An in vitro cell-free protein synthesis method based on hydrophobic interface, d2p kit and related applications

Abstract

The application provides an in-vitro cell-free synthesis method and kit of exogenous protein, and belongs to the technical field of protein synthesis. The in-vitro cell-free synthesis method of exogenous protein provided by the application provides an in-vitro cell-free protein synthesis system in an aqueous phase, a nucleic acid template encoding exogenous protein, and a hydrophobic interface in contact with the in-vitro cell-free protein synthesis system in the aqueous phase, and incubation reaction is carried out to synthesize exogenous protein, thereby improving the protein synthesis efficiency and protein expression amount of the cell-free system. The application further provides an in-vitro protein synthesis kit with higher efficiency and higher throughput. The application provides a simple and convenient hydrophobic interface optimization method, which is simple and convenient to operate and cost-saving.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of protein synthesis, in particular to the technical field of in vitro cell-free protein synthesis, and specifically to an in vitro cell-free protein synthesis method based on a hydrophobic interface, a D2P kit (DNA-to-Protein kit) and related applications. BACKGROUND

[0002] Proteins are important molecules in cells, and are involved in almost all functions of cells. Protein synthesis mainly includes traditional intracellular synthesis techniques and new generation of in vitro synthesis techniques. Traditional protein expression systems refer to a molecular biology technique for expressing foreign genes through model organisms such as bacteria, fungi, plant cells, insect cells or animal cells. In vitro protein synthesis system, also known as cell-free expression system, emerged in the 1960s. It uses exogenous mRNA or DNA as a template for protein synthesis, and realizes the synthesis of target proteins by artificially controlling the addition of substrates, energy and transcription and translation related protein factors required for protein synthesis. In vitro protein synthesis system generally refers to the addition of nucleic acid templates (mRNA templates or DNA templates), RNA polymerase, amino acids, ATP and other components in the lysate / extraction of bacteria, fungi, plant cells, insect cells or animal cells to complete the rapid and efficient translation of foreign proteins. In vitro protein synthesis system does not need to go through the steps of plasmid construction, transformation, cell culture, cell collection and crushing, and is a relatively fast, time-saving and convenient protein expression method, which is an important tool in the field of protein (―Garcia RA, Riley MR. Applied biochemistry and biotechnology. Humana Press. 1981, 263-264”;―Fromm HJ, Hargrove M. Essentials of Biochemistry. 2012”;CN109988801A;―Assenberg R, Wan PT, Geisse S, Mayr LM. Advances in recombinant protein expression for use in pharmaceutical research [J]. Current Opinion in Structural Biology. 2013, 23(3): 393-402”;―Anne Zemella, Lena Thoring, Christian Hoffmeister, and Stefan Kubick. Cell-free protein synthesis: pros and cons of prokaryotic and eukaryotic systems [J]. Chembiochem. 2015, 16: 2420-2431”). DSpecial proteins (e.g., amino acids) that can simultaneously and parallelly synthesize multiple proteins facilitate the development of high-throughput drug screening and proteomic research (Spirin AS, Swartz JR. Chapter 1. Cell-Free Protein Synthesis Systems: Historical Landmarks, Classification, and General Methods. Wiley-VCH Verlag GmbH & Co. KGaA, 2008: 1-34.). Protein products produced by in vitro synthesis systems can be widely used in various fields such as medicine, food, nutrition, dietary supplements, cosmetics, etc., including but not limited to the protein products of the applicant's PROTEINN TM , PROUDON TM , PUDON TM , etc.

[0003] Protein synthesis capacity is one of the key indicators to determine whether the in vitro protein synthesis system can be industrialized, mainly including protein synthesis efficiency and protein synthesis amount (protein expression amount). In order to improve the protein synthesis amount, more modifications are made from the aspects of cell extract, energy system, genetic template (nucleic acid template), reactor and operation form (Zhang X. Key technologies for high-efficiency synthesis of complex membrane proteins by cell-free system and industrial application exploration [D]. Zhejiang University, 2014). Among them, the reactor is modified, such as the continuous exchange type cell-free reaction developed by Kim et al. (Biotechnol. Prog., 1996, 12(5): 645-649), which uses a semi-permeable membrane with selective permeability to separate the reaction system into reaction zone and supplement zone two parts; the reaction zone is responsible for the transcription and reaction of the template molecule, and the target protein molecule is expressed; the supplement zone is responsible for supplementing the consumed energy and substrate; the energy and small molecule substrate in the supplement zone can penetrate the semi-permeable membrane into the reaction zone (maintain the reaction), and the inorganic phosphorus and other small molecules by-products in the reaction zone can also diffuse to the supplement zone through the semi-permeable membrane (reduce the inhibition), and the expressed target protein as a single molecule cannot diffuse to the supplement zone through the semi-permeable membrane; the duration of the cell-free reaction is prolonged from tens of minutes or even minutes to more than twenty hours. Sawasaki et al. also separate the reaction zone and the supplement zone by phase interface diffusion (FEBS Lett., 2002, 514(1): 102-105). Biyani et al. developed a solid-phase cell-free reaction system, which promotes the increase of local substrate concentration and increases the stability of the template (Nucleic Acids Res., 2006, 34:e140). Park et al. also use DNA hydrogel for in vitro protein synthesis, which significantly improves the reaction efficiency (Nat. Mater., 2009, 8:432-437). The above-mentioned modification methods of the reactor generally need to use specially designed or specially prepared reactor materials (selective semi-permeable membrane, hydrogel, solid-phase system, etc.), or the cost is high, or the steps are complicated, and moreover, it is not suitable for large-scale production.

[0004] In summary, it is still urgent to improve the reaction container to realize efficient and low-cost in vitro cell-free protein synthesis. SUMMARY

[0005] In view of the above technical problems, the present application discloses a simple, convenient, more efficient and higher flux in vitro cell-free synthesis method of exogenous protein, which can significantly improve the protein synthesis capacity by providing a hydrophobic interface and using a hydrophobic interface for face contact with the in vitro cell-free protein synthesis system of water phase.

[0006] The first aspect of the present application provides an in vitro cell-free synthesis method of exogenous protein, which comprises:

[0007] Step one, component ii is added to component i, and mixed;

[0008] wherein, component i is an in vitro cell-free protein synthesis system; the in vitro cell-free protein synthesis system is a water phase system, which is referred to as a water phase synthesis system, a water phase system, a water phase synthesis system, or a synthesis system;

[0009] wherein, component ii is a nucleic acid template encoding an exogenous protein;

[0010] Component i and component ii collectively provide translation-related elements required for synthesizing an exogenous protein;

[0011] Step two, under the assistance of a hydrophobic interface iii, an incubation reaction is carried out to synthesize the exogenous protein;

[0012] wherein, the hydrophobic interface iii is in surface contact with the water phase in vitro cell-free protein synthesis system.

[0013] Optionally, step three is included: separating or / and detecting the exogenous protein.

[0014] The hydrophobic interface is an interface formed by the hydrophobic phase in surface contact with the water phase synthesis system.

[0015] Hydrophobic phase: in the present application, the surface or interface in surface contact with the water phase synthesis system is hydrophobic. The hydrophobicity of the part of the substance or device constituting the hydrophobic phase that does not contact the water phase synthesis system is not particularly limited.

[0016] According to the physical state of the hydrophobic phase, the hydrophobic interface can be selected from a solid hydrophobic interface, a liquid hydrophobic interface, or a combination thereof. When the hydrophobic phase is a liquid phase, the entire liquid phase (including the internal and external surfaces / interfaces) is preferably hydrophobic.

[0017] Preferably, the hydrophobic interface is provided by a reactor inner surface, a hydrophobic outer surface of a hydrophobic built-in solid, a liquid interface of an oil phase system, or a combination thereof. The reactor inner surface, such as the inner surface of the side wall of the reactor, the inner surface of the bottom. The hydrophobic built-in solid refers to a solid with a hydrophobic outer surface, which can be deposited below the water phase synthesis system, suspended in the water phase synthesis system, or floated above the water phase synthesis system; preferably, the hydrophobic built-in solid is deposited below the water phase synthesis system or floated above the water phase synthesis system.

[0018] Preferably, the hydrophobic phase is provided by a hydrocarbon substance, and the main component is a hydrocarbon; the hydrocarbon substance is in the form of paste (semi-solid), solid (such as wax, granules, powder, etc.), liquid, or a mixture thereof.

[0019] Preferably, the hydrocarbon contained in the hydrocarbon substance is linear, branched, cyclic, or a combination thereof.

[0020] Preferably, the hydrocarbon substance has a carbon number of at least 6, more preferably, the hydrocarbon substance has a carbon number of 6-44.

[0021] Preferably, the hydrocarbon substance is selected from aliphatic hydrocarbons.

[0022] Preferably, the hydrocarbon substance is selected from alkanes.

[0023] Preferably, the alkane is linear, branched, cyclic, or a combination thereof.

[0024] Preferably, the hydrocarbon substance is selected from vaseline, mineral oil, or a combination thereof.

[0025] Preferably, the alkane has a carbon number of at least 6, more preferably, the alkane has a carbon number of 6-44. More preferably, the alkane is selected from cyclohexane, isooctane, decane, tetradecane, pentadecylcyclohexane, tetrafortyalkane, vaseline, or a combination thereof.

[0026] Preferably, the hydrophobic phase is provided by an alkane, which is in a paste, solid (e.g., wax, granule, powder, etc.), liquid, or a mixture thereof. More preferably, the alkane is selected from cyclohexane, isooctane, decane, tetradecane, pentadecylcyclohexane, tetrafortyalkane, vaseline, or a combination thereof.

[0027] The amount of the alkane capable of increasing the amount of synthesis of the exogenous protein is selected from Y PRT (C hc ) the interval of the amount of the alkane when the amount of expression of the exogenous protein is greater than Y0 in the curve. The above-mentioned optional amount interval can be continuous or discontinuous.

[0028] In the present application,

[0029] Q hc refers to the amount of the alkane capable of increasing the amount of synthesis of the exogenous protein covered by the present application.

[0030] C hc refers to the amount of the alkane.

[0031] Y PRT refers to the amount of expression of the exogenous protein;

[0032] Y PRT (C hc ) curve refers to the curve with the amount of the alkane as the independent variable and the amount of expression of the exogenous protein as the dependent variable when other reaction parameters are determined, which is also recorded as Y PRT ~ C hccurve. The "other reaction parameters" include, but are not limited to, other system components, the way of adding the reaction raw materials, the reaction temperature program, the reaction time length, the nature of the reaction vessel, the volume of the reaction system, etc. One component i system can correspond to multiple Y PRT ~ C hc curve. One component i system can be reacted under different reaction temperature programs, and can also be reacted for different time lengths, thus can produce several different Y PRT ~ C hc curve.

[0033] Y max , refers to the maximum expression amount of the exogenous protein in the Y PRT (C hc ) curve.

[0034] C max , refers to the alkane dosage when the maximum expression amount of the exogenous protein in the Y PRT (C hc ) curve.

[0035] Y min , refers to the minimum expression amount of the exogenous protein in the Y PRT (C hc ) concentration curve in the interval range of Y PRT > Y0.

[0036] Y0, refers to the expression amount of the exogenous protein when the C hc is 0.

[0037] Y Δ , is the difference between Y max and Y0, in the numerical value, Y Δ = Y max - Y0.

[0038] Preferably, the Q hc is selected from the interval range of the alkane dosage when the expression amount of the exogenous protein is at least Y0+ 50% Y Δ .

[0039] More preferably, the Q hc is selected from the interval range of the alkane dosage when the expression amount of the exogenous protein is at least Y0+ 60% Y Δ .

[0040] More preferably, the Q hc is selected from the interval range of the alkane dosage when the expression amount of the exogenous protein is at least Y0+ 70% Y Δ .

[0041] More preferably, the Q hc is selected from the interval range of the alkane dosage when the expression amount of the exogenous protein is at least Y0+ 80% Y Δ .

[0042] More preferably, the Q hc Selected from the group with an exogenous protein expression level of at least Y0+90%Y Δ The range of alkane dosage when

[0043] More preferably, the Q hc Selected from the group with an exogenous protein expression level of at least Y0+95%Y Δ The range of alkane dosage when

[0044] More preferably, the Q hc C is the alkane dosage when the exogenous protein is expressed at the highest level max .

[0045] The components of the in vitro cell-free protein synthesis system preferably include cell extracts.

[0046] Preferably, the in vitro cell-free protein synthesis system contains a system component capable of recognizing a promoter element on a nucleic acid template, so that the in vitro cell-free protein synthesis system can recognize the promoter element of a nucleic acid template encoding an exogenous protein, for example, the in vitro cell-free protein synthesis system contains an RNA polymerase corresponding to the promoter element.

[0047] The system components capable of recognizing the promoter element on the nucleic acid template (such as the corresponding RNA polymerase) can be provided by the cell extract, can be provided by other exogenous components, or can be provided by a combination of two or more methods.

[0048] Preferably, the gene transcription process of the exogenous protein is initiated by a promoter on the nucleic acid template, and the cell extract includes endogenously expressed RNA polymerase that recognizes the promoter on the nucleic acid template.

