CN113493813A - External source magnesium ion-containing in-vitro cell-free protein synthesis system and kit and application thereof - Google Patents

Abstract

The invention provides an in vitro cell-free Protein synthesis system containing exogenous magnesium ions, a kit and application thereof, and belongs to the technical field of Protein synthesis, wherein the system comprises a D2P system (a DNA-to-Protein system) and an mR2P system (an mRNA-to-Protein system). The in vitro cell-free protein synthesis system containing the exogenous magnesium ions adopts the magnesium gluconate as a novel magnesium ion supply source, and compared with the traditional magnesium ion supply source, the in vitro cell-free protein synthesis system containing the exogenous magnesium ions can obviously improve the protein synthesis efficiency and the protein expression quantity. Also provides a more efficient and higher-flux in-vitro protein synthesis kit and a synthesis method of the exogenous protein, and has the advantages of simplicity, convenience and low cost.

Description

External source magnesium ion-containing in-vitro cell-free protein synthesis system and kit and application thereof

Technical Field

The invention relates to the technical field of Protein synthesis, in particular to the technical field of in-vitro cell-free Protein synthesis, and specifically relates to an in-vitro cell-free Protein synthesis system containing exogenous magnesium ions, a kit and an application thereof, and more specifically relates to an in-vitro cell-free Protein synthesis system containing exogenous magnesium ions, which comprises a D2P system (DNA-to-Protein system), an mR2P system (mRNA-to-Protein system), an in-vitro Protein synthesis kit and an application thereof.

Background

Proteins are important molecules in cells, and are involved in performing almost all functions of cells. Protein synthesis mainly includes conventional intracellular synthesis techniques and a new generation of in vitro synthesis techniques. The conventional protein expression system refers to a molecular biological technique for expressing foreign genes by model organisms such as bacteria, fungi, plant cells or animal cells. In vitro protein Synthesis System, also known as SelenessThe cell expression system, which was developed in 1960, uses exogenous mRNA or DNA as a protein synthesis template, and artificially controls and adds substances such as substrates, energy, and transcription and/or translation-related protein factors required for protein synthesis to synthesize exogenous proteins. The in vitro protein synthesis system is generally characterized in that components such as a nucleic acid template (an mRNA template or a DNA template), RNA polymerase, amino acid, ATP and the like are added into a lysis system of bacteria, fungi, plant cells or animal cells to complete the rapid and efficient translation of the foreign protein. The protein in vitro synthesis system is a relatively fast, time-saving and convenient protein expression without plasmid construction, transformation, cell culture, cell collection and disruption steps, and is an important tool in the protein field (Garcia RA, Riley MR. applied biology and biotechnology. Humana Press, 263-264; -Fromm HJ, Hargreve M.essenties of biochemistry.2012;. CN 109988801A; Assenberg R, Wan PT, Geiss S, Mayr LM. Advances in recombinant protein expression for use in pharmaceutical research. Current expression in Structural biology.2013, 23; (3): 402. Zemb, Zembin, culture and culture of cells, and culture of cells of culture of cell culture of 14. 12. cell culture of cell of culture of cell 393. 12. and cell culture of cells of 14. 12. C. 3. the protein of culture of human of culture of human of culture of human of culture of human of culture of interest of culture of human of 3. 23, 23. 3. 23. 3. 12. 3. No. 3. 12. No. 3. In vitro protein synthesis systems may also express proteins that are toxic to cells or that contain unnatural amino acids (e.g._DAmino acids) capable of synthesizing a plurality of proteins simultaneously in parallel, facilitating the development of 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)&KGaA,2008: 1-34.). The protein product produced by the in vitro synthesis system can be widely applied to various fields such as medicine, food, nutriment, dietary supplement, cosmetics and the like, including but not limited to Proteinn of applicant^TMProlondon, Prolondon^TMGeneral, general^TMAnd the like.

The protein synthesis ability is one of the key indexes for determining whether an in vitro protein synthesis system can realize industrialization, and mainly comprises synthesis efficiency, protein synthesis amount (protein expression amount) and the like. In order to improve the yield of protein synthesis, the system is optimized and modified from the aspects of cell extracts, energy systems, genetic templates (nucleic acid templates), reactors, operation forms and the like (the key technology and industrial application of the cell-free system for efficiently synthesizing complex membrane protein are explored [ D ]. university of zhejiang, 2014), and various additives are explored and tried. Among them, the components which have a beneficial effect on intracellular protein synthesis are often the first choice for research. However, due to the large differences between the intracellular synthetic biological systems and the in vitro synthetic microenvironment, the effect is not simply predictable, but needs to be screened and verified with a large number of experiments.

The inorganic salt ions are common additives for the in vitro protein synthesis system and comprise magnesium ions, potassium ions and the like. Among them, magnesium ions play an important role in the protein translation process, which can promote the ribosome assembly process and improve the stability of RNA. In addition, magnesium ions also play a role in promoting polymerase binding and the like. Common compounds used as a source of magnesium ions include magnesium acetate, magnesium chloride, magnesium glutamate, and the like. References include WO2016005982A1, US20060211083A1, "L Kai, 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 ", and the like.

With the successful interpretation of a great deal of biological genetic information, how to realize more efficient and higher-flux protein synthesis in an in vitro system is an urgent need in the current field of protein synthesis.

Disclosure of Invention

Aiming at the technical problems, the invention discloses a simple, convenient, more efficient and higher-flux in-vitro cell-free protein synthesis system containing exogenous magnesium ions, which adopts magnesium gluconate as a novel magnesium ion supply source and can obviously improve the protein synthesis capacity compared with the traditional magnesium ion supply source.

1. In a first aspect, 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 as "CFPS (Mg +) system" in the present invention, and the CFPS (Mg +) system includes exogenous magnesium ions; the exogenous magnesium ions are derived from one or more sources and include at least magnesium gluconate (magnesium gluconate).

The CFPS (Mg +) system can provide translation related elements required by synthesizing the foreign protein together with a nucleic acid template for encoding the foreign protein, and the foreign protein is obtained by expression through in-vitro protein synthesis reaction.

The exogenous magnesium ions, including at least magnesium gluconate, may optionally also be one or more from the group: magnesium aspartate, magnesium acetate, magnesium glutamate, magnesium chloride, magnesium phosphate, magnesium sulfate, magnesium citrate, magnesium hydrogen phosphate, magnesium iodide, magnesium lactate, magnesium nitrate and magnesium oxalate.

In a preferred embodiment, the source of exogenous magnesium ions is a combination of magnesium gluconate and magnesium aspartate (preferably magnesium L-aspartate).

In a preferred embodiment, the source of exogenous magnesium ions is a combination of magnesium gluconate and magnesium glutamate (preferably magnesium L-glutamate).

In a preferred embodiment, the exogenous source of magnesium ions is a combination of magnesium gluconate, magnesium aspartate (independently preferably magnesium L-aspartate), and magnesium glutamate (independently preferably magnesium L-glutamate). More preferably, the source of exogenous magnesium ions is a combination of magnesium gluconate, magnesium L-aspartate, and magnesium L-glutamate.

Preferably, the magnesium gluconate provides exogenous magnesium ions in a molar percentage of the total exogenous magnesium ions selected from any one of the following percentage values, or a range of values 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 numerical ranges are inclusive of the two endpoints.

In one preferred form, at least 25 mol% of the exogenous magnesium ions are derived from magnesium gluconate.

In one preferred form, at least 30 mol% of the exogenous magnesium ions are derived from magnesium gluconate.

In one preferred form, at least 40 mol% of the exogenous magnesium ions are derived from magnesium gluconate.

In one preferred form, at least 50 mol% of the exogenous magnesium ions are derived from magnesium gluconate.

In one preferred form, at least 80 mol% of the exogenous magnesium ions are derived from magnesium gluconate.

In one preferred form, at least 90 mol% of the exogenous magnesium ions are derived from magnesium gluconate.

In one preferred embodiment, 100 mol% of the exogenous magnesium ions are derived from magnesium gluconate.

The "mol% of the exogenous magnesium ions" is calculated only by the molar amount of the exogenously added magnesium element, regardless of the difference in the free state or the chelated state. For example, when 1mol of magnesium chloride (completely ionized, magnesium ions are in a free state) and 1mol of magnesium gluconate (partially ionized, magnesium ions are partially in a free state and partially in a chelated state) are exogenously added, the magnesium gluconate provides 50 mol% of exogenous magnesium ions.

The magnesium gluconate capable of increasing the synthesis amount of foreign protein is selected from Y_PRT(C_MgP) The expression quantity of the foreign protein in the curve is more than Y₀The dosage range of the magnesium gluconate is within the time interval. The optional dosage intervals may be continuous or discontinuous.

In the present invention,

Q_MgPthe magnesium gluconate dosage capable of improving the synthesis amount of the foreign protein is covered by the invention.

The amount of the raw material to be used is generally referred to as the amount of the raw material to be added, unless otherwise specified, and may be represented by a concentration, a mass, an amount of a substance (or a molar amount), or the like.

C_MgPThe dosage of magnesium gluconate is indicated. The magnesium gluconate is preferably characterized by its amount in a concentration manner in the present invention.

Y_PRTThe expression level refers to the expression level of a foreign protein.

Y_PRT(C_MgP) The curve refers to the curve when the dosage of the magnesium gluconate is used as independent variable, the expression quantity of the foreign protein is used as dependent variable and other reaction parameters are determined, and is also marked as Y in the invention_PRT～C_MgPCurve line. The "other reaction parameters" include, but are not limited to: other system components, addition mode of reaction raw materials, reaction temperature program, reaction time length, properties of a reaction vessel, volume of the reaction system and the like. The CFPS (Mg +) system of a set of formulas can correspond to a plurality of Y_PRT～C_MgPCurve line. The CFPS (Mg +) systems of one set of formulations can be reacted under different reaction temperature programs and also for different time lengths, thus several different Y's can be generated_PRT～C_MgPCurve line.

Y_maxFinger Y_PRT(C_MgP) The highest expression level of the foreign protein in the curve.

C_maxFinger Y_PRT(C_MgP) And (3) the dosage of the magnesium gluconate when the foreign protein has the highest expression quantity in the curve.

Y_minFinger Y_PRT(C_MgP) In the concentration curve, Y_PRT>Y₀The lowest expression level of the foreign protein in the interval of (3).

Y₀Means that C is_MgPThe expression level of the foreign protein is 0.

Y_ΔIs Y_maxAnd Y₀Difference of (a), in value, Y_Δ＝Y_max-Y₀。

Preferably, said Q_MgPSelected from foreign proteins expressed in an amount of at least Y₀+50％Y_ΔThe dosage range of magnesium gluconate;

more preferably, said Q_MgPSelected from foreign proteins expressed in an amount of at least Y₀+60％Y_ΔThe dosage range of magnesium gluconate;

more preferably, said Q_MgPSelected from foreign proteins expressed in an amount of at least Y₀+70％Y_ΔThe dosage range of magnesium gluconate;

more preferably, said Q_MgPSelected from foreign proteins expressed in an amount of at least Y₀+80％Y_ΔThe dosage range of magnesium gluconate;

more preferably, said Q_MgPSelected from foreign proteins expressed in an amount of at least Y₀+90％Y_ΔThe dosage range of magnesium gluconate;

more preferably, said Q_MgPSelected from foreign proteins expressed in an amount of at least Y₀+95％Y_ΔThe dosage range of magnesium gluconate;

more preferably, said Q_MgPMagnesium gluconate dosage C when adopting the highest expression level of foreign protein_max。

In some preferred embodiments, the magnesium gluconate provides an exogenous magnesium ion at a concentration selected from any of the following concentrations, or a range of concentrations between any two of the following concentrations (the range of concentrations includes both endpoints): 0.1mM, 0.5mM, 1mM, 1.5mM, 2mM, 2.5mM, 3mM, 3.5mM, 4mM, 4.5mM, 5mM, 5.5mM, 6mM, 6.5mM, 7mM, 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, 80 mM.

The CFPS (Mg +) system, preferably, comprises a cell extract.

The CFPS (Mg +) system is capable of providing translation-related elements required for synthesis of a foreign protein in conjunction with a nucleic acid template encoding the foreign protein. Preferably, the CFPS (Mg +) system comprises a system component capable of recognizing a promoter element in a nucleic acid template such that the CFPS (Mg +) system is capable of recognizing the promoter element in the nucleic acid template encoding the foreign protein, e.g. the CFPS (Mg +) system comprises an RNA polymerase corresponding to the promoter element in the nucleic acid template.

The components of the system capable of recognizing the promoter element in the nucleic acid template, such as the corresponding RNA polymerase, may be provided by the cell extract, by other exogenous components, or by a combination of both.

Generally, the gene transcription process of the foreign protein is initiated by a promoter on the nucleic acid template. In one preferred mode, the gene transcription process of the foreign protein is initiated by the T7 promoter on the nucleic acid template.

In one preferred form, the cell extract contains an endogenously expressed RNA polymerase which recognizes a promoter in the nucleic acid template which initiates the gene transcription process of the foreign protein.

In order to obtain a cell extract containing endogenously expressed RNA polymerase, it is preferred that the source strain of said cell extract is genetically modified (endogenous strain modification) in the following manner: the coding sequence of RNA polymerase is inserted into an episomal plasmid in the cell, or the gene encoding RNA polymerase is integrated into the genome of the cell, or a combination of both. It should be noted that, in the case of the modification of the above-mentioned endogenous strain, in addition to the incorporation of the above-mentioned coding sequence/coding gene, it is also permissible to insert other nucleotide sequences such as a non-coding sequence, an enhancer sequence, a kozak sequence, a leader sequence or leader peptide sequence, a signal peptide sequence, a tag sequence, a codon sequence, and the like. The endogenous strain is modified, so that the modified strain can endogenously express RNA polymerase. The RNA polymerase is preferably T7RNA polymerase.

Preferably, the CFPS (Mg +) system comprises an RNA polymerase. Sources of the RNA polymerase include, but are not limited to: a cell extract comprising an endogenously expressed RNA polymerase, an exogenous RNA polymerase, a translation product of an exogenous nucleic acid template encoding the RNA polymerase, and combinations thereof. In each of the above embodiments, it is preferable that the RNA polymerase is T7RNA polymerase. The exogenous nucleic acid template encoding RNA polymerase can be translated into RNA polymerase by an in vitro protein synthesis reaction with the CFPS (Mg +) system.

Preferably, the CFPS (Mg +) system comprises a cell extract containing endogenously expressed T7RNA polymerase.

In one preferred embodiment, the CFPS (Mg +) system includes a DNA polymerase. Sources of the DNA polymerase include, but are not limited to: a cell extract comprising an endogenously expressed DNA polymerase, an exogenous DNA polymerase, a translation product of an exogenous nucleic acid template encoding the DNA polymerase, and combinations thereof. In each of the above embodiments, the DNA polymerase is preferably phi29 DNA polymerase independently. The exogenous nucleic acid template encoding the DNA polymerase can be translated into the DNA polymerase by an in vitro protein synthesis reaction with the CFPS (Mg +) system.

The RNA polymerase and the DNA polymerase, independently of each other, may be added directly by exogenous means or provided as reaction products or intermediates (e.g., addition of an exogenous nucleic acid template encoding the RNA polymerase or/and encoding the DNA polymerase).

In one preferred embodiment, the CFPS (Mg +) system includes exogenously added T7RNA polymerase.

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

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

In one preferred embodiment, the CFPS (Mg +) system includes an exogenous T7RNA polymerase and an exogenous phi29 DNA polymerase.

The cell extract is: a prokaryotic cell extract, a eukaryotic cell extract, or a combination thereof.

In a preferred embodiment, the cell extract is derived from any one of the following sources: prokaryotic cells, yeast cells, mammalian cells, plant cells, insect cells, or combinations thereof. The prokaryotic cell is preferably E.coli.

The yeast cell is preferably Kluyveromyces, Saccharomyces cerevisiae, Pichia pastoris, or a combination thereof.

The Kluyveromyces is further preferably Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces polybuhitensis, Kluyveromyces hainanensis, Kluyveromyces williamsii, Kluyveromyces fragilis, Kluyveromyces hubeiensis, Kluyveromyces polyspora, Kluyveromyces siamensis, Kluyveromyces lactis, or a combination thereof.

In a preferred embodiment, the cell extract is selected from any one of the following sources: coli, kluyveromyces lactis, wheat germ cells, Spodoptera frugiperda cells (Spodoptera frugiperda cells, an insect cell), Leishmania tarentolae cells (Leishmania tarentolae cells), rabbit reticulocyte, chinese hamster ovary cells (CHO cells), african green monkey kidney COS cells, african green monkey kidney VERO cells, baby hamster kidney cells (BHK cells), human Hela cells, human Hybridoma cells (human Hybridoma cells), human fibrosarcoma HT1080 cells, and any combination of the foregoing.

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

In one of the preferred modes, the CFPS (Mg +) system comprises a substrate for a synthetic protein; the substrate of the synthetic protein is preferably a mixture of amino acids, including at least the mixture of amino acids required for the synthesis of the foreign protein. Preferably, the amino acid mixture is a mixture of natural amino acids.

In one of the preferred modes, the CFPS (Mg +) system comprises a substrate for RNA synthesis; the substrate of the synthetic RNA is preferably a mixture of nucleotides, more preferably selected from: nucleoside monophosphates, nucleoside triphosphates, and combinations thereof. The substrate for the synthesis of RNA is more preferably a nucleoside triphosphate mixture.

In one of the preferred modes, the CFPS (Mg +) system comprises a substrate for DNA synthesis; the substrate for the synthesis of DNA is preferably a mixture of deoxynucleotides, more preferably a mixture of deoxynucleoside triphosphates.

In a preferred mode, the CFPS (Mg +) system comprises at least one externally added component as follows: translation-related elements, DNA amplification-related elements, RNA amplification-related elements, rnase inhibitors, crowding agents, potassium ions, antioxidants or reducing agents, cryoprotectants, trehalose, reaction promoters, antifoams, alkanes, buffers, aqueous solvents.

The translation-related element is preferably selected from: tRNA, ribosomes, other translation-related enzymes, initiation factors, elongation factors, termination factors, and combinations thereof. The translation-related element is preferably a purified translation-related element.

The crowding agent is preferably selected from: polyethylene glycol, polyvinyl alcohol, polystyrene, dextran, sucrose polymers (including Ficoll sucrose polymers, e.g., Ficoll sucrose polymers)

A reagent, a non-ionic synthetic sucrose polymer; also included are polysucrose), polyvinylpyrrolidone (PVP), albumin, the like, and combinations thereof.

The source of potassium ions 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, and combinations thereof.

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

The cryoprotectant may include, but is not limited to, trehalose.

Trehalose is useful as an antifreeze agent and as a component of the energy system.

The reaction promoter is preferably an aluminum salt, an aluminum oxide (e.g., alumina), an iron salt, an iron oxide, a calcium salt, or combinations thereof.

The alkane is preferably C₆～C₄₄A pure or mixture of alkanes; the alkane is further preferably cyclohexane, isooctane, decane, tetradecane, pentadecylcyclohexane, squalane, tetradecane, vaseline, or a combination thereof.

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

The aqueous solvent is preferably a buffer.

Any one of the components of the CFPS (Mg +) system to which the present invention relates allows two or more functions to be performed.

The CFPS (Mg +) system can be used for obtaining the foreign protein through in-vitro protein synthesis reaction with any one of a DNA template for coding the foreign protein, an mRNA template for coding the foreign protein or a combination thereof.

The above-described preferred modes may be combined in any suitable manner.

2. In a second aspect, the present invention provides an in vitro protein synthesis kit comprising:

(i) an in vitro cell-free protein synthesis system (CFPS (Mg +) system) containing exogenous magnesium ions according to the first aspect;

(ii) optionally including a nucleic acid template encoding a foreign protein;

(iii) a label or instructions.

The CFPS (Mg +) system is capable of providing translation-related elements required for synthesizing a foreign protein in conjunction with the nucleic acid template encoding the foreign protein.

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

Preferably, said (i) has a separate aliquot of the cell extract.

The in vitro protein synthesis kit can be used for carrying out in vitro protein synthesis reaction to synthesize exogenous protein.

3. The third aspect of the present invention provides a method for synthesizing a foreign protein, comprising the steps of:

(i) providing a CFPS (Mg +) system according to the first aspect of the invention;

(ii) adding a nucleic acid template for encoding the foreign protein, and carrying out incubation reaction to synthesize the foreign protein;

the CFPS (Mg +) system is capable of providing translation-related elements required for synthesizing a foreign protein together with the nucleic acid template encoding the foreign protein;

further optionally comprising step (iii): isolating or/and detecting the foreign protein.

In the second aspect and the third aspect, each independently includes but is not limited to the following preferred modes:

(1) in one preferred embodiment, the nucleic acid template encoding the foreign protein comprises a promoter element recognized by the CFPS (Mg +) system.

(2) In a preferred embodiment, the CFPS (Mg +) system comprises a cell extract, and the nucleic acid template encoding the foreign protein comprises a promoter element that is recognized by the cell extract. For example, the cell extract contains an endogenously expressed RNA polymerase which corresponds to the promoter element on the nucleic acid template.

(3) In one preferred embodiment, the nucleic acid template encoding the foreign protein comprises a T7 promoter, and the CFPS (Mg +) system comprises T7RNA polymerase.

(4) In one preferred embodiment, the nucleic acid template encoding the foreign protein comprises a T7 promoter, and the CFPS (Mg +) system comprises a cell extract comprising endogenously expressed T7RNA polymerase.

(5) Preferably, the nucleic acid template encoding the foreign protein contains a T7 promoter capable of initiating the gene transcription program of the foreign protein, i.e., the gene transcription process of the foreign protein is initiated by the T7 promoter on the nucleic acid template.

(6) In one preferred embodiment, the nucleic acid template encoding the foreign protein comprises a T7 promoter capable of initiating a gene transcription process for the foreign protein, and the CFPS (Mg +) system comprises T7RNA polymerase.

(7) In one preferred embodiment, the nucleic acid template encoding the foreign protein comprises a T7 promoter capable of initiating a gene transcription process for the foreign protein (T7 promoter is located upstream of the coding sequence for the foreign protein in the nucleic acid template, and the gene transcription process for the foreign protein is initiated by a T7 promoter), and the CFPS (Mg +) system comprises a cell extract comprising endogenously expressed T7RNA polymerase.

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.

4. The fourth aspect of the present invention provides an application of the in vitro cell-free protein synthesis system (CFPS (Mg +) system) containing exogenous magnesium ions according to the first aspect, in protein synthesis. The application of the method to protein synthesis includes, but is not limited to, application to protein manufacture, or application to detection based on protein synthesis and the like.

5. According to a fifth aspect of the present invention there is provided the use of magnesium gluconate in an in vitro cell-free protein synthesis system comprising exogenous magnesium ions according to the first aspect, or in an in vitro protein synthesis kit according to the second aspect, or in a method of synthesis of an exogenous protein according to the third aspect.

Has the advantages that:

the invention optimizes an in vitro cell-free protein synthesis system, provides an in vitro cell-free protein synthesis system (CFPS (Mg +) system) containing exogenous magnesium ions, adopts magnesium gluconate as a novel magnesium ion supply source, and can obviously improve the protein synthesis efficiency and the protein synthesis amount compared with the traditional magnesium ion supply source (particularly compared with the commonly used magnesium acetate and magnesium glutamate). Further provides a more efficient and higher-flux in-vitro cell-free protein synthesis kit (particularly a D2P kit) and a synthesis method of the exogenous protein, and has the advantages of simplicity, convenience and low cost. In addition, the invention can adopt extracts including but not limited to eukaryotic cells, can provide a better post-translational modification mechanism, is used for synthesizing complex functional proteins containing post-translational modifications (such as disulfide bonds, glycosylation and the like), and has wide application space.

We speculate that, in the reaction process, the nucleotide and the exogenous magnesium ion source have competitive binding effect on the metal magnesium ion (namely, the interaction between the nucleotide and the metal magnesium ion is competitive with the interaction between the nucleotide and the magnesium ion in the exogenous magnesium ion), and along with the dynamic concentration change of the nucleotide and the nucleic acid substance, the magnesium ion concentration of the system is in an unstable state, thereby affecting the protein synthesis efficiency and the protein expression amount. Compared with the traditional magnesium sources (magnesium acetate and magnesium glutamate), in the magnesium gluconate molecule, the gluconic acid residue part has relatively strong binding force to magnesium ions, a magnesium ion-gluconic acid complex can be formed in a solution, and a plurality of hydroxyl groups in the structure can also participate in stabilizing the magnesium ions, so that a more stable magnesium ion concentration is provided for a system, and the protein synthesis capability of the system is increased (refer to the following documents of Bailey GD and Car WR.13C NMR regeneration students of glucose and magnesium-glucose interactions [ J ]. Journal of organic Biochemistry,1993,52(2), 99-108).

When the in vitro protein synthesis is mainly regulated and controlled by the general attributes among different strains, the technical effect generated by the corresponding technical means can not be influenced by the difference of the strains and can be generally applied among the strains. The novel magnesium ion supply provided by the invention is not only suitable for optimizing an in vitro protein synthesis system of the kluyveromyces lactis cell extract, but also suitable for optimizing in vitro protein synthesis systems of other yeast systems and other eukaryotic systems; can also be used for optimizing an in vitro protein synthesis system of a prokaryotic system.

Drawings

FIG. 1 shows a schematic structure of a plasmid DNA encoding a foreign protein mEGFP, which is 6056bp in total and is designated as a plasmid D2P-mEGFP (abbreviated as pD 2P-mEGFP). The mEGFP is a mutant of enhanced green fluorescent protein. The plasmid DNA comprises the following elements: t7 promoter (recognized by T7RNA polymerase), 5 'noncoding region, leader sequence, purification tag (optional element), coding sequence of foreign protein mmefp, 3' noncoding region, LAC4 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, located downstream of ori, not shown in the figure), lacI promoter, and coding gene for LAC repressor (lacI).

FIG. 2, an exemplary structure of an exogenous nucleic acid template. The exogenous nucleic acid template in the figure is plasmid DNA encoding an exogenous protein.

FIG. 3, another exemplary structure of an exogenous nucleic acid template. The exogenous nucleic acid template in the figure is plasmid DNA encoding an exogenous protein.

FIG. 4, effect of magnesium gluconate and magnesium acetate concentrations on protein synthesis capacity of the in vitro cell-free protein synthesis system. The amount of synthesized foreign protein mEGFP is indicated by RFU value. The abscissa is marked as "group-magnesium ion concentration value", where the magnesium ion concentration value is in mM. The Gluconate group and the Acetate group respectively provide exogenous magnesium ions by magnesium Gluconate and magnesium Acetate. BC is blank control.

FIG. 5, effect of magnesium gluconate and magnesium acetate concentrations on protein synthesis capacity of the in vitro cell-free protein synthesis system. The amount of synthesized foreign protein mEGFP is indicated by RFU value. The abscissa is marked by the "total concentration of magnesium ions (percentage of magnesium gluconate)".

