synthesis reaction mixture composed of the CFPS(Mg+) system and the exogenous DNA template.

[0500] The four magnesium source reagents used in Examples 1-6 include magnesium gluconate (molecular weight 414.61), magnesium L-glutamate (molecular weight 388.61, tetrahydrate), magnesium acetate (molecular weight 214.45, tetrahydrate), and magnesium L-aspartate (molecular weight 324.54, dihydrate). Each molecule provides only one magnesium atom and two acid radical groups. It should be noted that the complexation mode of magnesium ions and acid radical residues in the reaction mixture is not limited to the proportions in the raw materials.

[0501] In Examples 1-6, a negative control group (NC group) was set up, in which no exogenous DNA template was added, and other reaction conditions were consistent with those of the experimental groups in the examples. After 3 hours and 18-24 hours of reaction, the RFU value did not exceed 20 and could be ignored. Some experimental results of the NC group are not shown in the figures.

[0502] Example 1: Effect of exogenous magnesium aspartate on the concentration curve of an in vitro protein synthesis system

[0503] 1.1 Raw material preparation: A plasmid vector expressing mEGFP was constructed, and DNA was amplified in vitro to prepare plasmid DNA encoding the exogenous protein mEGFP.

[0504] A mutant of enhanced green fluorescent protein (mEGFP) was selected as the exogenous protein and used as the target expression product. mEGFP is the A206K mutant of enhanced green fluorescent protein, and its amino acid sequence is shown in SEQ ID No. 2.

[0505] Plasmid vectors were selected. Artificially constructed plasmid vectors targeting Kluyveromyces lactis cell extract were used. These vectors contained functional elements such as the T7 promoter, LAC4 terminator, 5'UTR, and 3'UTR. This plasmid vector, combined with Kluyveromyces lactis cell extract containing endogenously expressed T7 RNA polymerase, could form an in vitro cell-free protein synthesis system for expressing various exogenous proteins in vitro.

[0506] Using PCR amplification and homologous fragment recombination, a DNA fragment containing the encoding gene of mEGFP was inserted into a plasmid vector to construct a plasmid vector expressing mEGFP, denoted as plasmid D2P-mEGFP (abbreviated as pD2P-mEGFP), with a total length of 6056 bp. The plasmid was confirmed to be correct by gene sequencing. The gene sequence encoding mEGFP is shown in SEQ ID No. 1. The map of the D2P plasmid is shown below. Figure 1 As shown in Table 2, its structural components are composed of elements.

[0507] Table 2. Structural elements of the plasmid (pD2P-mEGFP) encoding the exogenous protein mEGFP.

[0508]

[0509] DNA amplification (DNA replication process)

[0510] The final concentrations of each component in the amplification system were as follows: 1×phi29 reaction buffer (components included: 200mM Tris-HCl, 20mM MgCl2, 10mM (NH4)2SO4, 10mM KCl, pH 7.5), 4mM dithiothreitol (DTT), 0.1 mg / ml bovine serum albumin (BSA), 0.5mM deoxynucleotide phosphate mixture (dNTPs), 2–5 μM random primers, 0.004 mg / mL phi29 DNA polymerase, and 1.14 ng / μL of the above plasmid (pD2P-mEGFP, used as template). After mixing the above reaction system, the reaction was incubated overnight at room temperature for 22 hours, or incubated at 37°C for 2 hours to obtain the DNA template. The OD value at 260 nm was measured to calculate the nucleic acid concentration. The reaction solution was frozen or kept for later use as the nucleic acid template in subsequent examples.

[0511] 1.2 Raw material preparation: Preparation of cell extracts (specifically, Kluyveromyces lactis cell extract).

[0512] The cell extract was obtained from yeast cells, specifically Kluyveromyces lactis (K. lactis). A modified strain based on Kluyveromyces lactis strain ATCC8585 was used. The method described in CN109423496A was employed to integrate the coding gene for T7 RNA polymerase into the genome of Kluyveromyces lactis, resulting in a modified strain capable of endogenously expressing T7 RNA polymerase. Cellular material was cultured from the modified strain, and then the cell extract was prepared. Based on comparative experiments, the *Kluyveromyces lactis* system, lacking the encoding gene for endogenously integrated T7 RNA polymerase, was almost unable to perform in vitro protein synthesis without the addition of any exogenous RNA polymerase. After the aforementioned endogenous integration modification, efficient expression of exogenous proteins could be achieved without the addition of any exogenous RNA polymerase, serving as an alternative to exogenous addition methods and achieving the protein synthesis level of traditional in vitro protein synthesis systems (where cell extracts are prepared using strains without endogenous T7 RNA polymerase modification, and exogenous T7 RNA polymerase is added to the synthesis system). The method of adding magnesium aspartate as an optimized exogenous magnesium ion in this invention is also applicable to in vitro protein synthesis systems of strains without endogenous T7 RNA polymerase modification (including but not limited to *Kluyveromyces lactis* strains), achieving the same or similar optimization effects.

[0513] The preparation process of Kluyveromyces lactis cell extract adopts 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 up the cells; and collecting the supernatant by centrifugation to obtain the cell extract.

[0514] The protein concentration in the obtained Kluyveromyces lactis cell extract (sample number YY1908191) was 27 mg / mL. Unless otherwise specified, the amount of cell extract added in this example and the following examples refers to the volume percentage in the reaction mixture.

[0515] 1.3 In vitro cell-free protein synthesis system containing exogenous magnesium ions (without adding exogenous RNA polymerase).

[0516] Each system consisted of 100 μL of material, and the reaction was carried out in a flat-bottomed 96-well plate. Three replicates were prepared for each sample, and the mean and standard deviation (error bar) were calculated.