[0049] Preferably, the in vitro cell-free protein synthesis system further comprises an RNA polymerase. Sources of the RNA polymerase include, but are not limited to, a cell extract containing endogenously expressed RNA polymerase, an exogenous RNA polymerase, a translation product of an exogenous nucleic acid template encoding the RNA polymerase, or a combination thereof. In each of the above technical solutions, the RNA polymerase is preferably a T7 RNA polymerase, independently. The exogenous nucleic acid template encoding the RNA polymerase can be translated into the RNA polymerase through the in vitro protein synthesis reaction of the system. In one preferred mode, the in vitro cell-free protein synthesis system further comprises a DNA polymerase. Sources of the DNA polymerase include, but are not limited to, a cell extract containing endogenously expressed DNA polymerase, an exogenous DNA polymerase, a translation product of an exogenous nucleic acid template encoding the DNA polymerase, or a combination thereof. In each of the above technical solutions, the DNA polymerase is preferably a phi29 DNA polymerase, independently. The exogenous nucleic acid template encoding the DNA polymerase can be translated into the DNA polymerase through the in vitro protein synthesis reaction of the system. The RNA polymerase and the DNA polymerase can be added directly by an exogenous method, or provided in the form of a reaction product or an intermediate product (e.g., by adding an exogenous nucleic acid template encoding the RNA polymerase and / or the DNA polymerase).

[0050] In one preferred mode, the cell extract contains endogenously expressed RNA polymerase. More preferably, the cell extract is prepared by endogenous strain modification of the source cell, including but not limited to the following endogenous strain modification methods: inserting the coding sequence of the RNA polymerase into a free plasmid in the cell, or integrating the coding gene of the RNA polymerase into the genome of the cell, or using a combination of the above two methods. It should be noted that, in the above endogenous strain modification, in addition to the integration of the above coding sequence / coding gene, other nucleotide sequences such as non-coding sequences, enhancer sequences, kozak sequences, leader sequences, signal peptide sequences, tag sequences, codon sequences, etc. can also be inserted. Through the above endogenous strain modification, the modified strain can endogenously express the RNA polymerase. The RNA polymerase is preferably a T7 RNA polymerase.

[0051] In one preferred mode, the cell extract contains endogenously expressed RNA polymerase. More preferably, the cell extract is prepared by endogenous strain modification of the source cell, including but not limited to the following endogenous strain modification methods: inserting the coding sequence of the RNA polymerase into a free plasmid in the cell, or integrating the coding gene of the RNA polymerase into the genome of the cell, or using a combination of the above two methods. It should be noted that, in the above endogenous strain modification, in addition to the integration of the above coding sequence / coding gene, other nucleotide sequences such as non-coding sequences, enhancer sequences, kozak sequences, leader sequences, signal peptide sequences, tag sequences, codon sequences, etc. can also be inserted. Through the above endogenous strain modification, the modified strain can endogenously express the RNA polymerase. The RNA polymerase is preferably a T7 RNA polymerase.

[0052] In one preferred mode, the in vitro cell-free protein synthesis system comprises an exogenously added T7 RNA polymerase.

[0053] In one preferred mode, the in vitro cell-free protein synthesis system comprises at least one of the following components: an exogenous RNA polymerase, an exogenous DNA polymerase.

[0054] The cell extract is selected from the group consisting of: a prokaryotic cell extract, a eukaryotic cell extract, or a combination thereof.

[0055] In one preferred embodiment, the cell extract is selected from the group consisting of: a prokaryotic cell, a yeast cell, a mammalian cell, a plant cell, an insect cell, or a combination thereof. Preferably, the prokaryotic cell is E. coli.

[0056] The yeast cell is preferably selected from the group consisting of: Kluyveromyces lactis, Saccharomyces cerevisiae, Pichia pastoris, or a combination thereof.

[0057] The Kluyveromyces lactis is further preferably selected from the group consisting of: K. lactis, K. marxianus, K. dohrii, K. apiculatus, K. wickerhamii, K. fragilis, K. hubeiensis, K. polysporus, K. siamensis, K. yarrowii, or a combination thereof.

[0058] In one preferred embodiment, the cell extract is selected from the group consisting of: E. coli, K. lactis, wheat germ cells, Spodoptera frugiperda cells (Sf9 cells, an insect cell), Leishmania tarentolae cells (L. tarentolae cells), rabbit reticulocytes, 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, or a combination thereof.

[0059] Preferably, the nucleic acid template encoding the foreign protein contains a promoter element recognizable by component i; the system component refers to a component of the in vitro cell-free protein synthesis system in an aqueous phase.

[0060] Preferably, the nucleic acid template encoding the foreign protein contains a T7 promoter, and the in vitro cell-free protein synthesis system comprises a T7 RNA polymerase.

[0061] In one preferred embodiment, the transcription of the gene of the foreign protein is initiated by a T7 promoter on the nucleic acid template, and the in vitro cell-free protein synthesis system comprises a T7 RNA polymerase.

[0062] In one preferred embodiment, the nucleic acid template encoding the foreign protein contains a T7 promoter, and the in vitro cell-free protein synthesis system comprises a cell extract, and the endogenous expressed T7 RNA polymerase is provided by the cell extract.

[0063] In one preferred embodiment, the in vitro cell-free protein synthesis system further comprises an energy system; the energy system is preferably selected from the group consisting of sugar (e.g., monosaccharide, disaccharide, oligosaccharide, polysaccharide) and phosphate energy system, sugar and phosphagen energy system, phosphagen and phosphagen kinase system, phosphagen and phosphagen kinase system, glycolytic pathway and its intermediate energy system (e.g., monosaccharide and its glycolytic intermediate, glycogen and its glycolytic intermediate), or a combination thereof.

[0064] In one preferred embodiment, the in vitro cell-free protein synthesis system further comprises a substrate for protein synthesis; the substrate for protein synthesis is preferably an amino acid mixture, at least including an amino acid mixture required for synthesis of an exogenous protein. Preferably, the amino acid mixture is a mixture of natural amino acids.

[0065] In one preferred embodiment, the in vitro cell-free protein synthesis system further comprises a substrate for RNA synthesis; the substrate for RNA synthesis is preferably a nucleotide mixture, selected from the group consisting of nucleoside monophosphate, nucleoside triphosphate, or a combination thereof.

[0066] In one preferred embodiment, the in vitro cell-free protein synthesis system further comprises a substrate for DNA synthesis; the substrate for DNA synthesis is preferably a deoxynucleotide mixture, more preferably a deoxynucleotide triphosphate mixture.

[0067] In one preferred embodiment, the in vitro cell-free protein synthesis system further comprises at least one of the following components: a crowding agent, magnesium ion, potassium ion, an antioxidant or reducing agent, trehalose, a reaction promoter, a buffer, an aqueous solvent;

[0068] The crowding agent is preferably selected from the group consisting of polyethylene glycol, polyvinyl alcohol, polypropylene glycol, dextran, sucrose polymer (including Ficoll sucrose polymer, such as an agent, a non-ionic synthetic sucrose polymer), polyvinyl (vinyl pyrrolidone), albumin, etc., or a combination thereof;

[0069] The magnesium ion source is preferably selected from the group consisting of magnesium aspartate, magnesium acetate, magnesium glutamate, magnesium chloride, magnesium phosphate, magnesium sulfate, magnesium citrate, magnesium hydrogen phosphate, magnesium iodide, magnesium lactate, magnesium nitrate, magnesium oxalate, or a combination thereof;

[0070] The potassium ion source is preferably selected from the group consisting of potassium acetate, potassium glutamate, potassium chloride, potassium phosphate, potassium sulfate, potassium citrate, potassium hydrogen phosphate, potassium iodide, potassium lactate, potassium nitrate, potassium oxalate, or a combination thereof;

[0071] The antioxidant or reducing agent is preferably selected from the group consisting of dithiothreitol, 2-mercaptoethanesulfonic acid, 2-mercaptoethanol, reduced glutathione, tricarboxymethylphosphine, 3-mercapto-1,2-propanediol, or a combination thereof;

[0072] The reaction promoter is preferably alumina;

[0073] The buffer is preferably selected from the group consisting of Tris-HCl, Tris base, HEPES, or a combination thereof.

[0074] The aqueous solvent is preferably a buffer.

[0075] The in vitro cell-free protein synthesis system is capable of reacting with a DNA template or an mRNA template encoding an exogenous protein to synthesize the exogenous protein.

[0076] The above-mentioned preferred modes can be combined in any suitable manner.

[0077] The second aspect of the present application provides an in vitro protein synthesis kit, which comprises:

[0078] (i) the in vitro cell-free protein synthesis system in the method for in vitro cell-free synthesis of an exogenous protein according to the first aspect; the components of the in vitro cell-free protein synthesis system are placed in one or more containers in the form of dry powder, liquid, emulsion, suspension, or a combination thereof;

[0079] (ii) optionally, a nucleic acid template encoding an exogenous protein;

[0080] (iii) optionally, a reaction container;

[0081] (iv) a hydrophobic phase; which can include a substance or device for constructing a hydrophobic interface;

[0082] The hydrophobic phase is capable of face contact with the aqueous phase synthesis system to construct a hydrophobic interface;

[0083] The hydrophobic interface is selected from any of the hydrophobic interfaces in the method for in vitro cell-free synthesis of an exogenous protein according to the first aspect;

[0084] (v) a label or an instruction.

[0085] The (i) and (ii) collectively provide the translation-related elements required for the synthesis of an exogenous protein.

[0086] Using the in vitro protein synthesis kit, an in vitro protein synthesis reaction can be carried out to synthesize an exogenous protein.

[0087] The parts of the kit correspond to the components of the method for in vitro cell-free synthesis of an exogenous protein according to the first aspect.

[0088] Preferably, the nucleic acid template encoding an exogenous protein contains a promoter element that can be recognized by (i).

[0089] In one preferred embodiment, the nucleic acid template encoding the foreign protein contains a promoter element recognizable by the cell extract. For example, the cell extract contains an endogenously expressed RNA polymerase corresponding to the promoter element on the nucleic acid template.

[0090] In one preferred embodiment, the nucleic acid template encoding the foreign protein contains a T7 promoter, and the in vitro cell-free protein synthesis system contains a T7 RNA polymerase.

[0091] In one preferred embodiment, the nucleic acid template encoding the foreign protein contains a T7 promoter, and the cell extract contains an endogenously expressed T7 RNA polymerase.

[0092] Preferably, the transcription of the gene of the foreign protein is initiated by a T7 promoter on the nucleic acid template.

[0093] In one preferred embodiment, the transcription of the gene of the foreign protein is initiated by a T7 promoter on the nucleic acid template, and the in vitro cell-free protein synthesis system contains a T7 RNA polymerase.

[0094] In one preferred embodiment, the T7 promoter is located upstream of the coding sequence of the foreign protein in the nucleic acid template, and the transcription of the foreign protein is initiated by the T7 promoter, and the in vitro cell-free protein synthesis system contains a cell extract, and the cell extract provides an endogenously expressed T7 RNA polymerase.

[0095] The nucleic acid template encoding the foreign protein is a DNA template, an mRNA template, or a combination thereof; preferably, the nucleic acid template encoding the foreign protein is a DNA template.

[0096] The third aspect of the present application provides the use of an alkane in the in vitro cell-free synthesis method of the foreign protein according to the first aspect, or in the in vitro protein synthesis kit according to the second aspect, or in the in vitro protein synthesis; wherein the alkane is used to construct a hydrophobic interface.

[0097] Preferably, the use in the in vitro protein synthesis includes, but is not limited to, use in protein manufacturing, or use in detection based on protein synthesis, etc.

[0098] Advantages:

[0099] The in vitro cell-free synthesis method of the foreign protein provided by the present application provides an in vitro cell-free protein synthesis system in an aqueous phase and a nucleic acid template encoding the foreign protein, and introduces a hydrophobic interface in contact with the in vitro cell-free protein synthesis system in the aqueous phase, and performs an incubation reaction to synthesize the foreign protein, thereby improving the protein synthesis capacity (including at least the protein synthesis efficiency and the protein synthesis amount) of the cell-free system, and the improvement degree can reach 90%.

[0100] The hydrophobic interface, by using hydrophilic-hydrophobic interaction, on one hand, promotes the enrichment of hydrophobic components at the interface, increases the local concentration, improves the reaction efficiency and raw material utilization, and improves the reaction efficiency and the amount of protein synthesis; on the other hand, for the non-water-soluble protein product, the hydrophobic interface also plays an interface fixation role, so that the hydrophobic side of the protein product is attached to the interface by hydrophilic-hydrophobic interaction, and the hydrophilic end is dissolved in the in vitro cell-free protein synthesis system, which plays an interface solubilization role.

[0101] The present application constructs the hydrophobic interface by a simple way of adding alkanes, which is simpler in operation, cheaper in raw materials and lower in cost, compared with the existing complex modification of the reactor (such as constructing a selective semi-permeable membrane with complex structure, high cost and cumbersome replacement), compared with the need for prior polymerization reaction to construct a gel cross-linking system, and compared with the covalent fixation method of the solid-phase reaction system.

[0102] The further provided in vitro protein synthesis kit has the advantages of higher efficiency and higher throughput. BRIEF DESCRIPTION OF DRAWINGS

[0103] Figure 1 , the structure diagram of the plasmid DNA encoding the exogenous protein mEGFP, which is 6384 bp, and is denoted as plasmid D2P-mEGFP (abbreviated as pD2P-mEGFP). The mEGFP is a mutant of enhanced green fluorescent protein. The plasmid DNA includes the following elements: a T7 promoter (which can be recognized by T7 RNA polymerase), a 5' non-coding region (5'-UTR), a kozak sequence (which is located downstream of the 5' non-coding region and is an optional element), a leader sequence (an optional element), a purification tag (an optional element), a coding sequence of the exogenous protein mEGFP, a 3' non-coding region (3'-UTR), a T7 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 for controlling the copy number of the plasmid (rop gene), a coding gene of LacI repressor (lacI), and a LacI promoter.