FIG. 6, effect of magnesium gluconate and magnesium glutamate concentrations on protein synthesis capacity of in vitro cell-free protein synthesis system. The amount of synthesized foreign protein mEGFP is indicated by RFU value. The abscissa is marked as "group-magnesium ion concentration value", where the magnesium ion concentration value is in mM. The Gluconate group provides exogenous magnesium ions from magnesium Gluconate. BC is blank control. PC is a positive control, and magnesium glutamate is used for providing external magnesium ions.

FIG. 7, effect of magnesium gluconate and magnesium glutamate concentrations on protein synthesis capacity of in vitro cell-free protein synthesis system. The amount of synthesized foreign protein mEGFP is indicated by RFU value. The abscissa is marked as "group-magnesium ion concentration value", where the magnesium ion concentration value is in mM. The Gluconate group provides exogenous magnesium ions from magnesium Gluconate. BC is blank control. PC is a positive control, and magnesium glutamate is used for providing external magnesium ions.

FIG. 8, effect of magnesium gluconate, magnesium acetate, and magnesium glutamate concentrations on protein synthesis capacity of the in vitro cell-free protein synthesis system. The amount of synthesized foreign protein mEGFP is indicated by RFU value. The composition of the exogenous magnesium ions is shown in Table 4 of example 7.

FIG. 9, effect of magnesium gluconate, magnesium acetate, and magnesium glutamate concentrations on protein synthesis capacity of the in vitro cell-free protein synthesis system. The amount of synthesized foreign protein mEGFP is indicated by RFU value. The abscissa is marked as "group-magnesium ion concentration value", where the magnesium ion concentration value is in mM. BC is blank control. The Gluc group, the Ace group and the Glut group respectively adopt magnesium gluconate, magnesium acetate and magnesium glutamate to provide external magnesium ions.

FIG. 10 influence of magnesium gluconate, magnesium acetate, and magnesium glutamate concentrations on the protein synthesis capacity of the in vitro cell-free protein synthesis system (3 h). The amount of synthesized foreign protein mEGFP is indicated by RFU value. The Gluc group, the Ace group and the Glut group respectively adopt magnesium gluconate, magnesium acetate and magnesium glutamate to provide external magnesium ions.

FIG. 11, effect of magnesium gluconate, magnesium acetate, and magnesium glutamate concentrations on protein synthesis capacity of the in vitro cell-free protein synthesis system (20 h). The amount of synthesized foreign protein mEGFP is indicated by RFU value. The Gluc group, the Ace group and the Glut group respectively adopt magnesium gluconate, magnesium acetate and magnesium glutamate to provide external magnesium ions.

FIG. 12, effect of magnesium gluconate, magnesium acetate, and magnesium glutamate concentrations on protein synthesis capacity of in vitro cell-free protein synthesis system. The amount of synthesized foreign protein mEGFP is indicated by RFU value. The composition of the exogenous magnesium ions is shown in Table 5 of example 10.

FIG. 13, effect of magnesium gluconate, magnesium acetate, and magnesium glutamate concentrations on protein synthesis capacity of the in vitro cell-free protein synthesis system. The amount of synthesized foreign protein mEGFP is indicated by RFU value. The composition of the exogenous magnesium ions is shown in Table 6 of example 11.

FIG. 14, effect of magnesium gluconate, magnesium acetate, and magnesium glutamate concentrations on protein synthesis capacity of in vitro cell-free protein synthesis system. The amount of synthesized foreign protein mEGFP is indicated by RFU value. The composition of the exogenous magnesium ions is shown in Table 7 of example 12.

FIG. 15, effect of magnesium gluconate and magnesium aspartate concentrations on protein synthesis capacity of the in vitro cell-free protein synthesis system. The amount of synthesized foreign protein mEGFP is indicated by RFU value. The abscissa is marked by the "total concentration of magnesium ions (percentage of magnesium gluconate)".

FIG. 16, effect of magnesium gluconate, magnesium aspartate, and magnesium acetate concentrations on protein synthesis capacity of the in vitro cell-free protein synthesis system. The amount of synthesized foreign protein mEGFP is indicated by RFU value. The abscissa is marked by the "total concentration of magnesium ions (percentage of magnesium gluconate)".

Nucleotide and/or amino acid sequence listing

SEQ ID No.1, the gene sequence of the foreign protein mEGFP, the length of 714 bases.

SEQ ID No.2, amino acid sequence of foreign protein mEGFP, 238 amino acids in total.

Detailed Description

The meaning of the terms, nouns, phrases of the present invention.

The meaning of this section is to be interpreted as applying to the invention in its entirety, both as follows and as above. In the present invention, when a cited document is referred to, the definitions of the related terms, nouns and phrases in the cited document are also incorporated, but in case of conflict with the definitions in the present invention, the definitions in the present invention shall control. In the event that a definition in a cited reference conflicts with a definition in the present disclosure, the cited components, materials, compositions, materials, systems, formulations, species, methods, devices, etc. are not to be construed as limiting.

In vitro protein synthesis reaction refers to a reaction for synthesizing a protein in an in vitro cell-free synthesis system, and at least comprises a translation process. Including but not limited to IVT response (in vitro translation reaction), IVTT response (in vitro transcription translation reaction), IVDTT response (in vitro replication transcription translation reaction). In the present invention, IVTT reaction is preferred. Since the IVTT reaction, corresponding to the IVTT system, is a process of in vitro transcription and translation of DNA into Protein (Protein), we also refer to such in vitro Protein synthesis systems as the D2P system, the D-to-P system, the D _ to _ P system, and the DNA-to-Protein system; the corresponding in vitro Protein synthesis methods are also called D2P method, D-to-P method, D _ to _ P method, DNA-to-Protein method.

D2P, DNA-to-Protein, from DNA template to Protein product. For example, D2P technology, D2P system, D2P method, D2P kit, and the like.

mR2P, mRNA-to-Protein, from mRNA template to Protein product. For example, mR2P technology, mR2P system, mR2P method, mR2P kit, and the like.

IVTT, in vitro transcription translation.

IVDTT, in vitro replication transcription translation, replication transcription translation in vitro.

CFPS system: cell-free protein synthesis system, cell-free protein synthesis system.

CFPS (Mg +) system, a shorthand way of the 'external cell-free protein synthesis system containing exogenous magnesium ions' of the invention.

CFPS (Mg-) system refers to a system formed by other components except for the source magnesium ions in the CFPS (Mg +) system.

CFPS (MgP-) system, which refers to the system composed of other components except magnesium gluconate in the CFPS (Mg +) system.

"cell-free system" refers to a system in which protein synthesis is performed in vitro, but not by secretory expression from intact cells. It should be noted that, in the in vitro cell-free protein synthesis system of the present invention, it is also allowable to add cell components to promote the reaction, but the added cells do not mainly aim at secreting and expressing exogenous target proteins (exogenous target proteins). In addition, in the CFPS system without intact cells constructed under the guidance of the present invention, such a "evasion" manner that a small amount of intact cells is intentionally added (e.g., which provides a protein content of not more than 30 wt% compared to the protein content provided by the cell extract) is also included in the scope of the present invention.

The terms "expression system of the invention", "in vitro cell-free expression system" and "in vitro cell-free expression system" are used interchangeably and refer to in vitro protein expression systems of the invention, and can also be used in other descriptive ways, such as: protein in vitro synthesis system, in vitro protein synthesis system, cell-free protein synthesis system, cell-free in vitro protein synthesis system, in vitro cell-free synthesis system, CFS system (cell-free system), CFPS system (cell-free protein synthesis system), etc. According to the reaction mechanism, an in vitro translation system (abbreviated to IVT system, an mR2P system), an in vitro transcription translation system (abbreviated to IVTT system, a D2P system), an in vitro replication transcription translation system (abbreviated to IVDTT system, a D2P system) and the like can be included. In the present invention, the IVTT system is preferred. We also refer to the in vitro Protein synthesis system as a "Protein Factory" or "Protein Factory". The components of the in vitro protein synthesis system provided by the invention are described in an open mode.

In vitro protein synthesis reaction mixtures, also described as reaction mixtures, reaction mixing systems, in vitro protein synthesis reaction mixing systems, refer to mixing systems comprising in vitro protein synthesis systems, nucleic acid templates encoding foreign proteins; the system may be homogeneous or heterogeneous, and may be a liquid system such as a solution, an emulsion, or a suspension.

A magnesium source: the magnesium source of the exogenous magnesium ions of the present invention is classified by negative part, including but not limited to negative group, negative ion. The ratio between the magnesium atom and the negatively charged moiety is not particularly limited, unless otherwise specified. For example, the term "magnesium aspartate" used in the present invention defines that the magnesium source is a negative part provided by a residue of aspartic acid (specifically, a carboxyl group), and the chelating ratio between the magnesium source and magnesium is not particularly limited, and for example, one magnesium atom may chelate two aspartic acid molecules, or one magnesium atom may chelate one aspartic acid molecule by intramolecular chelation; the site of complexation, binding, or chelation with magnesium ions is not particularly limited, and may be a carboxyl group at the α -position, an carboxyl group at the e-position, or a combination of both. For another example, magnesium diglutamate clearly defines a magnesium atom chelating two glutamic acid molecules. In addition, there is no limitation as to whether the magnesium source has bound water. The invention quantifies the exogenous magnesium ions by controlling the content of magnesium atoms.

RFU, Relative Fluorescence Unit value (Relative Fluorescence Unit).

eGFP: enhanced green fluorescence protein (enhanced green fluorescence protein).

mEGFP: a206K mutant of eGFP.

mol%: mole percent, represents the amount of a substance as a percentage.

wt% or% (wt): are mass concentration units and all represent mass percent.

(v/v)% or% (v/v): all represent volume percent.

% (w/v): mass volume concentration units, corresponding to g/100 mL.

In the present invention, "protein" and "protein" have the same meaning, and are each translated into protein, and they can be used interchangeably.

In the present invention, both "system" and "system" are translated into system and used interchangeably.

In the present invention, "protein synthesis amount", "protein expression amount" and "protein expression yield" have the same meaning and are used interchangeably.

In the present invention, the cell extract, the cell lysate, the cell disruptant, and the cell lysate have the same meaning and can be used interchangeably, and english can adopt the descriptions of cell extract, cell lysate, and the like.

In the present invention, the energy system, and the energy supply system have equivalent meanings and can be used interchangeably. The energy regeneration system and the energy regeneration system have equivalent meanings and can be used interchangeably. The energy regeneration system is a preferred embodiment or component of the energy system.

Post-translational modification: also known as post-translational processing, post-translational modification, PTM. The PTM system plays a significant role in the normal folding, activity and stability of proteins.

In the present invention, "translation-related elements" refer to functional elements related to the synthesis of a protein product from a nucleic acid template, and are not limited to functional elements required for translation; when the nucleic acid template is DNA, functional elements required in the transcription process are also included in a broad sense. The translation-related elements can be provided by cell extracts (various endogenous factors), other exogenously added components of the in vitro protein synthesis system (e.g., translation-related elements such as exogenous RNA polymerase, tRNA, ribosomes, other translation-related enzymes, initiation factors, elongation factors, termination factors, or combinations thereof), functional elements on the nucleic acid template (e.g., functional elements that control transcription/translation of exogenous proteins, resistance gene translation systems, lac repressor translation systems, translation systems that control plasmid copy number, etc.), and the like. The functional elements for controlling transcription/translation of the foreign protein are exemplified by promoters, terminators, enhancers, IRES elements, kozak sequences, other elements for regulating the level of translation, signal sequences, leader sequences, functional tags (e.g., a selection marker tag, a tag for enhancing the level of translation), and the like.

DNA amplification related elements including at least a DNA polymerase. Other factors such as helicases (HDA amplification), recombinases and single stranded DNA binding proteins (RPA amplification), etc. may also be included, depending on the amplification mechanism.

Gene: including coding and non-coding regions.

The nucleotide sequence is as follows: a sequence consisting of nucleotide units.

Nucleic acid sequence: the sequence of the nucleic acid substance includes DNA sequence and RNA sequence.

A coding sequence: coding sequence, abbreviated CDS. A nucleotide sequence corresponding exactly to a codon of a protein, which sequence does not contain other sequences corresponding to the protein in between (irrespective of sequence changes during mRNA processing etc.).

The coding gene is as follows: the useful gene segments encoding the protein may be contiguous or non-contiguous. The coding gene necessarily includes a coding sequence.

Nucleic acid template: also referred to as 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 invention, the nucleic acid templates may each independently be DNA templates, mRNA templates, or a combination thereof. In any embodiment of the invention, the nucleic acid templates may each independently preferably be DNA templates. In the present invention, the nucleic acid template encoding the foreign protein is preferably a DNA template, unless otherwise specified.

"nucleic acid template encoding a protein X" refers to a nucleic acid template that contains a coding sequence for the protein X, on the basis of which the protein X can be synthesized by at least translation (e.g., by transcription and translation), and that allows the nucleic acid template to contain non-coding regions and also allows the nucleic acid template to contain coding sequences for polypeptides or proteins other than the protein X. For example, a "nucleic acid template encoding RNA polymerase" includes at least the coding sequence of RNA polymerase, and further allows the inclusion of other nucleic acid sequences such as non-coding regions, fusion tags, and the like; the corresponding expression product contains at least an RNA polymerase structure, and can be an RNA polymerase molecule or a fusion protein thereof, and can also be a mixed component comprising the RNA polymerase molecule or/and a fusion protein molecule thereof.

A reinforcing element: unless otherwise specified, the present invention refers to a sequence which functions to promote transcription or/and translation processes in a nucleic acid sequence located between a promoter and a coding sequence of a target protein, such as an omega sequence, a kozak sequence, an IRES sequence, and the like. Including transcription enhancing elements, translation enhancing elements.

Endogenous/endogenous: depending on the activity of the active cell metabolism. Endogenously expressed proteins are endogenously secreted by the cells as they grow and can be processed to be present in the cell extracts of the invention.

Exogenous/exogenous: independent of active cellular metabolic activity. The exogenous components are added directly to the in vitro protein synthesis system, rather than by way of adding cells or cell extracts. Such as: exogenous RNA polymerase can be added to the reaction system by exogenous means by adding a precursor (e.g., an inactive precursor that can be enzymatically or otherwise activated to produce RNA polymerase), a nucleic acid template (which can be translated into a protein by the system), a fusion protein, a pure substance, or a mixture. For another example: exogenous DNA polymerase can also be added to the reaction system by exogenous means as described above.

Foreign proteins: the expression product of interest of the in vitro protein synthesis system of the invention is not secreted and synthesized by the host cell. Can be a protein, a fusion protein, a mixture of protein-containing molecules or fusion protein molecules; also broadly included are polypeptides. The product obtained after the in vitro protein synthesis reaction based on the nucleic acid template encoding the foreign protein can be a single substance or a combination of two or more substances.

Exogenous RNA polymerase: has the same meaning as that of exogenous RNA polymerase.

Exogenous DNA polymerase: has the same meaning as that of an exogenous DNA polymerase.

"nucleic acid template encoding RNA polymerase (or nucleic acid template encoding DNA polymerase)" includes at least the coding sequence of RNA polymerase (or DNA polymerase), and further allows the inclusion of non-coding regions, fusion tags, and other nucleic acid sequences; accordingly, the expression product contains at least an RNA polymerase structure (or a DNA polymerase structure). Taking RNA polymerase as an example, the RNA polymerase can be an RNA polymerase molecule or a fusion protein thereof, and can also be a mixed component comprising the RNA polymerase molecule or/and a fusion protein molecule thereof.

A peptide is a compound formed by two or more amino acids linked together by peptide bonds. In the present invention, the peptide and the peptide fragment have the same meaning and may be used interchangeably.

Polypeptide, peptide composed of 10-50 amino acids.

Protein, peptide composed of more than 50 amino acids. The fusion protein is also a protein.

Derivatives of polypeptides, derivatives of proteins: any polypeptide or protein to which the present invention relates, unless otherwise specified (e.g., specifying a particular sequence), is understood to also include derivatives thereof. The derivatives of the polypeptide and the derivatives of the protein at least comprise C-terminal tags, N-terminal tags, C-terminal tags and N-terminal tags. Wherein, C terminal refers to COOH terminal, N terminal refers to NH₂The meaning of which is understood by those skilled in the art. The label can be a polypeptide label or a protein label. Some examples of tags include, but are not limited to, 6-histidine (6X-His, HHHHHHHH), Glu-Glu, c-myc epitopes (EQKLISEEDL),

Octapeptide (DYKDDDDK), protein C (EDQVDPRLIDGK), Tag-100(EETARFQPGYRS), V5 epitope Tag (V5 epitope, GKPIPNPLLGLDST), VSV-G (YTDIEMNRLGK), Xpress (DLYDDDDK), hemagglutinin (YPYD)VPDYA), β -galactosidase (β -galactosidase), thioredoxin (thioredoxin), His-site thioredoxin (His-batch thioredoxin), IgG binding domain (IgG-binding domain), intein-chitin binding domain (intein-chitin binding domain), T7 gene 10(T7 gene 10), glutathione S-transferase (GST), Green Fluorescent Protein (GFP), Maltose Binding Protein (MBP), and the like.

Homology (homology), unless otherwise specified, means 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; also such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology. The description object is exemplified by homologous sequences such as the omega sequences mentioned in the present description.

"variant," or "variant," refers to a substance that has a different structure (including, but not limited to, minor variations) but retains or substantially retains its original function or property. Such variants include, but are not limited to, nucleic acid variants, polypeptide variants, protein variants. Means for obtaining related variants include, but are not limited to, recombination, deletion or deletion, insertion, displacement, substitution, etc. of the building blocks. Such variants include, but are not limited to, modified products, genetically engineered products, fusion products, and the like. To obtain the gene modification product, the gene modification can be performed by, but not limited to, gene recombination (corresponding to the gene recombination product), gene deletion or deletion, insertion, frame shift, base substitution, and the like. Gene mutation products, also called gene mutants, belong to one type of gene modification products.

Modified product: including but not limited to chemically modified products, amino acid modifications, polypeptide modifications, protein modifications, and the like. The chemical modification product refers to a product modified by chemical synthesis methods such as organic chemistry, inorganic chemistry, polymer chemistry and the like. Examples of the modification method include ionization, salinization, desalinization, complexation, decomplexing, chelation, decomplexing, addition reaction, substitution reaction, elimination reaction, insertion reaction, oxidation reaction, reduction reaction, and post-translational modification, and specific examples thereof include oxidation, reduction, methylation, demethylation, amination, carboxylation, and vulcanization.

"mutant", mutant, as used herein, unless otherwise specified, refers to a mutant product that retains or substantially retains its original function or property, and the number of mutation sites is not particularly limited. Such mutants include, but are not limited to, gene mutants, polypeptide mutants, and protein mutants. Mutants are one type of variant. Means for obtaining relevant mutants include, but are not limited to, recombination, deletion or deletion of structural units, insertion, displacement, substitution, and the like. The structural unit of the gene is basic group, and the structural units of the polypeptide and the protein are amino acid. Types of gene mutations include, but are not limited to, gene deletions or deletions, insertions, frameshifts, base substitutions, and the like.

Amino acid mixture refers to a mixture containing at least two amino acids.

In the present invention, the amino acid may be a natural amino acid, an unnatural amino acid, or a mixture thereof, unless otherwise specified_L-an amino acid,_DAmino acids or combinations thereof, and may also be radiolabeled amino acids, modified amino acids, and the like. The modified amino acid refers to an amino acid to which a chemical modification group is attached, and the structure thereof is not particularly limited, including but not limited to modification by amino acid side groups. The above definition of amino acid encompasses any substance of the invention that includes an amino acid unit, including but not limited to: a polypeptide and a derivative thereof, a protein and a derivative thereof, a polypeptide tag, a protein tag, a polypeptide sequence, a protein sequence, an amino acid modification, a polypeptide modification, a protein modification, a partial domain of any of the foregoing, a subunit or a fragment of any of the foregoing (including a domain of any of the foregoing), and a variant of any of the foregoing (including a variant of a domain, a subunit, or a fragment of any of the foregoing). The "variant of any of the foregoing" includes, but is not limited to "a mutant of any of the foregoing. In the present invention, for compounds representing chiral types "_L-”、“_D- ", subscript form has the same meaning as non-subscript form.

Crowding agents, agents used to mimic the macromolecular environment of intracellular crowding in vitro. References "X Ge, D Luo and J xu. cell-free protein expression under macromolecular growth conditions [ J ]. PLoS One,2011,6(12): e 28707" and citations thereof, among others.

Sucrose polymer: refers to a polymer containing at least 2 sucrose units. Including but not limited to polysucrose.

Ficoll sucrose polymer: unless otherwise specified, refer in particular to

The reagent, a non-ionic synthetic sucrose polymer, is a highly branched polymer obtained by copolymerizing sucrose and epichlorohydrin, and can be selected from commercially available products. Examples are Ficoll-400 (Polysucrose 400, CAS:26873-85-8), Ficoll-70 (Polysucrose 70, CAS: 72146-89-5). Wherein the content of the first and second substances,

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.

The phosphoric acid compound comprises organic matters and inorganic matters.

The phosphate refers to an inorganic phosphate unless otherwise specified.

In the present invention, the "normal temperature" is preferably room temperature to 37 ℃, specifically, preferably 20 to 37 ℃, and more preferably 25 to 37 ℃.

In the present invention, the preferred embodiments such as "preferred", "more preferred" and "most preferred" are not intended to limit the embodiments of the present invention, but merely to provide examples of embodiments with better technical effects.

In the description of the present invention, for "one of the preferences", "one of the preferred embodiments", "preferred embodiment", "in a preferred embodiment", "preferably", "preferred", "preferably", "more preferred", "further preferred", "most preferred", etc. of the preferred embodiments, and "one of the embodiments", "example", "specific example", "by way of example", "for example", "such as", etc., of the illustrated exemplary embodiments, the specific features described in each embodiment are included in at least one embodiment of the present invention. The particular features described in connection with the various modes can be combined in any suitable manner in any one or more of the particular embodiments of the invention. In the invention, the technical schemes corresponding to the preferred modes can also be combined in any suitable mode; for example, an exogenous RNA polymerase and an exogenous DNA polymerase can be added simultaneously, see patent publication CN 108642076A.

In the present invention, "optionally" means either the presence or absence thereof. Whether the corresponding system is suitable or not is selected as the basis.

In the present invention, "any combination thereof" means "more than 1" in number, and means a group consisting of the following cases in an inclusive range: "optionally one of them, or optionally a group of at least two of them".

In the present invention, the description of "one or more", "one or more or all" and the like "has the same meaning as" at least one "," or a combination thereof "," and a combination thereof "," or any combination thereof "," and any combination thereof ", and the like, and may be used interchangeably to mean" equal to 1 or greater than 1 "in number.

In the present invention, "and/or" means "either one of them or any combination thereof, and also means at least one of them. By way of example, "comprising a substrate for a synthetic RNA and/or a substrate for a synthetic protein", it is meant that the substrate for a synthetic RNA alone may be included, the substrate for a synthetic protein alone may be included, and the substrate for a synthetic RNA and the substrate for a synthetic protein may be included at the same time.

The prior art means described in the modes of "usually", "conventionally", "generally", "often", and the like, are also referred to as the content of the present invention, and if not specifically stated, they are regarded as one of the preferred modes of the present invention.

All documents cited herein, and documents cited directly or indirectly by such documents, are hereby incorporated by reference into this application as if each were individually incorporated by reference.

It is understood that within the scope of the present invention, the above-mentioned technical features of the present invention and those specifically described below (including but not limited to the examples) can be combined with each other to constitute a new or preferred technical solution, as long as the foreign protein can be synthesized in vitro or, preferably, efficiently. Not described in detail.

1. The invention provides an external cell-free protein synthesis system containing exogenous magnesium ions, wherein the external cell-free protein synthesis system containing exogenous magnesium ions is also abbreviated as CFPS (Mg +) system in the invention and comprises exogenous magnesium ions; the exogenous magnesium ions come from one or more sources and at least comprise magnesium gluconate. The CFPS (Mg +) system is capable of providing translation-related elements required for synthesis of a foreign protein in conjunction with a nucleic acid template encoding the foreign protein.

The CFPS (Mg +) system can perform in-vitro protein synthesis reaction with the nucleic acid template for encoding the foreign protein so as to express the foreign protein.

Preferably, the CFPS (Mg +) system contains a system component capable of recognizing a promoter element in a nucleic acid template, such that the CFPS (Mg +) system is capable of recognizing a promoter element of a nucleic acid template encoding a foreign protein.

By limiting the technical functions of "expressing a foreign protein", the present invention covers only combinations of technical features that can achieve the above-described functions, and combinations of technical features that cannot achieve the above-described functions are, of course, excluded from the scope of the present invention. That is, the CFPS (Mg +) system should be a workable system in the first place, and a system capable of expressing a foreign protein.

1.1. External source magnesium ion-containing in-vitro cell-free protein synthesis system (CFPS (Mg +) system)

The in vitro protein synthesis reaction is carried out in an in vitro cell-free protein synthesis system containing exogenous magnesium ions.

The CFPS (Mg +) system is capable of providing various factors required for the in vitro synthesis of proteins. It can be provided in an integrated form by means of a cell extract, or in a separate addition form (e.g., the Japanese PURE system, such as the PURExpress kit).

The kind and content of each component of the CFPS (Mg +) system are not particularly limited as long as the system is constructed to be capable of reacting with a nucleic acid template encoding a foreign protein to synthesize the foreign protein, and a combination manner capable of efficiently expressing the foreign protein is preferred. Combinations that do not allow the expression of the foreign protein due to the concentration of certain components being too low or too high are, of course, excluded from the scope of the invention.

The order of addition of the components of the CFPS (Mg +) system is not particularly limited.

The concentrations of the individual components of the CFPS (Mg +) system, unless otherwise specified, refer to the initial concentrations in the in vitro protein synthesis reaction mixture.

Preferably, the CFPS (Mg +) system contains a system component capable of recognizing a promoter element on a nucleic acid template, such as an RNA polymerase corresponding to the promoter element.

The components of the system (e.g., the corresponding RNA polymerase) that are capable of recognizing the promoter element in the nucleic acid template may be provided by a cell extract in the system, may be provided by exogenous addition, or may be provided by a combination of the two.

In one embodiment, the CFPS (Mg +) system comprises a cell extract, exogenous magnesium ions (including at least magnesium gluconate).

In a preferred embodiment, the CFPS (Mg +) system comprises at least a cell extract. The cell extract is intended to provide a structure or biological factor for the transcription and translation of proteins. The selection criteria of the cell extract are as follows: can synthesize the exogenous protein through in vitro protein synthesis reaction based on a nucleic acid template for encoding the exogenous protein. The cell extract of the present invention may be derived from a wild type or a non-wild type. Non-wild-type modifications include, but are not limited to, gene modifications. The cell extract of the present invention is preferably derived from a eukaryotic cell, more preferably from a yeast cell, more preferably from a kluyveromyces lactis cell.