[0517] Experimental group (L-aspartic acid magnesium provides exogenous magnesium ions, denoted as Mg-Asp group): The final concentrations of each component were as follows: 9.78 mM pH 8.0 tris(hydroxymethyl)aminomethane (pH adjusted with HCl, Tris-HCl), 15 mM glucose, 320 mM maltodextrin (measured as glucose monomer), 24 mM potassium phosphate, 1.8 mM nucleoside triphosphate mixture (adenine nucleoside triphosphate, guanine nucleoside triphosphate, cytosine nucleoside triphosphate, and uracil nucleoside triphosphate, each with a final concentration of 1.8 mM), 0.7 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 (each amino acid at a final concentration of 0.7 mM), 80 mM potassium acetate, 2% (w / v) polyethylene glycol 8000, 0.06 g / ml trehalose (6% (w / v)), 80% (v / v) of Kluyveromyces lactis cell extract obtained in section 1.2 above, and 0–9 mM L-aspartate magnesium. Mix on a shaker.

[0518] Positive control group 1 (magnesium glutamate provides exogenous magnesium ions, denoted as Mg-Glu group): 0-9mM L-glutamate magnesium is used instead of L-aspartate magnesium to provide exogenous magnesium ions. The types and contents of other components and the experimental conditions for in vitro protein synthesis reactions are completely consistent with the experimental group.

[0519] Positive control group 2 (magnesium acetate provides exogenous magnesium ions, denoted as Mg-Ac group): 0-9mM magnesium acetate is used to replace L-aspartate magnesium to provide exogenous magnesium ions. The types and contents of other components and the experimental conditions for in vitro protein synthesis reaction are completely consistent with the experimental group.

[0520] Blank control group (BC group): No exogenous magnesium ions were added, and the types and contents of other components and the experimental conditions for in vitro protein synthesis were completely the same as those of the experimental group.

[0521] Negative control group (NC group): No exogenous magnesium ions were added, no exogenous DNA template was added subsequently, and the types and contents of other components and the experimental conditions for in vitro protein synthesis reaction were completely the same as those of the experimental group.

[0522] 1.4 Perform in vitro protein synthesis reaction: Add the DNA template encoding mEGFP prepared in step 1.1 above (the T7 promoter in the DNA template can be recognized by the T7 RNA polymerase expressed endogenously in the cell extract above, with a final concentration of 7.8 ng / μL) to each independent in vitro cell-free protein synthesis system of each group (except NC group). After mixing, all systems are placed at room temperature (18-25℃) and reacted overnight on a shaker at 200 rpm. Samples are taken at 3 h and 20-24 h for fluorescent protein activity testing.

[0523] It should be noted that the final concentrations described in 1.3 and 1.4 are based on the in vitro protein reaction mixture with added DNA template. Unless otherwise specified, the final concentrations in the following examples are interpreted in the same way.

[0524] 1.5 Fluorescent Protein Activity Assay: After the reaction, each sample was immediately placed in an Envision 2120 multi-functional microplate reader (Perkin Elmer) to detect the fluorescence signal intensity. The relative fluorescence unit (RFU) was used as the activity unit. The RFU value reflects the amount of mEGFP protein synthesized. The conversion relationship between the mEGFP mass-volume concentration C (unit μg / mL) and the RFU value is as follows: Within the testing scope of this invention, C and RFU exhibit a substantially linear relationship.

[0525] Fluorescence tests were performed on each reaction system. Sample preparation: Centrifuge at 4000 rpm for 1 minute at 4°C. Place the samples to be tested in an Envision 2120 multi-plate reader. The excitation / emission wavelengths (Ex / Em) used were 488 nm / 507 nm, and the relative fluorescence units (RFU) were measured.

[0526] 1.6 Experimental Results: (e.g.) Figure 2 (3h) Figure 3 As shown in (23h). At both sampling time points, the Mg-Asp group showed the highest protein expression level when 3mM L-aspartate magnesium was added, and this was significantly higher than the highest protein expression level when using the optimal magnesium ion concentration of traditional exogenous magnesium ion source magnesium glutamate (Mg-Glu group) and magnesium acetate (Mg-Ac group). 0mM in the figure corresponds to the blank control group.

[0527] At 3 h, the highest protein expression level in the Mg-Asp group (3 mM) was 60.7% higher than that in the Mg-Ac group (7 mM), and the corresponding RFU value was 102.4% higher than that in the Mg-Glu group (4.4 mM).

[0528] At 23 hours after incubation, the highest protein expression level in the Mg-Asp group (3 mM) was 45.8% higher than that in the Mg-Ac group (8 mM), and the corresponding RFU value was 133.6% higher than that in the Mg-Glu group (1.6 mM).

[0529] The RFU value of the negative control group is negligible (mean value is below 25), and is not shown in the figure.

[0530] Example 2: Effect of exogenous magnesium aspartate on the concentration curve of an in vitro protein synthesis system

[0531] Preparation of raw materials: Using the method in Example 1.1, a plasmid vector expressing mEGFP was constructed and in vitro DNA amplification was performed to prepare a DNA template encoding the exogenous protein mEGFP.

[0532] Using the modified strain obtained in Example 1.2, a Kluyveromyces lactis cell extract containing endogenously expressed T7 RNA polymerase was prepared. The protein concentration in the obtained Kluyveromyces lactis cell extract was 27 mg / mL.

[0533] An in vitro cell-free protein synthesis reaction system containing exogenous magnesium ions (without adding exogenous RNA polymerase).

[0534] The reaction system had a volume of 100 μL and was carried out in a flat-bottomed 96-well plate. Three parallel samples were prepared for each sample, and the mean and standard deviation were calculated.

[0535] The final concentrations of each component in the experimental group (L-aspartic acid magnesium provided exogenous magnesium ions, denoted as the Mg-Asp group) were as follows: 9.78 mM pH 8.0 tris(hydroxymethyl)aminomethane (pH adjusted with HCl, Tris-HCl), 15 mM glucose, 320 mM maltodextrin (measured as glucose monomer), 24 mM potassium phosphate, 1.8 mM nucleoside triphosphate mixture (adenine nucleoside triphosphate, guanine nucleoside triphosphate, cytosine nucleoside triphosphate, and uracil nucleoside triphosphate, each with a final concentration of 1.8 mM), and 0.7 mM amino acid mixture (glycine, alanine, and uridine). Ingredients: Acid, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine (each amino acid to a final concentration of 0.7 mM), 20 mM potassium acetate, 2% (w / v) polyethylene glycol 8000, 6% (w / v) trehalose, 80% (v / v) Kluyveromyces lactate cell extract, 0–9 mM L-aspartate magnesium. Mix on a shaker.