[0104] Figure 2 , an example structure of an exogenous nucleic acid template. The exogenous nucleic acid template in the figure is plasmid DNA encoding an exogenous protein.

[0105] Figure 3 , an example structure of an exogenous nucleic acid template. The exogenous nucleic acid template in the figure is plasmid DNA encoding an exogenous protein.

[0106] Figure 4, Effect of cyclohexane on in vitro protein synthesis reaction. Cyclohexane is liquid, and the amount used is 0-20% (v / v). Among them, 3h, 18h respectively represent the reaction time of 3h, 18h. Among them, the concentration "0" corresponds to the blank control (BC group). Alkanes form an oily hydrophobic interface above the in vitro protein synthesis system of the aqueous phase. The reaction was carried out in a 48-well cell culture plate (Example S3).

[0107] Figure 5 , Effect of isooctane on in vitro protein synthesis reaction. Isooctane is liquid, and the amount used is 0-5% (v / v). Among them, 3h, 18h respectively represent the reaction time of 3h, 18h. Among them, the concentration "0" corresponds to the blank control (BC group). Alkanes form an oily hydrophobic interface above the in vitro protein synthesis system of the aqueous phase. The reaction was carried out in a 48-well cell culture plate.

[0108] Figure 6 , Effect of decane on in vitro protein synthesis reaction. Among them, decane is a straight chain structure (n-decane), and the physical state is liquid, and the amount used is 0-5% (v / v). Among them, 3h, 18h respectively represent the reaction time of 3h, 18h. Among them, the concentration "0" corresponds to the blank control (BC group). Alkanes form an oily hydrophobic interface above the in vitro protein synthesis system of the aqueous phase. The reaction was carried out in a 48-well cell culture plate.

[0109] Figure 7 , Ten Effect of tetradecane on in vitro protein synthesis reaction. Among them, tetradecane is a straight chain structure (n-tetradecane), and the physical state is liquid, and the amount used is 0-2% (v / v). Among them, 3h, 18h respectively represent the reaction time of 3h, 18h. Among them, the concentration "0" corresponds to the blank control (BC group). Alkanes form an oily hydrophobic interface above the in vitro protein synthesis system of the aqueous phase. The reaction was carried out in a 48-well cell culture plate.

[0110] Figure 8 , Ten Effect of tetradecane on in vitro protein synthesis reaction. Among them, tetradecane is a straight chain structure (n-tetradecane), and the physical state is liquid, and the amount used is 2-20% (v / v). Among them, 3h, 18h respectively represent the reaction time of 3h, 18h. Among them, the concentration "0" corresponds to the blank control (BC group). Alkanes form an oily hydrophobic interface above the in vitro protein synthesis system of the aqueous phase. The reaction was carried out in a 48-well cell culture plate.

[0111] Figure 9 , TenEffect of pentylcyclohexane on in vitro protein synthesis reaction. Pentylcyclohexane is liquid, and its amount is 0-20% (v / v). 3h and 18h represent reaction time of 3h and 18h, respectively. The concentration of "0" corresponds to blank control (BC group), and NC is negative control. Alkanes form an oily hydrophobic interface above the in vitro protein synthesis system in aqueous phase. The reaction is carried out in a 48-well cell culture plate.

[0112] Figure 10 、 Four Effect of tetradecane on in vitro protein synthesis reaction. Tetradecane is solid, and its amount is 0-500 μg / μL. 3h and 18h represent reaction time of 3h and 18h, respectively. The concentration of "0" corresponds to blank control (BC group), and NC is negative control. Alkanes exist in the oily layer above the synthesis system in aqueous phase during in vitro protein synthesis. The reaction is carried out in a 48-well cell culture plate.

[0113] Figure 11 Effect of vaseline on in vitro protein synthesis reaction. Vaseline is a paste-like mixture of alkanes, and its amount is 0-0.09 g / well, which is smeared on the side wall and inner wall of the bottom of the well to construct a hydrophobic interface. The volume of in vitro protein synthesis system is 300 μL, and the reaction is carried out in a 48-well cell culture plate, as described in Example S10. BC group is blank control group.

[0114] Figure 12 Effect of vaseline on in vitro protein synthesis reaction. The volume of in vitro protein synthesis system is 300 μL, and the reaction is carried out in a 48-well plate, as described in Example S11. ZS01092 is the number of a modified strain for manufacturing cell extract, which is a modified Kluyveromyces lactis strain with endogenous integration of the coding gene of T7 RNA polymerase and can endogenously express T7 RNA polymerase. Vas indicates that vaseline is smeared on the side wall and inner wall of the bottom of the well to construct a hydrophobic interface; AC indicates that activated carbon is added; AC+Vas indicates that both vaseline is smeared and activated carbon is added; BC group is blank control group, neither vaseline is smeared nor activated carbon is added, but DNA template encoding exogenous protein is added; NC is negative control group, neither vaseline is smeared nor activated carbon is added, and DNA template encoding exogenous protein is not added. 3h, 6h and 21h represent reaction time of 3h, 6h and overnight (21h), respectively.

[0115] Figure 13, the influence of vaseline on the in vitro protein synthesis reaction. The volume of the in vitro protein synthesis system is 300 μL, and the reaction is carried out in a 48-well plate, and example S12. Among them, Vas represents that vaseline is used to smear the side wall of the hole and the inner wall of the bottom of the hole to construct a hydrophobic interface; AC represents the addition of activated carbon; ssDNA represents the addition of salmon sperm DNA in the in vitro protein synthesis system; Amp represents the addition of ampicillin in the in vitro protein synthesis system. Among them, AC+ssDNA+Amp represents the addition of activated carbon, salmon sperm DNA and ampicillin, which is the blank control group without using vaseline; Vas+AC+ssDNA+Amp represents that on the basis of the AC+ssDNA+Amp blank control group, vaseline is also smeared to construct a hydrophobic interface; NC is the negative control group, which does not smear vaseline and does not add DNA template encoding foreign protein. Among them, 3h, 6h, 21h represent the reaction time of 3h, 6h, and reaction overnight (21h) respectively.

[0116] Nucleotide and / or amino acid sequence table

[0117] SEQ ID No.: 1 is the gene sequence of the foreign protein mEGFP, which has a length of 717 bases.

[0118] SEQ ID No.: 2 is the amino acid sequence of the foreign protein mEGFP, which has a total of 239 amino acids. DETAILED DESCRIPTION

[0119] Meaning of terms, nouns and phrases in the present application. The meaning explanation in this part is applicable to the whole text of the present application, both to the following and to the preceding. In the present application, the definition of related terms, nouns and phrases in the cited literature is also cited, but if it conflicts with the definition in the present application, the definition in the present application shall prevail. When the definition in the cited literature conflicts with the definition in the present application, the cited components, substances, compositions, materials, systems, formulations, species, methods, equipment, etc. shall be subject to the content determined in the cited literature.

[0120] In the present application, the aqueous phase synthesis system refers to the in vitro cell-free protein synthesis system in aqueous phase.

[0121] In the present application, "synthesis system", "aqueous phase system", "aqueous phase synthesis system" and "aqueous phase synthesis system" have the same meaning and can be used interchangeably.

[0122] Interface: the interface between a substance phase and a substance phase.

[0123] Hydrophobic interface: an interface formed by the surface contact between a hydrophobic phase and the aqueous phase synthesis system.

[0124] Hydrophobic phase: The hydrophobic phase in the present application is hydrophobic on the surface or interface that is in contact with the aqueous phase synthetic system. The hydrophobicity of the hydrophobic phase refers to the hydrophobicity relative to the aqueous phase synthetic system. The hydrophobicity of the hydrophobic phase refers to the hydrophobicity of the outer surface or outer interface. The hydrophobicity of the part of the substance or device that constitutes the hydrophobic phase and is not in contact with the aqueous phase synthetic system is not particularly limited. The hydrophobic phase can be a substance phase or device with a hydrophobic outer surface or outer interface. According to the physical state of the hydrophobic phase, the hydrophobic interface can be selected from: a solid hydrophobic interface, a liquid hydrophobic interface, or a combination thereof. When the hydrophobic phase is a liquid phase, the entire liquid phase (including the inner and outer surfaces / interfaces) is preferably hydrophobic.

[0125] In the present application, "protein synthesis amount", "protein expression amount" and "protein expression yield" have the same meaning and can be used interchangeably.

[0126] D2P, DNA-to-Protein, from DNA template to protein product. For example, D2P technology, D2P system, D2P method, D2P kit, etc.

[0127] IVTT, in vitro transcription translation.

[0128] RFU, Relative Fluorescence Unit.

[0129] eGFP: enhanced green fluorescence protein.

[0130] mEGFP: A206K mutant of eGFP.

[0131] wt% or % (wt): mass concentration unit, both represent mass percentage.

[0132] (v / v)% or % (v / v): both represent volume percentage.

[0133] %(w / v): mass volume concentration unit, corresponding to g / 100 mL.

[0134] g / well, represents grams per well.

[0135] Sucrose polymer: refers to a polymer containing at least 2 sucrose units. It includes but is not limited to polysucrose.

[0136] Ficoll sucrose polymer: refers to Reagent, a non-ionic synthetic sucrose polymer, is a highly branched polymer copolymerized from sucrose and epichlorohydrin, which can be selected from commercially available products. For example, Ficoll-400 (poly sucrose 400, CAS: 26873-85-8), Ficoll-70 (poly sucrose 70, CAS: 72146-89-5). Among them, PM 400 (Sigma Aldrich) is a highly branched polymer copolymerized from sucrose and epichlorohydrin, with an average molecular weight of 400 kg / mol; the average molecular weight of Ficoll PM 70 (Sigma Aldrich) is 70 kg / mol.

[0137] Hydrocarbon substance, the main component is hydrocarbon, and the impurity is generated during the preparation process, not added and introduced. For example, vaseline, mineral oil.

[0138] Hydrocarbon, a carbon-hydrogen compound, only includes carbon atoms and hydrogen atoms. According to the degree of unsaturation, hydrocarbons are divided into saturated hydrocarbons and unsaturated hydrocarbons. According to the aromaticity, hydrocarbons are divided into aromatic hydrocarbons and aliphatic hydrocarbons.

[0139] Aliphatic hydrocarbon, a hydrocarbon without aromatic ring.

[0140] Alkane, that is, saturated hydrocarbon, can be open chain or contain cyclic structure. The open chain includes but is not limited to straight chain and branched chain; the branched chain includes but is not limited to ordinary branched structure with low degree of branching, and also includes comb-shaped and dendritic structure with special structure, and also includes hyperbranched structure with high degree of branching. The cyclic structure includes but is not limited to cyclic structure and combination structure of cyclic structure and open chain structure. The alkane suitable for the present application has at least 6 carbon atoms, so as to form liquid, paste, solid (such as wax, particles, powder, etc.), or a mixture thereof, and then construct a hydrophobic interface. When containing cyclic structure, the number of cyclic structure can be 1, 2 or more.

[0141] Unsaturated hydrocarbon, including but not limited to olefin, diene, alkyne, etc., each independently can be open chain structure or contain cyclic structure (for example, the olefin includes but is not limited to open chain olefin and cyclic olefin). The olefin only includes one carbon-carbon double bond. The diene includes and only includes two carbon-carbon double bonds, which can be conjugated diene or non-conjugated structure.

[0142] Cyclic structure, all carbon atoms participate in forming a cyclic skeleton. Including but not limited to monocyclic, bicyclic, polycyclic, bridged ring, spiro ring, nested cyclic structure, etc. The bridged ring shares two carbon atoms between adjacent rings, and the shared carbon atom is called a bridgehead carbon atom; the two bridgehead carbon atoms can be directly bonded by a chemical bond or can be separated by other carbon atoms without being directly connected. The spiro ring shares one carbon atom between adjacent rings.

[0143] Open-chain hydrocarbon, hydrocarbon without cyclic structure, including straight-chain hydrocarbon, branched-chain hydrocarbon.

[0144] Straight-chain hydrocarbon, open-chain hydrocarbon with all carbon atoms connected in sequence to form a chain structure, for example, n-hexane, n-decane, n-tetradecane.

[0145] Branched-chain hydrocarbon, open-chain hydrocarbon with at least one carbon atom in a side chain, wherein at least one carbon atom is simultaneously connected to at least three carbon atoms. For example, isooctane.

[0146] Cyclic hydrocarbon, hydrocarbon containing cyclic structure, for example, cyclohexane (containing only cyclic structure), pentadecylcyclohexane (combination of cyclic structure and open-chain structure), etc. The cyclic hydrocarbon can be saturated hydrocarbon or unsaturated hydrocarbon.

[0147] The “expression system of the present invention”, “in vitro expression system of the present invention”, “in vitro cell-free expression system”, “in vitro cell-free expression system” are used interchangeably, referring to the in vitro protein expression system of the present invention, and other description methods can also be used, such as: protein in vitro synthesis system, in vitro protein synthesis system, cell-free system, cell-free system, cell-free protein synthesis system, cell-free in vitro protein synthesis system, cell-free in vitro protein synthesis system, CFS system (cell-free system), CFPS system (cell-free protein synthesis system), etc. Description methods include in vitro translation system, in vitro transcription translation system (IVTT system), etc. In the present invention, IVTT system is preferred. We also call the in vitro protein synthesis system as “protein synthesis factory” (“Protein Factory” or “protein factory”). The in vitro protein synthesis system provided by the present invention is described in an open manner.