In a preferred embodiment, the CFPS (Mg +) system comprises a cell extract containing an endogenously expressed RNA polymerase corresponding to a promoter element on a nucleic acid template. Specifically, for example, the kluyveromyces lactis cell extract contains endogenously expressed T7RNA polymerase, which can recognize the T7 promoter on the nucleic acid template.

In a preferred embodiment, the Protein synthesis Using Recombinant Elements (PURE) system developed by Japanese scientists is used to provide various factors required for the in vitro Protein synthesis process, but not to provide the various factors integrally by cell extraction. Reference is made to the introduction of the PURE system in the publications "Lu, Y.Advances in Cell-Free Biosynthetic technology.Current Developments in Biotechnology and Bioengineering,2019, Chapter 2, 23-45.", "Y Shimizu, A Inoue, Y Tomari, et al.cell-Free transformed with purified components [ J ]. Nature Biotechnology,2001,19(8):751 755", et al.

The in vitro cell-free protein synthesis system comprises exogenous magnesium ions, and the CFPS (Mg +) system at least comprises magnesium gluconate; preferably at least 25 mol%, more preferably at least 30 mol%, more preferably at least 40 mol%, more preferably at least 50 mol% of the exogenous magnesium ions are provided by magnesium gluconate.

The process of in vitro synthesis of proteins includes at least a translation process and optionally also a transcription process.

The transcription process to convert DNA into mRNA is not isolated from RNA polymerase. In this case, the corresponding CFPS (Mg +) system preferably further comprises an RNA polymerase, and the source of the RNA polymerase may be selected from: endogenously expressed RNA polymerase (provided via cell extract), exogenously added RNA polymerase, translation products of an exogenous nucleic acid template encoding RNA polymerase, and combinations thereof.

The endogenously expressed RNA polymerase is not added separately but is present in the cell extract.

In order to achieve the inclusion of endogenously expressed RNA polymerase in the cell extract, the coding sequence/gene for the RNA polymerase is preferably integrated into the host cell from which the cell extract is prepared; particularly preferably by: inserting the coding sequence/gene of RNA polymerase into cell free plasmid, or integrating the coding gene of RNA polymerase into cell genome, or performing strain modification by adopting the combination of the two ways, and then preparing cell extract. Such means of integrating the coding sequence/gene of the RNA polymerase into the genome of the cell include, but are not limited to: insertion into the genome of a cell, in situ replacement of portions of the genome, and combinations thereof.

The exogenously added or endogenously expressed RNA polymerase is each independently preferably T7RNA polymerase.

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

The CFPS (Mg +) system, optionally including an exogenous RNA polymerase or/and a nucleic acid template encoding an RNA polymerase.

The CFPS (Mg +) system, optionally includes an exogenous DNA polymerase or/and a nucleic acid template encoding a DNA polymerase.

In one preferred embodiment, the CFPS (Mg +) system includes an exogenous RNA polymerase and an exogenous DNA polymerase. Reference CN 108642076A.

In one preferred form, the CFPS (Mg +) system includes an energy system.

In one preferred form, the CFPS (Mg +) system includes a substrate for RNA synthesis.

In one preferred form, the CFPS (Mg +) system includes a substrate for a synthetic protein.

In one preferred embodiment, the CFPS (Mg +) system comprises a DNA polymerase, a substrate for DNA synthesis.

In a preferred embodiment, the CFPS (Mg +) system comprises a cell extract, exogenous magnesium ions (including at least magnesium gluconate), an energy system, a substrate for RNA synthesis, and a substrate for protein synthesis.

In one preferred embodiment, the CFPS (Mg +) system comprises a cell extract, exogenous magnesium ions (at least including magnesium gluconate; one preferred is magnesium gluconate), an energy system, a substrate for protein synthesis, RNA polymerase (contained in the cell extract or independently added exogenously), and a substrate for RNA synthesis.

In one preferred embodiment, the CFPS (Mg +) system comprises a cell extract, magnesium gluconate, an energy system, a substrate for protein synthesis, an RNA polymerase (contained in the cell extract or separately added exogenously), a substrate for RNA synthesis, a DNA polymerase (contained in the cell extract or separately added exogenously), and a substrate for DNA synthesis.

In one preferred embodiment, the CFPS (Mg +) system comprises kluyveromyces lactis cell extract (containing endogenously expressed T7RNA polymerase), magnesium gluconate, an energy system, a substrate for RNA synthesis, and a substrate for protein synthesis.

In one preferred embodiment, the CFPS (Mg +) system comprises kluyveromyces lactis cell extract (the host cell does not endogenously incorporate the coding gene for RNA polymerase), magnesium gluconate, an energy system, an exogenous RNA polymerase, a substrate for RNA synthesis, and a substrate for protein synthesis.

The CFPS (Mg +) system also optionally includes at least one of the following exogenously added components: translation-related elements, DNA amplification-related elements, RNA amplification-related elements, rnase inhibitors, crowding agents, potassium ions, antioxidants or reducing agents, cryoprotectants, trehalose, reaction promoters, antifoams, alkanes, buffers, aqueous solvents.

1.1.1. External source of magnesium ions

The in vitro cell-free protein synthesis system comprises exogenous magnesium ions to form a CFPS (Mg +) system.

The supply source of the exogenous magnesium ions at least comprises magnesium gluconate.

The supply of exogenous magnesium ions, optionally also from the group of: magnesium aspartate (_L-The model is,_D-Or combinations thereof, preferably_L-Type (III)), magnesium acetate, magnesium glutamate: (_L-Type or_D-Or combinations thereof, preferably_L-Type), magnesium chloride, magnesium phosphate, magnesium sulfate, magnesium citrate, magnesium hydrogen phosphate, magnesium iodide, magnesium lactate, magnesium nitrate, magnesium oxalate, and combinations thereof.

In the present invention, unless otherwise specified, the "magnesium ion source" means "a source for supplying an external magnesium ion".

In one preferred embodiment, the source of magnesium ions comprises magnesium gluconate and further comprises any one, two or three of magnesium aspartate (independently preferably magnesium L-aspartate), magnesium glutamate (independently preferably magnesium L-glutamate) and magnesium acetate.

In one preferred embodiment, the magnesium ion source is a combination of magnesium gluconate, magnesium aspartate (independently preferably magnesium L-aspartate), and magnesium glutamate (independently preferably magnesium L-glutamate). More preferably, the source of magnesium ions is a combination of magnesium gluconate, magnesium L-aspartate and magnesium L-glutamate.

In a preferred embodiment, the magnesium ion source is a combination of magnesium gluconate and magnesium aspartate, preferably magnesium L-aspartate.

Magnesium gluconate dosage (Q) capable of increasing synthesis amount of foreign protein_MgP) According to Y_PRT(C_MgP) Determining the expression level of the foreign protein in the curve, selected from Y_PRT(C_MgP) The expression quantity of the foreign protein in the curve is more than Y₀The interval of time of use. Said Y is_PRT(C_MgP) The curves can be obtained by preliminary experiments.

Said Q_MgPPreferably, the expression level of the foreign protein is at least Y₀+50％Y_ΔThe dosage range of magnesium gluconate;

said Q_MgPMore preferably, the expression level of the foreign protein is at leastY₀+60％Y_ΔThe dosage range of magnesium gluconate;

said Q_MgPMore preferably, the expression level of the foreign protein is at least Y₀+70％Y_ΔThe dosage range of magnesium gluconate;

said Q_MgPMore preferably, the expression level of the foreign protein is at least Y₀+80％Y_ΔThe dosage range of magnesium gluconate;

said Q_MgPMore preferably, the expression level of the foreign protein is at least Y₀+90％Y_ΔThe dosage range of magnesium gluconate;

said Q_MgPMore preferably, the expression level of the foreign protein is at least Y₀+95％Y_ΔThe dosage range of magnesium gluconate;

more preferably, said Q_MgPMagnesium gluconate dosage C when adopting the highest expression level of foreign protein_max。

Preferably, said Q_MgP、C_MgP、C_maxThe same characterization methods are adopted, for example, all the characterization methods adopt a concentration mode, all the characterization methods adopt a mass mode, or all the characterization methods adopt a molar quantity mode. For example, the amount of magnesium gluconate (C) can be characterized by concentration_MgP) Adding different amounts of magnesium gluconate into the CFPS (MgP-) system respectively to obtain a series of CFPS (Mg +) systems with different magnesium gluconate concentrations; performing in vitro protein synthesis reaction, and testing to obtain exogenous protein expression level (Y)_PRT) Obtaining the expression level (Y) of the foreign protein_PRT) And magnesium gluconate addition concentration (C)_MgP) Curve of relationship between (Y)_PRT(C_MgP) Curves); determining the maximum expression level (Y) of the foreign protein from the curve_max) Corresponding to the added concentration of magnesium gluconate (C)_max) Further, the dosage (Q) of magnesium gluconate capable of improving the expression level of the foreign protein, which is protected by the invention, is determined in a concentration characterization mode_MgP) The dosage interval of the concentration mode of (1).

In the present invention, the amount of magnesium gluconate to be used is preferably measured and controlled by the amount of the exogenous reagent to be added.

For the amount of foreign protein expression (Y)_PRT) Suitable assay methods may be optional, and suitable characterization means may be optional. Methods for measuring the amount of protein expression include, but are not limited to, ultraviolet absorption method, biuret method, BCA method, Lowry method, Coomassie Brilliant blue method, Kjeldahl method, and the like. The concentration, mass or amount of substance of the protein may be indicated by different characterization methods, such as, but not limited to, absorbance value (OD value) of the fluorescent-like protein, relative fluorescence unit value (RFU value) of the fluorescent-like protein, etc.

The magnesium gluconate is preferably characterized by its amount in a concentration manner in the present invention.

In one embodiment, a mutant mEGFP having a green fluorescence-enhanced protein or a fusion protein thereof, in which the mEGFP portion fluoresces, is used as a foreign Protein (PRT); after the in vitro protein synthesis reaction is finished, an ultraviolet absorption method is adopted to test the RFU value of the solution sample under the conditions of 488nm of excitation wavelength and 507nm of emission wavelength. Expression level (Y) of foreign protein product having mEGFP Structure_PRT) The method can be converted according to the following formula:

wherein RFU is the relative fluorescence unit value reading, C_PRTConcentration of the foreign protein product (in. mu.g/mL), M_mEGFPRelative molecular weight, M, of the mEGFP fluorescent protein Structure_PRTIs the relative molecular weight of the foreign protein product. In the conventional test range, C_PRTCan conform to a substantially linear relationship with the RFU. When mEGFP is directly used as a foreign protein, M_PRTAnd M_mEGFPAnd so on as in examples 3-15. And then combining the volume of the solution of the foreign protein product, and calculating to obtain the mass and molar weight of the foreign protein product.

In a preferred embodiment, the method for determining the magnesium gluconate dosage comprises: when the types and the contents of the components of the CFPS (Mg +) system are determined, the using amount of the magnesium gluconate is adjusted within a wider concentration range, and under the specified reaction conditions (reaction temperature, reaction time and the like), the using amount C of the magnesium gluconate when the expression amount of the foreign protein is the highest is determined_maxThe optimum dosage of the magnesium gluconate under the technical scheme is obtained.

Said Y is_PRT(C_MgP) Curve, Q_MgP，C_MgP，Y_PRT，Y_max，C_max，Y₀，Y_ΔThe definitions of (a) and (b) are consistent with the above.

In one preferred form, at least 25 mol% of the exogenous magnesium ions are provided by magnesium gluconate.

In one preferred form, at least 30 mol% of the exogenous magnesium ions are provided by magnesium gluconate.

In one preferred form, at least 40 mol% of the exogenous magnesium ions are provided by magnesium gluconate.

In one preferred form, at least 50 mol% of the exogenous magnesium ions are provided by magnesium gluconate.

In one preferred form, at least 80 mol% of the exogenous magnesium ions are provided by magnesium gluconate.

In one preferred form, at least 90 mol% of the exogenous magnesium ions are provided by magnesium gluconate.

In one preferred embodiment, 100 mol% of the exogenous magnesium ions are provided by magnesium gluconate.

The magnesium gluconate provides a mole percentage of exogenous magnesium ions to total exogenous magnesium ions, such as any one of the following concentration values, or a range of values between any two of the following concentration values (the range of values includes both endpoints): 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, 100%.

In a preferred embodiment, the concentration of the exogenous magnesium ions is 0.1 to 50 mM.

In another preferred embodiment, the concentration of the exogenous magnesium ions is 0.5 to 20 mM.

In another preferred embodiment, the concentration of the exogenous magnesium ions is 1 to 10 mM.

In another preferred embodiment, the concentration of the exogenous magnesium gluconate is 0.1 to 50mM, more preferably 0.5 to 20mM, and still more preferably 1 to 10 mM.

The concentration of the exogenous magnesium ion provided by the magnesium gluconate 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 two endpoints): 0.1mM, 0.5mM, 1mM, 1.5mM, 2mM, 2.5mM, 3mM, 3.5mM, 4mM, 4.5mM, 5mM, 5.5mM, 6mM, 6.5mM, 7mM, 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, 80 mM.

The protection scope of the invention only covers the technical scheme corresponding to the dosage of the magnesium gluconate capable of improving the expression quantity of the foreign protein. In the invention, for a CFPS (Mg +) system with any system formula, at least one magnesium gluconate dosage is required to be present to improve the expression quantity of the foreign protein, and all magnesium gluconate dosages are not required to be improved.

1.1.2. Cell extract

The cell extract should be capable of expressing the nucleic acid template encoding the foreign protein, i.e., capable of synthesizing the foreign protein encoded by the nucleic acid template encoding the foreign protein.

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

Cell extracts can provide many key translation-related elements required for synthesis of foreign proteins; this is the endogenous way of supply.

The cell extract, including yeast cell extracts, is typically used to provide ribosomes, transfer RNA (tRNA), aminoacyl tRNA synthetases, initiation and elongation factors for protein synthesis, and stop release factors, and may also be engineered to provide other enzymatic materials, such as polymerases (RNA polymerases and/or DNA polymerases), endogenously.

The cell extract is in principle free of intact cells, because the preparation method of the cell extract contains a step of disrupting cells (also referred to as a cell disruption treatment, a lysis step, etc.). In contrast to the traditional synthetic approach of secreting expressed proteins from intact cells, the in vitro protein synthesis system thus constructed is referred to as a cell-free system.

The cell extract may also contain some other proteins, especially soluble proteins, originating from the cytoplasm of the cell.

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

The related coding gene can be naturally present in the genome of the cell, or can be integrated into the genome of the cell (integrated into a chromosome), or can be inserted into an episomal plasmid of the cell. Taking RNA polymerase and DNA polymerase as examples, in one preferred embodiment, the cell extract contains endogenously expressed RNA polymerase and/or DNA polymerase.

The CFPS (Mg +) system optionally includes purified translation-related elements. When the cell extract is insufficient to provide all the translation-related elements necessary for synthesis of the foreign protein (in deficient species and/or in deficient amounts), the missing translation-related elements may also be added by means of exogenous additions. In particular, when the endogenous secretion product of the source strain is deficient in a component for the expression of the heterologous protein, it can be supplemented by exogenous addition. For example, the purified translation-related elements, including but not limited to, may be selected from any one or a combination of the following groups: tRNA, ribosomes, other translation-related enzymes, initiation factors, elongation factors, termination factors. Such translation-related enzymes include, but are not limited to, various aminoacyl-tRNA synthetases, peptidyl transferases, and the like.

Endogenous integration of the coding sequence or genes encoding the heterologous proteins into the cell from which the cell extract is derived may allow the engineered strain to endogenously express the heterologous proteins which may include, but are not limited to: RNA polymerase, DNA polymerase, etc. Methods for endogenous integration of the coding sequence or genes encoding the heterologous proteins can be referred to methods provided in the prior art documents including, but not limited to, documents CN109423496A, CN10697843A, CN2018116198190, "Molecular and Cellular Biology,1990,10(1): 353-360" and the like, and the references cited therein, and specifically, include, but are not limited to: the methods include inserting a coding sequence into an episomal intracellular plasmid, inserting a coding gene into a cellular genome, substituting a part of a gene of a cellular genome with a coding gene in situ, and the like, and combinations thereof.

In a preferred mode, the source cell of the cell extract is endogenously integrated with the coding gene of RNA polymerase, can endogenously express the RNA polymerase, can perform in-vitro cell-free protein synthesis under the condition of not adding exogenous RNA polymerase, replaces an exogenous addition mode, simplifies a formula, improves the operation convenience and saves the cost. Implementations of the endogenous integrated RNA polymerase include, but are not limited to: inserting a gene encoding RNA polymerase into a cellular plasmid or into the genome of a cell, replacing a portion of a gene or sequence of the genome with a gene encoding RNA polymerase in situ (i.e., including a step of knocking out the original portion of the gene or sequence), knocking out the original portion of the gene and inserting a gene encoding RNA polymerase, and combinations thereof. Further preferably, the source of the cell extract is yeast. Still more preferably, the source of the cellular extract is kluyveromyces lactis. In examples 2-15, the gene encoding T7RNA polymerase was integrated into the genome of a Kluyveromyces lactis cell that endogenously expresses T7RNA polymerase, and the cell extract thus prepared contained endogenously expressed T7RNA polymerase, without the additional addition of RNA polymerase to the in vitro cell-free protein synthesis system. In other embodiments, the cellular extract is prepared by inserting the coding sequence for RNA polymerase into an intracellular free plasmid of Kluyveromyces lactis. Refer specifically to the preparation method of CN 109423496A.

Other genetic modification methods can be adopted to modify the source cells so as to improve the activity of cell extracts and better promote in vitro protein synthesis, such as the genetic knockout methods of CN2018116083534, CN2019107298813 and CN108949801A, and the genetic modification method of CN 2018112862093.

The preparation method of the cell extract can adopt the reported technical means. In brief summary, the following steps may generally be included: providing sufficient amount of cells, quick freezing the cells with liquid nitrogen, breaking the cells, centrifuging and collecting supernatant to obtain cell extract. Reference is made to documents CN106978349A, CN108535489A, CN108642076A, CN109593656A, CN109971783A and the like. The seed cells may be subjected to fermentation culture, centrifuged, and the culture solution removed to collect a sufficient amount of cells to prepare a cell extract.

The cell extract prepared by the method can ensure that in vitro protein synthesis reaction is normally carried out, and contains necessary components required by protein synthesis such as tRNA with amino acid transport function, aminoacyl tRNA synthetase and the like. In some embodiments, the cell extract is a yeast cell extract, prepared using a method comprising: (i) providing a source cell; (ii) washing the yeast cells to obtain washed yeast cells; (iii) performing cell breaking treatment on the washed yeast cells to obtain a yeast crude extract; and (iv) performing solid-liquid separation on the yeast crude extract, wherein the collected supernatant part is the cell extract. The yeast cell extract is preferably a Kluyveromyces lactis cell extract.

In the present invention, one of preferable modes of the content of the protein contained in the cell extract is 20 to 100 mg/mL. Another preferred embodiment is 20 to 50 mg/mL. Another preferred embodiment is 50 to 100 mg/mL. Another preferred embodiment is a concentration of any one of 25mg/mL, 30mg/mL, 35mg/mL, 40mg/mL, 45mg/mL, 50mg/mL, 60mg/mL, 70mg/mL, 80mg/mL, 90mg/mL, or a range of concentrations between any two concentrations, inclusive. Methods for determining protein content include, but are not limited to, ultraviolet absorption, biuret, BCA, Lowry, Coomassie Brilliant blue, Kjeldahl, and the like. In a preferred embodiment, the method for determining the protein content is a coomassie brilliant blue assay.

The concentration of the cell extract in the in vitro protein synthesis reaction mixture is not particularly limited. The volume ratio and the weight ratio can be adopted; unless otherwise specified, the final volume ratio is meant. In a preferred embodiment, the concentration of the cell extract is 20% to 80% (v/v); in another preferred embodiment, the concentration of the cell extract is 20% to 70% (v/v); in another preferred embodiment, the concentration of the cell extract is from 30% to 60% (v/v); in another preferred embodiment, the concentration of the cell extract is from 40% to 50% (v/v); in another preferred embodiment, the concentration of the cell extract is 80% (v/v); all based on the total volume of the in vitro cell-free protein synthesis system. Examples of the concentration of the cell extract also include, but are not limited to, any one of the following volume percentages, or a numerical range between any two of the following volume percentages (the numerical range may or may not include both of the following endpoints): 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%.

The cell extract is derived from a prokaryotic cell, a eukaryotic cell, or a combination thereof.

One of the preferred modes of the cell extract of the present invention is derived from prokaryotic cells, more preferably E.coli (E.coli cells) or Bacillus.

One of the preferred embodiments of the cell extract of the present invention is derived from eukaryotic cells.

Prokaryotic cells and eukaryotic cells have fundamental differences in translation initiation and regulation mechanisms, and prokaryotic expression systems lack various post-translational protein processing mechanisms. Typically, cell-free systems based on E.coli cells lack the ability to perform translation or post-translational modifications that can only be performed by eukaryotic cell-free systems, making many eukaryotic proteins unsuitable for expression in E.coli systems; the synthesized protein process contains incomplete new polypeptide. The cell-free protein synthesis system based on the prokaryotic system is far from the cell-free protein synthesis system based on the eukaryotic system in terms of synthesis mechanism, and particularly relates to various factors in cell extracts. References include, but are not limited to, the following: "Nicole E.Gregorio, Max Z.Levine and Javin P.Oza.methods protocols.2019, 2, 24", "Edited by Alexander S.spirin and James R.Swartz.cell-free protein synthesis: methods and protocols [ M ] 2008, p.5", "Zhang Shu. The in vitro cell-free protein synthesis system of the present invention preferably employs eukaryotic cell extracts.

The cell source of the cell extract may be selected from, including but not limited to, eukaryotic cells from the group consisting of: mammalian cells (e.g., rabbit reticulocyte, HF9, Hela, CHO, K562, HEK293), plant cells (e.g., wheat germ cells, tobacco BY-2 cells), yeast cells, insect cells, nematode cells, and combinations thereof. Sources of such mammalian cells include, but are not limited to, murine, rabbit, monkey, human, ovine, porcine, bovine, and the like.

The cell source of the cell extract and the preparation method thereof can also be reported by reference to the prior documents, and the cell sources reported by the following documents are all taken as references and are included in the invention: "Nicole E.Gregorio, Max Z.Levine and Javin P.Oza.A. User's Guide to Cell-Free Protein Synthesis [ J ]. Methods protocol.2019, 2, 24", "Y Lu.Advances in Cell-Free biochemical technology [ J ]. Current Developments in Biotechnology and Bioengineering,2019, Chapter 2, 23-45" and the like and documents cited directly or indirectly. For example, prokaryotic sources include, but are not limited to, e.coli (e.coli); eukaryotic cell sources include, but are not limited to, Saccharomyces cerevisiae (Saccharomyces cerevisiae), Streptomyces lividans (Streptomyces lividans), wheat germ cells (steamed gem), tobacco BY-2 cells (tobaco BY-2 cells), Spodoptera frugiperda cells (sf cells), an insect cell), Trichoplusia ni cells (Trichoplusia cells), rabbit reticulocyte (rabbitreticulocyte), CHO cell (Chinese hamster ovary cell), human K562 cell, HEK293 cell, HeLa cell, mouse fibroblast (mouse fibroblast cell), Leishmania tarentolae cell (Leishmania tarania, yeast cell, single cell), and the like.

The yeast cell is preferably one of the embodiments, preferably Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces, or a combination thereof; the Kluyveromyces is further preferably Kluyveromyces lactis (K.lactis), Kluyveromyces lactis var. drosophilarium, Kluyveromyces lactis var. lactis, Kluyveromyces marxianus var. ceratus, Kluyveromyces marxianus, Kluyveromyces marxianus var. vannudenii, Kluyveromyces polyvidus (Kluyveromyces dozshanensis), Kluyveromyces marinus (Kluyveromyces aestivus), Kluyveromyces marxianus (Kluyveromyces marxianus), Kluyveromyces marxianus (Zymyces marxianus), Kluyveromyces lactis, Kluyveromyces lactis (Kluyveromyces), Kluyveromyces lactis Kluyveromyces, Kluyveromyces lactis (Kluyveromyces), Kluyveromyces lactis, Kluyveromyces (Kluyveromyces, Kluyveromyces lactis, Kluyveromyces, or the like; references include, but are not limited to, the following: EP1197560A1, "Marc-Andre Lachance, the Yeast (Fifth edition), Chapter 35, Kluyveromyces van der Walt (1971) 2011, Pages 471-.

Kluyveromyces (Kluyveromyces) is a species of ascosporogenous yeast, and among them, Kluyveromyces marxianus (Kluyveromyces marxianus) and Kluyveromyces lactis (Kluyveromyces lactis) are industrially widely used yeasts. In comparison with other yeasts, kluyveromyces lactis has many advantages such as superior secretion ability, better large-scale fermentation characteristics, a level of food safety, and also the ability of post-translational modification of proteins. The genome of the wild-type strain of Kluyveromyces lactis does not contain a gene encoding T7RNA polymerase.

In a preferred embodiment, the source of the cellular extract is kluyveromyces lactis, and any one or a combination of the following gene sequences is endogenously integrated: a gene encoding RNA polymerase and a gene encoding DNA polymerase. Preferably, the endogenous integration is into an episomal plasmid or into the genome of the cell.

In a preferred embodiment, the source of the cellular extract is kluyveromyces lactis, and any one or a combination of the following gene sequences is endogenously integrated: a gene encoding T7RNA polymerase and a gene encoding phi29 DNA polymerase. Preferably, the endogenous integration is into an episomal plasmid or into the genome of the cell.

In a preferred embodiment of the cell extract according to the present invention, the cell extract may be selected from any of the following sources: escherichia coli, yeast cells, mammalian cells, plant cells, insect cells, and combinations thereof. More preferably, the yeast cell is a Kluyveromyces, Saccharomyces cerevisiae, Pichia pastoris, or a combination thereof; the Kluyveromyces is further preferably Kluyveromyces lactis var. drosophilarium, Kluyveromyces lactis var. lactis, Kluyveromyces marxianus var. lactis, Kluyveromyces marxianus, Kluyveromyces marxianus, Kluyveromyces marxianus vanduli, Kluyveromyces polybubali, Kluyveromyces amabilis, Kluyveromyces thermotolerans, Kluyveromyces fragilis, Kluyveromyces hupehensis, Kluyveromyces polyspora, Kluyveromyces siae, Kluyveromyces lactis, or a combination thereof.

In another preferred embodiment, the cell extract is a yeast cell extract, more preferably a kluyveromyces marxianus cell extract or a kluyveromyces lactis cell extract.