[0536] In vitro protein synthesis reaction: The DNA template encoding mEGFP (final concentration 17.8 ng / μL) prepared above was added to the aforementioned in vitro cell-free protein synthesis reaction system containing exogenous magnesium ions. After mixing, all systems were placed at room temperature (18℃~25℃) and reacted overnight on a shaker at 200 rpm. Samples were taken for testing at 3h and 20~24h.

[0537] Positive control group 1 (magnesium glutamate provides exogenous magnesium ions, denoted as Mg-Glu group): 0-9mM L-glutamate magnesium is used instead of L-aspartate magnesium to provide exogenous magnesium ions. The types and contents of other components and the experimental conditions for in vitro protein synthesis reactions are completely consistent with the experimental group.

[0538] Positive control group 2 (magnesium acetate provides exogenous magnesium ions, denoted as Mg-Ac group): 0-9mM magnesium acetate is used to replace L-aspartate magnesium to provide exogenous magnesium ions. The types and contents of other components and the experimental conditions for in vitro protein synthesis reaction are completely consistent with the experimental group.

[0539] Blank control group (BC group): No exogenous magnesium ions were added, and the types and contents of other components and the experimental conditions for in vitro protein synthesis were completely the same as those of the experimental group.

[0540] Negative control group (NC group): No exogenous magnesium ions or exogenous DNA template were added. The types and contents of other components and the experimental conditions for in vitro protein synthesis were completely the same as those of the experimental group.

[0541] Fluorescent protein activity assay: The RFU value of mEGFP fluorescent protein was determined using the method described in 1.5 of Example 1.

[0542] Experimental results: such as Figure 4 (3h) Figure 5 As shown in (21h). At both sampling time points, the Mg-Asp group showed the highest protein expression level when 2 mM magnesium aspartate was added, and this was significantly higher than the highest protein expression level when using the optimal magnesium ion concentration of traditional exogenous magnesium ion source magnesium glutamate (Mg-Glu group) and magnesium acetate (Mg-Ac group). 0 mM in the figure corresponds to the blank control group.

[0543] At 3 h, the highest protein expression level in the Mg-Asp group (2 mM) was 223.2% higher than that in the Mg-Ac group (8 mM), and the corresponding RFU value was 74.2% higher than that in the Mg-Glu group (6 mM).

[0544] At 21 hours after incubation, the highest protein expression level in the Mg-Asp group (2 mM) was 159.3% higher than that in the Mg-Ac group (8 mM), and the corresponding RFU value was 94.9% higher than that in the Mg-Glu group (6 mM).

[0545] The RFU value of the negative control group is negligible (mean value is below 25), and is not shown in the figure.

[0546] Example 3: Effect of exogenous magnesium aspartate on the concentration curve of an in vitro protein synthesis system

[0547] Preparation of raw materials: Using the method in Example 1.1, a plasmid vector encoding mEGFP was constructed and amplified in vitro to prepare a DNA template encoding the exogenous protein mEGFP.

[0548] Kluyveromyces lactis cell extract was prepared using the method described in Example 1.2; it contained endogenously expressed T7 RNA polymerase; the amino acid sequence of the T7 RNA polymerase remained unchanged, and the optimized gene sequence was inserted into the genome of Kluyveromyces lactis strain ATCC8585. The protein concentration in the obtained Kluyveromyces lactis cell extract (CM1910291) was 36 mg / mL.

[0549] An in vitro cell-free protein synthesis reaction system containing exogenous magnesium ions (without adding exogenous RNA polymerase).

[0550] The reaction system had a volume of 100 μL and was carried out in a flat-bottomed 96-well plate. Three parallel samples were prepared for each sample, and the mean and standard deviation were calculated.

[0551] Experimental group (exogenous magnesium ions provided by L-aspartate magnesium, denoted as Mg-Asp group): The final concentrations of each component were as follows: 9.78 mM pH 8.0 tris(hydroxymethyl)aminomethane (pH adjusted with HCl, Tris-HCl), 15 mM glucose, 320 mM maltodextrin (measured as glucose monomer), 18 mM potassium phosphate, 1.8 mM nucleoside triphosphate mixture (adenine nucleoside triphosphate, guanine nucleoside triphosphate, cytosine nucleoside triphosphate, and uracil nucleoside triphosphate, each with a final concentration of 1.8 mM), and 0.7 mM amino acid mixture (glycine, alanine, and uridine). Ingredients: Acid, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine (each amino acid having a final concentration of 0.7 mM), 30 mM potassium acetate, 2% (w / v) polyethylene glycol 8000, 6% (w / v) trehalose, 80% (v / v) Kluyveromyces lactis cell extract, and 0–9 mM L-aspartate magnesium. Mix on a shaker.

[0552] In vitro protein synthesis reaction: The prepared DNA template encoding mEGFP (final concentration 6.6 ng / μL) was added to the aforementioned in vitro cell-free protein synthesis reaction system containing exogenous magnesium ions. After mixing, all systems were placed at room temperature (18–25 °C) and reacted overnight on a shaker at 200 rpm. Samples were taken for testing at 3 h and 20–24 h. Three replicates were set up for each sample.

[0553] Positive control group 1 (magnesium glutamate provides exogenous magnesium ions, denoted as Mg-Glu group): 0-10mM L-glutamate magnesium is used to replace L-aspartate magnesium to provide exogenous magnesium ions. The types and contents of other components and the experimental conditions for in vitro protein synthesis reaction are completely consistent with the experimental group.

[0554] Positive control group 2 (magnesium acetate provides exogenous magnesium ions, denoted as Mg-Ac group): 0-13mM magnesium acetate is used to replace L-aspartate magnesium to provide exogenous magnesium ions. The types and contents of other components and the experimental conditions for in vitro protein synthesis reaction are completely consistent with the experimental group.

[0555] Blank control group (BC group): No exogenous magnesium ions were added, and the types and contents of other components and the experimental conditions for in vitro protein synthesis were completely the same as those of the experimental group.

[0556] Negative control group (NC group): No exogenous magnesium ions or exogenous DNA template were added. The types and contents of other components and the experimental conditions for in vitro protein synthesis were completely the same as those of the experimental group.