[0148] “Cell-free system” refers to the process of in vitro protein synthesis, which is not performed by complete cell secretion expression. It should be noted that in the in vitro cell-free protein synthesis system of the present invention, cell components can also be added to promote the reaction, but the added cells are not the main purpose of secreting exogeneous target protein (exogeneous target protein). In addition, in the CFPS system without complete cells constructed under the guidance of the present invention, a small amount of complete cells are intentionally added (for example, the protein content provided by the cells does not exceed 30wt% compared with the protein content provided by the cell extract), and such “avoidance” method is also included in the protection scope of the present invention.

[0149] In vitro protein synthesis reaction refers to a reaction for synthesizing a protein in an in vitro cell-free synthesis system, and at least includes a translation process. It includes but is not limited to an IVTT reaction (in vitro transcription translation reaction). In the present application, the IVTT reaction is preferred. The IVTT reaction, corresponding to the IVTT system, is a process for transcribing and translating DNA into a protein in vitro, and therefore, the in vitro protein synthesis system of this type is also referred to as a D2P system, a D-to-P system, a D_to_P system, a DNA-to-Protein system; and the corresponding in vitro protein synthesis method is also referred to as a D2P method, a D-to-P method, a D_to_P method, a DNA-to-Protein method.

[0150] Post-translational modification: also referred to as post-translational processing, post-translational modification, PTM. The PTM system plays a significant role in the normal folding, activity and stability of a protein.

[0151] In the present application, “translation-related elements” refer to related functional elements required in the process of synthesizing a protein product from a nucleic acid template, and are not limited to functional elements required in the translation process; when the nucleic acid template is DNA, the functional elements required in the transcription process are also broadly included. The translation-related elements can be provided by means of cell extracts (various endogenous factors), other exogenous components of an in vitro protein synthesis system (such as exogenous RNA polymerase, auxiliary factors, etc.), functional elements on a nucleic acid template (such as functional elements for controlling the transcription / translation of an exogenous protein, a resistance gene translation system, a Lac repressor translation system, a translation system for controlling the copy number of a plasmid, etc.), etc. The functional elements for controlling the transcription / translation of an exogenous protein include, for example, a promoter, a terminator, an enhancer, an IRES element, a kozak sequence, other elements for regulating the translation level, a signal sequence, a leader sequence, a functional tag (such as a screening marker tag, a tag for enhancing the translation level), etc.

[0152] Gene: includes a coding region and a non-coding region.

[0153] Nucleotide sequence: a sequence composed of nucleotide units.

[0154] Nucleic acid sequence: a sequence of nucleic acid substances, including a DNA sequence and an RNA sequence.

[0155] Coding sequence: coding sequence, abbreviated as CDS. A nucleotide sequence completely corresponding to the codons of a protein, and the sequence does not contain other sequences not corresponding to the protein (without considering the sequence changes in the process of mRNA processing, etc.).

[0156] Coding gene: a functional gene segment encoding a protein, which can be continuous or discontinuous. The coding gene must include a coding sequence.

[0157] Nucleic acid template: also known as genetic template, refers to a nucleic acid sequence serving as a template for protein synthesis, including DNA template and mRNA template. In any embodiment of the present application, the nucleic acid template can be independently a DNA template, an mRNA template or a combination thereof. In any embodiment of the present application, the nucleic acid template can be independently preferably a DNA template. In the present application, unless otherwise specified, the nucleic acid template encoding a foreign protein is preferably a DNA template.

[0158] "Nucleic acid template encoding X protein" refers to a nucleic acid template containing a coding sequence of the X protein, which can be used to synthesize the X protein through a translation process or a transcription-translation process, and allows the nucleic acid template to contain a non-coding region, and also allows to contain a coding sequence of other polypeptides or proteins other than the X protein. For example, "nucleic acid template encoding RNA polymerase" at least includes a coding sequence of RNA polymerase, and in addition, other nucleic acid sequences such as non-coding regions, fusion tags, etc. are allowed; the corresponding expression product at least contains RNA polymerase structure, which can be an RNA polymerase molecule or a fusion protein thereof, and can also be a mixed component including the RNA polymerase molecule or / and the fusion protein thereof.

[0159] Enhancement element: unless otherwise specified, in the present application, refers to a sequence in a nucleic acid sequence between a promoter and a coding sequence of a target protein, which plays a role in promoting transcription or / and translation process, such as Ω sequence, kozak sequence, IRES sequence, etc. including transcription enhancement element, translation enhancement element.

[0160] Endogenous / endogenously: dependent on active cellular metabolic activity. The protein expressed endogenously is secreted by the cell culture, and can exist in the cell extract of the present application after treatment.

[0161] Exogenous / exogenously: not dependent on active cellular metabolic activity. The exogenous component is directly added to the in vitro protein synthesis system, rather than by adding cells or cell extracts. For example, exogenous RNA polymerase can be added to the reaction system by exogenous means of adding precursors (such as inactive precursors, which can be activated by enzyme digestion or other means to generate RNA polymerase), nucleic acid templates (which can be translated to synthesize proteins by the system), fusion proteins, pure substances or mixtures. For another example, exogenous DNA polymerase can also be added to the reaction system by the above-mentioned exogenous means.

[0162] Exogenous protein: The target expression product of the in vitro protein synthesis system of the present invention, which is not synthesized and secreted by the host cell. It can be a protein, a fusion protein, or a mixture containing a protein or fusion protein; it also broadly includes polypeptides. The product obtained by the in vitro protein synthesis reaction based on the nucleic acid template encoding the exogenous protein can be a single substance or a mixture.

[0163] Exogenous RNA polymerase: has the same meaning as exogenous RNA polymerase.

[0164] Exogenous DNA polymerase: has the same meaning as exogenous DNA polymerase.

[0165] A "nucleic acid template encoding an RNA polymerase (or a nucleic acid template encoding a DNA polymerase)" includes at least the coding sequence of the RNA polymerase (or DNA polymerase) and may also include other nucleic acid sequences such as non-coding regions and fusion tags; accordingly, the expression product contains at least the RNA polymerase structure (or DNA polymerase structure). Taking RNA polymerase as an example, it can be an RNA polymerase molecule or a fusion protein thereof, or a mixed component including an RNA polymerase molecule and / or a fusion protein thereof.

[0166] A peptide is a compound composed of two or more amino acids linked by peptide bonds. In the present invention, peptide and peptide segment have the same meaning and can be used interchangeably.

[0167] Polypeptide, a peptide composed of 10 to 50 amino acids.

[0168] Proteins are peptides consisting of more than 50 amino acids. Fusion proteins are also a type of protein.

[0169] Derivatives of polypeptides and proteins: Any polypeptide or protein involved in the present invention, unless otherwise specified (for example, a specific sequence is specified), should be understood to include derivatives thereof. The derivatives of the polypeptide and protein include at least C-terminal tags, N-terminal tags, and C-terminal and N-terminal tags. Among them, the C-terminus refers to the COOH-terminus, and the N-terminus refers to the NH2-terminus. Those skilled in the art understand their meanings. The tag can be a polypeptide tag or a protein tag. Some examples of tags include, but are not limited to, 6-histidine (6×-His, HHHHHH), Glu-Glu, c-myc epitope (EQKLISEEDL), Octapeptide (DYKDDDDK), Protein C (EDQVDPRLIDGK), Tag-100 (EETARFQPGYRS), V5 epitope tag (V5 epitope, GKPIPNPLLGLDST), VSV-G (YTDIEMNRLGK), Xpress (DLYDDDDK), hemagglutinin (YPYDVPDYA), β-galactosidase, thioredoxin, His-patch thioredoxin, IgG-binding domain, intein-chitin binding domain, T7 gene 10, glutathione-S-transferase (GST), green fluorescent protein (GFP), maltose binding protein (MBP), etc.

[0170] Homology, if not 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; and also 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%, 100% homology. For example, homologous sequences of the omega sequences mentioned herein.

[0171] "Variant", means a substance having different structure (including but not limited to making minor changes), but still maintaining or substantially maintaining the original function or performance. The variant includes but is not limited to nucleic acid variant, polypeptide variant, protein variant. The way to obtain the relevant variant includes but is not limited to recombination, deletion or deletion, insertion, translocation, substitution of structural units, etc. The variant includes but is not limited to modified products, genetically modified products, fusion products, etc. In order to obtain genetically modified products, the way to carry out genetic modification includes but is not limited to genetic recombination (corresponding to genetic recombination products), gene deletion or deletion, insertion, frame shift, base substitution, etc. Genetic mutation products, also known as genetic mutants, are a type of genetically modified products.

[0172] Modified product: including but not limited to chemical modification product, amino acid modification, polypeptide modification, protein modification, etc. The chemical modification product refers to the modified product by chemical synthesis method of organic chemistry, inorganic chemistry, polymer chemistry, etc. The chemical modification method includes ionization, salification, desalination, complexation, decomplexation, chelation, dechelation, addition reaction, substitution reaction, elimination reaction, etc. The modification method includes oxidation, reduction, methylation, demethylation, amination, carboxylation, sulfurization, etc.

[0173] Mutant, mutant, in the present application, if no special instructions, refers to the mutant product which still maintains or basically maintains the original function or performance, and the number of mutation sites is not particularly limited. The mutant includes but is not limited to gene mutant, polypeptide mutant, protein mutant. Mutant is a type of variant. The way to obtain the related mutant includes but is not limited to recombination, deletion or deletion, insertion, translocation, substitution, etc. The structural unit of gene is base, and the structural unit of polypeptide and protein is amino acid. The type of gene mutation includes but is not limited to gene deletion or deletion, insertion, frame shift, base substitution, etc.

[0174] In the present application, the meanings of cell extract, cell extract, cell lysate, cell broken product, cell lysis product are the same, and can be used interchangeably. English can use cell extract, cell lysate, etc. Description method.

[0175] In the present application, energy system, energy system, energy supply system have the same meaning, and can be used interchangeably. Energy regeneration system, energy regeneration system have the same meaning, and can be used interchangeably. Energy regeneration system is the preferred embodiment or component of energy system.

[0176] Amino acid mixture refers to a mixture containing at least two or more amino acids.

[0177] In the present application, if no special instructions, the amino acid can be natural amino acid, also can be non-natural amino acid, can be L amino acid, D- amino acid or a combination thereof, and can also be a radioisotope-labeled amino acid, a modified amino acid, etc. The modified amino acid refers to an amino acid connected with a chemical modification group, and the structure thereof is not particularly limited, including but not limited to modification by amino acid side groups. The definition range of the above-mentioned amino acid covers any substance including an amino acid unit in the present application, 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 modifier, a polypeptide modifier, a protein modifier, 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 a variant of a domain, a subunit, a fragment of any of the foregoing). The "variant of any of the foregoing" includes but is not limited to "mutant of any of the foregoing". In the present application, "R" represents a chiral type, and "S" represents a chiral type. L -”, “ D -”, the subscript form has the same meaning as the non-subscript form.

[0178] Crowding agent, a reagent for simulating the macromolecular environment of crowded cells in vitro. Reference "X Ge, D Luo and J Xu. Cell-free protein expression under macromolecular crowding conditions [J]. PLoS One, 2011, 6(12): e28707" and its cited references, etc.

[0179] Phosphorus compound, including organic and inorganic.

[0180] Phosphate, if not specified, refers to inorganic phosphate.

[0181] In the present application, "room temperature" is preferably room temperature to 37℃, specifically, preferably 20℃-37℃, more preferably 25℃-37℃.

[0182] In the present application, "preferably", "more preferably", "even more preferably", "most preferably" and the like preferred embodiments are not used to limit the embodiments of the present application, but only to provide examples of embodiments with better technical effects.

[0183] In the description of the present invention, for preferred modes such as "one of the preferred modes", "one of the preferred embodiments", "one of the preferred examples", "preferred example", "in a preferred embodiment", "preferably", "preferably", "preferably", "more preferably", "more preferably", "further preferably", "most preferably", and schematic enumeration modes such as "one of the embodiments", "one of the modes", "example", "specific example", "for example", "as an example", "for example", "such as", "such as", the specific features described in each mode are included in at least one specific embodiment of the present invention. In the present invention, the specific features described in each mode can be combined in a suitable manner in any one or more specific embodiments. In the present invention, the technical solutions corresponding to the preferred modes can also be combined in any suitable manner; for example, exogenous RNA polymerase and exogenous DNA polymerase can be added at the same time, refer to patent document CN108642076A.

[0184] In the present invention, “optionally” means it may or may not be present.

[0185] In the present invention, “any combination thereof” means “equal to 1 or greater than 1” in terms of quantity, and means a group consisting of the following situations in terms of scope: “any one of them, or a combination of at least two of them”.

[0186] In the present invention, the descriptions of "one or more", "one or more" and "one or more" have the same meaning as "at least one", "at least one", "any combination", etc., and can be used interchangeably to indicate "equal to 1 or greater than 1" in terms of quantity.