In another preferred embodiment, the cell extract may be selected from any one of the following sources: escherichia coli, kluyveromyces lactis, wheat germ cells, Spodoptera frugiperda cells (sf cells, an insect cell), leishmania tarentolae cells, rabbit reticulocyte, chinese hamster ovary cells (CHO cells), african green monkey kidney COS cells, african green monkey kidney VERO cells, baby hamster kidney cells (BHK cells), human Hela cells, human Hybridoma cells (human Hybridoma cells), human fibrosarcoma HT1080 cells, and combinations thereof.

1.1.3. Exogenous RNA polymerase and exogenous DNA polymerase

When the genome of the cell from which the cell extract is derived does not contain the gene encoding RNA polymerase, nor does it endogenously integrate the coding sequence/gene encoding RNA polymerase, it is usually necessary to add an additional exogenous RNA polymerase to facilitate the reaction. For example, when a cell extract of a wild-type Kluyveromyces lactis strain is used, the T7 promoter cannot be recognized by the cell extract prepared from the wild-type Kluyveromyces lactis strain.

The addition of exogenous RNA polymerase to an in vitro protein synthesis system is a conventional technical approach. In vitro protein synthesis systems with the addition of exogenous RNA polymerase, reported in the prior art, are all included in the present invention as an alternative to the CFPS (MgP-) system of the present invention. The CFPS (MgP-) system refers to a system formed by other components except magnesium gluconate in the CFPS (Mg +) system. For example, the in vitro protein synthesis system of Kluyveromyces lactis with exogenous RNA polymerase (such as T7RNA polymerase) added in CN108535489A is included in the present invention as an alternative to CFPS (MgP-).

The CFPS (Mg +) system can also comprise at least one of the following components: exogenous RNA polymerase, exogenous nucleic acid template for coding RNA polymerase, exogenous DNA polymerase and exogenous nucleic acid template for coding DNA polymerase.

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

In one preferred embodiment, the CFPS (Mg +) system includes an exogenous T7RNA polymerase and an exogenous phi29 DNA polymerase.

The exogenous RNA polymerase may be added directly, an exogenous nucleic acid template encoding RNA polymerase may be added, or a combination thereof. The coding sequence of RNA polymerase may be constructed together with the nucleic acid template encoding the foreign protein or separately from the foreign nucleic acid template.

Similarly, the DNA polymerase may be added directly, or an exogenous nucleic acid template containing its coding sequence may be added, or a combination thereof. Can be a nucleic acid template for encoding the exogenous protein or can be an independent exogenous nucleic acid template.

When the nucleic acid template for encoding the foreign protein is a DNA template, the amplification process of the DNA can be included, or the amplification process of the DNA can not be included; if the in vitro protein synthesis reaction also includes a DNA amplification process, especially when the amount of the DNA template is insufficient, the system needs to contain endogenously expressed or/and exogenously added DNA polymerase, for example, exogenous phi29 DNA polymerase is added to CN 108642076A. In examples 1 to 15 of the present invention, after the DNA encoding the foreign protein mmefp was amplified in vitro, the amplification product was added to the reaction system as a foreign DNA template, and the in vitro protein synthesis reaction did not need to include a DNA amplification process. When a DNA polymerase is added to the system, that is, when the in vitro reaction process includes a DNA amplification process, it is usually necessary to add a substrate for synthesizing DNA.

The DNA polymerase may be a polymerase derived from a eukaryote or a prokaryote. Examples of eukaryotic polymerases are any one or any combination of the following: pol- α, pol- β, pol- δ, pol- ε, and the like, fragments of any of the foregoing, and variants of any of the foregoing (including variants of any of the foregoing fragments). Prokaryotic polymerases are exemplified by any one or any combination of the following: coli (e.coli) DNA polymerase I (e.g., 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 (Bacillus stearothermophilus) polymerase I, Phi29 DNA polymerase, T7 DNA polymerase, Bacillus subtilis Pol I, Staphylococcus aureus (Staphylococcus aureus) Pol I, etc., a partial domain of any of the foregoing, a subunit or fragment of any of the foregoing, a variant of any of the foregoing (including variants of any of the foregoing fragments). Such variants include, but are not limited to, mutants.

The polymerase (exogenous RNA polymerase, exogenous DNA polymerase) is preferably a polymerase capable of performing normal-temperature amplification, and the normal temperature is preferably room temperature to 37 ℃, specifically preferably 20-37 ℃, and more preferably 25-37 ℃. The polymerase capable of carrying out normal-temperature amplification can be selected according to an exogenous nucleic acid template; the room temperature amplification polymerases that can be used in vitro cell-free systems are all included as reference in the scope of the present invention, and include, but are not limited to, phi29 DNA polymerase, T4 DNA polymerase, T7 DNA polymerase, exo-klenow DNA polymerase, Bsu DNA polymerase, Pol III DNA polymerase, T7RNA polymerase, T3 RNA polymerase, T4 RNA polymerase, T5 RNA polymerase, etc., partial domains of any of the foregoing polymerases, subunits or fragments of any of the foregoing, variants of any of the foregoing, and any combination of the foregoing polymerases and partial domains, subunits, fragments, variants (including, but not limited to, mutants) thereof. The present invention may also employ other DNA polymerases such as Taq DNA polymerase, Pfu DNA polymerase, Pol I DNA polymerase, Pol II DNA polymerase, and the like.

In some preferred embodiments, the DNA polymerase has a strand displacement function.

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

The amplification techniques, particularly the normal temperature amplification method, which can be used in the present invention are not particularly limited, and the normal temperature amplification techniques which can be used in vitro cell-free systems are all included in the scope of the present invention by reference.

1.1.4. Energy system/energy regeneration system

An energy system/energy regeneration system is used to provide the energy required for the protein synthesis process.

Energy systems/energy regeneration systems reported for cell-free in vitro protein synthesis systems can provide energy for the in vitro protein synthesis system of the invention. Including but not limited to the literature: CN109988801A, CN2018116198186, CN2018116198190, US20130316397A, US20150376673A, "MJ Anderson, JC Stark, CE Hodgman and MC Jewett. engineering Cell-Free protein synthesis with glucose synthesis [ J ]. FEBS Letters,2015,589(15):1723 and 1727", "Y Lu. Advances in Cell-Free biosynetic Technology [ J ]. Current Developments in Biotechnology and Bioengineering,2019, Chapter 2, 23-45", "P reset, MT and BC, Cell-Free amino acid regeneration systems [ J ]. and 201434 are all cited as an indirect energy regeneration system [ 28, 2014-34, et al, for the present invention and for the references cited therein.

In a preferred embodiment, the energy system is a sugar (e.g., a monosaccharide, disaccharide, oligosaccharide or polysaccharide) and phosphate energy system, a sugar and phosphocreatine energy system, a phosphocreatine and phosphocreatine enzyme system, a phosphocreatine and phosphocreatine kinase system, a glycolytic pathway and its intermediate energy systems (a monosaccharide and its glycolytic intermediate energy system, a glycogen and its glycolytic intermediate energy system), or a combination thereof. Specifically, the phosphate refers to an inorganic phosphate, preferably orthophosphate, dihydrogen phosphate, metaphosphate, pyrophosphate or a combination thereof. The polysaccharide may be selected from polysaccharides including, but not limited to, starch, glycogen, dextrins (e.g., maltodextrin, corn dextrin, cyclodextrin), and the like. Examples of the disaccharide include sucrose, maltose and the like. The monosaccharide can be a six-carbon sugar or a five-carbon sugar. Examples of such monosaccharides include: glucose, mannose, lactose, and the like. The glycolytic pathway and its intermediate energy systems include, but are not limited to, glucose-based energy systems.

In one of the preferred embodiments, the energy system is a sugar and phosphate energy system, and the sugar may be selected from the group including but not limited to: glucose, fucose, mannose, galactose, lactose, xylose, arabinose, sucrose, maltose, starch, glycogen, dextrins (such as maltodextrin, corn dextrin, cyclodextrin), and any combination thereof.

The concentration of each component in the energy system is not particularly limited, including but not limited to, the use of the presently reported protocols and equivalents thereof. Examples 3-15 use energy systems a complex energy system of monosaccharides (glucose), polysaccharides (maltodextrin or corn dextrin) and phosphates.

1.1.5. Substrate for RNA synthesis

The substrate for RNA synthesis refers to a starting material capable of providing a structural unit of RNA. The substrate for the synthetic RNA is preferably a mixture of nucleotides. In one embodiment, the substrate for the synthesis of RNA is a nucleoside monophosphate, a nucleoside triphosphate, or a combination thereof. The substrate for the synthetic RNA is preferably a nucleoside triphosphate mixture (NTPs). The nucleoside triphosphate mixture is preferably a mixture of adenosine triphosphate, guanosine triphosphate, cytidine triphosphate or/and uridine triphosphate; more preferably, a mixture of the four nucleoside triphosphates is used. In the present invention, the concentration of each mononucleotide is not particularly limited, and it is measured as a nucleotide necessary for synthesizing a protein, and in one of the generally preferred embodiments, the concentration of each mononucleotide is 0.5 to 5mM, and in another preferred embodiment, the concentration of each mononucleotide is 1.0 to 2.0 mM. The concentration of each single nucleotide is each independently exemplified by any one of the following concentrations, or a range of concentrations between any two of the following concentrations (the range of concentrations includes both endpoints): 0.5mM, 1.0mM, 1.5mM, 2.0mM, 2.5mM, 3.0mM, 3.5mM, 4.0mM, 4.5mM, 5.0mM, 5.5mM, 6.0 mM. The above concentrations refer to the initial concentrations in the in vitro protein synthesis reaction mixture.

1.1.6. Substrate for DNA synthesis

In the case of DNA amplification or in vitro protein synthesis reactions involving DNA replication, it is often necessary to add substrates for DNA synthesis. The substrate for synthesizing DNA refers to a raw material capable of providing a structural unit of DNA. The substrate for synthesizing DNA is preferably a mixture of deoxynucleotides, and more preferably a mixture of deoxynucleoside triphosphates (dNTPs).

When the CFPS (Mg +) system contains a DNA polymerase, it preferably also contains a substrate for synthesizing DNA.

1.1.7. Substrates for synthetic proteins

The substrate of the synthetic protein refers to a raw material capable of providing amino acid units constituting the protein. The substrate of the synthetic protein is preferably a mixture of amino acids. The amino acids required for the synthesis of the protein are metered in.

The different kinds of amino acids supplied as the substrate raw materials of the synthetic protein, wherein the amounts of any two kinds of amino acids may be the same or different from each other independently of each other.

The concentration of each amino acid is, independently of the other, 0.01 to 5mM in one of the general preferred embodiments, and 0.1 to 1mM in another preferred embodiment. The concentration of each amino acid is, independently, exemplified by any one of the following concentrations, or a range of concentrations between any two of the following concentrations (the range of concentrations includes both endpoints): 0.1mM, 0.2mM, 0.4mM, 0.5mM, 1.0mM, 1.2mM, 1.5mM, 1.8mM, 2.0mM, 2.5mM, 3.0mM, 3.5mM, 4.0mM, 4.5mM, 5.0mM, 5.5mM, 6.0 mM. The above concentrations refer to the initial concentrations in the in vitro protein synthesis reaction mixture.

The amino acid mixture at least comprises amino acid mixtures required by the process of synthesizing the foreign protein, and is selected from the group consisting of 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 of the preferred embodiments is a mixture of the aforementioned twenty amino acids. The amino acid mixture required in the process of synthesizing the foreign protein not only comprises the amino acid forming the primary sequence of the foreign protein, but also comprises other amino acids involved in the synthesis process.

The amino acid mixture may include natural amino acids, unnatural amino acids.

The amino acid mixture may comprise_L-an amino acid,_D-amino acids, and combinations thereof.

The amino acid mixture may include, in addition to natural amino acids, unnatural amino acids,_D-amino acids, radioisotope labelled amino acids, modified amino acids, etc. The unnatural amino acid is not particularly limited and may be selected from the group consisting of: including but not limited to unnatural amino acids reported or cited in the following documents: "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 the like, and documents cited directly or indirectly. The radioisotope-labeled amino acid is notSpecific limitations include, but are not limited to, isotopic labeling employed in the reported field of protein synthesis. The modified amino acid is not particularly limited, including but not limited to modification by amino acid side groups.

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

In a preferred embodiment, the amino acid mixture is a mixture of twenty natural amino acids.

1.1.8. Other additive Components

The CFPS (Mg +) system can also comprise at least one of the following exogenous addition components: translation-related elements, DNA amplification-related elements, RNA amplification-related elements, RNase inhibitors, crowding agents (preferably one of polyethylene glycol and/or the like), potassium ions, antioxidants or reducing agents, antifreeze agents, trehalose, reaction promoters, antifoam agents, alkanes, buffers, aqueous solvents. Reference may be made to WO2016005982A1, US20060211083A1, "L Kai, 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 the like and documents cited directly or indirectly thereof.

The relevant elements required for translation of the foreign protein may also be provided or supplemented by means of exogenously added translation-related elements. The translation-related element is preferably selected from: tRNA, ribosomes, other translation-related enzymes, initiation factors, elongation factors, termination factors, and combinations thereof. The translation-related element is preferably a purified translation-related element.

When the protein synthesis process involves DNA amplification, elements associated with DNA amplification may be added by exogenous means in addition to the means provided endogenously. The DNA amplification-related elements may include, in addition to DNA polymerase, other factors such as helicase (HDA amplification), recombinase, and single-stranded DNA binding protein (RPA amplification), depending on different amplification mechanisms.

When the protein synthesis process involves RNA amplification, elements associated with RNA amplification may be added by exogenous means in addition to the means provided endogenously.

The RNA inhibitor may function to stabilize RNA.

In some preferred embodiments, the CFPS (Mg +) system also contains crowding agents (crowding agents) for mimicking the crowded macromolecular environment within the cell. The structure of the crowding agent is not particularly limited, and may be linear or non-linear, and the non-linear structure includes, but is not limited to, branched, multi-armed, cyclic, comb-shaped, tree-shaped, star-shaped, and other structural types. In some preferred examples, the crowding agent may be selected from the group consisting of: polyethylene glycol, polyvinyl alcohol (PVA), polystyrene (polystyrene), dextran (dextran), sucrose polymers (e.g., Ficoll sucrose polymers, such as Ficoll-400), polyvinylpyrrolidone (PVP, poly (vinylpyrrolidone), albumin, the like, any combination thereof. Sources of albumin 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 (human serum albumin). The crowding agents can also be referred to as crowding agents disclosed in the following documents: the document "X Ge, D Luo and J xu. cell-free protein expression under macromolecular growth conditions [ J ]. PLoS One,2011,6(12): e 28707" and references cited therein. In some preferred embodiments, the concentration of crowding agent in the in vitro protein synthesis reaction mixture is sufficient to increase the amount of protein synthesis.

In some preferred embodiments, the crowding agent has a molecular weight of no more than 400 kDa. In some preferred embodiments, the crowding agent has a molecular weight of no more than 200 kDa. Generally, the molecular weight specification preferably has a molecular weight distribution of. + -. 10% or less. In one preferred embodiment, the amount of the crowding agent is selected from 0.5% to 15%, more preferably from 1% to 12%, in terms of the weight percent (wt%) or volume percent (% (v/v)) or mass volume concentration (% (w/v)) of the crowding agent in the in vitro protein synthesis reaction mixture; for example, any one 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%.

In a preferred embodiment, the CFPS (Mg +) system further comprises polyethylene glycol and/or analogues thereof, which act as crowding agents. Such as polyethylene glycol, among others, can also adjust the system viscosity. Polyethylene glycol having CH₂CH₂Repeating units of O (EO units), commonly known as peg (polyethylene glycol), PEO (poly (ethylene oxide), poe (polyoxyethyleneene). Analogs of the polyethylene glycol include, but are not limited to, copolymers rich in EO units, polyethylene glycol derivatives, other polyoxyalkylenes that can act as crowding agents (e.g., polyoxypropylene, POP), derivatives of the other polyoxyalkylenes, and the like; the derivatives, taking polyethylene glycol derivatives as examples, include, but are not limited to, chemical modifications (such as methoxy polyethylene glycol, amino modifications, carboxyl modifications, etc.), amino acid modifications, polypeptide modifications, protein modifications, block polymers containing polyethylene glycol blocks, polymers containing polyethylene glycol side chains, etc. The concentration of polyethylene glycol or an analog thereof is not particularly limited, and generally, the concentration of polyethylene glycol or an analog thereof is 0.1% to 10%, preferably 0.1% to 8%, more preferably 0.5% to 4%, more preferably 1% to 2%, in terms of mass volume concentration in the in vitro protein synthesis reaction mixture (% (w/v)) or in terms of total weight (% by weight); unless otherwise specified, the present invention refers to the mass volume concentration in% (w/v), e.g., 2%, which means 2% (w/v), corresponding to 2g/100mL, 20 mg/mL. In some preferred embodiments, the molecular weight of the polyethylene glycol and/or the analog thereof is no more than 40000Da, and representative molecular weights are, for example, any one of the following molecular weights or a numerical interval between any two of the following molecular weights (inclusive): 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. Generally, the molecular weight specification preferably has a molecular weight distribution of. + -. 10% or less. The polyethylene glycol and/or the analogue thereof preferably has a molecular weight of 200Da to 10000Da, more preferably 3000Da to 10000 Da. Another preferred embodiment is 200Da to 8000 Da. Another preferred embodiment is 2000Da to 8000 Da. Another preferred embodiment is 3000Da to 8000 Da. In the present invention, the molecular weight of polyethylene glycol or the like refers to the weight average molecular weight M unless otherwise specified_w. Representative PEGs are selected from the group consisting of: PEG200, PEG400, PEG1000, PEG1500, PEG2000, PEG3000, PEG3350, PEG5000, PEG6000, PEG8000, PEG10000, and the like, combinations thereof; wherein the number of 3350 and the like is numerically equal to the weight average molecular weight.

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

The optimization and optimization of polyethylene glycol, magnesium ion and potassium ion reported in WO2016005982A1 are incorporated herein by reference.

The antioxidant, which may also be referred to as a reducing agent. May include, but is not limited to, Dithiothreitol (DTT), 2-mercaptoethanesulfonic acid, 2-mercaptoethanol, reduced Glutathione (GSH), tricarboxymethylphosphonic acid (TCEP), 3-mercapto-1, 2-propanediol (MPD), and the like. One of the preferred embodiments is dithiothreitol. The DTT is obtained by adopting the conventional use concentration; one embodiment is 0.5 to 10 mM; in another embodiment, the concentration is 0 to 1.7 mM.

The anti-freezing agent may be selected from anti-freezing agents used for solid formulations including, but not limited to, WO2018138195A1 and references cited therein. For example, trehalose. The purpose of adding the antifreezing agent is mainly to enable the whole system or partial subpackaged components of the system to be stored at low temperature, particularly when the components are stored in a kit mode. The added antifreezing agent allows the function of regulating the in vitro protein synthesis reaction.

Some cryoprotectants, including but not limited to trehalose, may also be a constituent component of the energy system.

The reaction promoter includes, but is not limited to, a reaction promoter (e.g., aluminum salt) as provided in CN 109971783A. In one preferred embodiment, the reaction promoter is an aluminum salt, an aluminum oxide (e.g., alumina), an iron salt, an iron oxide, a calcium salt, or a combination thereof.

Such anti-foaming agents are exemplified by those provided in CN1934276A and its cited references. Specific examples include, but are not limited to, alkyl polyoxyalkylene glycol ethers (alkyl polyoxyalkyllene glycol ethers), esters, silicones, polysiloxanes, sulfites, sulfonates, fatty acids and derivatives thereof, and the like.

The alkane may function to provide a hydrophobic interface or to mimic a hydrophobic environment. The relevant content of patent application CN202010179689.4 is incorporated herein by reference. E.g. C_6～44Pure or mixed alkanes, such as cyclohexane, isooctane, decane, tetradecane, pentadecylcyclohexane, squalane, tetradecane, vaseline, etc.

The buffer is mainly used for maintaining the pH environment of the system. One of the preferred embodiments is selected from any one or a combination of the following: Tris-HCl, Tris base, HEPES (4-hydroxyethylpiperazine ethanesulfonic acid system).

The aqueous solvent is preferably a buffer.

It is noted that any of the components of the CFPS (Mg +) system to which the present invention relates allows for the addition of other functions or purposes to the system than those previously described.

Any one of the components of the CFPS (Mg +) system to which the present invention relates allows two or more functions to be performed. For example, some sugar components may serve as both components of the energy system and functional components such as crowding agents, freezing point depressants, and the like.

1.1.9. Specific embodiment of the in vitro protein synthesis system (CFPS (Mg +) system) containing exogenous magnesium ions is exemplified

The concentrations of the components in the following embodiments are final concentrations (relative to the mother liquor) corresponding to the initial concentrations in the in vitro protein synthesis reaction mixture.

In a preferred embodiment, the CFPS (Mg +) system comprises a cell extract, magnesium gluconate, an endogenously expressed RNA polymerase (contained in the cell extract) or an exogenously added RNA polymerase, an energy system, a substrate for RNA synthesis, a substrate for protein synthesis, a crowding agent, potassium ions, a buffer, and optionally any one of the following exogenous components: other sources of magnesium ions, exogenous nucleic acid templates (independently preferably DNA templates) encoding RNA polymerases, endogenously expressed DNA polymerases or exogenously added DNA polymerases, exogenous nucleic acid templates (independently preferably DNA templates) encoding DNA polymerases, other DNA amplification related elements, substrates for synthesizing DNA, translation related elements, RNA amplification related elements, rnase inhibitors, antioxidants or reducing agents, cryoprotectants, trehalose, reaction promoters, antifoams, alkanes, aqueous solvents. The cell extract is preferably a eukaryotic cell extract, more preferably a yeast cell extract, and more preferably in one of the ways is a kluyveromyces lactis cell extract.

In a preferred embodiment, the CFPS (Mg +) system comprises a cell extract (the cell source has been modified by a strain to integrate the gene encoding RNA polymerase into the cell genome or into an intracellular episomal plasmid), magnesium gluconate, and further comprises one or more or all of the following exogenous components selected from the group consisting of: potassium 4-hydroxyethylpiperazine ethanesulfonate (HEPES-K) or Tris (hydroxymethyl) aminomethane (Tris), potassium acetate, potassium glutamate (preferably potassium L-glutamate), potassium chloride, magnesium acetate, magnesium glutamate (preferably magnesium L-glutamate), magnesium aspartate (preferably magnesium L-aspartate), nucleoside triphosphate mixtures (NTPs), amino acid mixtures, phosphocreatine, phosphocreatinase, phosphocreatine kinase, glucose, L-arabinose, sucrose, maltose, starch, glycogen, dextrin, corn dextrin, maltodextrin, cyclodextrin, phosphates (such as potassium phosphate), DNA amplification related elements, deoxynucleoside triphosphate mixtures, RNA amplification related elements, rnase inhibitors, polyethylene glycols, dextran, sucrose polymers, Dithiothreitol (DTT). The cell extract is preferably a eukaryotic cell extract, more preferably a yeast cell extract, and more preferably in one of the ways is a kluyveromyces lactis cell extract.

In a preferred embodiment, the CFPS (Mg +) system comprises a cell extract, magnesium gluconate, and one or more or all exogenous components selected from the group consisting of: HEPES-K or Tris, potassium acetate, potassium glutamate (preferably L-potassium glutamate), potassium chloride, magnesium acetate, magnesium glutamate (preferably L-magnesium glutamate), magnesium aspartate, nucleoside triphosphate mixtures (NTPs), amino acid mixtures, creatine phosphate, phosphocreatine kinase, 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 T7RNA polymerase, exogenous phi29 DNA polymerase, other DNA amplification related elements, deoxynucleoside triphosphate mixtures, RNA amplification related elements. The cell extract is preferably a eukaryotic cell extract, more preferably a yeast cell extract, and more preferably in one of the ways is a kluyveromyces lactis cell extract.

In a preferred embodiment, the CFPS (Mg +) system comprises a cell extract (the source cell is optionally strain-engineered, optionally incorporates a gene encoding an RNA polymerase into the cell genome or into an intracellular episomal plasmid), magnesium gluconate, and further comprises one or more or all exogenous components selected from the group consisting of: HEPES-K or Tris (HCl), potassium acetate, potassium glutamate (preferably L-potassium glutamate), potassium chloride, magnesium acetate, magnesium glutamate (preferably magnesium L-glutamate), magnesium aspartate (preferably magnesium L-aspartate), nucleoside triphosphate mixtures (NTPs), amino acid mixtures, creatine phosphate, creatinase phosphate, creatine phosphate kinase, glucose, L-arabinose, sucrose, maltose, starch, glycogen, dextrin, corn dextrin, maltodextrin, cyclodextrin, potassium phosphate, RNase inhibitor, polyethylene glycol, dextran, sucrose polymers, dithiothreitol, trehalose, alumina promoters, antifoams, alkanes, exogenous T7RNA polymerase, exogenous phi29 DNA polymerase, a DNA template encoding T7RNA polymerase, a DNA template encoding phi29 DNA polymerase, Other DNA amplification related elements, deoxynucleoside triphosphate mixtures, RNA amplification related elements. The cell extract is preferably a eukaryotic cell extract, more preferably a yeast cell extract, and more preferably in one of the ways is a kluyveromyces lactis cell extract.

In another preferred embodiment, the CFPS (Mg +) system comprises a cell extract, magnesium gluconate, and further comprises one or more or all exogenous components selected from the group consisting of: Tris-HCl (pH8.0), potassium acetate, potassium glutamate (preferably potassium L-glutamate), potassium chloride, magnesium acetate, magnesium glutamate (preferably magnesium L-glutamate), magnesium aspartate (preferably magnesium L-aspartate), glucose, L-arabinose, sucrose, maltose, maltodextrin, corn dextrin, a mixture of nucleoside triphosphates (a mixture of four nucleoside triphosphates, wherein the concentrations of the single nucleoside triphosphates are 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 concentrations of the single amino acids may be the same), Potassium phosphate, exogenous T7RNA polymerase, exogenous phi29 DNA polymerase, other DNA amplification related elements, deoxynucleoside triphosphate mixture, RNA amplification related elements, RNase inhibitor, polyethylene glycol, dextran, sucrose polymer, dithiothreitol. The cell extract is preferably a eukaryotic cell extract, more preferably a yeast cell extract, and more preferably in one of the ways is a kluyveromyces lactis cell extract.