[0557] Fluorescent protein activity assay: The RFU value of mEGFP fluorescent protein was determined using the method described in 1.5 of Example 1.

[0558] Experimental results: such as Figure 6 (3h) Figure 7 As shown in (22h). At both sampling time points, the Mg-Asp group showed the highest protein expression level when 4 mM Mg-aspartate magnesium was added, and this was significantly higher than the highest protein expression level when using the optimal magnesium ion concentration of traditional exogenous magnesium ion source magnesium glutamate (Mg-Glu group) and magnesium acetate (Mg-Ac group). 0 mM in the figure corresponds to the blank control group.

[0559] At 3 h, the highest protein expression level in the Mg-Asp group (4 mM) was 167.9% higher than that in the Mg-Ac group (12 mM), and the corresponding RFU value was 98.5% higher than that in the Mg-Glu group (5 mM).

[0560] At 22 hours after incubation, the highest protein expression level in the Mg-Asp group (4 mM) was 170.3% higher than that in the Mg-Ac group (6 mM), and the corresponding RFU value was 110.8% higher than that in the Mg-Glu group (5 mM).

[0561] The RFU value of the negative control group is negligible (mean value is below 25), and is not shown in the figure.

[0562] In summary, in Examples 1-3, using L-aspartate magnesium to provide exogenous magnesium ions, after overnight reaction, the RFU value corresponding to the highest protein expression level can be increased by more than 45% compared to magnesium acetate, and even by more than 170%; compared to L-glutamate magnesium, it can be increased by more than 90%, and even by more than 130%.

[0563] In the early stage of in vitro protein synthesis reaction, at 3 hours: the RFU value corresponding to the highest protein expression level can be increased by more than 60% compared to magnesium acetate, and even more than 220%; compared to magnesium L-glutamate, it can be increased by more than 70%, and even more than 100%.

[0564] Exogenous magnesium ions can be provided by L-aspartate magnesium, which significantly improves the early reaction rate compared to magnesium acetate, by more than three times; compared to L-glutamate magnesium, both the early reaction rate and the final protein synthesis are significantly improved, by more than two times.

[0565] Example 4 investigated the effects of magnesium aspartate and magnesium gluconate on the protein synthesis capacity of an in vitro protein synthesis system.

[0566] 4.1 In vitro cell-free protein synthesis system containing exogenous magnesium ions (without adding exogenous RNA polymerase)

[0567] Each system consisted of 100 μL cells, and the reaction was carried out in a flat-bottomed cell culture plate. Three replicates were prepared for each sample, and the mean and standard deviation (error bar) were calculated.

[0568] Experimental group: The final concentrations of each component were as follows: 9.78 mM pH 8.0 tris(hydroxymethyl)aminomethane (pH adjusted with HCl, Tris-HCl), 80 mM potassium acetate, 15 mM glucose, 320 mM maltodextrin (measured in glucose units), 24 mM potassium phosphate, 1.8 mM nucleoside triphosphate mixture (adenine nucleoside triphosphate, guanine nucleoside triphosphate, cytosine nucleoside triphosphate, and uracil nucleoside triphosphate, each with a final concentration of 1.8 mM), and 0.7 mM amino acid mixture (glycine, alanine, valine, leucine, and isoleucine). The ingredients included: acid, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine (each amino acid having a final concentration of 0.7 mM), 80 mM potassium acetate, 2% (w / v) polyethylene glycol 8000, 0.06 g / mL trehalose, 80% (v / v) Kluyveromyces lactis cell extract (CM191107), and 6 mM exogenous magnesium ions; the method of providing exogenous magnesium ions is shown in Table 3.

[0569] The Kluyveromyces lactis cell extract was prepared using the method described in Example 1, Section 1.2, and had a protein content of approximately 35 mg / mL.

[0570] Table 3. Methods of Providing Exogenous Magnesium Ions

[0571]

[0572]

[0573] Mix is ​​a mixed magnesium source of L-aspartic acid magnesium and magnesium gluconate.

[0574] In the NC group, no exogenous magnesium ions or DNA template were added; in the BC group, no exogenous magnesium ions were added, but an equal amount of DNA template as in the other systems was added.

[0575] In the Mix group, the numbers in parentheses correspond to the molar percentage content of magnesium aspartate.

[0576] Negative control group (NC group): No exogenous magnesium ions were added, no exogenous DNA template was added subsequently, and the types and contents of other components and the experimental conditions for in vitro protein synthesis reaction were completely the same as those of the experimental group.

[0577] 4.2 In vitro protein synthesis reaction: No exogenous DNA template was added to the NC group; DNA template encoding mEGFP (obtained by in vitro amplification using the method in 1.1 of Example 1) was added to each independent in vitro cell-free protein synthesis system of the above experimental groups at a final concentration of 40.4 ng / μL. After mixing, all systems were placed at room temperature (18-30℃) and reacted overnight on a shaker. Samples were taken at 3h and 20-24h for fluorescent protein activity testing.

[0578] 4.3 Fluorescent protein activity assay: The RFU value of the synthesized exogenous fluorescent protein mEGFP in the sample was determined using the method in 1.5 of Example 1.

[0579] 4.4 Experimental Results: The experimental results are as follows Figure 8 As shown. When the exogenous magnesium ions were 2–8 mM L-aspartate magnesium, the highest RFU value was at 5 mM at 3 h (643±16) and at 5 mM at 22 h (1846±134). When the exogenous magnesium ions were 2–8 mM gluconate magnesium, the highest RFU value was at 6 mM at 3 h (446±49) and at 7 mM at 22 h (1545±20). The peak RFU value corresponding to L-aspartate magnesium was higher than that of gluconate magnesium, increasing by 44.2% at 3 h and by 21.4% at 22 h.

[0580] In this embodiment, L-aspartic acid magnesium has a better promoting effect on the protein synthesis ability of the system.