[0187] In the present invention, "or / and" and "and / or" are used to mean "optionally one or a combination thereof", and also mean at least one. For example, "comprising a substrate for synthesizing RNA and / or a substrate for synthesizing protein" means that only a substrate for synthesizing RNA, only a substrate for synthesizing protein, or both a substrate for synthesizing RNA and a substrate for synthesizing protein are included.

[0188] The prior art means described in the present invention in terms of “usually”, “conventional”, “generally”, “frequently”, “often”, etc. are also cited as a reference for the content of the present invention and, unless otherwise specified, can be regarded as one of the preferred aspects of the present invention.

[0189] All documents mentioned in the present invention and documents directly or indirectly cited by these documents are incorporated into this application by reference, just as if each document was individually incorporated by reference.

[0190] It should be understood that, within the scope of the present application, the above technical features of the present application and the technical features specifically described hereinafter (including but not limited to the embodiments) can be combined with each other to form new or preferred technical solutions, as long as the exogenous protein can be synthesized in vitro or preferably synthesized in vitro with high efficiency. Due to the limited space, they will not be listed one by one here.

[0191] The first aspect of the present application provides a method for in vitro cell-free synthesis of an exogenous protein, which comprises:

[0192] Step one, adding component ii to component i and mixing;

[0193] Wherein, component i is an in vitro cell-free protein synthesis system; the in vitro cell-free protein synthesis system is an aqueous phase system;

[0194] Wherein, component ii is a nucleic acid template encoding an exogenous protein;

[0195] Step two, incubating the reaction with the aid of a hydrophobic interface iii to synthesize the exogenous protein;

[0196] Wherein, the hydrophobic interface iii is in surface contact with the aqueous phase in vitro cell-free protein synthesis system.

[0197] Optionally, a step of separating or / and detecting the exogenous protein is further included.

[0198] The component i and component ii can collectively provide the translation-related elements required for the synthesis of the exogenous protein. Preferably, the nucleic acid template encoding the exogenous protein contains a promoter element that can be recognized by component i.

[0199] Through the limitation of being able to achieve the technical function of "expressing exogenous proteins", the present application only covers the combination of technical features that can achieve the above function, and the combination of technical features that cannot achieve the above function is of course excluded from the scope of the present application. That is, the in vitro cell-free protein synthesis system (component i system) should first be a working system, that is, a system capable of expressing exogenous proteins.

[0200] Hydrophobic interface

[0201] The hydrophobic interface is an interface formed by the surface contact of the hydrophobic phase with the aqueous phase synthesis system.

[0202] The hydrophobic interface provides local enrichment of raw materials by hydrophobic interaction, and also provides interface solubilization for non-water-soluble protein products. In addition, the translation process from mRNA to protein is carried out in ribosomes, and the membrane surface of the endoplasmic reticulum (ER) of the ribosome has a hydrophobic structure and hydrophobic properties; the hydrophobic interface provides a hydrophobic structure similar to that of the ribosome, and it is speculated that the hydrophobic interface constructed in the present application may also have a promoting effect on the folding of newly synthesized proteins.

[0203] According to the physical state of the hydrophobic phase, the hydrophobic interface can be selected from a solid-phase hydrophobic interface, a liquid-phase hydrophobic interface, or a combination thereof. When the hydrophobic phase is a liquid phase, the entire liquid phase (including the internal and external surfaces / interfaces) is preferably hydrophobic. Preferably, the hydrophobic interface is provided by the inner surface of a reactor, the outer hydrophobic surface of a hydrophobic built-in solid, the liquid interface of an oil phase system, or a combination thereof. The inner surface of the reactor, such as the inner surface of the side wall and the bottom of the reactor. The hydrophobic built-in solid refers to a solid whose outer surface is hydrophobic, and its position is not particularly limited, which can be deposited below the aqueous phase synthesis system, suspended in the aqueous phase synthesis system, or floated above the aqueous phase synthesis system; preferably, the hydrophobic built-in solid is deposited below the aqueous phase synthesis system or floated above the aqueous phase synthesis system.

[0204] Preferably, the hydrophobic phase is provided by a hydrocarbon substance. The main component of the hydrocarbon substance is a hydrocarbon, and the physical state is paste, solid (such as wax, particles, powder, etc.), liquid, or a mixed form thereof. The number of carbon atoms of the hydrocarbon contained in the hydrocarbon substance is at least 6, and one of the preferred modes is 6-44. The hydrocarbon contained in the hydrocarbon substance can be a saturated hydrocarbon or an unsaturated hydrocarbon. According to the degree of unsaturation, the hydrocarbon contained in the hydrocarbon substance includes but is not limited to alkanes, alkenes, dienes, and alkynes; according to the structural type, the hydrocarbon contained in the hydrocarbon substance includes but is not limited to open-chain hydrocarbons and cyclic hydrocarbons.

[0205] The hydrocarbon substance is preferably selected from aliphatic hydrocarbons.

[0206] The hydrocarbon substance is preferably selected from aliphatic hydrocarbons.

[0207] The hydrocarbon substance is preferably selected from aliphatic hydrocarbons.

[0208] The hydrocarbon substance is preferably selected from aliphatic hydrocarbons.

[0209] The hydrocarbon substance is preferably selected from aliphatic hydrocarbons.

[0210] The hydrocarbon substance is preferably selected from aliphatic hydrocarbons. n H 2n+2, the molecular formula of the cyclic structure is typically C n H 2n ( single ring ).

[0211] Preferably, the number of carbon atoms of the alkane is 6-44.

[0212] More preferably, the alkane is selected from the group consisting of cyclohexane, isooctane, decane, tetradecane, pentadecylcyclohexane, tetranonadecane, vaseline, or a combination thereof.

[0213] Preferably, the hydrocarbon substance is selected from the group consisting of vaseline, mineral oil, or a combination thereof.

[0214] The amount of the alkane capable of increasing the amount of synthesis of the exogenous protein (Q hc ) is determined according to the amount of expression of the exogenous protein in the Y PRT (C hc ) curve, and is selected from the interval of the amount of the alkane when the amount of expression of the exogenous protein is greater than Y0 in the Y PRT (C hc ) curve.

[0215] Preferably, the Q hc is selected from the interval of the amount of the alkane when the amount of expression of the exogenous protein is at least Y0+50%Y Δ .

[0216] More preferably, the Q hc is selected from the interval of the amount of the alkane when the amount of expression of the exogenous protein is at least Y0+60%Y Δ .

[0217] More preferably, the Q hc is selected from the interval of the amount of the alkane when the amount of expression of the exogenous protein is at least Y0+70%Y Δ .

[0218] More preferably, the Q hc is selected from the interval of the amount of the alkane when the amount of expression of the exogenous protein is at least Y0+80%Y Δ .

[0219] More preferably, the Q hc is selected from the interval of the amount of the alkane when the amount of expression of the exogenous protein is at least Y0+90%Y Δ .

[0220] More preferably, the Q hc is selected from the interval of the amount of the alkane when the amount of expression of the exogenous protein is at least Y0+95%Y Δ .

[0221] More preferably, the Q hc is the amount of the alkane when the amount of expression of the exogenous protein is the highest, C max .

[0222] In one preferred embodiment, the method for determining the amount of alkane is as follows: when the types and amounts of the components of the in vitro cell-free protein synthesis system are determined, the amount of alkane is adjusted in a wide range of concentrations, and the amount of alkane C is determined under the specified reaction conditions (reaction temperature, reaction time, etc.) when the expression amount of the exogenous protein is the highest. max That is, the optimal amount of alkane under the technical solution.

[0223] The Y PRT (C hc ) curve, Q hc , C hc , Y PRT , Y max , C max , Y0, Y Δ The definitions are the same as described above.

[0224] The protection scope of the present application only covers those technical solutions corresponding to the amount of alkane that can improve the expression amount of the exogenous protein. In the present application, for any component i of the in vitro cell-free protein synthesis system, it is only required that at least one amount of alkane can improve the expression amount of the exogenous protein, and it is not required that all amounts of alkane have the promoting effect.

[0225] In vitro cell-free protein synthesis reaction and in vitro cell-free protein synthesis system

[0226] The in vitro protein synthesis reaction of the present application is carried out in the in vitro cell-free protein synthesis system in an aqueous phase.

[0227] The in vitro cell-free protein synthesis system can provide various factors required for the in vitro synthesis of proteins. It can be provided in an integrated manner by means of cell extract, or it can be provided by separate addition.

[0228] The types and amounts of the components of the in vitro cell-free protein synthesis system are not particularly limited, as long as the system formed can react with the nucleic acid template encoding the exogenous protein to synthesize the exogenous protein, and preferred are those combined modes that can efficiently express the exogenous protein. Those combined modes in which the exogenous protein cannot be expressed due to too low or too high concentration of some components are of course excluded from the scope of the present application.

[0229] The order of addition of the components of the in vitro cell-free protein synthesis system is not particularly limited.

[0230] Preferably, the in vitro cell-free protein synthesis system contains a component that can recognize the promoter element on the nucleic acid template, such as an RNA polymerase corresponding to the promoter element.

[0231] The system component capable of recognizing the promoter element on the nucleic acid template (e.g. the corresponding RNA polymerase) can be provided by the cell extract, can be provided by exogenous addition, or can be provided by a combination of the two.

[0232] In one preferred mode, the in vitro cell-free protein synthesis system comprises at least a cell extract. The cell extract is intended to provide structural or biological factors for protein transcription and translation. The selection criteria for the cell extract is that it can synthesize an exogenous protein based on a nucleic acid template encoding the exogenous protein through in vitro protein synthesis reaction. The cell extract of the present application can be derived from a wild type or a non-wild type. The modification of the non-wild type includes but is not limited to genetic modification. The cell extract of the present application is preferably derived from a eukaryotic cell, more preferably derived from a yeast cell, and more preferably derived from a Kluyveromyces lactis cell.

[0233] The components of the in vitro cell-free protein synthesis system preferably include a cell extract. More preferably, the cell extract contains an endogenously expressed RNA polymerase corresponding to the promoter element on the nucleic acid template. Specifically, for example, the Kluyveromyces lactis cell extract contains an endogenously expressed T7 RNA polymerase capable of recognizing the T7 promoter on the nucleic acid template.

[0234] In one preferred mode, the PURE system (Protein synthesis Using Recombinant Elements (PURE) system) developed by Japanese scientists is used to provide various factors required for in vitro protein synthesis. For details, please refer to the literature “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 translation reconstituted with purified components [J]. Nature Biotechnology, 2001, 19(8): 751-755” and the references cited therein.

[0235] The process of in vitro protein synthesis at least includes the translation process, and optionally includes the transcription process.

[0236] The transcription process of converting DNA into mRNA cannot be carried out without RNA polymerase. The in vitro cell-free protein synthesis system preferably further comprises RNA polymerase. The RNA polymerase can be selected from the group consisting of endogenously expressed RNA polymerase (provided via cell extract), exogenously added RNA polymerase, or a combination thereof.

[0237] The endogenously expressed RNA polymerase is not added independently but is present in the cell extract.

[0238] In order to achieve the endogenously expressed RNA polymerase in the cell extract, the coding sequence / coding gene of the RNA polymerase is preferably integrated into the host cell from which the cell extract is prepared, and is more preferably achieved by inserting the coding sequence of the RNA polymerase into a free plasmid in the cell, or integrating the coding gene of the RNA polymerase into the genome of the cell, or a combination of the above two methods, and then preparing the cell extract. The method of integrating the coding sequence / coding gene of the RNA polymerase into the genome of the cell includes but is not limited to inserting into the genome of the cell, replacing part of the genome in situ, or a combination thereof.

[0239] The exogenously added or endogenously expressed RNA polymerase is each independently preferably T7 RNA polymerase.

[0240] The in vitro cell-free protein synthesis system preferably comprises DNA polymerase, which can be selected from the group consisting of endogenously expressed DNA polymerase (provided via cell extract), exogenously added DNA polymerase, or a combination thereof.

[0241] The in vitro cell-free protein synthesis system can optionally comprise exogenous RNA polymerase or / and nucleic acid template encoding RNA polymerase.

[0242] The in vitro cell-free protein synthesis system can optionally comprise exogenous DNA polymerase or / and nucleic acid template encoding DNA polymerase.

[0243] In one preferred manner, the in vitro cell-free protein synthesis system comprises exogenous RNA polymerase, exogenous DNA polymerase. Reference CN108642076A, WO2018171747A1 (CN201710176691.4).

[0244] In one preferred manner, the in vitro cell-free protein synthesis system comprises an energy system.

[0245] In one preferred manner, the in vitro cell-free protein synthesis system comprises a substrate for synthesizing RNA.

[0246] In one preferred embodiment, the in vitro cell-free protein synthesis system comprises a substrate for synthesizing a protein.

[0247] In one preferred embodiment, the in vitro cell-free protein synthesis system comprises a DNA polymerase and a substrate for synthesizing DNA.

[0248] In one preferred embodiment, the in vitro cell-free protein synthesis system comprises a cell extract, an energy system, a substrate for synthesizing a protein, an RNA polymerase (either included in the cell extract or added exogenously), and a substrate for synthesizing RNA.

[0249] In one preferred embodiment, the in vitro cell-free protein synthesis system comprises a cell extract, an energy system, a substrate for synthesizing a protein, an RNA polymerase (either included in the cell extract or added exogenously), a substrate for synthesizing RNA, a DNA polymerase (either included in the cell extract or added exogenously), and a substrate for synthesizing DNA.