Specifically, in a preferred embodiment, the CFPS (Mg +) system comprises 50% to 80% (v/v) of a cell extract, 1.5 to 12mM of magnesium gluconate, and further comprises one or more or all of the following components: 9.78mM Tris-HCl (pH8.0), 20-80 mM potassium acetate, 2-10 mM magnesium acetate, 1.5-6 mM L-aspartic acid magnesium, 0.5-5 mM four nucleoside triphosphates (the concentrations of the four nucleoside triphosphates are the same, such as 1.8mM), 0.1-1 mM twenty amino acid mixtures (glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine, the concentrations of the single amino acids are the same, such as 0.5mM), 10-40 mM glucose, 5-110 mM L-arabinose, 200-400 mM maltodextrin (measured as glucose monomer, such as about 52mg/mL at 320 mM), 10-40 mM potassium phosphate, 0.5-5% (w/v) polyethylene glycol (such as 2% (w/v)) 0.4-5 mM dithiothreitol (e.g., 0.44 mM). The cell extract is preferably a eukaryotic cell extract, more preferably a yeast cell extract, and more preferably in one of the ways is a kluyveromyces lactis cell extract.

One embodiment of the CFPS (MgP-) system without magnesium gluconate addition also includes, but is not limited to, the E.coli-based cell-free protein synthesis system described in, for example, WO2016005982A 1. Other citations of the present invention, including but not limited to in vitro cell-free protein synthesis systems based on wheat germ cells, rabbit reticulocytes, Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces marxianus, as described in direct and indirect citations thereof, are also included as alternative embodiments of the CFPS (MgP-) system of the present invention. For example, the in vitro Cell-Free protein synthesis system described in the "Lu, Y.Advances in Cell-Free biosynthestic technology. Current Developments in Biotechnology and Bioengineering,2019, Chapter 2, 23-45" section, including but not limited to the "2.1 Systems and Advantages" section, pages 27-28, is an alternative embodiment to the CFPS (MgP-) system of the present invention to which magnesium gluconate has not been added. For example, in vitro cell-free protein synthesis systems described in documents CN106978349A, CN108535489A, CN108690139A, CN108949801A, CN108642076A, CN109022478A, CN109423496A, CN109423497A, CN109837293A, CN109971783A, CN109988801A, CN110551700A, CN109971775A, CN110551745A, CN110551700A, CN2018116083534, CN2018116198186, CN2018116198190, CN2019102128619, CN2019102355148, CN 20191072813, CN 2011209166163, CN 2018108848, CN2018109550734, CN2018111131300, CN 2018123277, CN 2018162093, CN201911418151.8, CN202010069383.3, CN202010179689.4 and references cited therein are all alternative embodiments of the CFPS (MgP-) system of the present invention.

1.2. Foreign proteins

The foreign protein suitable for the CFPS (Mg +) system of the present invention is not particularly limited as long as it can be synthesized in vitro based on a cell extract (including a prokaryotic cell extract, a eukaryotic cell extract; particularly a eukaryotic cell extract, more particularly a yeast cell extract, more particularly a kluyveromyces lactis cell extract). The exogenous proteins disclosed in the prior art for use in vitro protein synthesis systems for prokaryotic cell extracts, eukaryotic cell extracts (preferably yeast cell extracts, more preferably from kluyveromyces lactis), or endogenous proteins for use in prokaryotic cell systems or eukaryotic cell systems (preferably yeast cell systems, more preferably kluyveromyces lactis) synthesized in cells can also be synthesized using the system of the present invention, or in vitro protein synthesis systems provided by the present invention are used for synthesis.

The application fields of the exogenous protein include but are not limited to the fields of biomedicine, molecular biology, medicine, in vitro detection, medical diagnosis, regenerative medicine, bioengineering, tissue engineering, stem cell engineering, genetic engineering, polymer engineering, surface engineering, nano engineering, cosmetics, food additives, nutritional agents, agriculture, feed, living goods, washing, environment, chemical dyeing, fluorescent labeling and the like.

The foreign protein can be natural protein or its modified product, or can be artificially synthesized sequence. The source of the native protein is not particularly limited, including but not limited to: eukaryotic cells, prokaryotic cells; wherein eukaryotic cell sources include, but are not limited to: mammalian cells, plant cells, yeast cells, insect cells, nematode cells, and combinations thereof; sources of such mammalian cells include, but are not limited to, murine, rabbit, monkey, human, porcine, ovine, bovine, and the like.

Types of foreign proteins include, but are not limited to, polypeptides ("foreign proteins" in the present invention broadly include polypeptides), fluorescent proteins, enzymes and corresponding zymogens, antibodies and fragments thereof, antigens, immunoglobulins, hormones, collagens, polyamino acids, vaccines and the like, partial domains of any of the foregoing, subunits or fragments of any of the foregoing, and variants of any of the foregoing. The "subunit or fragment of any one of the aforementioned proteins" includes a subunit or fragment of "a partial domain of any one of the aforementioned proteins". The "variant of any one of the aforementioned proteins" includes a variant of "a partial domain of any one of the aforementioned proteins, a subunit or fragment of any one of the aforementioned proteins". Such "variants of any of the foregoing proteins" include, but are not limited to, mutants of any of the foregoing proteins. In the present invention, the meanings of two or more "preceding" cases in succession in other positions are similarly explained.

The structure of the foreign protein can be a complete structure, and can also be selected from corresponding partial domains, subunits, fragments, dimers, multimers, fusion proteins, glycoproteins and the like. For example, a nanobody (heavy chain antibody lacking a light chain) is an incomplete antibody structure.

For example, the exogenous protein synthesized by the CFPS (Mg +) system of the present invention may be selected from the group consisting of, but not limited to, any one of the following proteins, fusion proteins in any combination, and 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 (Catalase, e.g., murine Catalase), actin, the variable region of an antibody (e.g., the single chain variable region of an antibody, scFV), the single chain of an antibody and fragments thereof (e.g., the heavy chain of an antibody, the light chain of an antibody), alpha-amylase, enteromycin A, hepatitis C virus E2 glycoprotein, insulin and precursors thereof, glucagon-like peptide (GLP-1), interferons (including but not limited to interferon alpha, such as interferon alpha A, interferon beta, interferon gamma, etc.), interleukins (such as interleukin-1 beta, interleukin 2, interleukin 12, etc.), lysozyme, serum albumin (including but not limited to human serum albumin, including but not limited to human serum albumin, Bovine serum albumin), transthyretin, tyrosinase, xylanase, beta-galactosidase (beta-galactosidase, LacZ, e.g., e.coli beta-galactosidase), and the like, partial domains of any of the foregoing, subunits or fragments of any of the foregoing, or variants of any of the foregoing (as defined above, including mutants, e.g., luciferase mutants, eGFP mutants). Examples of the aminoacyl tRNA synthetase include human lysine-tRNA synthetase (lysine-tRNA synthetase), human leucine-tRNA synthetase (leucine-tRNA synthetase), and the like. Examples of the glyceraldehyde-3-phosphate dehydrogenase include Arabidopsis glyceraldehyde-3-phosphate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase. Reference may also be made to patent document CN 109423496A. The mixture in any combination may include any one of the proteins described above, and may also include any combination of the fusion proteins described above.

In a preferred embodiment, the protein synthesis ability of the CFPS (Mg +) system is evaluated by using a fluorescent foreign protein such as GFP, eGFP, a similar substance, or a mutant thereof.

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

The exogenous nucleic acid template of the present invention refers to a nucleic acid template encoding an exogenous protein, unless otherwise specified. In addition, the exogenous nucleic acid template of the present invention, where indicated, may also include nucleic acid templates encoding protein factors or proteases required for in vitro protein synthesis processes, such as, for example, exogenous nucleic acid templates encoding RNA polymerase, exogenous nucleic acid templates encoding DNA polymerase.

If the synthesis system does not have a nucleic acid template for encoding the foreign protein, the in vitro synthesis reaction of the foreign protein cannot be carried out.

The nucleic acid templates encoding the foreign protein in any of the embodiments of the invention may each independently be DNA templates, mRNA templates, or combinations thereof.

The nucleic acid templates encoding the foreign protein in any of the embodiments of the present invention may each independently preferably be DNA templates.

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.

The nucleic acid template encoding the foreign protein may include a non-coding region. The expression product can be polypeptide or protein, and can also be fusion protein. One translation (or transcription translation) process is performed on one nucleic acid template molecule, allowing the number of polypeptide or protein molecules synthesized to be 1,2, or more.

The transcription and translation mode protein synthesis process takes a DNA template as an indirect template, and the translation mode protein synthesis process can only adopt an mRNA template as a direct template.

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

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

In any embodiment of the present invention, it is preferred that the nucleic acid template encoding the foreign protein further comprises a promoter element recognized by the cell extract.

In one preferred embodiment, the nucleic acid template encoding the foreign protein contains a promoter element recognized by the cell extract.

In one preferred embodiment, the nucleic acid template encoding the foreign protein contains a T7 promoter capable of initiating the gene transcription process of the foreign protein, i.e., the gene transcription process of the foreign protein is initiated by the T7 promoter on the nucleic acid template.

In a preferred embodiment, the nucleic acid template encoding the foreign protein contains a T7 promoter capable of promoting a gene transcription process for the foreign protein (in this case, the T7 promoter is located upstream of the coding sequence for the foreign protein in the nucleic acid template, and the T7 promoter promotes the gene transcription process for the foreign protein), and the cell extract in the CFPS (Mg +) system contains an endogenously expressed T7RNA polymerase.

In one preferred embodiment, the nucleic acid template encoding the foreign protein comprises a foreign protein translation system, a resistance gene translation system, a lac repressor translation system; the translation systems each include a corresponding promoter.

In a preferred embodiment, the nucleic acid template encoding the foreign protein further comprises a gene controlling the copy number of the plasmid.

In one preferred embodiment, the nucleic acid template encoding the foreign protein further comprises a transcription enhancing element, such as a kozak sequence.

In one preferred embodiment, the nucleic acid template encoding the foreign protein further comprises a translation enhancing element, such as a translation enhancer element, an IRES element, a kozak sequence, or the like.

1.3.1. Exogenous DNA template (including DNA template encoding exogenous protein)

The foreign DNA template of the present invention refers to a DNA template encoding a foreign protein, unless otherwise specified.

The exogenous DNA template of the present invention may be DNA, cDNA, methylated DNA, or a combination thereof. Wherein, the cDNA can be obtained by reverse transcription of RNA or miRNA. miRNA (MicroRNA) is a non-coding single-stranded RNA molecule which is coded by endogenous genes and has the length of about 20-25 nucleotides.

The DNA template for coding the foreign protein contains a coding sequence of the foreign protein.

Preferably, the DNA template for encoding the foreign protein contains a gene encoding the foreign protein.

The DNA template for encoding the foreign protein is determined according to the foreign protein.

The DNA template encoding the foreign protein may further contain other functional elements selected from the group consisting of promoters, terminators, enhancers (for example, CN109423497A, CN109022478A, CN109837293A (CN201711194355.9), CN109971775A) and the like, and enhancer elements described in the cited documents thereof, such as omega sequences and their homologous sequences, combined enhancer elements, kozak sequences (refer to CN109022478A, CN109837293A, CN109971775A and the like and the cited documents thereof), IRES elements (refer to internal ribosome entry sequences, refer to CN109022478A, CN109423497A and the like and the cited documents thereof), Multiple Cloning Sites (MCS), genes controlling plasmid copy number, and the like. It may also contain coding sequence for other amino acid chains such as signal peptide (for signal sequence), leader peptide (for leader sequence), functional tags (such as purification tag and solubilization tag), and linker peptide. It may further contain a 5 'untranslated sequence and a 3' untranslated sequence. The solubilization tags disclosed directly or indirectly in patent application CN201911204796.1 and the references cited therein are also incorporated herein by reference.

The DNA template encoding the foreign protein preferably contains a promoter element. The promoter element is required to be recognized by the cell extract used or other components of the CFPS (Mg +) system; it may be a promoter recognized by a wild-type cell extract, or a strain from which a cell extract is derived may be modified to recognize the promoter. The promoter in the DNA template of the invention may be selected from the group consisting of: AOD1, MOX, AUG1, AOX1, GAP, FLD1, PEX8, YPT1, LAC4, PGK, ADH4, AMY1, GAM1, XYL1, XPR2, TEF, RPS7, T7, and combinations thereof. References include, but are not limited to, the following and citations thereof: "Cereghino G. applications of yeast in Biotechnology: protein production and genetic analysis. Current operation in Biotechnology,1999,10(5), 422-" 427 ".

In examples 3-15, the foreign DNA template uses the T7 promoter to initiate the transcription process of the foreign protein; the T7 promoter is a strong promoter capable of specifically reacting to T7RNA polymerase.

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

Regarding the concentration of the exogenous DNA template, the amount of the exogenous protein to be expressed is determined according to the experimental protocol. In a preferred embodiment, the concentration of the exogenous DNA template is 1 to 400 ng/. mu.L. In another preferred embodiment, the concentration of the exogenous DNA template is 1 to 80 ng/. mu.L. In another preferred embodiment, the concentration of the exogenous DNA template is 5 to 50 ng/. mu.L. In another preferred embodiment, the concentration of the exogenous DNA template is 1 to 50 ng/. mu.L. In the present invention, the DNA template is added at a final concentration, which is the initial concentration in the reaction mixture for in vitro protein synthesis, unless otherwise specified.

The exogenous DNA template can be circular DNA or linear DNA; may be single-stranded or double-stranded. The gene encoding the foreign protein may be selected from the group including, but 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.

In a preferred embodiment, the exogenous DNA template further comprises 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 (signal sequences), leader sequences (for example, as described in CN109022478A and cited therein), genes controlling plasmid copy number (rop genes), tags enhancing translation level (for example, polypeptide tags as described in CN 2019112066163), other functional tags (for example, purification tags, fluorescence tags, solubilization tags, etc.), and the like. Reference may be made to US20060211083a1 and the like.

The exogenous DNA template may also be constructed in an expression vector. One of ordinary skill in the art can construct an expression vector containing a gene encoding a foreign protein using well-known methods. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.

For example, the nucleic acid construct of the "Z1-Z2" structure is inserted into the cloning site of a plasmid vector as plasmid DNA; wherein Z1 is a promoter, "-" is a covalent bond or a nucleotide fragment, and Z2 is a coding sequence of a foreign protein. Among them, one of preferable modes for Z1 is the T7 promoter.

In a preferred embodiment, the exogenous DNA template is a circular DNA, more preferably a plasmid DNA. The corresponding plasmid DNA is not particularly limited as long as it can react with a cell extract of the system to synthesize a foreign protein. Generally, the plasmid contains functional elements such as a promoter, a terminator, and an untranslated region (UTR). In a preferred embodiment, the plasmid contains a promoter recognized by an in vitro protein synthesis system; in a particularly preferred embodiment, the plasmid contains a promoter recognized by a cell extract. For example, a plasmid containing the T7 promoter can be theoretically used as an expression vector for the foreign DNA template used in examples 3 to 15. For example, pET series plasmids of Escherichia coli, pGEM series plasmids, etc. may be used in place of the plasmid vectors of the Kluyveromyces lactis extract of examples 3-15 to practice the present invention. In another preferred form, the plasmid contains a promoter that is recognized by an exogenously added component.

Taking the transcription process of foreign protein initiated by foreign DNA template using T7 promoter as an example, the T7 promoter can be initiated by recognition of endogenously expressed T7RNA polymerase in cell extract or by recognition of exogenously added T7RNA polymerase.

Linear DNA can be obtained by in vitro nucleic acid amplification techniques. The amplification techniques that can be used are not particularly limited and include, but are not limited to, PCR amplification techniques, isothermal amplification techniques, room temperature amplification techniques, and the like. Wherein the constant temperature amplification technology is preferably a normal temperature amplification technology.

In a preferred embodiment, the exogenous DNA template is a linear DNA and is a PCR linear fragment. The PCR linear fragment can be obtained by reported PCR technology.

In another preferred embodiment, the exogenous DNA template is a linear DNA and is a double-stranded linear DNA obtained by an amplification system. The amplification system is not particularly limited, and may be selected from the group consisting of, but not limited to, existing commercial kits, and amplification systems reported in the literature, as long as it can amplify the DNA template encoding the foreign protein of the present invention. Examples include, but are not limited to, commercial DNA amplification systems provided by Biomatch, Neta Scientific Inc., ABM, Thermo Fisher Scientific, Expedeon, Vivantis, and the like.

In another preferred embodiment, double-stranded DNA is used as the foreign DNA template and is constructed in a circular plasmid vector. The plasmid vector used contains, as one of typical structures, functional elements such as a T7 promoter, a T7 or LAC4 terminator, 5 'UTR, 3' UTR and the like.

As a preferred embodiment, in examples 3 to 15, double-stranded DNA was used as a template for foreign DNA and constructed in a circular plasmid vector; these plasmids contain the T7 promoter as a promoter for transcription and translation of foreign proteins; in examples 2-15, the modified kluyveromyces lactis endogenously expressed T7RNA polymerase, the modified strain was used to prepare cell extracts, and an in vitro cell-free protein synthesis system was constructed, in which the T7 promoter was suitable for in vitro cell-free expression of various proteins. The plasmid also contains functional elements such as a terminator and UTR.

In some embodiments, the following functional elements are included in the plasmid DNA: a promoter, a 5 'non-coding region, a coding sequence of a foreign protein, a 3' non-coding region, a terminator, a replication initiation site (f1 ori), an AmpR promoter, an ampicillin resistance gene (AmpR gene), a high copy number replication initiation site (ori), a gene controlling the copy number of a plasmid (rop gene), a lacI promoter, a coding sequence of lacI.

In one embodiment, the plasmid DNA comprises at least the structural elements identified in Table 1. Specific examples thereof include the plasmid structure shown in FIG. 2.

TABLE 1 description of the structural elements of the plasmid DNA encoding mEGFP (pD2P) indicated in FIG. 2

In other embodiments, in addition to the functional elements identified in FIG. 2, a purification tag, such as a polyhistidine tag (His-tag), is provided between the 5' UTR and the coding sequence of mEGFP. As shown for example in figure 3.

In other embodiments, in addition to the functional elements identified in FIG. 2, a kozak sequence is present downstream of the 5' UTR to increase translation levels. As shown for example in figure 3.

In other embodiments, in addition to the functional elements indicated in fig. 2, there is a coding sequence for a signal peptide (signal sequence) between the 5 'UTR and the coding sequence for the mmefp, downstream of the 5' UTR.

In other embodiments, the following functional elements are included in the plasmid DNA: a promoter, a 5 'non-coding region, a leader sequence, a coding sequence of a foreign protein, a 3' non-coding region, a terminator, a replication initiation site (f1 ori), an AmpR promoter, an AmpR gene, a high copy number replication initiation site (ori), a gene controlling the copy number of a plasmid (rop gene), a lacI promoter, a coding sequence of lacI. Examples are the plasmid structures shown in FIG. 1. Among them, rop gene is not indicated, and a T7 promoter and a LAC4 terminator are used in a translation system of a foreign protein.

In other embodiments, the following functional elements are included in the plasmid DNA: a promoter, a 5 'non-coding region, a coding sequence of a signal peptide, a coding sequence of a foreign protein, a 3' non-coding region, a terminator, an ori of f1, an AmpR promoter, an AmpR gene, an ori, a rop gene, a lacI promoter, a coding sequence of lacI. Specifically, for example, the following functional elements are included in the plasmid DNA: a T7 promoter, a 5 'non-coding region, a coding sequence of a signal peptide, a coding sequence of a foreign protein mEGFP, a 3' non-coding region, a T7 terminator, f1 ori, an AmpR promoter, an AmpR gene, ori, a rop gene, a lacI promoter and a coding sequence of lacI.

In other embodiments, the following functional elements are included in the plasmid DNA: a promoter, a 5 'non-coding 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' non-coding region, a terminator, f1 ori, an AmpR promoter, an AmpR gene, ori, a rop gene, a lacI promoter, a coding sequence for lacI. Specifically, for example, the following functional elements are included in the plasmid DNA: a T7 promoter, a 5 'noncoding region, a coding sequence for a signal peptide, a coding sequence for a purification tag, MCS, a coding sequence for the foreign protein mmefp, a 3' noncoding region, a LAC4 terminator or a T7 terminator, f1 ori, AmpR promoter, AmpR gene, ori, rop gene, lacI promoter, a coding sequence for lacI.

The basic structure of the plasmid and the method for inserting the coding gene of the foreign protein into the plasmid vector can adopt the conventional technical means in the field, and are not described in detail herein. For example, patent documents CN108690139A, CN107574179A, CN108949801A and the like can be referred to. For example, the basic structure of the plasmid can be referred to the attached drawings of the Chinese patent application CN 201910460987.8.

In the present invention, the concentration of the DNA template encoding a non-foreign protein can be determined according to the desired expression level of the non-foreign protein, with reference to the amount of the above-mentioned DNA template encoding a foreign protein. The non-foreign protein refers to a translation product that is not intended to be expressed but synthesized to facilitate the reaction.

1.3.2. Exogenous mRNA template

The invention can also adopt exogenous mRNA template to replace exogenous DNA template, or adopt the mixture of exogenous mRNA template and exogenous DNA template, add into above-mentioned CFPS (Mg +) system, carry on the synthetic reaction of in vitro protein, synthesize the exogenous protein encoded by mRNA template.

1.3.3. In vitro nucleic acid amplification (in vitro nucleic acid amplification technique, in vitro nucleic acid amplification method)

"in vitro nucleic acid amplification" is the process of replicating nucleic acids in vitro.

The nucleic acid templates used in the in vitro protein synthesis system of the present invention, including nucleic acid templates encoding foreign proteins and optionally nucleic acid templates encoding other proteins, can be used to prepare template materials using in vitro nucleic acid amplification techniques, or can include nucleic acid amplification during in vitro protein synthesis reactions.

The in vitro nucleic acid amplification technique that can be used is not particularly limited, and may be non-isothermal amplification or isothermal amplification (also referred to as isothermal amplification). Including but not limited to Polymerase Chain Reaction (PCR) technology, isothermal amplification technology, room temperature amplification technology, etc. Wherein the constant temperature amplification technology is preferably a normal temperature amplification technology.

Among them, isothermal amplification techniques can be referred to those disclosed in the following documents: "J Kim et al, Isothermal DNA Amplification in biochemical analysis: templates and applications [ J ]. Bioanalysis,2011,3(2): 227-. Specifically, nucleic acid isothermal amplification methods that can be used in the technical means of the present invention include, but are not limited to: loop-mediated isothermal amplification method/loop-mediated isothermal amplification (LAMP), strand displacement amplification method/strand displacement amplification method (SDA), nucleic acid sequence-dependent amplification method (NASBA), rolling circle amplification method (RCA), nicking enzyme isothermal amplification of nucleic acids (nicking enzyme amplification reaction, NEAR), helicase-dependent isothermal amplification method (HDA), transcription-dependent amplification method, hybrid capture method, transcription-mediated amplification method (TMA), recombinase-mediated amplification method (RAA), recombinase polymerase amplification method (RPA), and the like. One of the preferred modes is a rolling circle amplification method.

The in vitro nucleic acid amplification method, particularly the normal temperature amplification method, which can be used in the present invention is not particularly limited, and the normal temperature amplification techniques that can be used in the in vitro cell-free system in the prior art are all included in the scope of the present invention by reference, including but not limited to Rolling Circle Amplification (RCA), polymerase amplification with combinatorial enzymes (RPA), Strand Displacement Amplification (SDA), Helicase Dependent Amplification (HDA), 3SR (self-sustained sequence amplification), and the like. In vitro nucleic acid amplification methods (particularly, ambient temperature amplification methods) disclosed in the following references, including but not limited to: "Nicole E.Gregorio, Max Z.Levine and Javin P.Oza.A. User's Guide to Cell-Free Protein Synthesis [ J ]. Methods protocol.2019, 2, 24", "Y Lu.Advances in Cell-Free biosynthesis Technology [ J ]. Current Developments in Biotechnology and Bioengineering,2019, Chapter 2, 23-45", "Y Lu.cell-Free Synthesis biology: Engineering in an open world System [ J ]. Synthesis and Biotechnology,2017,2, 23-27" and the like and direct or indirect citations thereof.

In vitro nucleic acid amplification of the invention may also employ amplification techniques such as SMART amplification method (SMAP), Single Primer Isothermal Amplification (SPIA), exponential amplification reaction (EXPAR), thermostable HDA (tHDA), Multiple Displacement Amplification (MDA), restriction assisted RCA, and the like.

The in vitro nucleic acid amplification reaction of the present invention may be carried out continuously at a specific temperature or temperature range which is advantageous for the reaction. Any of the ambient amplification techniques of the invention also allows for performance under conditions of small temperature fluctuations. The reaction conditions of any one of the ambient amplification techniques of the invention are also allowed to fluctuate within an acceptable temperature range.

1.4. Incubation reaction (in vitro protein synthesis reaction)

Adding a nucleic acid template (preferably a DNA template) for encoding the foreign protein into the CFPS (Mg +) system, and incubating for a period of time to express and synthesize the foreign protein.

The conditions for carrying out the in vitro protein synthesis reaction are determined according to a specific in vitro cell-free protein synthesis system, and reference may be made to reported reaction conditions including, but not limited to, the reaction conditions described in documents CN106978349A, CN108535489A, CN108642076A, and the like. The in vitro protein synthesis reaction may be continued at a specific temperature or temperature range that facilitates the reaction. In one preferred embodiment, the temperature of the mixture varies by less than 25% (e.g., less than 20%, less than 15%, less than 10%, less than 5%) throughout the reaction time and/or the temperature of the mixture varies by less than 15 ℃ (e.g., less than 10 ℃, less than 5 ℃, less than 2 ℃, or less than 1 ℃) throughout the reaction time. Preferably, the in vitro protein synthesis reaction is carried out under normal temperature conditions. The normal temperature is preferably between room temperature and 37 ℃, and particularly preferably between 20 and 37 ℃. One of the preferable modes is 25 to 37 ℃. Another preferred embodiment is 20 to 30 ℃. The reported normal-temperature protein synthesis method or isothermal protein synthesis method suitable for normal-temperature conditions can be used for implementing the technical scheme of the invention.

The reaction time can be determined comprehensively according to the factors such as the amount of raw materials used (such as the amount of reaction substrate, the expected protein expression amount and the like), the protein synthesis reaction efficiency and the like.

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

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

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

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

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

For the determination of the expression level of the foreign protein, a method for testing the protein content in the cell extract can be used for reference, and a suitable method can be selected according to the characteristics of the foreign protein. For example, in examples 3 to 15, the amount of synthesis of the foreign protein was measured by measuring the RFU value by the ultraviolet absorption method.

2. In a second aspect, the present invention provides an in vitro protein synthesis kit comprising:

(i) an in vitro cell-free protein synthesis system (CFPS (Mg +) system) containing exogenous magnesium ions according to the first aspect;

(ii) optionally including a nucleic acid template encoding a foreign protein;

(iii) a label or instructions.

The CFPS (Mg +) system is capable of providing translation-related elements required for synthesizing a foreign protein in conjunction with the nucleic acid template encoding the foreign protein.