[0581] For the mixed magnesium source system of L-aspartic magnesium and magnesium gluconate, the RFU values ​​were all higher than the peak value of magnesium gluconate, and the higher the proportion of L-aspartic magnesium, the more significant the increase in RFU value. In the mixed magnesium source systems with aspartic magnesium proportions of 25%, 50%, and 75%, the peak values ​​were 26.6%, 24.5%, and 27.0% higher than those of magnesium gluconate at 3 h, respectively, and 3.2%, 18.0%, and 20.9% higher at 22 h, respectively. Adding aspartic magnesium to magnesium gluconate can improve both the synthesis efficiency and the synthesis yield.

[0582] Example 5 investigated the effect of a mixed magnesium source of magnesium aspartate and magnesium gluconate on the protein synthesis capacity of an in vitro protein synthesis system.

[0583] 5.1 In vitro cell-free protein synthesis system containing exogenous magnesium ions (without adding exogenous RNA polymerase)

[0584] Each system consisted of 100 μL cells, and the reaction was carried out in a flat-bottomed cell culture plate. Three replicates were prepared for each sample, and the mean and standard deviation (error bar) were calculated.

[0585] Experimental group: The final concentrations of each component were as follows: 25.08 mM pH 8.0 tris(hydroxymethyl)aminomethane (pH adjusted with HCl), 34.2 mM potassium acetate, 0.99 mM dithiothreitol, 1.6% (w / v) PFG8000, 4.58 mM glucose, 0.058 g / mL corn dextrin, 1.23 mM nucleoside triphosphate mixture (adenine, guanine, cytosine, and uracil triphosphates, each with a final concentration of 1.23 mM), 1.37 mM potassium bicarbonate, and a mixture of twenty amino acids (final concentrations of glycine 4.18 mM, alanine 0.70 mM, valine 3.02 mM, leucine 0.52 mM, and isoleucine 1.3 mM). 6 mM, phenylalanine 1.06 mM, proline 0.49 mM, tryptophan 0.46 mM, serine 0.23 mM, tyrosine 0.06 mM, cysteine ​​1.75 mM, methionine 0.78 mM, asparagine 0.95 mM, glutamine 0.18 mM, threonine 0.23 mM, aspartic acid 0.93 mM, glutamic acid 0.41 mM, lysine 2.53 mM, arginine 1.27 mM and histidine 1.27 mM), 41.1 mg / mL trehalose, 6.0% (v / v) squalane, 60% (v / v) lactic acid Kluyveromyces cell extract (YY1904281), 28.5 mM tripotassium phosphate, 0–9 mM L-aspartate magnesium, 1.8 mM magnesium gluconate.

[0586] The Kluyveromyces lactis cell extract was prepared using the method described in Example 1.2, and had a protein content of approximately 22 mg / mL.

[0587] Negative control group (NC group): No exogenous magnesium ions were added, no exogenous DNA template was added subsequently, and the types and contents of other components and the experimental conditions for in vitro protein synthesis reaction were completely the same as those of the experimental group.

[0588] 5.2 In vitro protein synthesis reaction: No exogenous DNA template was added to the NC group; DNA template encoding mEGFP (obtained by in vitro amplification using the method in 1.1 of Example 1) was added to each independent in vitro cell-free protein synthesis system of the above experimental groups at a final concentration of 34.2 ng / μL. After mixing, all systems were placed at room temperature (18-30℃) and reacted overnight on a shaker. Samples were taken at 3h and 20-24h for fluorescent protein activity testing.

[0589] 5.3 Fluorescent protein activity assay: The RFU value of the synthesized exogenous fluorescent protein mEGFP in the sample was determined using the method in 1.5 of Example 1.

[0590] 5.4 Experimental Results: (e.g.) Figure 9 As shown in the figure. The area outside the brackets on the horizontal axis represents the total magnesium ion concentration, and the area inside the brackets represents the molar percentage of magnesium aspartate. Protein synthesis was highest when the total magnesium ion concentration was 3.8 mM, at which point the proportion of magnesium L-aspartate was 52.6%; the RFU value was 1805±186 at 3 h and 3762±381 at 22 h.

[0591] The RFU value of the negative control group is negligible (mean value is below 20), and is not shown in the figure.

[0592] Example 6 investigated the effect of a mixed magnesium source of magnesium aspartate, magnesium gluconate, and magnesium acetate on the protein synthesis capacity of an in vitro protein synthesis system.

[0593] 6.1 In vitro cell-free protein synthesis system containing exogenous magnesium ions (without adding exogenous RNA polymerase)

[0594] Each system consisted of 100 μL cells, and the reaction was carried out in a flat-bottomed cell culture plate. Three replicates were prepared for each sample, and the mean and standard deviation (error bar) were calculated.

[0595] Experimental group: The final concentrations of each component were as follows: 21.48 mM pH 8.0 tris(hydroxymethyl)aminomethane (pH adjusted with HCl), 29.3 mM potassium acetate, 1.4% (w / v) PFG8000, 3.92 mM glucose, 0.050 g / mL corn dextrin, 1.05 mM nucleoside triphosphate mixture (adenine, guanine, cytosine, and uracil triphosphates, each with a final concentration of 1.05 mM), 1.37 mM potassium bicarbonate, and a mixture of twenty amino acids (final concentrations were: glycine 9.66 mM, alanine 1.63 mM, valine 6.99 mM, leucine 1.20 mM, isoleucine 3.13 mM, phenylalanine 2.46 mM, and proline 1.14 mM). The following ingredients were added: tryptophan 1.07 mM, serine 0.54 mM, tyrosine 0.13 mM, cysteine ​​5.86 mM, methionine 1.81 mM, asparagine 2.02 mM, glutamine 0.41 mM, threonine 0.54 mM, aspartic acid 0.61 mM, glutamic acid 0.94 mM, lysine 5.86 mM, arginine 2.94 mM, and histidine 2.93 mM; 50% (v / v) Kluyveromyces lactate cell extract (YY1904281, same as Example 5); 20.99 mM tripotassium phosphate; 36.3 mg / mL trehalose; 2.99% (v / v) squalane; 1.87 mM magnesium aspartate; 1.54 mM magnesium gluconate; and 1–5 mM magnesium acetate.

[0596] Negative control group (NC group): No exogenous magnesium ions were added, no exogenous DNA template was added subsequently, and the types and contents of other components and the experimental conditions for in vitro protein synthesis reaction were completely the same as those of the experimental group.