[0250] In one preferred embodiment, the in vitro cell-free protein synthesis system comprises a cell extract of Kluyveromyces lactis (containing an endogenously expressed RNA polymerase), an energy system, a substrate for synthesizing RNA, and a substrate for synthesizing a protein.

[0251] The in vitro cell-free protein synthesis system can optionally comprise at least one of the following components: a crowding agent, magnesium ions, potassium ions, an antioxidant or reducing agent, trehalose, a reaction accelerator, a buffer, and an aqueous solvent.

[0252] Cell extract

[0253] The cell extract should be capable of expressing a nucleic acid template encoding a foreign protein in the in vitro cell-free protein synthesis system, i.e., capable of synthesizing the foreign protein encoded by the nucleic acid template based on the nucleic acid template.

[0254] The cell extract is intended to provide structural factors or / and biological factors for protein transcription and translation.

[0255] The cell extract can provide many key translation-related elements required for the synthesis of a foreign protein; this is an endogenous provision.

[0256] The cell extract is typically used to provide ribosomes, transfer RNAs (tRNAs), aminoacyl tRNA synthetases, initiation factors and elongation factors required for protein synthesis, and termination release factors, and can also provide other enzyme substances such as polymerases (RNA polymerase and / or DNA polymerase) endogenously after strain modification.

[0257] The cell extract is in principle free of intact cells, because the preparation method of the cell extract comprises a step of disrupting the cells (also referred to as cell disruption treatment, lysis step, etc.). Compared with the traditional synthesis of expressed proteins by intact cells, the in vitro protein synthesis system thus constructed is referred to as a cell-free system. The cell extract can also contain some other proteins derived from the cytoplasm of the cells, especially soluble proteins.

[0258] Preferably, the cell extract contains various factors required for protein synthesis.

[0259] The various protein factors provided by the above-mentioned cell extract can be naturally present in the genome of the cells, or the relevant coding genes can be integrated into the genome of the cells (integrated into the chromosome), or the relevant genes or gene fragments or coding sequences can be inserted into the free plasmid in the cells. Taking RNA polymerase and DNA polymerase as examples, one of the preferred modes is that the cell extract contains endogenously expressed RNA polymerase and / or DNA polymerase.

[0260] For the source cells of the cell extract, the coding sequence or coding gene of the heterologous protein can be endogenously integrated to endogenously express the heterologous protein, which can include but is not limited to RNA polymerase, DNA polymerase, etc. The method of endogenously integrating the coding sequence or coding gene of the heterologous protein can refer to the methods provided in the existing documents including but not limited to patent application documents CN109423496A, CN10697843A, CN2018116198190, “Molecular and Cellular Biology, 1990, 10(1): 353-360” and the cited documents thereof, specifically including but not limited to the following methods: inserting the coding sequence into the free plasmid, inserting the coding gene into the genome of the cells, replacing part of the genome of the cells with the coding gene in situ, etc., or a combination thereof.

[0261] In one preferred embodiment, the source cell of the cell extract has endogenous integration of a gene encoding RNA polymerase, and can endogenously express RNA polymerase, and can perform in vitro cell-free protein synthesis without the addition of exogenous RNA polymerase. The implementation of endogenous integration of RNA polymerase includes, but is not limited to, inserting a gene encoding RNA polymerase into a cell plasmid or into a cell genome, replacing part of a genome or a sequence in situ with a gene encoding RNA polymerase (i.e., including the step of knocking out the original part of the gene or sequence), knocking out the original part of the gene and inserting a gene encoding RNA polymerase, or a combination thereof. In Example S2, a gene encoding T7 RNA polymerase is integrated into the genome of a Kluyveromyces lactis cell, which endogenously expresses T7 RNA polymerase. The cell extract prepared in this way contains endogenously expressed T7 RNA polymerase, and the in vitro cell-free protein synthesis system does not require additional RNA polymerase. In some other specific embodiments, the coding sequence of the RNA polymerase is inserted into a free plasmid in the cell of Kluyveromyces lactis, and then a cell extract is prepared. For specific preparation methods, refer to CN109423496A.

[0262] Other genetic modification methods can also be used to modify the source cell to promote the activity of the cell extract in promoting in vitro protein synthesis, such as the gene knockout methods of CN2018116083534, CN2019107298813, CN108949801A, and the genetic modification methods of 2018112862093.

[0263] The preparation method of the cell extract can use the reported technical means. In brief, it generally includes the following steps: quick-freezing the cells with liquid nitrogen, breaking the cells, and centrifuging to collect the supernatant to obtain the cell extract. For reference, CN106978349A, CN108535489A, CN108642076A, CN109593656A, CN109971783A, and other documents. The seed cells can be fermented, centrifuged, and the culture solution is removed to collect the cells, and then the cell extract is prepared.

[0264] The cell extract prepared by the method provided in the present application can make in vitro protein synthesis reaction proceed normally, and contains necessary components such as tRNA with amino acid transport function and aminoacyl tRNA synthetase required for protein synthesis. In some embodiments, the cell extract is a yeast cell extract, which is prepared by a method comprising the following steps: (i) providing a source cell; (ii) washing the yeast cell to obtain a washed yeast cell; (iii) performing cell disruption treatment on the washed yeast cell to obtain a yeast crude extract; and (iv) performing solid-liquid separation on the yeast crude extract, and the supernatant part collected is the cell extract. The yeast cell extract is preferably a Kluyveromyces lactis cell extract.

[0265] In the present application, one of the preferred ways of the protein content contained in the cell extract is 20 mg / mL-100 mg / mL. Another one of the preferred ways is 20 mg / mL-50 mg / mL. Another one of the preferred ways is 50 mg / mL-100 mg / mL. Another one of the preferred ways is any one of 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, or a concentration range (including both endpoints) between any two of the concentrations. The method for determining the protein content can use the Coomassie brilliant blue determination method.

[0266] The concentration of the cell extract in the in vitro protein synthesis system is not particularly limited. In one of the preferred embodiments, the concentration of the cell extract is 20%-80% (v / v); in another preferred embodiment, the concentration of the cell extract is 20%-70% (v / v); in another preferred embodiment, the concentration of the cell extract is 30%-60% (v / v); in another preferred embodiment, the concentration of the cell extract is 40%-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. The concentration of the cell extract also includes, but is not limited to, any one of the following volume percentages, or a range (which can or can not include both endpoints) between any two of the following volume percentages: 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%.

[0267] The cell extract is derived from prokaryotic cells, eukaryotic cells, or a combination thereof.

[0268] The cell extract of the present application is preferably derived from prokaryotic cells, more preferably E. coli cells or Bacillus.

[0269] The cell extract of the present application is preferably derived from eukaryotic cells.

[0270] Prokaryotic cells and eukaryotic cells have fundamental differences in translation initiation and regulation mechanisms. The prokaryotic expression system lacks a variety of post-translational processing mechanisms for proteins. Typically, a cell-free system of E. coli cells lacks the ability to perform translation or post-translational modification that can only be performed by eukaryotic cell-free systems, making many eukaryotic proteins unsuitable for expression in this system; the process of synthesizing proteins contains incomplete new polypeptides. The synthesis mechanism of a cell-free protein synthesis system based on a prokaryotic system is very different from that of a cell-free protein synthesis system based on a eukaryotic system. For reference, including but not limited to the following documents: “Nicole E. Gregorio, Max Z. Levine and Javin P. Oza. Methods Protoc. 2019, 2, 24”, “Edited by Alexander S. Spirin and James R. Swartz. Cell-free protein synthesis: methods and protocols [M]. 2008, p. 5”, “Zhang Xu. Key technologies for efficient synthesis of complex membrane proteins in cell-free systems and industrial application exploration [D]. Zhejiang University, 2014.”, etc. The in vitro cell-free protein synthesis system of the present application preferably uses eukaryotic cell extract.

[0271] The cell source of the cell extract can be selected from, including but not limited to, eukaryotic cells in the following group: mammalian cells (such as rabbit reticulocytes, HF9, Hela, CHO, K562, HEK293), plant cells (such as wheat germ cells, tobacco BY-2 cells), yeast cells, insect cells, nematode cells, or combinations thereof. The mammalian cell source includes but is not limited to mouse origin, rabbit origin, monkey origin, human origin, etc.

[0272] The cell sources of the cell extract and its preparation method can also be referred to in the existing literature, 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 Protoc. 2019, 2, 24", "Y Lu. Advances in cell-free biosynthetic technology [J]. Current Developments in Biotechnology and Bioengineering, 2019, Chapter 2, 23-45" and other documents and their direct or indirect references reported in the literature. For example, the prokaryotic cell sources include but are not limited to E. coli; the eukaryotic cell sources include but are not limited to Saccharomyces cerevisiae, Streptomyces lividans, wheat germ, tobacco BY-2 cell, Spodoptera frugiperda cell (sf cell, an insect cell), Trichoplusiani cell (an insect cell), rabbit reticulocyte, CHO cell (Chinese hamster ovary cell), human K562 cell, HEK293 cell, HeLa cell, mouse embryonic fibroblast, Leishmania tarentolae cell (protozoan, single-cell organism), etc.

[0273] The yeast cell is preferably one of the following embodiments, preferably selected from Saccharomyces cerevisiae, Saccharomyces boulardii, Kluyveromyces, or a combination thereof; the Kluyveromyces is further preferably Kluyveromyces lactis (K. lactis), Kluyveromyces lactis var. drosophilarum, Kluyveromyces lactis var. lactis, Kluyveromyces marxianus, Kluyveromyces marxianus var. lactis, Kluyveromyces marxianus var. marxianus, Kluyveromyces marxianus var. vanudenii, Kluyveromyces dobzhanskii, Kluyveromyces aestuarii, Kluyveromyces nonfermentans, Kluyveromyces wickerhamii, Kluyveromyces thermotolerans, Kluyveromyces fragilis, Kluyveromyces hubeiensis, Kluyveromyces polysporus, Kluyveromyces siamensis, Kluyveromyces yarrowii, etc., or a combination thereof; reference documents include but are not limited to the following documents: EP1197560A1, "Marc-André Lachance. The Yeasts (Fifth Edition), Chapter 35, Kluyveromyces van der Walt (1971). 2011, Pages 471-481", "JL Souciet, B Dujon, C Gaillardin, M Johnston et al. Comparative genomics of protoploid Saccharomycetaceae [J]. Genome Res. 2009, 19: 1696-1709".

[0274] Kluyveromyces is a group of ascosporogenous yeasts, among which Kluyveromyces marxianus and Kluyveromyces lactis are widely used in industry. Compared with other yeasts, Kluyveromyces lactis has many advantages, such as super strong secretion ability, better large-scale fermentation characteristics, food safety level, and the ability of post-translational modification of proteins. The genome of wild strains of Kluyveromyces lactis does not contain the coding gene of T7 RNA polymerase.

[0275] In one preferred embodiment, the cell extract is derived from Kluyveromyces lactis, and the following gene sequence or a combination thereof is endogenously integrated: a coding gene of RNA polymerase, a coding gene of DNA polymerase. Preferably, the endogenous integration is into a cell plasmid or into a cell genome.

[0276] In one preferred embodiment, the cell extract is derived from Kluyveromyces lactis, and the following gene sequence or a combination thereof is endogenously integrated: a coding gene of T7 RNA polymerase, a coding gene of phi29 DNA polymerase. Preferably, the endogenous integration is into a cell plasmid or into a cell genome.

[0277] In one preferred embodiment of the cell extract of the present application, the cell extract can be selected from any of the following sources: Escherichia coli, yeast cells, mammalian cells, plant cells, insect cells, or a combination thereof. The yeast cells are more preferably selected from Kluyveromyces, Saccharomyces cerevisiae, Pichia, or a combination thereof; the Kluyveromyces is further preferably Kluyveromyces lactis, Kluyveromyces lactis var. drosophilarum, Kluyveromyces lactis var. lactis, Kluyveromyces marxianus, Kluyveromyces marxianus var. lactis, Kluyveromyces marxianus var. marxianus, Kluyveromyces marxianus var. vanudenii, Kluyveromyces dobzhanskii, Kluyveromyces drosophilarum, Kluyveromyces lactis, Kluyveromyces thermotolerans, Kluyveromyces fragilis, Kluyveromyces herberianus, Kluyveromyces polysporus, Kluyveromyces siamensis, Kluyveromyces yarrowii, or a combination thereof.

[0278] In another preferred embodiment, the cell extract is a yeast cell extract, more preferably a Kluyveromyces cell extract, more preferably a Kluyveromyces marxianus cell extract or a Kluyveromyces lactis cell extract.

[0279] In another preferred embodiment, the cell extract can be selected from any one of the following sources: Escherichia coli, Kluyveromyces lactis, wheat germ cell, Spodoptera frugiperda cell (sf cell, an insect cell), Lizard Leishmania cell, rabbit reticulocyte cell, Chinese hamster ovary cell (CHO cell), African green monkey kidney COS cell, African green monkey kidney VERO cell, baby hamster kidney cell (BHK cell), human Hela cell, human Hybridoma cell (human hybridoma cell), human fibrosarcoma HT1080 cell, or a combination thereof.

[0280] Exogenous RNA polymerase, exogenous DNA polymerase

[0281] When the cell genome as the source of the cell extract does not contain the gene of the RNA polymerase, and there is no endogenous integrated coding sequence / coding gene of the RNA polymerase, it is generally necessary to additionally add an exogenous RNA polymerase to promote the reaction. For example, when the cell extract of wild-type Kluyveromyces lactis is used.