The nucleic acid template for encoding the foreign protein can be provided by a user after matched design, and is matched with a CFPS (Mg +) system provided by the kit (i) for application.

In one preferred embodiment, the nucleic acid template encoding the foreign protein serves as a reference control.

Preferably, the components of the CFPS (Mg +) system are placed in one or more containers as solids, semisolids, liquids, emulsions, suspensions, or combinations thereof. One of the preferred modes of the dry powder is freeze-dried powder or vacuum-dried powder. The liquid comprises a pure substance and a solution.

In one preferred embodiment, the (k1) and the (k2) are packaged separately.

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

The semi-solid, such as a paste.

The liquid can be pure or a mixture.

The emulsion refers to a mixed system of incompatible liquid phases, and is also called emulsion.

The suspension refers to a mixed system of an incompatible liquid phase and a solid.

Preferably, said (i) has a separate aliquot of the cell extract.

The in vitro protein synthesis kit can be used for carrying out in vitro protein synthesis reaction to synthesize exogenous protein.

In one preferred form, the components of the kit together comprise an aqueous solution. The kit includes a container for the aqueous solution.

In a preferred mode, each component of the kit is divided into a dry powder (such as freeze-dried powder and vacuum-dried powder) and a liquid reagent. The kit comprises two containers, one for containing the dry powder component and one for containing the liquid reagent component. The liquid reagent includes all systems containing liquid phase, and can be homogeneous system or mixed system, including but not limited to pure substance, solution, emulsion, suspension, and combination thereof.

In a preferred form, the kit comprises separate containers each comprising: (a) a cell extract; (b) an energy system; (c) optionally, a nucleic acid template; (d) a buffer solution; (e) optionally, a pH adjusting component; (f) optionally, a number of other solid components; (h) optionally, several other liquid components. Wherein components (a), (b), (c) are each independently a dry powder or an aqueous solution. Wherein components (c), (e), (f) are each independently present or absent. The "number" herein means 1,2 or more. The exogenous magnesium ions can be packaged in one or more containers of (d), (e), (f) and (h), and can also be packaged in other containers.

When the kit contains a liquid reagent, it is preferred to include a cryoprotectant component therein.

In one preferred mode, each component of the kit is divided into dry powder, buffer solution and other liquid reagents, and the reagent kit optionally comprises solvent water.

In one of the preferred modes, the following components can be respectively dispensed or dispensed in different containers in a proper combination mode: cell extract (containing endogenously expressed RNA polymerase, optionally containing endogenously expressed DNA polymerase), exogenous magnesium ions (including at least magnesium gluconate), an energy system, a substrate for RNA synthesis, a substrate for protein synthesis, a crowding agent, exogenous potassium ions, a buffer, and optionally a packaging container comprising any one of the following exogenous components or suitable combinations thereof: a nucleic acid template encoding a foreign protein, other foreign magnesium ions, a foreign added RNA polymerase, a foreign DNA template encoding an RNA polymerase, a foreign added DNA polymerase, a foreign DNA template encoding an RNA polymerase, other DNA amplification related elements, a substrate for synthesizing DNA, translation related elements, RNA amplification related elements, an RNase inhibitor, an antioxidant or reducing agent, a cryoprotectant, trehalose, a reaction promoter, an antifoaming agent, an alkane, an aqueous solvent. The cell extract preferably contains transfer rna (trna), ribosomes (ribosomes). The RNA polymerase is independently more preferably T7RNA polymerase. The DNA polymerase is independently more preferably phi29 DNA polymerase. The cell extract is preferably a eukaryotic cell extract, more preferably a kluyveromyces lactis cell extract. When a DNA polymerase is provided, a substrate for synthesizing DNA may be provided endogenously, exogenously or in combination thereof, usually together.

In one of the preferred modes, the following components can be respectively dispensed or dispensed in different containers in a proper combination mode: a cell extract (the source cell has no endogenous coding sequence/coding gene for integrated RNA polymerase, nor endogenous coding sequence/coding gene for integrated DNA polymerase), exogenously added RNA polymerase, an energy system, substrates for RNA synthesis, substrates for protein synthesis, crowding agents, exogenous magnesium ions (including at least magnesium gluconate), exogenous potassium ions, buffers, and optionally further comprising any one of the following components or suitable combinations thereof: a nucleic acid template encoding a foreign protein, other foreign magnesium ions, a foreign DNA template encoding an RNA polymerase, a foreign added DNA polymerase, a foreign DNA template encoding a DNA polymerase, other DNA amplification related elements, substrates for synthesizing DNA, translation related elements, RNA amplification related elements, RNase inhibitors, antioxidants or reducing agents, cryoprotectants, trehalose, reaction promoters, antifoams, alkanes, aqueous solvents. The cell extract preferably contains transfer RNA, ribosomes. The RNA polymerase is independently more preferably T7RNA polymerase. The DNA polymerase is independently more preferably phi29 DNA polymerase. The cell extract is preferably a eukaryotic cell extract, more preferably a kluyveromyces lactis cell extract.

3. The third aspect of the present invention provides a method for synthesizing a foreign protein, comprising the steps of:

(i) providing the CFPS (Mg +) system provided in the first aspect of the invention;

(ii) adding a nucleic acid template for encoding the foreign protein, and carrying out incubation reaction to synthesize the foreign protein;

the CFPS (Mg +) system is capable of providing translation-related elements required for synthesizing a foreign protein together with the nucleic acid template encoding the foreign protein;

further optionally comprising step (iii): isolating or/and detecting the foreign protein.

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

In one preferred embodiment, a DNA template encoding a foreign protein is used, and accordingly, the incubation reaction includes transcription and translation processes.

Separation or/and detection step

The method for synthesizing the foreign protein may further optionally comprise the step of isolating or/and detecting the foreign protein. The separation or/and detection method can be realized by adopting a conventional technical method.

By detecting and calculating, the results including but not limited to yield, purity, molecular weight, protein function and the like can be obtained.

4. In the second aspect and the third aspect, each independently includes but is not limited to the following preferred modes:

(1) in one preferred embodiment, the nucleic acid template encoding the foreign protein comprises a promoter element recognized by the CFPS (Mg +) system.

(2) In a preferred embodiment, the CFPS (Mg +) system comprises a cell extract, and the nucleic acid template encoding the foreign protein comprises a promoter element that is recognized by the cell extract. For example, the cell extract contains an endogenously expressed RNA polymerase which corresponds to the promoter element on the nucleic acid template.

(3) In one preferred embodiment, the nucleic acid template encoding the foreign protein contains a T7 promoter, and the CFPS (Mg +) system includes T7RNA polymerase.

(4) In one preferred embodiment, the nucleic acid template encoding the foreign protein comprises a T7 promoter, and the CFPS (Mg +) system comprises a cell extract comprising endogenously expressed T7RNA polymerase.

(5) Preferably, the nucleic acid template encoding the foreign protein contains a T7 promoter capable of initiating the gene transcription program of the foreign protein, i.e., the gene transcription process of the foreign protein is initiated by the T7 promoter on the nucleic acid template.

(6) In one preferred embodiment, the nucleic acid template encoding the foreign protein contains a T7 promoter capable of initiating a gene transcription process for the foreign protein, and the CFPS (Mg +) system includes T7RNA polymerase.

(7) In one preferred mode, the T7 promoter is located upstream of the coding sequence for the foreign protein of the nucleic acid template to initiate the transcription process of the foreign protein, and the CFPS (Mg +) system comprises a cell extract containing endogenously expressed T7RNA polymerase.

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 proteins are each independently preferably DNA templates.

5. The fourth aspect of the present invention provides an application of the in vitro cell-free protein synthesis system (CFPS (Mg +) system) containing exogenous magnesium ions according to the first aspect, in protein synthesis. The application of the method to protein synthesis includes, but is not limited to, application to protein manufacture, or application to detection based on protein synthesis and the like.

The application fields of the CFPS (Mg +) system include but are not limited to the fields of biomedicine, molecular biology, medicine, in vitro detection, medical diagnosis, regenerative medicine, bioengineering, tissue engineering, stem cell engineering, genetic engineering, polymer engineering, surface engineering, nano engineering, cosmetics, food additives, nutritional agents, agriculture, feed, living goods, washing, environment, chemical dyeing, fluorescent labeling and the like.

6. According to a fifth aspect of the present invention there is provided the use of magnesium gluconate in an in vitro cell-free protein synthesis system comprising exogenous magnesium ions according to the first aspect, or in an in vitro protein synthesis kit according to the second aspect, or in a method of synthesis of an exogenous protein according to the third aspect.

7. The invention will be further elucidated with reference to the specific embodiments and the accompanying figures 1-16. It should be understood that these examples are illustrative only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, preferably according to, with reference to, the conditions as directed by the specific embodiments described above, may then be generally followed by conventional conditions, e.g. "Sambrook et al, molecular cloning: a laboratory Manual (New York: Cold Spring Harbor laboratory Press,1989), "A laboratory Manual for cell-free protein Synthesis" Experimental Manual for ethylene by Alexander S.Spirin and James R.Swartz.cell-free protein synthesis: methods and protocols [ M ].2008 ", etc., or according to the conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts referred to in this invention are percentages and parts by weight.

Unless otherwise specified, the materials and reagents used in the examples of the present invention are commercially available products.

Kluyveromyces lactis (abbreviated as K.lactis or kl) was used as a source of the cell extract in examples 2 to 15. Examples 3-15 of the present invention all used the modified strain of kluyveromyces lactis to prepare the cell extract. It is to be understood that the same design and analysis and experimental procedures are also applicable to other cell extract sources described herein, including, but not limited to, prokaryotic cells (e.g., E.coli), such as other yeast cells, such as plant cells, insect cells, animal cells (e.g., mammalian cells, specifically, murine, rabbit, monkey, human, etc.), and other eukaryotic cells.

The plasmid expression vectors used in the examples of the present invention are only for specifically illustrating the embodiments of the present invention and do not limit the scope of the present invention; other plasmid vectors useful in the practice of the present invention include, but are not limited to, common plasmid vectors commercially available at the present time, such as, for example: pET series plasmids, pGEM series plasmids, and the like.

The concentrations of the components of the in vitro cell-free protein synthesis system of examples 3-15, and the concentrations of the exogenous DNA template, not specifically indicated, were final concentrations; 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.

The four magnesium source reagents used in examples 3-15, including magnesium gluconate (molecular weight 414.61), magnesium L-glutamate (molecular weight 388.61, tetrahydrate), magnesium acetate (molecular weight 214.45, tetrahydrate), magnesium L-aspartate (molecular weight 324.54, dihydrate), each provided only one magnesium atom and two acid groups. In the reaction mixture system, the complexing manner of the magnesium ion and the acid residue is not limited to the ratio in the raw materials.

In the examples of the present invention, negative control groups (NC groups) were provided, at least no exogenous DNA template was added, and other reaction conditions were consistent with those in the experimental groups in the examples. After reaction for 3h and 18-24 h, the RFU value is not more than 20 and can be ignored, and the experimental result data of part of NC groups are not shown in the chart.

Example 1 preparation of nucleic acid template encoding foreign protein mEGFP

1.1 plasmid construction: and constructing a plasmid vector for expressing the mEGFP, and performing in-vitro DNA amplification to prepare a plasmid for encoding the exogenous protein mEGFP.

Selecting a mutant (mEGFP) of the enhanced green fluorescent protein as a foreign protein to be used as a target expression product. Wherein, the mEGFP is an A206K mutant of the enhanced green fluorescent protein, and the amino acid sequence of the mEGFP is shown as SEQ ID No. 2.

And selecting a plasmid vector. The artificial plasmid vector designed aiming at the kluyveromyces lactis cell extract is adopted, and the artificial plasmid vector contains functional elements such as a T7 promoter, a LAC4 terminator, a 5 'UTR and a 3' UTR. The plasmid vector can be combined with a Kluyveromyces lactis cell extract containing endogenously expressed T7RNA polymerase to construct an in vitro cell-free protein synthesis system, and various exogenous proteins are expressed in vitro.

The DNA fragment containing the mEGFP coding gene is inserted into a plasmid vector by adopting a PCR amplification and homologous fragment recombination method to construct a plasmid vector for expressing the mEGFP, which is marked as a plasmid D2P-mEGFP (abbreviated as pD2P-mEGFP) with 6056 bp. The plasmid was confirmed to be correct by gene sequencing. Wherein, the gene sequence of the code mEGFP is shown as SEQ ID No. 1.

The map of the pD2P-mEGFP plasmid is shown in FIG. 1, and the structural element composition is shown in Table 2.

TABLE 2 structural element description of plasmid encoding foreign protein mEGFP (pD2P-mEGFP)

1.2 in vitro DNA amplification (DNA replication Process)

DNA amplification was performed. The final concentrations of the components of the amplification system were: 1 XPhi 29 reaction buffer (composition including 200mM Tris-HCl,20mM MgCl)₂,10mM(NH₄)₂SO₄10mM KCl, pH7.5), 4mM Dithiothreitol (DTT), 0.1mg/mL Bovine Serum Albumin (BSA), 0.5mM deoxynucleoside triphosphate mixture (dNTPs), 2-5. mu.M random primers, 0.004-0.006 mg/mL phi29 DNA polymerase, 1.14ng/μ L of the above plasmid (pD2P-mEGFP, as template). The reaction system is mixed evenly and placed at room temperature for reaction overnight for 20 hours, or placed at 37 ℃ for reaction for 2 hours, and the DNA template is obtained. OD at 260nm was measured, the concentration of nucleic acid was calculated, and the reaction solution was frozen or refrigerated for use as a nucleic acid template in the subsequent examples.

Example 2 preparation of cell extracts

Examples 2-15 of the present invention all used the modified strain of kluyveromyces lactis to prepare the cell extract.

The source of the cell extract is selected from yeast cells, specifically Kluyveromyces lactis (Kluyveromyces lactis, K.lactis). Adopting a modified strain based on a Kluyveromyces lactis strain ATCC 8585; integrating a coding gene of T7RNA polymerase into a genome of Kluyveromyces lactis by adopting the method described in CN109423496A to obtain a modified strain, so that the modified strain can endogenously express T7RNA polymerase; culturing cell material with the modified strain, and preparing cell extract. According to comparison of control experiments, the Kluyveromyces lactis system without endogenously integrating the coding gene of T7RNA polymerase can hardly perform in-vitro protein synthesis reaction under the condition of not adding any exogenous RNA polymerase; after the endogenous integration and transformation, the high-efficiency expression of the exogenous protein can be realized without adding any exogenous RNA polymerase, the exogenous protein can be used as a substitution mode of an exogenous addition mode, and the protein synthesis level of a traditional in-vitro protein synthesis system can be reached (in the traditional in-vitro protein synthesis system, a bacterial strain which is not subjected to endogenous transformation of T7RNA polymerase is adopted to prepare a cell extract, and exogenous T7RNA polymerase is added in the synthesis system). The optimization method of adding magnesium gluconate as the exogenous magnesium ions is also applicable to an in-vitro protein synthesis system of strains (including but not limited to Kluyveromyces lactis strains) which are not subjected to endogenous modification of T7RNA polymerase, and can achieve the same or similar optimization effect.

The preparation process of the kluyveromyces lactis cell extract adopts conventional technical means, and refers to the method recorded in CN 109593656A. The preparation steps, in summary, include: providing appropriate amount of raw materials of Kluyveromyces lactis cells cultured by fermentation, quickly freezing the cells with liquid nitrogen, crushing the cells, centrifuging, and collecting supernatant to obtain cell extract.

The protein concentration of the obtained kluyveromyces lactis cell extract is 20-40 mg/mL. In examples 3 to 15, the amount of the cell extract added is, unless otherwise specified, the volume percentage in the reaction mixture system.

Table 3 examples 3-15 the following kluyveromyces lactis cell extracts (k. lactis lysate) were used

Example numbering	Source	Cell extract numbering
			Example 3	Kluyveromyces lactis	YY1904291
Example 4	Kluyveromyces lactis	YY1904291
			Example 5	Kluyveromyces lactis	CM191107
Example 6	Kluyveromyces lactis	CM191205
			Example 7	Kluyveromyces lactis	CM191107
Example 8	Kluyveromyces lactis	CM191107
			Example 9	Kluyveromyces lactis	CM191107
Example 10	Kluyveromyces lactis	CM191107
			Example 11	Kluyveromyces lactis	CM191107
Example 12	Kluyveromyces lactis	CM191107
			Example 13	Kluyveromyces lactis	YY1904281
Example 14	Kluyveromyces lactis	YY1904281
			Example 15	Kluyveromyces lactis	YY1904281

Example 3 comparison of the Effect of magnesium gluconate and magnesium acetate on the protein Synthesis Capacity of an in vitro protein Synthesis System

3.1 in vitro cell-free protein Synthesis System containing exogenous magnesium ions (without addition of exogenous RNA polymerase)

Each system was 100. mu.L in volume and the reactions were performed in flat-bottom cell culture plates. 3 replicates were set up for each sample and the mean and standard deviation (error bar) were calculated.

Experimental group (magnesium Gluconate provides exogenous magnesium ion, noted as Gluconate group): the final concentration of each component is as follows: 28.3mM pH8.0 Tris (HCl pH adjusted), 45.0mM potassium acetate, 1.30mM Dithiothreitol (DTT), 2.1% (w/v) PFG8000, 6.0mM glucose, 0.076g/mL corn dextrin, 1.62mM nucleoside triphosphate mixture (adenine nucleoside triphosphate, guanine nucleoside triphosphate, cytosine nucleoside triphosphate and uracil nucleoside triphosphate, each at a final concentration of 1.62mM), 1.80mM potassium bicarbonate, twenty amino acid mixtures (glycine 14.7mM, alanine 2.48mM, valine 10.66mM, leucine 1.83mM, isoleucine 4.77mM, phenylalanine 3.75mM, proline 1.74mM, tryptophan 1.64mM, serine 0.82mM, tyrosine 0.20mM, cysteine 6.17mM, methionine 2.76mM, asparagine 3.36mM, aspartic acid 0.43 mM, aspartic acid 0.93mM, glutamic acid 0.93mM, 1.93 mM, 1.9 mM, 3mM, glutamic acid, 3mM, 2.3 mM, 3mM, glutamic acid, 3mM, D, 3mM, and 1.3 mM, 3mM, 2mM, 3mM, 2.3 mM, 2mM, 2.3 mM, 2mM, 2.3 mM, 2mM, 2.3 mM, 2mM, 2mM, 2, 1.3 mM, 2.3 mM, 2, 2.3 mM, 2, 2.3 mM, 2, Lysine 8.93mM, arginine 4.49mM and histidine 4.47mM), Kluyveromyces lactis cell extract (YY1904291) 60% (v/v), tripotassium phosphate 22.4mM, and magnesium gluconate 0.25-32 mM.

Positive control group (magnesium Acetate provides exogenous magnesium ion, noted Acetate group): magnesium acetate with the final concentration of 1-6 mM replaces magnesium gluconate in the experimental group to provide exogenous magnesium ions, and the types and contents of other components and experimental conditions for carrying out in-vitro protein synthesis reaction are completely consistent with those of the experimental group.

Blank control group (Blank control group, BC group): no external magnesium ion is added, and the types and contents of other components and the experimental conditions for carrying out the in vitro protein synthesis reaction are completely consistent with those of the experimental group.

Negative control group (Negative control group, NC group): no exogenous magnesium ion is added, no exogenous DNA template is added subsequently, and the types and contents of other components and the experimental conditions for carrying out in-vitro protein synthesis reaction are completely consistent with those of the experimental group.

3.2 in vitro protein Synthesis reactions: the NC group is not added with an exogenous DNA template; adding a DNA template (obtained by performing in-vitro amplification by 1.2 in the example 1 and having a final concentration of 56.9 ng/. mu.L) for encoding mEGFP into each independent in-vitro cell-free protein synthesis system of the experimental group, the positive control group and the blank control group, uniformly mixing, placing all the systems in a room-temperature environment (18-30 ℃), performing shaking table reaction overnight, and sampling at time points of 3 hours and 20-24 hours respectively to perform fluorescent protein activity test.

Note that the final concentrations described in 3.1 and 3.2 are based on the in vitro protein reaction mixture with the DNA template added. The final concentrations in examples 4 to 15 were also explained in the same manner unless otherwise specified.

3.3 fluorescent protein Activity assay: after the reaction is finished, the reaction solution is added,immediately placing a sample to be detected in an infinite F200TECAN multifunctional microplate reader, detecting the intensity of a Fluorescence signal, and taking a Relative Fluorescence Unit (RFU) as an activity Unit. The size of the RFU value can reflect the synthesis amount of the mEGFP protein, and the conversion relation between the mass volume concentration C (unit mu g/mL) of the mEGFP and the RFU value is as follows:

within the scope of the present invention, a substantially linear relationship is observed between C and RFU.

The fluorescence test was performed on each reaction system sample. Sample treatment: at 4000 rpm, the mixture was centrifuged at 4 ℃ for 1 minute. The sample to be detected is placed in an infinite F200TECAN multifunctional microplate reader, the adopted detection wavelength excitation wavelength/emission wavelength (Ex/Em) is 488nm/507nm, and the relative fluorescence unit value (RFU) is determined.

3.4 Experimental results: as shown in FIG. 4, the abscissa is labeled as "group-magnesium ion concentration value", wherein the magnesium ion concentration value is in mM. In fig. 4, at two sampling time points of 3h and 22h, the Gluconate group had higher protein expression when 2mM magnesium Gluconate was added, and both were significantly higher than the highest protein expression when the optimal magnesium ion concentration of the conventional magnesium Acetate (Acetate group) as a source of exogenous magnesium ions was used.

At 3h, the RFU value of the highest protein expression level of the Gluconate group (2mM, RFU value of 1496. + -. 113) was increased by 20.2% as compared with the RFU value of the highest protein expression level of the Acetate group (3mM, RFU value of 1245. + -. 76).

The RFU value of the highest protein expression level of the Gluconate group (2mM, 1587 + -328) was improved by 9.6% compared with the RFU value of the highest protein expression level of the Acetate group (3mM, RFU value of 1448 + -266) at 22h overnight.

The negative control group showed negligible RFU values (mean value below 20), not shown.

Example 4 examination of the Effect of magnesium gluconate content on the protein Synthesis Capacity of in vitro protein Synthesis System

4.1 in vitro cell-free protein Synthesis System containing exogenous magnesium ions (without addition of exogenous RNA polymerase)

Each system was 100. mu.L in volume and the reactions were performed in flat-bottom cell culture plates. 3 replicates were set up for each sample and the mean and standard deviation (error bar) were calculated.

Single magnesium source experimental set (single magnesium source, noted as Gluconate set): the final concentration of each component is as follows: 28.3mM pH8.0 Tris (HCl-adjusted pH), 38.6mM potassium acetate, 1.12mM dithiothreitol, 1.8% (w/v) PFG8000, 6.0mM glucose, 0.065g/mL corn dextrin, 1.39mM nucleoside triphosphate mixture (adenine nucleoside triphosphate, guanine nucleoside triphosphate, cytosine nucleoside triphosphate and uracil nucleoside triphosphate, each at a final concentration of 1.39mM), 1.54mM potassium bicarbonate, twenty amino acid mixtures (final concentrations: glycine 13.18mM, alanine 2.22mM, valine 9.53mM, leucine 1.64mM, isoleucine 4.27mM, phenylalanine 3.35mM, proline 1.55mM, tryptophan 1.46mM, serine 0.73mM, tyrosine 0.18mM, cysteine 5.52mM, methionine 2.47mM, asparagine 3.00mM, asparagine 0.74mM, aspartic acid 0.83mM, glutamic acid 0.83mM, aspartic acid 0.83mM, glutamic acid 0.27 mM, glutamic acid 0.9 mM, glutamic acid 0.27 mM, and the like, Lysine 7.99mM, arginine 4.01mM and histidine 4.00mM), Kluyveromyces lactis cell extract 70% (v/v) (YY1904291), tripotassium phosphate 20.4mM, magnesium gluconate 2.6-6.1 mM; the mole percentage of the magnesium gluconate in the exogenous magnesium ions is 100 percent.

Mixed magnesium source experimental group (magnesium gluconate and magnesium acetate together provide exogenous magnesium ion, noted as Mix + acetate group): the exogenous magnesium ions are a mixture of magnesium gluconate and magnesium acetate, the final concentration of the magnesium gluconate is fixed to be 1.61mM, the final concentration of the magnesium acetate is 0.25-4.0 mM, and correspondingly, the molar percentage of the magnesium gluconate is 86.6-28.7%; the species and content of other components and the experimental conditions for carrying out the in vitro protein synthesis reaction are completely consistent with those of the experimental group.

Negative control group (Negative control group, NC group): no exogenous magnesium ion is added, no exogenous DNA template is added subsequently, and the types and contents of other components and the experimental conditions for carrying out in-vitro protein synthesis reaction are completely consistent with those of the experimental group.

4.2 in vitro protein Synthesis reactions: the NC group is not added with an exogenous DNA template; adding DNA templates (obtained by performing in-vitro amplification by the method of 1.2 in the example 1) of encoding mEGFP with the final concentration of 56.9 ng/. mu.L into each independent in-vitro cell-free protein synthesis system of the single magnesium source experimental group and the mixed magnesium source experimental group respectively, uniformly mixing, placing all the systems in a room temperature environment (18-30 ℃), performing shaking table reaction overnight, and sampling at the time points of 3 hours and 20-24 hours respectively to perform fluorescent protein activity test.

4.3 fluorescent protein Activity assay: the RFU value of the exogenous fluorescent protein, mmefp, synthesized in the sample was determined using the method of 3.3 in example 3.

4.4 results of the experiment: as shown in fig. 5, the abscissa is plotted in terms of "total concentration of magnesium ions (percentage of magnesium gluconate)". In FIG. 5, both the Gluconate group of a single magnesium source had high protein expression at the addition of 3.6mM magnesium Gluconate at both sampling time points of 3h and 22h, RFU values of 2404. + -. 161 at 3h and 3436. + -. 25 at 22 h; the mixed magnesium source experimental group has the highest protein expression amount when the total concentration of magnesium ions is 3.6mM and the mole percentage of magnesium gluconate is 44.6%, the RFU value is 1650 +/-90 at 3h and 2491 +/-226 at 22 h.

When the concentration of the exogenous magnesium ions is 3.6mM, the protein synthesis amount of the Gluconate group (magnesium Gluconate concentration is 100%) of a single magnesium source is higher than that of the mixed magnesium source experimental group (magnesium Gluconate concentration is 44.6%), the RFU value is improved by 45.7% at 3h, and the RFU value is improved by 37.9% at 22 h. It can be seen that for the mixed magnesium source of magnesium gluconate and magnesium acetate, the increase of the content of magnesium gluconate is beneficial to the synthesis of protein. In addition, according to experimental data of the mixed magnesium source, the optimal condition of the synthesis amount (indicated by an RFU value) of the exogenous protein requires a certain total concentration of magnesium ions and a certain proportion of magnesium gluconate; too low or too high total concentration of magnesium ions and too low proportion of magnesium gluconate all result in reduced protein synthesis capacity.