[0597] 6.2 In vitro protein synthesis reaction: No exogenous DNA template was added to the NC group; DNA template encoding mEGFP (obtained by in vitro amplification using the method in 1.1 of Example 1) was added to each independent in vitro cell-free protein synthesis system of the above experimental groups at a final concentration of 34.2 ng / μL. After mixing, all systems were placed at room temperature (18-30℃) and reacted overnight on a shaker. Samples were taken at 3h and 20-24h for fluorescent protein activity testing.

[0598] 6.3 Fluorescent protein activity assay: The RFU value of the synthesized exogenous fluorescent protein mEGFP in the sample was determined using the method in 1.5 of Example 1.

[0599] 6.4 Experimental Results: (e.g.) Figure 10As shown in the figure. The area outside the parentheses on the horizontal axis represents the total magnesium ion concentration, and the area inside the parentheses represents the molar percentage of L-aspartate magnesium. With the total magnesium ion concentration of L-aspartate magnesium and magnesium gluconate reaching 3.41 mM, the synthesis of exogenous protein was inhibited with the addition of magnesium acetate. Within the concentration range shown for the mixed system, the higher the L-aspartate magnesium content, the higher the protein synthesis. The highest RFU value was observed at 3.41 mM, with an RFU value of 1992 ± 133 at 3 h and 4359 ± 383 at 22 h.

[0600] Compare Figure 9 and Figure 10 . Figure 9 Even at a total magnesium ion concentration of 4.8 mM (62.5%), protein expression remained considerably high. At 3 h, the RFU value (1580.5 ± 131) reached 87.6% of the RFU value (1805 ± 186) at 3.8 mM (52.6%), and at 22 h, the RFU value (3350 ± 108) reached 89.1% of the RFU value at 3.8 mM (52.6%). Meanwhile... Figure 10 In this study, the addition of just 1 mM magnesium acetate, increasing the total magnesium ion concentration to 4.41 mM, significantly inhibited protein synthesis. At 3 h, the RFU value was only 35.6% of that at 3.41 mM, and at 22 h, it was only 30.9% of that at 3.41 mM. This indicates that the system is more tolerant of magnesium aspartate and magnesium gluconate than magnesium acetate, and that the system is more sensitive to magnesium acetate concentration.

[0601] The above are merely some preferred embodiments of the present invention, and the present invention is not limited to the contents of the above embodiments. For those skilled in the art, various changes and modifications can be made within the scope of the concept of the technical solution of the present invention or under its guidance or inspiration. Any changes and modifications with equivalent technical effects are within the protection scope of the present invention. sequence list <110> Kangma (Shanghai) Biotechnology Co., Ltd. <120> An in vitro cell-free protein synthesis system and kit containing exogenous magnesium ions and its applications <130> 2020 <141> 2020-04-08 <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 714 <212> DNA <213> Artificial sequence <400> 1 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc 60 gacgtaaacg gccacaagtt cagcgtgcgc ggcgagggcg agggcgatgc caccaacggc 120 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 180 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag 240 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catctcttc 300 areacgacg gcacctacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg 360 aaccgcatcg agctgaaggg catcgacttc aaggaggagg gcaacatcct ggggcacaag 420 ctggagtaca acttcaacag ccacaacgtc tatatcacgg ccgacaagca gaaacggc 480 atcaaggcga acttcaagat ccgccacaac gtcgaggacg gcagcgtgca gctcgccgac 540 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac 600 ctgagcaccc agtccaagct gagcaaagac cccaacgaga agcgcgatca catggtcctg 660 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta rubber 714 <210> 2 <211> 238 <212> PRT <213> Artificial sequence <400> 2 Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val 1 5 10 15 Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Arg Gly Glu 20 25 30 Gly Glu Gly Asp Ala Thr Asn Gly Lys Leu Thr Leu Lys Phe Ile Cys 35 40 45 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu 50 55 60 Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln 65 70 75 80 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 Thr Ile Ser Phe Lys Asp Asp Gly Thr Tyr Lys Thr Arg Ala Glu Val 100 105 110 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile 115 120 125 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn 130 135 140 Phe Asn Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly 145 150 155 160 Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp Gly Ser Val 165 170 175 Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Lys Leu Ser 195 200 205 Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val 210 215 220 Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys 225 230 235

Claims

1. An in vitro cell-free protein synthesis system containing exogenous magnesium ions, characterized in that, The CFPS (Mg+) system comprises the following components: eukaryotic cell extract and exogenous magnesium ions; the exogenous magnesium ions are from one or more sources, at least including magnesium aspartate; the CFPS (Mg+) system can perform in vitro protein synthesis reaction with a DNA template encoding an exogenous protein to synthesize the exogenous protein; The source of the exogenous magnesium ions is magnesium aspartate or a combination of magnesium aspartate and magnesium gluconate; The magnesium aspartate is L-magnesium aspartate; The concentration of the exogenous magnesium ions provided by the magnesium aspartate is 2 mM to 4 mM; When the source of the exogenous magnesium ions is magnesium aspartate and magnesium gluconate, the proportion of the exogenous magnesium ions provided by the magnesium aspartate is greater than 25% and less than 100%; and the eukaryotic cell extract is derived from Kluyveromyces lactis cells; The eukaryotic cell extract contains RNA polymerase, or the CFPS (Mg+) system further comprises exogenous RNA polymerase.

2. The in vitro cell-free protein synthesis system comprising exogenous magnesium ions according to claim 1, characterized in that, In one molecule of magnesium aspartate, the ratio of magnesium atoms to aspartic acid residues is 1:

2.

3. The CFPS (Mg+) system according to claim 1 or 2, characterized in that: The amount of magnesium aspartate capable of increasing the amount of synthesized exogenous protein is denoted as QAsp; The QAsp is selected from the range of magnesium aspartate amounts in which the YPRT is greater than Y0 in the YPRT (CAsp) curve; wherein CAsp refers to the amount of magnesium aspartate, YPRT refers to the amount of expressed exogenous protein, YPRT (CAsp) curve refers to a curve with the amount of magnesium aspartate as the independent variable and the amount of expressed exogenous protein as the dependent variable when other reaction parameters are determined, and Y0 refers to the amount of expressed exogenous protein corresponding to CAsp of 0; The QAsp is selected from the range of magnesium aspartate amounts in which the amount of expressed exogenous protein is at least Y0+50% YΔ; YΔ = Ymax-Y0; Ymax refers to the maximum amount of expressed exogenous protein in the YPRT (CAsp) curve.