[0282] Adding an exogenous RNA polymerase to the in vitro protein synthesis system is a traditional technical means. The in vitro protein synthesis system reported in the prior art by adding an exogenous RNA polymerase (such as T7 RNA polymerase) is included in the present application as an optional way of component i system. For example, the Kluyveromyces lactis in vitro protein synthesis system in CN108535489A by adding an exogenous RNA polymerase (such as T7 RNA polymerase) is included in the present application as an optional way of component i system.

[0283] The in vitro cell-free protein synthesis system can further comprise at least one of the following components to optimize the reaction system: exogenous RNA polymerase, nucleic acid template encoding exogenous RNA polymerase, exogenous DNA polymerase, nucleic acid template encoding exogenous DNA polymerase.

[0284] The exogenous RNA polymerase can be directly added, or an exogenous nucleic acid template encoding the RNA polymerase can be added, or a combination thereof. The coding sequence of the RNA polymerase can be constructed in the nucleic acid template encoding the exogenous protein, or can be constructed in a separate exogenous nucleic acid template.

[0285] Similarly, the DNA polymerase can be directly added, or an exogenous nucleic acid template containing its coding sequence can be added, or a combination thereof. It can be a nucleic acid template encoding an exogenous protein, or a separate exogenous nucleic acid template.

[0286] When the nucleic acid template encoding the exogenous protein is a DNA template, the DNA amplification process can or can not be included. If the DNA amplification process is included in the in vitro protein synthesis reaction, the system needs to contain endogenous expressed or / and exogenously added DNA polymerase, such as the exogenous phi29 DNA polymerase added in CN108642076A. In embodiments S1-S2 of the present application, the DNA encoding the exogenous protein mEGFP is amplified in vitro, and the amplification product is added to the reaction system as an exogenous DNA template. The in vitro protein synthesis reaction needs to include the DNA amplification process. When DNA polymerase is added to the system, i.e., when the DNA amplification process is included, the substrate for synthesizing DNA also needs to be added.

[0287] The DNA polymerase can be a polymerase derived from eukaryotes or prokaryotes. Examples of eukaryotic polymerases include any one of the following or any combination thereof: pol-α, pol-β, pol-δ, pol-ε, etc., fragments of any of the foregoing, variants of any of the foregoing (including variants of fragments of any of the foregoing). Examples of prokaryotic polymerases include any one of the following or any combination thereof: E. coli DNA polymerase I (such as Klenow fragment), E. coli DNA polymerase II, E. coli DNA polymerase III, E. coli DNA polymerase IV, E. coli DNA polymerase V, bacteriophage T4 DNA polymerase, Bacillus stearothermophilus polymerase I, Phi29 DNA polymerase, T7 DNA polymerase, Bacillus subtilis Pol I, Staphylococcus aureus Pol I, etc., partial domains of any of the foregoing, subunits or fragments of any of the foregoing, variants of any of the foregoing (including variants of any of the foregoing domains, subunits, fragments). The variants include but are not limited to mutants.

[0288] The polymerase (exogenous RNA polymerase, exogenous DNA polymerase) is preferably a polymerase that can perform room temperature amplification, and the room temperature is preferably room temperature to 37°C, specifically, preferably 20°C to 37°C, more preferably 25°C to 37°C. The polymerase that can perform room temperature amplification can be selected according to the exogenous nucleic acid template, and the polymerase that can be used for room temperature amplification in an in vitro cell-free system is also included in the scope of the present application, including but not limited to phi29 DNA polymerase, T4 DNA polymerase, T7 DNA polymerase, exo-klenow DNA polymerase, Bsu DNA polymerase, Pol III DNA polymerase, T7 RNA polymerase, T3 RNA polymerase, T4 RNA polymerase, T5 RNA polymerase, etc., a partial domain of any of the above polymerases, a subunit or fragment of any of the above, a variant of any of the above, and any combination of the above polymerases and their partial domains, subunits, fragments, variants (including but not limited to mutants). The present application can also use Taq DNA polymerase, Pfu DNA polymerase, Pol I DNA polymerase, Pol II DNA polymerase and other DNA polymerases.

[0289] In some preferred examples, the DNA polymerase has strand displacement function.

[0290] In some preferred examples, the DNA polymerase lacks 3'-5' exonuclease activity.

[0291] The amplification technology, especially the room temperature amplification method, that can be used in the present application is not particularly limited, and the room temperature amplification technology that can be used in an in vitro cell-free system is also included in the scope of the present application.

[0292] Energy system / energy regeneration system

[0293] The energy system / energy regeneration system is used to provide the energy required for protein synthesis.

[0294] The reported energy systems / energy regeneration systems for cell-free in vitro protein synthesis systems can provide energy for the in vitro protein synthesis system of the present application. Including but not limited to: documents CN109988801A, CN2018116198186, CN2018116198190, US20130316397A, US20150376673A, “MJ Anderson, JC Stark, CE Hodgman and MC Jewett. Energizing eukaryotic cell-free protein synthesis with glucose metabolism [J]. FEBS Letters, 2015, 589(15): 1723-1727”, “Y Lu. Advances in Cell-Free Biosynthetic Technology [J]. Current Developments in Biotechnology and Bioengineering, 2019, Chapter 2, 23-45”, “P Shrestha, MT Smith and BC Bundy. Cell-free unnatural amino acid incorporation with alternative energy systems and linear expression templates [J]. New Biotechnology, 2014, 31(1): 28-34” and other documents and their direct or indirect cited documents reported in the energy systems / energy regeneration systems, which are incorporated herein by reference.

[0295] In one of the preferred embodiments, the energy system is a sugar (monosaccharide, disaccharide, oligosaccharide or polysaccharide) and phosphate energy system, a sugar and phosphagen energy system, a phosphagen and phosphagen kinase system, a phosphagen and phosphagen kinase system, a glycolysis pathway and its intermediate energy system (monosaccharide and its glycolysis intermediate energy system, glycogen and its glycolysis intermediate energy system), or a combination thereof. Specifically, the phosphate refers to inorganic phosphate, preferably selected from orthophosphate, dihydrogen phosphate, hydrogen dihydrogen phosphate, metaphosphate, pyrophosphate or a combination thereof. The polysaccharide can be selected from polysaccharides including but not limited to starch, malt dextrin, corn dextrin and the like. The disaccharide, for example, sucrose, maltose and the like. The glycolysis pathway and its intermediate energy system includes but is not limited to a glucose-based energy system.

[0296] The concentration of each component in the energy system is not particularly limited, including but not limited to the use of existing reported technical solutions and their equivalent technical solutions. The energy system used in Example S3 is a monosaccharide (glucose), polysaccharide (maltodextrin) and phosphate energy system; the polysaccharide and phosphate energy system used in Examples S11 and S12, wherein the polysaccharide is maltodextrin, and the phosphate is tri-potassium phosphate (potassium phosphate is equivalent to tri-potassium phosphate).

[0297] Substrate for synthesizing RNA

[0298] The substrate for synthesizing RNA is a nucleotide mixture, one of the embodiments, selected from: nucleoside monophosphate, nucleoside triphosphate, or a combination thereof. Preferably, it is a nucleoside triphosphate mixture (NTP). The nucleoside triphosphate mixture is preferably a mixture of adenine nucleoside triphosphate, guanine nucleoside triphosphate, cytosine nucleoside triphosphate and uracil nucleoside triphosphate. In the present application, the concentration of each single nucleotide is not particularly limited, and the amount of nucleotide required for protein synthesis is generally preferred. One of the preferred embodiments, the concentration of each single nucleotide is 0.5 mM to 5 mM, and another preferred embodiment is 1.0 mM to 2.0 mM. The concentration of each single nucleotide is independently selected from any of the following concentrations, or a range of concentrations between any two of the following concentrations (the range includes both endpoints): 0.5 mM, 1.0 mM, 1.5 mM, 2.0 mM, 2.5 mM, 3.0 mM, 3.5 mM, 4.0 mM, 4.5 mM, 5.0 mM, 5.5 mM, 6.0 mM.

[0299] Substrate for synthesizing DNA

[0300] During the DNA amplification or in vitro protein synthesis reaction process containing the step of DNA replication, the substrate for synthesizing DNA needs to be added. The substrate for synthesizing DNA is preferably a deoxyribonucleotide mixture, and further preferably a deoxyribonucleotide triphosphate mixture (dNTP).

[0301] When the in vitro cell-free protein synthesis system contains DNA polymerase, it is preferably also contains the substrate for synthesizing DNA.

[0302] Substrate for synthesizing protein

[0303] The substrate for the synthetic protein is a mixture of amino acids. The nucleotides required for the synthesis of the protein are dosed. The concentration of each amino acid is typically one of the preferred embodiments 0.01 mM to 5 mM, and one of the other preferred embodiments 0.1 mM to 1 mM. The concentration of each amino acid is independently of the other, for example, one of the following concentrations, or a range of concentrations between any two of the following values (the range includes the two endpoints): 0.1 mM, 0.2 mM, 0.4 mM, 0.5 mM, 1.0 mM, 1.2 mM, 1.5 mM, 1.8 mM, 2.0 mM, 2.5 mM, 3.0 mM, 3.5 mM, 4.0 mM, 4.5 mM, 5.0 mM, 5.5 mM, 6.0 mM.

[0304] The mixture of amino acids comprises at least the mixture of amino acids required for the synthesis of the foreign protein, selected from the group comprising, 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 and histidine. The mixture of amino acids required for the synthesis of the foreign protein comprises not only the amino acids that make up the primary sequence of the foreign protein, but also other amino acids involved in the synthesis process.

[0305] The mixture of amino acids can comprise natural amino acids, non-natural amino acids.

[0306] The mixture of amino acids can comprise L - amino acids, D - amino acids, or combinations thereof.

[0307] In addition to natural amino acids, the mixture of amino acids can comprise non-natural amino acids, D- amino acids, radioisotope-labeled amino acids, modified amino acids, and the like components. The unnatural amino acids are not particularly limited, and can be selected from the unnatural amino acids reported or cited in the literatures such as "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 the directly or indirectly cited literatures thereof. The radioisotope-labeled amino acids are not particularly limited, and include but are not limited to the isotope labels reported to be used in the field of protein synthesis. The modified amino acids are not particularly limited, and include but are not limited to the modifications by amino acid side groups.

[0308] Preferably, the amino acid mixture is a mixture of natural amino acids.

[0309] Other additive components

[0310] The in vitro cell-free protein synthesis system can further include at least one of the following components: polyethylene glycol and / or its analogues, magnesium ions, potassium ions, antioxidants or reducing agents, trehalose, reaction promoters, buffers, aqueous solvents. Reference can be made to the literatures 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 the directly or indirectly cited literatures thereof.

[0311] In some preferred embodiments, the in vitro cell-free protein synthesis system further comprises a crowding agent for mimicking the crowded macromolecular environment in cells. The structure of the crowding agent is not particularly limited, and can be linear or non-linear, including but not limited to branched, multi-armed, cyclic, comb-like, dendritic, star-shaped, etc. In some preferred embodiments, the crowding agent can be selected from the group consisting of polyethylene glycol, polyvinyl alcohol (PVA), polypropylene glycol, dextran, sucrose polymer (such as Ficoll sucrose polymer), polyvinyl (pyrrolidone) (PVP), albumin, etc., and any combination thereof. The source of the albumin includes but is not limited to human serum albumin, bovine serum albumin, porcine serum albumin; preferably, the albumin is human serum albumin. The crowding agent can also refer to the crowding agents disclosed in the document “X Ge, D Luo and J Xu. Cell-free protein expression under macromolecular crowding conditions [J]. PLoS One, 2011, 6(12): e28707” and the cited documents thereof. In some preferred embodiments, the concentration of the crowding agent in the in vitro protein synthesis reaction mixture is sufficient to increase the amount of protein synthesis.

[0312] In some preferred embodiments, the molecular weight of the crowding agent is not more than 400 kDa. In some preferred embodiments, the molecular weight of the crowding agent is not more than 200 kDa. Generally, the molecular weight specification is preferably a molecular weight distribution of ±10% or narrower. In one preferred manner, the amount of the crowding agent is selected from 0.5% to 15%, further preferably from 1% to 12%, in terms of weight percentage (wt%) or volume percentage (% (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: 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%.