The negative control group showed negligible RFU values (mean value below 20), not shown.

Example 5 comparison of the Effect of magnesium gluconate and magnesium glutamate on the protein Synthesis Capacity of an in vitro protein Synthesis System

5.1 in vitro cell-free protein Synthesis System containing exogenous magnesium ions (without addition of exogenous RNA polymerase)

Each system was 100. mu.L in volume and the reactions were performed in flat-bottom cell culture plates. 3 replicates were set up for each sample and the mean and standard deviation (error bar) were calculated.

Experimental groups (noted as Gluconate group): the final concentration of each component is as follows: 9.78mM pH8.0 Tris (pH adjusted with HCl, Tris-HCl), 80mM potassium acetate, 15mM glucose, 320mM maltodextrin (measured in glucose units), 24mM potassium phosphate, 1.8mM nucleoside triphosphate mixture (adenosine triphosphate, guanosine triphosphate, cytosine nucleoside triphosphate and uracil nucleoside triphosphate, each at a final concentration of 1.8mM), 0.7mM 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 at a final concentration of 0.7mM), 80mM potassium acetate, 2% (w/v) polyethylene glycol 8000, trehalose 0.06g/mL, Kluyveromyces lactis cell extract 80% (v/v) (CM191107), and magnesium gluconate 0.5-8 mM.

Positive control group (Positive group, denoted PC group, also denoted Glutamate group): magnesium glutamate with the final concentration of 2-9 mM replaces magnesium gluconate in the experimental group to provide exogenous magnesium ions, and the types and contents of other components and experimental conditions for carrying out in-vitro protein synthesis reaction are completely consistent with those of the experimental group.

Blank control group (Blank control group, BC group): no external magnesium ion is added, and the types and contents of other components and the experimental conditions for carrying out the in vitro protein synthesis reaction are completely consistent with those of the experimental group.

Negative control group (Negative control group, NC group): no exogenous magnesium ion is added, no exogenous DNA template is added subsequently, and the types and contents of other components and the experimental conditions for carrying out in-vitro protein synthesis reaction are completely consistent with those of the experimental group.

5.2 in vitro protein Synthesis reactions: the NC group is not added with an exogenous DNA template; adding a DNA template (obtained by performing in-vitro amplification by the method of 1.2 in the example 1) for encoding mEGFP with the final concentration of 11.9 ng/. mu.L into each independent in-vitro cell-free protein synthesis system of the experimental group, the positive control group and the blank control group respectively, uniformly mixing, placing all the systems in a room-temperature environment (18-30 ℃), performing shaking table reaction overnight, and sampling at the time points of 3 hours and 20-24 hours respectively to perform fluorescent protein activity test.

5.3 fluorescent protein Activity assay: the RFU value of the exogenous fluorescent protein, mmefp, synthesized in the sample was determined using the method of 3.3 in example 3.

5.4 Experimental results: as shown in FIG. 6, the abscissa is labeled as "group-magnesium ion concentration value", wherein the magnesium ion concentration value is in mM. In the PC group using magnesium glutamate as a magnesium source, the protein synthesis amount peaked at a magnesium ion concentration of 8 mM. When magnesium gluconate is used as a magnesium source, the protein synthesis amount is higher than the peak value of the PC group within the range of 5-8 mM, the RFU value is improved by 16.9-52.4% compared with the peak value of the PC group within 3 hours, and the RFU value is improved by 15.9-57.7% compared with the peak value of the PC group within 20 hours. In addition, when magnesium gluconate was used as the magnesium source, the RFU value reached substantially half of the peak value of the PC group at 4mM (45.3% at 3h and 53.4% at 20h), and the RFU value reached substantially 80% or more of the peak value of the PC group at 4.5mM (83.3% and 83.6% at 3h and 20h, respectively).

Example 6 comparison of the Effect of magnesium gluconate and magnesium glutamate on the protein Synthesis Capacity of an in vitro protein Synthesis System

6.1 in vitro cell-free protein Synthesis System containing exogenous magnesium ions (without addition of exogenous RNA polymerase)

Each system was 100. mu.L in volume and the reactions were performed in flat-bottom cell culture plates. 3 replicates were set up for each sample and the mean and standard deviation (error bar) were calculated.

Experimental groups (noted as Gluconate group): the final concentration of each component is as follows: 9.78mM pH8.0 Tris (pH adjusted with HCl, Tris-HCl), 80mM potassium acetate, 15mM glucose, 320mM maltodextrin (measured in glucose units), 24mM potassium phosphate, 1.8mM nucleoside triphosphate mixture (adenosine triphosphate, guanosine triphosphate, cytosine nucleoside triphosphate and uracil nucleoside triphosphate, each at a final concentration of 1.8mM), 0.7mM 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 at a final concentration of 0.7mM), 80mM potassium acetate, 2% (w/v) polyethylene glycol 8000, 0.06g/mL trehalose, 80% (v/v) Kluyveromyces lactis cell extract (CM191205), and 1-10 mM magnesium gluconate.

Positive control group (Positive group, denoted PC group, also denoted Glutamate group): magnesium glutamate with the final concentration of 2-11 mM replaces magnesium gluconate in the experimental group to provide external magnesium ions, and the types and contents of other components and experimental conditions for carrying out in-vitro protein synthesis reaction are completely consistent with those of the experimental group.

Blank control group (Blank control group, BC group): no external magnesium ion is added, and the types and contents of other components and the experimental conditions for carrying out the in vitro protein synthesis reaction are completely consistent with those of the experimental group.

Negative control group (Negative control group, NC group): no exogenous magnesium ion is added, no exogenous DNA template is added subsequently, and the types and contents of other components and the experimental conditions for carrying out in-vitro protein synthesis reaction are completely consistent with those of the experimental group.

6.2 in vitro protein Synthesis reactions: the NC group is not added with an exogenous DNA template; adding a DNA template (obtained by performing in-vitro amplification by the method of 1.2 in the example 1) encoding mEGFP with the final concentration of 11.9 ng/. mu.L into each independent in-vitro cell-free protein synthesis system of the experimental group, the positive control group and the blank control group respectively, uniformly mixing, placing all the systems in a room-temperature environment (18-30 ℃), performing shaking table reaction overnight, and sampling at the time points of 3h and 19h respectively to perform fluorescent protein activity test.

6.3 fluorescent protein Activity assay: the RFU value of the exogenous fluorescent protein, mmefp, synthesized in the sample was determined using the method of 3.3 in example 3.

6.4 Experimental results: as shown in FIG. 7, the abscissa is labeled as "group-magnesium ion concentration value", wherein the magnesium ion concentration value is in mM. The RFU values peaked in the Gluconate group at 3mM magnesium Gluconate (peak values for 3h and 19 h), and in the PC group at 4mM magnesium glutamate (peak values for 3h and 19 h). The RFU value of the Gluconate group can be improved by more than 30% compared with the highest RFU value of the PC group. The peak RFU value of the Gluconate group was increased by 26.7% at 3h compared to the peak of the PC group and by 33.4% at 19h compared to the peak of the PC group. In addition, at 19h, the RFU value can be improved by 20.9% compared with the peak value of the PC group at the concentration of 2mM magnesium Gluconate in the Gluconate group.

Example 7 examination of the Effect of different magnesium sources on the protein Synthesis Capacity of in vitro protein Synthesis System

7.1 in vitro cell-free protein Synthesis System containing exogenous magnesium ions (without addition of exogenous RNA polymerase)

Each system was 100. mu.L in volume and the reactions were performed in flat-bottom cell culture plates. 3 replicates were set up for each sample and the mean and standard deviation (error bar) were calculated.

Experimental groups (noted as Gluconate group): the final concentration of each component is as follows: 9.78mM pH8.0 Tris (pH adjusted with HCl, Tris-HCl), 80mM potassium acetate, 15mM glucose, 320mM maltodextrin (measured in glucose units), 24mM potassium phosphate, 1.8mM nucleoside triphosphate mixture (adenosine triphosphate, guanosine triphosphate, cytosine nucleoside triphosphate and uracil nucleoside triphosphate, each at a final concentration of 1.8mM), 0.7mM 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 at a final concentration of 0.7mM), 80mM potassium acetate, 2% (w/v) polyethylene glycol 8000, trehalose 0.06g/mL, Kluyveromyces lactis cell extract 80% (v/v) (CM191107), total exogenous magnesium ion concentration 3 mM; the formulation of the foreign magnesium ions is shown in table 4.

TABLE 4 mode of supply of exogenous magnesium ions

Wherein, Mix-I is a mixed magnesium source of magnesium gluconate and magnesium acetate, Mix-II is a mixed magnesium source of magnesium gluconate and magnesium glutamate, and Mix-III is a mixed magnesium source of magnesium acetate and magnesium glutamate. The numbers in parentheses for Mix-I (25%), Mix-I (50%), etc. correspond to the molar percentage of magnesium gluconate.

Blank control group (Blank control group, BC group): no external magnesium ion is added, and the types and contents of other components and the experimental conditions for carrying out the in vitro protein synthesis reaction are completely consistent with those of the experimental group.

Negative control group (Negative control group, NC group): no exogenous magnesium ion is added, no exogenous DNA template is added subsequently, and the types and contents of other components and the experimental conditions for carrying out in-vitro protein synthesis reaction are completely consistent with those of the experimental group.

7.2 in vitro protein Synthesis reactions: the NC group is not added with an exogenous DNA template; adding a DNA template (obtained by performing in-vitro amplification by the method of 1.2 in the example 1) encoding mEGFP with the final concentration of 16.6 ng/. mu.L into each independent in-vitro cell-free protein synthesis system of the experimental group and the blank control group respectively, uniformly mixing, placing all the systems in a room temperature environment (18-30 ℃), performing shaking table reaction overnight, and sampling at the time points of 3 hours and 20-24 hours respectively to perform fluorescent protein activity test.

7.3 fluorescent protein Activity assay: the RFU value of the exogenous fluorescent protein, mmefp, synthesized in the sample was determined using the method of 3.3 in example 3.

7.4 Experimental results: as shown in fig. 8. Using a single magnesium source, RFU values of 3mM magnesium Gluconate (Gluconate) were 14.3 times and 2.9 times higher than those of magnesium Acetate (Acetate) and glutamate (glutamate), respectively, at 3 h; at 20h, the RFU value of 3mM magnesium Gluconate (Gluconate) was 18.9 times and 2.8 times that of magnesium Acetate (Acetate) and glutamate (glutamate), respectively. For the mixed magnesium source of magnesium gluconate and magnesium acetate, the higher the content of magnesium gluconate, the higher the protein synthesis amount (indicated by RFU value) of the system at 3h and 20 h. At 20h, for the mixed magnesium source of magnesium gluconate and magnesium glutamate, the higher the content of magnesium gluconate, the higher the protein synthesis amount of the system. At 3h, the RFU values of the Mix-I (25%) group, Mix-II (25%) group, Mix-I (50%) group and Mix-II (50%) group reached 27.5%, 66.6%, 56.5% and 80.2% of the Gluconate group, respectively. At 20h, the RFU values of the Mix-I (25%) group, Mix-II (25%) group, Mix-I (50%), Mix-II (50%), Mix-I (75%) group and Mix-II (75%) group reached 25.5%, 62.4%, 53.5%, 79.8%, 58.9% and 82.4% of the Gluconate group, respectively.

Example 8 examination of the Effect of different magnesium sources on the protein Synthesis Capacity of in vitro protein Synthesis System

8.1 in vitro cell-free protein Synthesis System containing exogenous magnesium ions (without addition of exogenous RNA polymerase)

Each system was 100. mu.L in volume and the reactions were performed in flat-bottom cell culture plates. 3 replicates were set up for each sample and the mean and standard deviation (error bar) were calculated.

Experimental groups: the final concentration of each component is as follows: 9.78mM pH8.0 Tris (pH adjusted with HCl, Tris-HCl), 80mM potassium acetate, 15mM glucose, 320mM maltodextrin (measured in glucose units), 24mM potassium phosphate, 1.8mM nucleoside triphosphate mixture (adenosine triphosphate, guanosine triphosphate, cytosine nucleoside triphosphate and uracil nucleoside triphosphate, each at a final concentration of 1.8mM), 0.7mM 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 at a final concentration of 0.7mM), 80mM potassium acetate, 2% (w/v) polyethylene glycol 8000, 0.06g/mL trehalose, 80% (v/v) Kluyveromyces lactis cell extract (CM191107), 2-12 mM magnesium gluconate.

Positive control group 1(Positive group, denoted PC1 group, further denoted Ace group): magnesium acetate with the final concentration of 2-16 mM replaces magnesium gluconate in the experimental group to provide exogenous magnesium ions, and the types and contents of other components and experimental conditions for carrying out in-vitro protein synthesis reaction are completely consistent with those of the experimental group.

Positive control group 2(Positive group, scored as PC2 group, also scored as Glut group): magnesium glutamate with the final concentration of 2-10 mM replaces magnesium gluconate in the experimental group to provide external magnesium ions, and the types and contents of other components and experimental conditions for carrying out in-vitro protein synthesis reaction are completely consistent with those of the experimental group.

Blank control group (Blank control group, BC group): no external magnesium ion is added, and the types and contents of other components and the experimental conditions for carrying out the in vitro protein synthesis reaction are completely consistent with those of the experimental group.

Negative control group (Negative control group, NC group): no exogenous magnesium ion is added, no exogenous DNA template is added subsequently, and the types and contents of other components and the experimental conditions for carrying out in-vitro protein synthesis reaction are completely consistent with those of the experimental group.

8.2 in vitro protein Synthesis reactions: the NC group is not added with an exogenous DNA template; adding a DNA template (obtained by performing in-vitro amplification by the method of 1.2 in the example 1) encoding mEGFP with the final concentration of 40.4 ng/. mu.L into each independent in-vitro cell-free protein synthesis system of the experimental group, the PC1 group, the PC2 group and the blank control group, mixing uniformly, placing all the systems in a room-temperature environment (18-30 ℃), performing shaking table reaction overnight, and sampling at the time points of 3 hours and 20-24 hours respectively to perform fluorescent protein activity test.

8.3 fluorescent protein Activity assay: the RFU value of the exogenous fluorescent protein, mmefp, synthesized in the sample was determined using the method of 3.3 in example 3.

8.4 results of the experiment: as shown in fig. 9, the abscissa is labeled as "group-magnesium ion concentration value", wherein the magnesium ion concentration value is in mM. In FIG. 9, the Gluc group showed the highest protein expression level at the addition of 4mM magnesium gluconate at both sampling time points of 3h and 22h (RFU 910. + -. 14 at 3h and RFU 1430. + -. 56 at 22 h).

At 3h, the highest RFU value of the Gluc group was increased by 38.1% compared with the highest RFU value of the Ace group (14mM, RFU values of 659 + -21), and by 12.4% compared with the highest RFU value of the Glut group (6mM, RFU values of 810 + -55).

At 20h, the highest RFU value of the Gluc group is increased by 42.2 percent compared with the RFU value of 16mM (1006 +/-52) of the Ace group and 30.6 percent compared with the highest RFU value of the Glut group (6mM, the RFU value is 1095 +/-34).

The negative control group showed negligible RFU values (mean value below 15), not shown.

Example 9 examination of the Effect of different magnesium sources on the protein Synthesis Capacity of in vitro protein Synthesis System

9.1 in vitro cell-free protein Synthesis System containing exogenous magnesium ions (without addition of exogenous RNA polymerase)

Each system was 100. mu.L in volume and the reactions were performed in flat-bottom cell culture plates. 3 replicates were set up for each sample and the mean and standard deviation (error bar) were calculated.

Experimental groups (noted as Gluc group): the final concentration of each component is as follows: 9.78mM pH8.0 Tris (pH adjusted with HCl, Tris-HCl), 80mM potassium acetate, 15mM glucose, 320mM maltodextrin (measured in glucose units), 24mM potassium phosphate, 1.8mM nucleoside triphosphate mixture (adenosine triphosphate, guanosine triphosphate, cytosine nucleoside triphosphate and uracil nucleoside triphosphate, each at a final concentration of 1.8mM), 0.7mM 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 at a final concentration of 0.7mM), 80mM potassium acetate, 2% (w/v) polyethylene glycol 8000, 0.06g/mL trehalose, 80% (v/v) Kluyveromyces lactis cell extract (CM191107), 2-10 mM magnesium gluconate.

Positive control group 1(Positive group, denoted PC1 group, further denoted Ace group): magnesium acetate with the final concentration of 6-24 mM replaces magnesium gluconate in the experimental group to provide exogenous magnesium ions, and the types and contents of other components and experimental conditions for carrying out in-vitro protein synthesis reaction are completely consistent with those of the experimental group.

Positive control group 2(Positive group, scored as PC2 group, also scored as Glut group): magnesium glutamate with the final concentration of 2-12 mM replaces magnesium gluconate in the experimental group to provide external magnesium ions, and the types and contents of other components and experimental conditions for carrying out in-vitro protein synthesis reaction are completely consistent with those of the experimental group.

Blank control group (Blank control group, BC group): no external magnesium ion is added, and the types and contents of other components and the experimental conditions for carrying out the in vitro protein synthesis reaction are completely consistent with those of the experimental group.

Negative control group (Negative control group, NC group): no exogenous magnesium ion is added, no exogenous DNA template is added subsequently, and the types and contents of other components and the experimental conditions for carrying out in-vitro protein synthesis reaction are completely consistent with those of the experimental group.

9.2 in vitro protein Synthesis reactions: the NC group is not added with an exogenous DNA template; adding a DNA template (obtained by performing in-vitro amplification by the method of 1.2 in the example 1) encoding mEGFP with the final concentration of 40.4 ng/. mu.L into each independent in-vitro cell-free protein synthesis system of the experimental group, the PC1 group, the PC2 group and the blank control group, mixing uniformly, placing all the systems in a room-temperature environment (18-30 ℃), performing shaking table reaction overnight, and sampling at the time points of 3 hours and 20-24 hours respectively to perform fluorescent protein activity test.

9.3 fluorescent protein Activity assay: the RFU value of the exogenous fluorescent protein, mmefp, synthesized in the sample was determined using the method of 3.3 in example 3.

9.4 results of the experiment: as shown in fig. 10-11, where 0mM corresponds to BC group. At 3h in FIG. 10, the Gluc group had the highest protein expression at the time of addition of 6mM magnesium gluconate, and the RFU value was 829. + -. 6; in FIG. 11, the Gluc group had the highest protein expression at 20 hours when 6mM magnesium gluconate was added, and the RFU value was 1333. + -. 12.

At 3h, the highest RFU value of the Gluc group was increased by 62.3% compared with the highest RFU value of the Ace group (17mM, RFU value of 511. + -. 19), and by 39.5% compared with the highest RFU value of the Glut group (7mM, RFU value of 594. + -. 33). The RFU value of Gluc group at 5-6 mM magnesium gluconate is higher than that of Ace group and that of Glut group.

At 20h, the highest RFU value of the Gluc group was increased by 34.3% compared with the RFU value of 17mM (993. + -. 26) in the Ace group and 39.3% compared with the highest RFU value of the Glut group (7mM, RFU value of 957. + -. 37). RFU values of Gluc group at 4.5-6 mM magnesium gluconate are higher than those of Ace group and Glut group.

The negative control group showed negligible RFU values (mean value below 15), not shown.

Example 10 examination of the Effect of different magnesium sources on the protein Synthesis Capacity of in vitro protein Synthesis System

10.1 in vitro cell-free protein Synthesis System containing exogenous magnesium ions (without addition of exogenous RNA polymerase)

Each system was 100. mu.L in volume and the reactions were performed in flat-bottom cell culture plates. 3 replicates were set up for each sample and the mean and standard deviation (error bar) were calculated.

Experimental groups: the final concentration of each component is as follows: 9.78mM pH8.0 Tris (pH adjusted with HCl, Tris-HCl), 80mM potassium acetate, 15mM glucose, 320mM maltodextrin (measured in glucose units), 24mM potassium phosphate, 1.8mM nucleoside triphosphate mixture (adenosine triphosphate, guanosine triphosphate, cytosine nucleoside triphosphate and uracil nucleoside triphosphate, each at a final concentration of 1.8mM), 0.7mM 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 at a final concentration of 0.7mM), 80mM potassium acetate, 2% (w/v) 8000 polyethylene glycol, 0.06g/mL trehalose, 80% (v/v) kluyveromyces lactis cell extract (CM191107), 6mM exogenous magnesium ions; the manner of supplying the foreign magnesium ions is shown in table 5.

TABLE 5 mode of supply of exogenous magnesium ions

Wherein, Mix-I is a mixed magnesium source of magnesium gluconate and magnesium acetate, and Mix-II is a mixed magnesium source of magnesium gluconate and magnesium glutamate. The numbers in parentheses for Mix-I (25%), Mix-I (50%), etc. correspond to the molar percentage of magnesium gluconate.

Negative control group (Negative control group, NC group): no exogenous magnesium ion is added, no exogenous DNA template is added subsequently, and the types and contents of other components and the experimental conditions for carrying out in-vitro protein synthesis reaction are completely consistent with those of the experimental group.

10.2 in vitro protein Synthesis reactions: the NC group is not added with an exogenous DNA template; adding a DNA template (obtained by performing in-vitro amplification by the method of 1.2 in the example 1) for encoding mEGFP with the final concentration of 40.4 ng/. mu.L into each independent in-vitro cell-free protein synthesis system of the experimental group, uniformly mixing, placing all the systems in a room temperature environment (18-30 ℃), performing shaking table reaction overnight, and sampling at the time points of 3 hours and 20-24 hours respectively to perform fluorescent protein activity test.

10.3 fluorescent protein Activity assay: the RFU value of the exogenous fluorescent protein, mmefp, synthesized in the sample was determined using the method of 3.3 in example 3.

10.4 results of the experiment: as shown in fig. 12. At 20h, for the mixed magnesium source of magnesium gluconate and magnesium acetate, the higher the magnesium gluconate content, the higher the protein synthesis capacity (indicated by RFU value) of the system. At 3h and 20h, for the mixed magnesium source of the magnesium gluconate and the magnesium glutamate, the higher the content of the magnesium gluconate is, the higher the protein synthesis amount of the system is.

Example 11 examination of the Effect of different magnesium sources on the protein Synthesis Capacity of in vitro protein Synthesis System

The experimental parameters of example 11 were the same as those of example 10, except that the total concentration of magnesium ions was 5mM and the supply of exogenous magnesium ions was varied.

Table 6 shows the manner of providing the external magnesium ions in this embodiment.

TABLE 6 mode of supply of exogenous magnesium ions

Wherein, Mix-I is a mixed magnesium source of magnesium gluconate and magnesium acetate, Mix-II is a mixed magnesium source of magnesium gluconate and magnesium glutamate, and Mix-III is a mixed magnesium source of magnesium acetate and magnesium glutamate. The numbers in parentheses for Mix-I (25%), Mix-I (50%), etc. correspond to the molar percentage of magnesium gluconate.

Wherein, the NC group is not added with a DNA template; the BC group was added with the same amount of DNA template as the other system.

The results of the experiment are shown in FIG. 13. For the mixed magnesium source of magnesium gluconate and magnesium acetate, the higher the content of magnesium gluconate, the higher the protein synthesis amount (indicated by RFU value) of the system at 3h and 22 h. At 22h, for the mixed magnesium source of magnesium gluconate and magnesium glutamate, the higher the content of magnesium gluconate, the higher the protein synthesis amount of the system.

When a single magnesium source is adopted, the RFU value of 3mM magnesium Gluconate (Gluconate) is 65.5 times and 3.45 times that of magnesium Acetate (Acetate) and magnesium glutamate (glutamate) respectively at 3 h; at 22h, the RFU value of 3mM magnesium Gluconate (Gluconate) was 67.1 times and 3.65 times that of magnesium Acetate (Acetate) and magnesium glutamate (glutamate), respectively.

Comparing 3 groups Mix-I (50%), Mix-III, Mix-II (50%) containing 50% magnesium glutamate, the protein synthesis (RFU value) increases with the increase of magnesium glutamate content when 0%, 25%, 50%, 3h and 22h respectively contain magnesium glutamate.

Also, two groups Mix-I (25%), Mix-II (25%) containing 25% magnesium gluconate and a group Mix-I (75%), Mix-II (75%) containing 75% magnesium gluconate were compared, and the results showed that the protein synthesis amount of the Mix-II mixed system was higher than that of the Mix-I mixed system.

At 3h, the RFU values of the Mix-I (25%) group, Mix-II (25%) group, Mix-I (50%) group and Mix-II (50%) group reached 10.4%, 58.4%, 28.1% and 93.7% of the Gluconate group, respectively. At 20h, the RFU values of the Mix-I (25%) group, Mix-II (25%) group, Mix-I (50%), Mix-II (50%), Mix-I (75%) group and Mix-II (75%) group reached 20.1%, 52.3%, 38.8%, 74.9%, 66.9% and 80.9% of the Gluconate group, respectively.

Example 12 examination of the Effect of magnesium gluconate and magnesium aspartate on the protein Synthesis ability of the in vitro protein Synthesis System

The remaining experimental parameters of example 12 were the same as those of example 10, except that the supply of the exogenous magnesium ions was different.

Table 7 shows the manner of providing the external magnesium ions in this embodiment.

TABLE 7 mode of supply of exogenous magnesium ions

Wherein the data of Gluconate and Gluc-6 are the same data.

Wherein, Mix is a mixed magnesium source of magnesium gluconate and magnesium L-aspartate.

Wherein, the NC group is not added with exogenous magnesium ions and DNA templates; no exogenous magnesium ions were added to the BC group, but equal amounts of DNA template to the other systems were added.

Wherein, the numbers in brackets of Mix-I (25%), Mix-I (50%), etc. correspond to the molar percentage of the magnesium gluconate.

The results of the experiment are shown in FIG. 14. When the exogenous magnesium ions are 2-8 mM magnesium gluconate, the RFU value is highest (446 +/-49) at 6mM for 3h, and the RFU value is highest (1545 +/-20) at 7mM for 22 h. When the exogenous magnesium ions are 2-8 mM magnesium L-aspartate, the RFU value is highest (643 +/-16) at 5mM for 3h, and the RFU value is highest (1846 +/-134) at 5mM for 22 h. The peak RFU value of the magnesium L-aspartate is higher than that of the magnesium gluconate.

In this example, magnesium L-aspartate has a better effect of promoting the protein synthesis ability of the system.

Example 13 examination of the Effect of a magnesium gluconate-magnesium aspartate mixture on the protein Synthesis ability of an in vitro protein Synthesis System

13.1 in vitro cell-free protein Synthesis System containing exogenous magnesium ions (without addition of exogenous RNA polymerase)

Each system was 100. mu.L in volume and the reactions were performed in flat-bottom cell culture plates. 3 replicates were set up for each sample and the mean and standard deviation (error bar) were calculated.