4. The CFPS (Mg+) system according to claim 3, characterized in that: The QAsp is selected from the range of magnesium aspartate amounts in which the amount of expressed exogenous protein is at least Y0+60% YΔ.

5. The CFPS (Mg+) system according to claim 4, characterized in that: The QAsp is selected from the range of magnesium aspartate amounts in which the amount of expressed exogenous protein is at least Y0+70% YΔ.

6. The CFPS (Mg+) system according to claim 5, characterized in that: The QAsp is selected from the range of magnesium aspartate amounts in which the amount of expressed exogenous protein is at least Y0+80% YΔ.

7. The CFPS (Mg+) system according to claim 6, characterized in that: The QAsp is selected from the range of magnesium aspartate amounts in which the amount of expressed exogenous protein is at least Y0+90% YΔ.

8. The cell-free protein synthesis system of claim 7, wherein the CFPS(Mg+) system comprises a magnesium ion. The QAsp is selected from the magnesium aspartate dosage range at which the exogenous protein expression is at least Y0+95% YΔ.

9. The in vitro cell-free protein synthesis system comprising exogenous magnesium ions according to claim 8, characterized in that: The QAsp is selected from the magnesium aspartate dosage range at which the exogenous protein expression is at least Y0+95% YΔ.

10. The in vitro cell-free protein synthesis system comprising exogenous magnesium ions according to claim 1 or 2, characterized in that: The CFPS(Mg+) system is capable of providing, in combination with a nucleic acid template encoding an exogenous protein, translation-related elements required for synthesis of the exogenous protein, and expressing the exogenous protein through an in vitro protein synthesis reaction. The nucleic acid template encoding the exogenous protein is a DNA template.

11. The in vitro cell-free protein synthesis system comprising exogenous magnesium ions according to claim 10, characterized in that: The gene transcription process of the exogenous protein is initiated by a T7 promoter on the nucleic acid template.

12. The in vitro cell-free protein synthesis system comprising exogenous magnesium ions according to claim 1 or 2, characterized in that: The RNA polymerase is selected from any one or a combination of the following: a cell extract containing endogenously expressed RNA polymerase, an exogenous RNA polymerase, and a translation product of an exogenous nucleic acid template encoding the RNA polymerase. The RNA polymerase is a T7 RNA polymerase.

13. The in vitro cell-free protein synthesis system comprising exogenous magnesium ions according to claim 1 or 2, characterized in that, The CFPS(Mg+) system further comprises a DNA polymerase.

14. The in vitro cell-free protein synthesis system comprising exogenous magnesium ions according to claim 13, characterized in that, The DNA polymerase is a phi 29 DNA polymerase.

15. The cell-free protein synthesis system of claim 1 or 2, wherein the CFPS(Mg+) system comprises an exogenous RNA polymerase and an exogenous DNA polymerase. The CFPS(Mg+) system comprises an exogenous T7 RNA polymerase and an exogenous phi 29 DNA polymerase.

16. The in vitro cell-free protein synthesis system comprising exogenous magnesium ions according to claim 1 or 2, characterized in that, The eukaryotic cell extract contains an endogenously expressed RNA polymerase.

17. The in vitro cell-free protein synthesis system comprising exogenous magnesium ions according to claim 1 or 2, characterized in that, The endogenously expressed RNA polymerase is capable of recognizing a promoter in the nucleic acid template that initiates the gene transcription process of the exogenous protein.

18. The cell-free protein synthesis system of claim 17, wherein the eukaryotic cell extract is derived from a strain that has been genetically modified by inserting a coding sequence or a coding gene of the RNA polymerase into a free plasmid in the cell, or by integrating the coding gene of the RNA polymerase into the genome of the cell, or by using a combination of the above two methods.

19. The cell-free protein synthesis system of claim 18, wherein the endogenously expressed RNA polymerase is an endogenously expressed T7 RNA polymerase. The CFPS(Mg+) system further comprises an energy system. The energy system is selected from any one or a combination of the following: a sugar and phosphate energy system, a sugar and phosphocreatine energy system, a phosphocreatine and creatine phosphokinase system, a phosphocreatine and phosphocreatine kinase system, a monosaccharide and its catabolic intermediates, and a glycogen and its catabolic intermediates. The CFPS(Mg+) system further comprises a substrate for protein synthesis.

20. The in vitro cell-free protein synthesis system comprising exogenous magnesium ions according to claim 1 or 2, characterized in that, The substrate for protein synthesis is an amino acid mixture, which at least comprises amino acids that can be used to synthesize the exogenous protein. ​ 21. The in vitro cell-free protein synthesis system comprising exogenous magnesium ions according to claim 1 or 2, characterized in that, ​ ​ 22. The in vitro cell-free protein synthesis system comprising exogenous magnesium ions of claim 21, wherein: the amino acid mixture is a mixture of natural amino acids.

23. The in vitro cell-free protein synthesis system comprising exogenous magnesium ions according to claim 1 or 2, characterized in that, the CFPS(Mg+) system further comprises a substrate for synthesizing RNA; the substrate for synthesizing RNA is a mixture of nucleotides selected from the group consisting of nucleoside monophosphates, nucleoside triphosphates, or a combination thereof; the CFPS(Mg+) system further comprises a substrate for synthesizing DNA; the substrate for synthesizing DNA is a mixture of deoxynucleotides.