Experimental group I (noted as Gluc group): the final concentration of each component is as follows: 25.0mM pH8.0 Tris (HCl-adjusted pH), 34.1mM potassium acetate, 1.0mM Dithiothreitol (DTT), 1.6% (w/v) PFG8000, 4.5mM glucose, 0.058g/mL corn dextrin, 1.2mM nucleoside triphosphate mixture (adenine nucleoside triphosphate, guanine nucleoside triphosphate, cytosine nucleoside triphosphate and uracil nucleoside triphosphate, each at a final concentration of 1.2mM), 1.4mM potassium bicarbonate, twenty amino acid mixtures (glycine 5.12mM, alanine 0.86mM, valine 3.70mM, leucine 0.64mM, isoleucine 1.66mM, phenylalanine 1.30mM, proline 0.60mM, tryptophan 0.57mM, serine 0.29mM, tyrosine 0.07mM, cysteine 2.14mM, methionine 0.96mM, asparagine 1.17mM, aspartic acid 0.17 mM, glutamic acid 0.32mM, 1.12mM, 1.6% and the like, Lysine 3.10mM, arginine 1.56mM and histidine 1.55mM), trehalose 41.0mg/mL, squalane 5.8% (v/v), Kluyveromyces lactis cell extract 63% (v/v) (YY1904291), tripotassium phosphate 24.5mM, magnesium gluconate 1.80 mM.

Experimental group II (noted as Asp group): except that magnesium ions adopt the magnesium L-aspartate with the final concentration of 1-28 mM, the types and the contents of other components and the experimental conditions for carrying out in-vitro protein synthesis reaction are completely consistent with those of the experimental group I.

Negative control group (Negative control group, NC group): no exogenous magnesium ion is added, no exogenous DNA template is added subsequently, and the types and the contents of other components and the experimental conditions for carrying out in-vitro protein synthesis reaction are completely consistent with those of the experimental group I and the experimental group II.

13.2 in vitro protein Synthesis reactions: the NC group is not added with an exogenous DNA template; adding a DNA template (obtained by performing in-vitro amplification by the method of 1.2 in the example 1) encoding mEGFP with a final concentration of 33 ng/. mu.L into each independent in-vitro cell-free protein synthesis system of the experiment group I and the experiment group II respectively, uniformly mixing, placing all the systems in a room temperature environment (18-30 ℃), performing shaking table reaction overnight, and sampling at time points of 3 hours and 20-24 hours respectively to perform fluorescent protein activity test.

13.3 fluorescent protein Activity assay: the RFU value of the exogenous fluorescent protein, mmefp, synthesized in the sample was determined using the method of 3.3 in example 3.

13.4 results of the experiment (not shown): the highest RFU value (2030 + -44) was obtained at 4mM when 1-28 mM magnesium L-aspartate was used at 21 h. And the RFU value of the magnesium gluconate with 1.80mM is 2970 +/-32 at 21h, which is 46.3 percent higher than the highest value of the magnesium L-aspartate.

Example 14 examination of the Effect of a magnesium gluconate-magnesium aspartate mixture on the protein Synthesis ability of an in vitro protein Synthesis System

14.1 in vitro cell-free protein Synthesis System containing exogenous magnesium ions (without addition of exogenous RNA polymerase)

Each system was 100. mu.L in volume and the reactions were performed in flat-bottom cell culture plates. 3 replicates were set up for each sample and the mean and standard deviation (error bar) were calculated.

Experimental groups: the final concentration of each component is as follows: 25.08mM pH8.0 Tris (HCl-adjusted pH), 34.2mM potassium acetate, 0.99mM dithiothreitol, 1.6% (w/v) PFG8000, 4.58mM glucose, 0.058g/mL corn dextrin, 1.23mM nucleoside triphosphate mixture (adenine nucleoside triphosphate, guanine nucleoside triphosphate, cytosine nucleoside triphosphate and uracil nucleoside triphosphate, each at a final concentration of 1.23mM), 1.37mM potassium bicarbonate, twenty amino acid mixtures (final concentrations: glycine 4.18mM, alanine 0.70mM, valine 3.02mM, leucine 0.52mM, isoleucine 1.36mM, phenylalanine 1.06mM, proline 0.49mM, tryptophan 0.46mM, serine 0.23mM, tyrosine 0.06mM, cysteine 1.75mM, methionine 0.78mM, threonine 0.95mM, asparagine 0.95mM, glutamine 0.23mM, aspartic acid 0.41mM, glutamic acid 0.23mM, glutamic acid 0.41mM, 1.23mM, Lysine 2.53mM, arginine 1.27mM and histidine 1.27mM), trehalose 41.1mg/mL, squalane 6.0% (v/v), Kluyveromyces lactis cell extract 60% (v/v) (YY1904281), tripotassium phosphate 28.5mM, magnesium gluconate 1.8mM, magnesium L-aspartate 0-9 mM.

Negative control group (Negative control group, NC group): no exogenous magnesium ion is added, no exogenous DNA template is added subsequently, and the types and contents of other components and the experimental conditions for carrying out in-vitro protein synthesis reaction are completely consistent with those of the experimental group.

14.2 in vitro protein Synthesis reactions: the NC group is not added with an exogenous DNA template; adding a DNA template (obtained by performing in-vitro amplification by the method of 1.2 in the example 1) encoding mEGFP with the final concentration of 34.2 ng/. mu.L into each independent in-vitro cell-free protein synthesis system of the experimental group, uniformly mixing, placing all the systems in a room temperature environment (18-30 ℃), performing shaking table reaction overnight, and sampling at the time points of 3 hours and 20-24 hours respectively to perform fluorescent protein activity test.

14.3 fluorescent protein Activity assay: the RFU value of the exogenous fluorescent protein, mmefp, synthesized in the sample was determined using the method of 3.3 in example 3.

14.4 results of the experiment: as shown in fig. 15. The outside of the abscissa is the total concentration of magnesium ions, and the inside of the parenthesis is the mole percentage of magnesium gluconate. The protein synthesis amount is highest when the total concentration of magnesium ions is 3.8mM, the concentrations of magnesium gluconate and magnesium L-aspartate are close to each other, and the RFU value is 1805 +/-186 at 3h and 3762 +/-381 at 22 h. Comparing RFU values of 1.8 (100%) and 2.8 (64.3%), while the total concentration of magnesium ions is increasing, RFU values are decreasing instead as the proportion of magnesium gluconate decreases.

The negative control group showed negligible RFU values (mean value below 20), not shown.

Example 15 examination of the Effect of a magnesium mixture of magnesium gluconate, magnesium aspartate and magnesium acetate on the protein Synthesis ability of an in vitro protein Synthesis System

15.1 in vitro cell-free protein Synthesis System containing exogenous magnesium ions (without addition of exogenous RNA polymerase)

Each system was 100. mu.L in volume and the reactions were performed in flat-bottom cell culture plates. 3 replicates were set up for each sample and the mean and standard deviation (error bar) were calculated.

Experimental groups: the final concentration of each component is as follows: 21.48mM pH8.0 Tris (HCl pH adjusted), 29.3mM potassium acetate, 1.4% (w/v) PFG8000, 3.92mM glucose, 0.050g/mL corn dextrin, 1.05mM nucleoside triphosphate mixture (adenosine triphosphate, guanosine triphosphate, cytosine triphosphate and uracil nucleoside triphosphate, each at a final concentration of 1.05mM), 1.37mM potassium bicarbonate, twenty amino acid mixtures (final concentrations: glycine 9.66mM, alanine 1.63mM, valine 6.99mM, leucine 1.20mM, isoleucine 3.13mM, phenylalanine 2.46mM, proline 1.14mM, tryptophan 1.07mM, serine 0.54mM, tyrosine 0.13mM, cysteine 5.86mM, methionine 1.81mM, asparagine 2.02mM, glutamine 0.41mM, threonine 0.54mM, aspartic acid 0.94mM, lysine 0.93mM, arginine 0.93mM, and histidine 0.93 mM), 50% (v/v) Kluyveromyces lactis cell extract (YY1904281), 20.99mM tripotassium phosphate, 36.3mg/mL trehalose, 2.99% (v/v) squalane, 1.54mM magnesium gluconate, 1.87mM magnesium aspartate, and 1-5 mM magnesium acetate.

Negative control group (Negative control group, NC group): no exogenous magnesium ion is added, no exogenous DNA template is added subsequently, and the types and contents of other components and the experimental conditions for carrying out in-vitro protein synthesis reaction are completely consistent with those of the experimental group.

15.2 in vitro protein Synthesis reactions: the NC group is not added with an exogenous DNA template; adding a DNA template (obtained by performing in-vitro amplification by the method of 1.2 in the example 1) encoding mEGFP with the final concentration of 34.2 ng/. mu.L into each independent in-vitro cell-free protein synthesis system of the experimental group, uniformly mixing, placing all the systems in a room temperature environment (18-30 ℃), performing shaking table reaction overnight, and sampling at the time points of 3 hours and 20-24 hours respectively to perform fluorescent protein activity test.

15.3 fluorescent protein Activity assay: the RFU value of the exogenous fluorescent protein, mmefp, synthesized in the sample was determined using the method of 3.3 in example 3.

15.4 results of the experiment: as shown in fig. 16. The outside of the abscissa is the total concentration of magnesium ions, and the inside of the parenthesis is the mole percentage of magnesium gluconate. On the basis that the total concentration of magnesium ions of the magnesium gluconate and the magnesium L-aspartate reaches 3.41mM, the synthesis of foreign protein is inhibited along with the addition of magnesium acetate. RFU values were highest at 3.41mM, 1992. + -. 133 at 3h and 4359. + -. 383 at 22 h.

Compare fig. 15 and fig. 16. In FIG. 15, the total concentration of magnesium ions was 4.8mM (37.5%), which still gave a relatively high protein expression, and the RFU values (1580.5. + -. 131) reached 87.6% of the RFU values (1805. + -. 186) at 3h and 3.8 (47.4%) at 3h, and 89.1% of the RFU values (3350. + -. 108) reached 3.8 (47.4%) at 22 h. In FIG. 15, the amount of protein synthesis was considerably suppressed by increasing the total concentration of magnesium ions to 4.41mM by adding only 1mM magnesium acetate, and the RFU value was 35.6% at 3.41mM at 3h and 30.9% at 3.41mM at 22 h. Therefore, the tolerance of the system to magnesium gluconate and magnesium aspartate is higher than that of magnesium acetate, and the system is more sensitive to the concentration of magnesium acetate.

In conclusion, magnesium gluconate is used for providing exogenous magnesium ions, and after the overnight reaction: the highest protein expression can be improved by more than 40 percent relative to magnesium acetate and can be improved by more than 55 percent relative to magnesium L-glutamate. Early in the initiation of the in vitro protein synthesis reaction, at 3 h: the highest protein expression quantity can be improved by more than 60 percent relative to magnesium acetate; can be improved by more than 50 percent relative to magnesium L-glutamate. Compared with the traditional magnesium acetate and magnesium glutamate, the magnesium gluconate has the effect of generally improving the protein synthesis capacity; in certain embodiments, the magnesium gluconate also corresponds to a protein synthesis greater than 45% higher than magnesium L-aspartate. In a mixed magnesium source system, particularly in a mixed system of magnesium gluconate, traditional magnesium acetate and magnesium glutamate, the protein synthesis amount is increased along with the increase of the content of magnesium gluconate. Furthermore, in some embodiments, the mixed magnesium source system of magnesium gluconate and magnesium glutamate is superior to the mixed magnesium source system of magnesium gluconate and magnesium acetate.

The above is only a part of the preferred embodiments of the present invention, and the present invention is not limited to the contents of the above embodiments. It will be apparent to those skilled in the art that various changes and modifications can be made which will achieve the same technical effects within the spirit or scope of the invention and the scope of the invention is to be determined by the appended claims.

Sequence listing

<110> Kangma (Shanghai) Biotech Co., Ltd

<120> exogenous magnesium ion-containing in-vitro cell-free protein synthesis system and kit and application thereof

<130> 2020

<141> 2020-04-08

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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

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His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg

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Thr Ile Ser Phe Lys Asp Asp Gly Thr Tyr Lys Thr Arg Ala Glu Val

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Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile

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Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn

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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

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Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro

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Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Lys Leu Ser

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Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val

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Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys

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Claims (16)

1. An in vitro cell-free protein synthesis system containing exogenous magnesium ions is characterized in that the in vitro cell-free protein synthesis system containing the exogenous magnesium ions is marked as a CFPS (Mg +) system and comprises the exogenous magnesium ions; the exogenous magnesium ions come from one or more sources and at least comprise magnesium gluconate; the CFPS (Mg +) system can provide translation related elements required by synthesizing the foreign protein together with a nucleic acid template for encoding the foreign protein, and the foreign protein is obtained by in vitro protein synthesis reaction expression.

2. The in vitro cell-free protein synthesis system according to claim 1, wherein the source of exogenous magnesium ions further optionally comprises at least one of: magnesium aspartate, magnesium acetate, magnesium glutamate, magnesium chloride, magnesium phosphate, magnesium sulfate, magnesium citrate, magnesium hydrogen phosphate, magnesium iodide, magnesium lactate, magnesium nitrate, and magnesium oxalate;

preferably, the supply source of the exogenous magnesium ions is a combination of magnesium gluconate and magnesium aspartate;

more preferably, the source of exogenous magnesium ions is a combination of magnesium gluconate and magnesium L-aspartate;

preferably, the supply source of the exogenous magnesium ions is a combination of magnesium gluconate and magnesium glutamate;

more preferably, the source of exogenous magnesium ions is a combination of magnesium gluconate and magnesium L-glutamate;

preferably, the supply source of the exogenous magnesium ions is a combination of magnesium gluconate, magnesium aspartate and magnesium glutamate;

more preferably, the source of exogenous magnesium ions is a combination of magnesium gluconate, magnesium L-aspartate and magnesium L-glutamate;

preferably, the magnesium gluconate provides exogenous magnesium ions in a molar percentage of the total exogenous magnesium ions selected from any one of the following percentage values, or a range of values 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 numerical ranges are inclusive of the two endpoints.

3. The in vitro cell-free protein synthesis system comprising an exogenous magnesium ion according to any one of claims 1-2, wherein: the amount of magnesium gluconate capable of increasing the amount of synthesized foreign protein is recorded as Q_MgP；

Said Q_MgPIs selected from Y_PRT(C_MgP) In curve Y_PRTGreater than Y₀The dosage range of magnesium gluconate; wherein, C_MgPThe amount of magnesium gluconate, Y_PRTMeans the amount of expression of foreign protein, Y_PRT(C_MgP) The curve is determined by taking the dosage of the magnesium gluconate as an independent variable, the expression level of the foreign protein as a dependent variable and other reaction parameters; y is₀Means said C_MgPThe corresponding foreign protein expression level is 0;

preferably, said Q_MgPSelected from foreign proteins expressed in an amount of at least Y₀+50％Y_ΔThe dosage range of magnesium gluconate;

more preferably, said Q_MgPSelected from foreign proteinsThe expression amount is at least Y₀+60％Y_ΔThe dosage range of magnesium gluconate;

more preferably, said Q_MgPSelected from foreign proteins expressed in an amount of at least Y₀+70％Y_ΔThe dosage range of magnesium gluconate;

more preferably, said Q_MgPSelected from foreign proteins expressed in an amount of at least Y₀+80％Y_ΔThe dosage range of magnesium gluconate;

more preferably, said Q_MgPSelected from foreign proteins expressed in an amount of at least Y₀+90％Y_ΔThe dosage range of magnesium gluconate;

more preferably, said Q_MgPSelected from foreign proteins expressed in an amount of at least Y₀+95％Y_ΔThe dosage range of magnesium gluconate;

more preferably, said Q_MgPMagnesium gluconate dosage C when adopting the highest expression level of foreign protein_max；

Wherein, the Y is_Δ＝Y_max-Y₀(ii) a Wherein, the Y is_maxFinger Y_PRT(C_MgP) The highest expression level of the foreign protein in the curve.

4. The in vitro cell-free protein synthesis system comprising an exogenous magnesium ion according to any one of claims 1 to 3, wherein: the magnesium gluconate provides exogenous magnesium ions at a concentration selected from any one of the following concentrations, or a range of concentrations between any two of the following concentrations: 0.1mM, 0.5mM, 1mM, 1.5mM, 2mM, 2.5mM, 3mM, 3.5mM, 4mM, 4.5mM, 5mM, 5.5mM, 6mM, 6.5mM, 7mM, 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, 80 mM; the concentration ranges are inclusive of the two endpoints.

5. The in vitro cell-free protein synthesis system comprising an exogenous magnesium ion according to any one of claims 1 to 4, wherein: the CFPS (Mg +) system includes a cell extract;

preferably, the source strain of the cell extract is genetically modified in the following manner: inserting the coding sequence of RNA polymerase into the cell episome plasmid, or integrating the coding gene of RNA polymerase into the cell genome, or adopting the combination mode of the two modes;

preferably, the endogenously expressed RNA polymerase is endogenously expressed T7RNA polymerase.

6. The in vitro cell-free protein synthesis system according to any one of claims 1-5, wherein the CFPS (Mg +) system further comprises RNA polymerase;

the source of the RNA polymerase is selected from any one or combination of the following: a translation product of a cell extract comprising an endogenously expressed RNA polymerase, an exogenous nucleic acid template encoding the RNA polymerase;

preferably, the RNA polymerase is T7RNA polymerase;

preferably, the CFPS (Mg +) system comprises a cellular extract containing an endogenously expressed RNA polymerase capable of recognizing a promoter in the nucleic acid template that initiates a gene transcription process of the foreign protein;

further preferably, the CFPS (Mg +) system comprises a cell extract containing endogenously expressed T7RNA polymerase.

7. The in vitro cell-free protein synthesis system according to any one of claims 1 to 6, wherein the CFPS (Mg +) system further comprises a DNA polymerase; the source of the DNA polymerase is selected from any one or combination of the following: a translation product of a cell extract comprising an endogenously expressed DNA polymerase, an exogenous nucleic acid template encoding the DNA polymerase; preferably, the DNA polymerase is phi29 DNA polymerase;

preferably, the CFPS (Mg +) system comprises an exogenous RNA polymerase and an exogenous DNA polymerase;

preferably, the CFPS (Mg +) system comprises an exogenous T7RNA polymerase and an exogenous phi29 DNA polymerase.

8. The in vitro cell-free protein synthesis system comprising an exogenous magnesium ion according to any one of claims 5-7, wherein the cellular extract is a prokaryotic cellular extract, a eukaryotic cellular extract, or a combination thereof;

preferably, the cell extract is selected from any one or a combination of the following sources: escherichia coli, yeast cells, mammalian cells, plant cells, insect cells;

more preferably, the yeast cells are selected from any one or a combination of the following sources: kluyveromyces, Saccharomyces cerevisiae, Pichia pastoris;

further preferably, the kluyveromyces is selected from any one or a combination of the following: kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces polybuvinsis, Kluyveromyces hainanensis, Kluyveromyces wakakii, Kluyveromyces fragilis, Kluyveromyces hubeiensis, Kluyveromyces polyspora, Kluyveromyces siamensis, and Kluyveromyces lactis;

preferably, the source of the cell extract is selected from: any one of escherichia coli, kluyveromyces lactis, wheat germ cells, spodoptera frugiperda cells, leishmania cells, rabbit reticulocytes, chinese hamster ovary cells, african green monkey kidney COS cells, african green monkey kidney VERO cells, baby hamster kidney cells, human Hela cells, human hybridoma cells, human fibrosarcoma HT1080 cells, and any combination of the foregoing.

9. The in vitro cell-free protein synthesis system comprising an exogenous magnesium ion according to any one of claims 1-8, wherein the CFPS (Mg +) system further comprises an energy system;

preferably, the energy system is selected from any one or combination of the following: sugar and phosphate energy system, sugar and phosphocreatine energy system, phosphocreatine and phosphocreatine enzyme system, phosphocreatine and phosphocreatine kinase system, monosaccharide and glycolysis intermediate thereof, glycogen and glycolysis intermediate thereof.

10. The in vitro cell-free protein synthesis system comprising an exogenous magnesium ion according to any one of claims 1 to 9, wherein the CFPS (Mg +) system further comprises a substrate for RNA synthesis and/or a substrate for protein synthesis;

preferably, the substrate of the synthetic RNA is a mixture of nucleotides, more preferably selected from: nucleoside monophosphates, nucleoside triphosphates, and combinations thereof; the substrate for the synthesis of RNA is more preferably a nucleoside triphosphate mixture;

preferably, the substrate of the synthetic protein is an amino acid mixture at least comprising the amino acid mixture required by the process of synthesizing the foreign protein; more preferably, the amino acid mixture is a mixture of natural amino acids;

the CFPS (Mg +) system also optionally includes a substrate for the synthesis of DNA; the substrate for the synthesis of DNA is preferably a mixture of deoxynucleotides, more preferably a mixture of deoxynucleoside triphosphates.

11. The in vitro cell-free protein synthesis system according to any one of claims 1 to 10, wherein the CFPS (Mg +) system further comprises at least one of the following exogenously added components: translation-related elements, DNA amplification-related elements, RNA amplification-related elements, rnase inhibitors, crowding agents, potassium ions, antioxidants or reducing agents, cryoprotectants, trehalose, reaction promoters, antifoams, alkanes, buffers, aqueous solvents;

the translation-related element is preferably selected from: trnas, ribosomes, other translation-related enzymes, initiation factors, elongation factors, termination factors, and combinations thereof;

the crowding agent is preferably polyethylene glycol, polyvinyl alcohol, polystyrene, dextran, sucrose polymer, polyvinylpyrrolidone, albumin, or a combination thereof;

the potassium ions are preferably derived 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 antioxidant or reducing agent is preferably dithiothreitol, 2-mercaptoethanesulfonic acid, 2-mercaptoethanol, reduced glutathione, tricarboxymethylphosphonic acid, 3-mercapto-1, 2-propanediol, or a combination thereof;

the reaction promoter is preferably an aluminum salt, aluminum oxide, iron salt, iron oxide, calcium salt, or a combination thereof;

the alkane is preferably cyclohexane, isooctane, decane, tetradecane, pentadecylcyclohexane, squalane, tetradecane, petrolatum, or a combination thereof;

the buffer is preferably Tris-HCl, Tris base, HEPES, or a combination thereof;

the aqueous solvent is preferably a buffer.

12. The in vitro cell-free protein synthesis system according to any one of claims 1 to 11, wherein the exogenous protein is selected from any one of the following proteins, fusion proteins in any combination, and mixtures in any combination: luciferase, green fluorescent protein, enhanced green fluorescent protein, yellow fluorescent protein, aminoacyl tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, variable regions of an antibody, single-chain and single-chain fragments of an antibody, alpha-amylase, enteromycin A, hepatitis C virus E2 glycoprotein, insulin and precursors thereof, glucagon-like peptide, interferon, interleukin, lysozyme, serum albumin, transthyretin, tyrosinase, xylanase, beta-galactosidase, a partial domain of any of the foregoing, a subunit or fragment of any of the foregoing, a variant of any of the foregoing;

preferably, the variant is a mutant.

13. An in vitro protein synthesis kit, comprising:

(i) the CFPS (Mg +) system of any one of claims 1-12;

preferably, the components of the CFPS (Mg +) system are placed in one or more containers as a solid, semi-solid, liquid, emulsion, suspension, or combination thereof;

(ii) optionally including a nucleic acid template encoding a foreign protein;

(iii) a label or instructions;

(ii) capable of providing, together with (ii), translation-related elements required for synthesis of the foreign protein;

preferably, said (i) has a separate aliquot of the cell extract;

preferably, said (ii) nucleic acid template encoding a foreign protein comprises a promoter element capable of being recognized by (i) said CFPS (Mg +) system;

preferably, the nucleic acid template encoding the foreign protein comprises a foreign protein translation system, a resistance gene translation system, a lac repressor translation system; the translation systems respectively comprise corresponding promoters;

preferably, the nucleic acid template encoding the foreign protein further contains a gene controlling the copy number of the plasmid;

preferably, the nucleic acid template encoding the foreign protein further comprises a transcription enhancing element or/and a translation enhancing element;

preferably, the nucleic acid template encoding the foreign protein contains a T7 promoter, and the CFPS (Mg +) system comprises T7RNA polymerase; more preferably, the CFPS (Mg +) system comprises a cellular extract containing endogenously expressed T7RNA polymerase;

preferably, the nucleic acid template encoding the foreign protein contains a T7 promoter capable of initiating a gene transcription process for the foreign protein, and the CFPS (Mg +) system comprises T7RNA polymerase; more preferably, the CFPS (Mg +) system comprises a cellular extract containing endogenously expressed T7RNA polymerase;

the nucleic acid template encoding the foreign protein is a DNA template, an mRNA template, or a combination thereof.

14. A method for synthesizing a foreign protein, comprising the steps of:

(i) providing a CFPS (Mg +) system as defined in any one of claims 1-12;

(ii) adding a DNA template for encoding the exogenous protein, and carrying out incubation reaction to synthesize the exogenous protein;

further optionally comprising step (iii): isolating or/and detecting the foreign protein;

(i) the CFPS (Mg +) system of (i) is capable of providing translation-related elements required for synthesizing a foreign protein together with the DNA template encoding a foreign protein of (ii);

preferably, said nucleic acid template encoding a foreign protein comprises a promoter element capable of being recognized by the CFPS (Mg +) system of (i);

preferably, the nucleic acid template encoding the foreign protein comprises a foreign protein translation system, a resistance gene translation system, a lac repressor translation system; the translation systems respectively comprise corresponding promoters;

preferably, the nucleic acid template encoding the foreign protein further contains a gene controlling the copy number of the plasmid;

preferably, the nucleic acid template encoding the foreign protein further comprises a transcription enhancing element or/and a translation enhancing element;

preferably, the nucleic acid template encoding the foreign protein contains a T7 promoter capable of initiating a gene transcription program for the foreign protein, and the CFPS (Mg +) system comprises T7RNA polymerase;

more preferably, the nucleic acid template encoding the foreign protein comprises a T7 promoter capable of initiating a gene transcription process for the foreign protein, and the CFPS (Mg +) system comprises a cell extract comprising endogenously expressed T7RNA polymerase.

15. Use of an in vitro cell-free protein synthesis system comprising an exogenous magnesium ion according to any one of claims 1 to 12, in protein synthesis;

preferably, it is applied to protein manufacturing, or to assays based on protein synthesis.

16. Use of magnesium gluconate in a system for the in vitro cell-free protein synthesis comprising exogenous magnesium ions according to any one of claims 1 to 12, or in a kit for the in vitro protein synthesis according to claim 13, or in a method for the synthesis of an exogenous protein according to claim 14.

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