24. The in vitro cell-free protein synthesis system comprising exogenous magnesium ions according to claim 1 or 2, characterized in that, the CFPS(Mg+) system further comprises at least one exogenously added component selected from the group consisting of other soluble amino acid salts, translation-related elements, DNA amplification-related elements, RNA amplification-related elements, RNase inhibitors, crowding agents, potassium ions, antioxidants or reducing agents, antifreezes, trehalose, reaction accelerators, antifoaming agents, alkanes, buffers; the translation-related elements are selected from the group consisting of tRNAs, ribosomes, other translation-related enzymes, initiation factors, elongation factors, termination factors, and combinations thereof; The crowding agent is polyethylene glycol, polyvinyl alcohol, polystyrene, dextran, sucrose polymer, polyvinylpyrrolidone, albumin, or a combination thereof; the sucrose polymer is reagent; the potassium ions are from potassium acetate, potassium glutamate, potassium chloride, potassium phosphate, potassium sulfate, potassium citrate, potassium hydrogen phosphate, potassium iodide, potassium lactate, potassium nitrate, potassium oxalate, and combinations thereof; the antioxidants or reducing agents are dithiothreitol, 2-mercaptoethanesulfonic acid, 2-mercaptoethanol, reduced glutathione, tricarbomethylphosphate, 3-mercapto-l,2-propanediol, or combinations thereof; the reaction accelerators are aluminum salts, aluminum oxides, iron salts, iron oxides, calcium salts, or combinations thereof; the alkanes are cyclohexane, isooctane, decane, tetradecane, pentadecylcyclohexane, squalane, tetrafortyalkane, vaseline, or combinations thereof; the buffers are selected from the group consisting of Tris-HCl, Tris base, HEPES, or combinations thereof.

25. The in vitro cell-free protein synthesis system comprising exogenous magnesium ions according to claim 1 or 2, characterized in that, the exogenous proteins are selected from the group consisting of luciferase, green fluorescent protein, enhanced green fluorescent protein, yellow fluorescent protein, aminoacyl tRNA synthetase, glycerolaldehyde-3-phosphate dehydrogenase, catalase, actin, variable regions of antibodies, single chains of antibodies and fragments of single chains, alpha-amylase, enterotoxin A, hepatitis C virus E2 glycoprotein, insulin and precursors thereof, glucagon-like peptide, interferon, interleukin, lysozyme, serum albumin, transthyretin, tyrosinase, xylanase, beta-galactosidase, variants of any of the foregoing, in any combination.

26. The in vitro cell-free protein synthesis system comprising exogenous magnesium ions of claim 25, wherein: the aminoacyl tRNA synthetase is human lysine-tRNA synthetase, human leucine-tRNA synthetase, or a combination thereof; and / or the glycerolaldehyde-3-phosphate dehydrogenase is Arabidopsis thaliana glycerolaldehyde 3-phosphate dehydrogenase; and / or the catalase is murine catalase.

27. An in vitro protein synthesis kit, characterized in that, the in vitro protein synthesis kit comprises: (i) the CFPS(Mg+) system of any one of claims 1-26; The components of the CFPS(Mg+) system are placed in one or more containers in solid, semi-solid, liquid, emulsion, suspension, or a combination thereof; (ii) including or not including a nucleic acid template encoding an exogenous protein; (iii) a label or an instruction; The (i) can provide the translation-related elements required for the synthesis of the exogenous protein together with the (ii).

28. The in vitro protein synthesis kit of claim 27, wherein, The cell extract in the (i) is separately packaged; And / or, the nucleic acid template encoding the exogenous protein contains a promoter element recognizable by the CFPS(Mg+) system in the (i); And / or, the nucleic acid template encoding the exogenous protein includes an exogenous protein translation system, a resistance gene translation system, a lac repressor translation system, each of which includes a corresponding promoter; And / or, the nucleic acid template encoding the exogenous protein further contains a gene for controlling the copy number of plasmid; And / or, the nucleic acid template encoding the exogenous protein further contains a transcription enhancer element or / and a translation enhancer element; And / or, the nucleic acid template encoding the exogenous protein contains a T7 promoter, and the CFPS(Mg+) system includes a T7 RNA polymerase; And / or, the eukaryotic cell extract contains an endogenously expressed T7 RNA polymerase; And / or, the nucleic acid template encoding the exogenous protein contains a T7 promoter for initiating the gene transcription program of the exogenous protein, and the CFPS(Mg+) system includes a T7 RNA polymerase; And / or, the nucleic acid template encoding the exogenous protein contains a T7 promoter, and the eukaryotic cell extract contains an endogenously expressed T7 RNA polymerase; The nucleic acid template encoding the exogenous protein is a DNA template.

29. A method of synthesizing an exogenous protein, comprising: The method for synthesizing the exogenous protein comprises the following steps: (i) providing the CFPS(Mg+) system of any one of claims 1-26; (ii) adding a DNA template encoding an exogenous protein, incubating the reaction, and synthesizing the exogenous protein; Further comprising the step (iii) isolating or / and detecting the exogenous protein; The CFPS(Mg+) system in the (i) can provide the translation-related elements required for the synthesis of the exogenous protein together with the DNA template encoding the exogenous protein in the (ii); The nucleic acid template encoding the exogenous protein contains a promoter element recognizable by the CFPS(Mg+) system; The nucleic acid template encoding the exogenous protein includes an exogenous protein translation system, a resistance gene translation system, a lac repressor translation system, each of which includes a corresponding promoter; The nucleic acid template encoding the exogenous protein further contains a gene for controlling the copy number of plasmid; The nucleic acid template encoding the exogenous protein further contains a transcription enhancer element or / and a translation enhancer element; The nucleic acid template encoding the exogenous protein contains a T7 promoter for initiating the gene transcription program of the exogenous protein, and the CFPS(Mg+) system includes a T7 RNA polymerase.

30. The method for synthesizing the exogenous protein of claim 29, wherein, The nucleic acid template encoding the foreign protein contains a T7 promoter capable of initiating the transcription program of the gene of the foreign protein, and the eukaryotic cell extract contains an endogenously expressed T7 RNA polymerase.

31. Use of the in vitro cell-free protein synthesis system comprising exogenous magnesium ions according to any one of claims 1 to 26, characterized in that, for protein synthesis; or for protein production, or for detection based on protein synthesis.

32. Use of L-magnesium aspartate in an in vitro cell-free protein synthesis system containing exogenous magnesium ions according to any one of claims 1 to 26, or in an in vitro protein synthesis kit according to claim 27, or in a method for the synthesis of a foreign protein according to claim 29 or 30.

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