[0313] In one preferred embodiment, the in vitro cell-free protein synthesis system further comprises polyethylene glycol and / or its analogues as crowding agents and / or to stabilize mRNA (ref. Yin G, Swartz J R. Enhancing multiple disulfide bonded protein folding in a cell-free system [J]. Biotechnology and bioengineering, 2004, 86(2): 188-195.). The polyethylene glycol, which has repeating units of CH2CH2O (EO units), is commonly referred to as PEG (polyethylene glycol), PEO (poly(ethylene oxide)), or POE (polyoxyethylene). The analogues of polyethylene glycol include, but are not limited to, copolymers rich in EO units, derivatives of polyethylene glycol, other polyalkylene oxides (e.g., polyoxypropylene, POP) that can function as crowding agents, derivatives of the other polyalkylene oxides, etc.; the derivatives, exemplified by derivatives of polyethylene glycol, include, but are not limited to, chemical modifications (e.g., methoxypolyethylene glycol, amino modifications, carboxyl modifications, etc.), amino acid modifications, polypeptide modifications, protein modifications, block polymers with polyethylene glycol blocks, polymers with polyethylene glycol side chains, etc. The concentration of polyethylene glycol or its analogues is not particularly limited, and generally, the concentration of polyethylene glycol or its analogues is 0.1%-10%, preferably 0.1%-8%, more preferably 0.5%-4%, and even more preferably 1%-2%, based on the mass concentration of the protein synthesis system (%(w / v)) or based on the total weight (wt%). Unless otherwise specified, the concentration in the present application is the mass concentration, with the unit of %(w / v), e.g., 2% refers to 2%(w / v), which corresponds to 2g / 100mL or 20mg / mL. In some preferred embodiments, the molecular weight of polyethylene glycol and / or its analogues is no more than 40,000 Da, and representative molecular weights include, but are not limited to, 200, 400, 500, 600, 800, 1,000, 1,200, 1,400, 1,450, 1,500, 1,600, 1,800, 2,000, 2,500, 3,000, 3,350, 4,000, 4,500, 5,000, 5,500, 6,000, 6,500, 7,000, 7,500, 8,000, 8,500, 9,000, 9,500, 10,000, 12,000, 14,000, 15,000, 16,000, 18,000, 20,000, 25,000, 30,000, 35,000, 40,000, combinations thereof, etc.; the unit is Da; each of the above numbers is equal in value to the weight average molecular weight or the number average molecular weight. Generally, the molecular weight specifications are preferably within ±10% or narrower.The polyethylene glycol and / or its analogues preferably have a molecular weight of 200 Da to 10,000 Da, more preferably a molecular weight of 3,000 Da to 10,000 Da. In another preferred embodiment, the polyethylene glycol and / or its analogues have a molecular weight of 200 Da to 8,000 Da. In another preferred embodiment, the polyethylene glycol and / or its analogues have a molecular weight of 200 Da to 8,000 Da. In the present application, the molecular weight of the polyethylene glycol and / or its analogues, if not specified, refers to the weight average molecular weight M. w Representative PEGs are selected from the group consisting of PEG 200, PEG 400, PEG 1,000, PEG 1,500, PEG 2,000, PEG 3,000, PEG 3,350, PEG 5,000, PEG 6,000, PEG 8,000, PEG 10,000, and the like, combinations thereof; wherein the numbers 3,350 and the like refer to the weight average molecular weight.

[0314] The magnesium ions are derived from a magnesium ion source, which is not particularly limited and can be selected from the group consisting of magnesium acetate, magnesium glutamate (preferably L-magnesium glutamate), magnesium aspartate (preferably L-magnesium aspartate), magnesium chloride, magnesium phosphate, magnesium sulfate, magnesium citrate, magnesium hydrogen phosphate, magnesium iodide, magnesium lactate, magnesium nitrate, magnesium oxalate, combinations thereof. In one preferred embodiment, the concentration ranges from 0.1 mM to 50 mM. In another preferred embodiment, the concentration ranges from 0.5 mM to 20 mM. In another preferred embodiment, the concentration ranges from 1 mM to 10 mM. In one preferred embodiment, the magnesium ion source is any one or a mixture of any combination of magnesium aspartate, magnesium acetate, and magnesium glutamate.

[0315] The potassium ions are derived from a potassium ion source, which is not particularly limited and can be selected from the group consisting of 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, combinations thereof. In one preferred embodiment, the concentration ranges from 0 to 500 mM. In another preferred embodiment, the concentration ranges from 1 mM to 250 mM. In another preferred embodiment, the concentration ranges from 5 mM to 200 mM. In another preferred embodiment, the concentration ranges from 10 mM to 100 mM.

[0316] The optimization and preferred embodiments of the polyethylene glycol, the magnesium ions, and the potassium ions reported in patent document WO2016005982A1 can also be incorporated into the present application by reference.

[0317] The antioxidant, also known as reducing agent, can be selected from, but not limited to, dithiothreitol (DTT), 2-mercaptoethanesulfonic acid, 2-mercaptoethanol, reduced glutathione (GSH), tricarboxymethyl phosphine (TCEP), 3-mercapto-1,2-propanediol (MPD), and the like. In one preferred embodiment, the antioxidant is dithiothreitol. DTT can be used at its conventional concentration; in one embodiment, the concentration is 0.5 mM to 10 mM; in another embodiment, the concentration is 0 to 1.7 mM.

[0318] The reaction accelerator includes, but is not limited to, the reaction accelerator (e.g., an aluminum salt) provided in CN109971783A.

[0319] The buffer is mainly used to maintain the pH environment of the system. In one preferred embodiment, the buffer is selected from any one or a combination of the following: Tris-HCl, Tris base, HEPES (4-hydroxyethylpiperazine ethanesulfonic acid system).

[0320] The aqueous solvent is preferably a buffer.

[0321] Examples of embodiments of in vitro protein synthesis system

[0322] In one preferred embodiment, the in vitro protein synthesis system contains a cell extract, an endogenously expressed RNA polymerase (contained in the aforementioned cell extract) or an exogenously added RNA polymerase, an energy system, a substrate for synthesizing RNA, a substrate for synthesizing protein, a crowding agent, magnesium ions, potassium ions, a buffer, and can optionally include any one of the following components: an exogenous nucleic acid template (preferably a DNA template) encoding an RNA polymerase, an endogenously expressed DNA polymerase or an exogenously added DNA polymerase, an exogenous nucleic acid template (preferably a DNA template) encoding a DNA polymerase, a substrate for synthesizing DNA, an antioxidant or reducing agent, trehalose, a reaction accelerator, an aqueous solvent. The cell extract is preferably a eukaryotic cell extract, more preferably a yeast cell extract, and in one more preferred embodiment, the cell extract is a Kluyveromyces lactis cell extract.

[0323] One of the preferred embodiments is that the in vitro protein synthesis system contains a cell extract (the strain is modified by integrating the coding gene of RNA polymerase into the genome of the cell or inserting into the free plasmid in the cell), and further contains one or more or all components selected from the group consisting of: potassium 4-hydroxyethylpiperazineethanesulfonate (HEPES-K) or tris(hydroxymethyl)aminomethane (Tris), potassium acetate, magnesium acetate, magnesium glutamate, magnesium aspartate, nucleotide triphosphate mixture (NTP), amino acid mixture, creatine phosphate, creatine phosphokinase, creatine phosphokinase, dithiothreitol (DTT), RNAase inhibitor, L-arabinose, glucose, sucrose, starch, dextrin, corn dextrin, malt dextrin, phosphate (such as potassium phosphate). The cell extract is preferably a eukaryotic cell extract, more preferably a yeast cell extract, and one of the more preferred ways is a Kluyveromyces lactis cell extract.

[0324] One of the preferred embodiments is that the in vitro protein synthesis system contains a cell extract, and further contains one or more or all components selected from the group consisting of: HEPES-K or Tris, potassium acetate, magnesium acetate, magnesium glutamate, magnesium aspartate, nucleotide triphosphate mixture, amino acid mixture, creatine phosphate, creatine phosphokinase, creatine phosphokinase, DTT, RNAase inhibitor, L-arabinose, glucose, sucrose, starch, dextrin, corn dextrin, malt dextrin, phosphate (such as potassium phosphate), exogenous T7 RNA polymerase, exogenous phi29 DNA polymerase, deoxynucleotide triphosphate mixture. The cell extract is preferably a eukaryotic cell extract, more preferably a yeast cell extract, and one of the more preferred ways is a Kluyveromyces lactis cell extract.

[0325] One of the preferred embodiments is that the in vitro protein synthesis system contains a cell extract (optionally, the strain is modified by integrating the coding gene of RNA polymerase into the genome of the cell or inserting into the free plasmid in the cell), and further contains one or more or all components selected from the group consisting of: HEPES-K or tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), potassium acetate, magnesium acetate, magnesium glutamate (preferably L-magnesium glutamate), magnesium aspartate (preferably L-magnesium aspartate), nucleotide triphosphate mixture, amino acid mixture, creatine phosphate, creatine phosphokinase, creatine phosphokinase, DTT, RNAase inhibitor, L-arabinose, glucose, sucrose, starch, dextrin, corn dextrin, malt dextrin, potassium phosphate, polyethylene glycol, dextran, sucrose polymer, alumina promoter, exogenous T7 RNA polymerase, exogenous phi29 DNA polymerase, DNA template encoding T7 RNA polymerase, DNA template encoding phi29 DNA polymerase, deoxynucleotide triphosphate mixture. The cell extract is preferably a eukaryotic cell extract, more preferably a yeast cell extract, and one of the more preferred ways is a Kluyveromyces lactis cell extract.

[0326] In another preferred embodiment, the in vitro protein synthesis system comprises a cell extract, and further comprises one or more or all of the following components: Tris-HCl (pH 8.0), potassium acetate, magnesium acetate, magnesium glutamate, magnesium aspartate (preferably L-magnesium aspartate), dithiothreitol, polyethylene glycol, dextran, sucrose polymer, glucose, L-arabinose, nucleotide triphosphate mixture (a mixture of four nucleotide triphosphates, same concentration of each single nucleotide triphosphate), 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; same concentration of each single amino acid), potassium phosphate, exogenous T7 RNA polymerase, exogenous phi29 DNA polymerase, deoxynucleotide triphosphate mixture. The cell extract is preferably a eukaryotic cell extract, more preferably a yeast cell extract, and more preferably a cell extract of Kluyveromyces lactis.

[0327] In particular, in one preferred embodiment, the in vitro protein synthesis system comprises 50% to 80% (v / v) of a cell extract, and further comprises one or more or all of the following components: 9.78 mM Tris-HCl (pH 8.0), 20 mM to 80 mM potassium acetate, 2 mM to 10 mM magnesium acetate, 1.5 mM to 6 mM L-magnesium aspartate, 0.4 mM to 5 mM dithiothreitol (e.g. 0.44 mM), 0.5% to 5% (w / v) polyethylene glycol (e.g. 2% (w / v)), 0.5 mM to 5 mM of a mixture of four nucleotide triphosphates (same concentration of each single nucleotide triphosphate, e.g. 1.8 mM), 0.1 mM to 1 mM of an 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; same concentration of each single amino acid, e.g. 0.5 mM), 5 mM to 110 mM L-arabinose, 200 mM to 400 mM maltodextrin (as glucose monomers, e.g. 320 mM corresponds to about 52 mg / mL), 10 mM to 40 mM potassium phosphate. The cell extract is preferably a eukaryotic cell extract, more preferably a yeast cell extract, and more preferably a cell extract of Kluyveromyces lactis.

[0328] One of the specific embodiments of the in vitro protein synthesis system also includes, but is not limited to, for example, the E. coli-based cell-free protein synthesis system described in WO2016005982A1. The in vitro cell-free protein synthesis systems described in other references of the present application, direct and indirect references thereof, including but not limited to the wheat germ cell, rabbit reticulocyte, S. cerevisiae, P. pastoris, K. marxianus-based in vitro cell-free protein synthesis systems, are also included as embodiments of the present application. For example, the in vitro cell-free protein synthesis systems described in the literature "Lu, Y. Advances in Cell-Free Biosynthetic Technology. Current Developments in Biotechnology and Bioengineering, 2019, Chapter 2, 23-45", including but not limited to the in vitro cell-free protein synthesis systems described in the references cited in "2.1 Systems and Advantages" section on pages 27-28, can be used as in vitro cell-free protein synthesis systems for practicing the present application. For example, the in vitro cell-free protein synthesis systems described in the literature CN106978349A, CN108535489A, CN108690139A, CN108949801A, CN108642076A, CN109022478A, CN109423496A, CN109423497A, CN109837293A, CN109971783A, CN109988801A, CN110551700A, CN109971775A, CN110551745A, CN110551700A, CN2018116083534, CN2018116198186, CN2018116198190, CN2019102128619, CN2019102355148, CN2019107298813, CN2019112066163, CN2018108881848, CN2018109550734, CN2018111131300, CN2018111423277, CN2018112862093, CN201911418151.8, CN202010069383.3 and the references cited therein can be used as in vitro cell-free protein synthesis systems for practicing the present application.

[0329] Exogenous protein

[0330] The foreign protein suitable for the in vitro protein synthesis system of the present application 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 foreign protein suitable for the in vitro protein synthesis system of the prior art using a prokaryotic cell extract, a eukaryotic cell extract (preferably a yeast cell extract, more preferably a Kluyveromyces lactis cell extract), or an endogenous protein of a prokaryotic cell system or a eukaryotic cell system (preferably a yeast cell system, more preferably a Kluyveromyces lactis system) for intracellular synthesis can also be synthesized using the system of the present application, or attempts can be made to synthesize it using the in vitro protein synthesis system of the present application.

[0331] The application field of the foreign protein includes, but is not limited to, biological medicine, molecular biology, medicine, in vitro detection, medical diagnosis, regenerative medicine, bioengineering, tissue engineering, stem cell engineering, genetic engineering, polymer engineering, surface engineering, nanotechnology, cosmetics, food, food additives, nutritional agents, agriculture, feed, household goods, washing, environment, chemical dyeing, fluorescence labeling, etc.

[0332] The type of foreign protein includes, but is not limited to, polypeptides (the "foreign protein" in the present application broadly includes polypeptides), fluorescent proteins, enzymes and corresponding proenzymes, antibodies and fragments thereof, antigens, immunoglobulins, hormones, collagens, polyamino acids, vaccines, etc., partial domains of any of the foregoing proteins, subunits or fragments of any of the foregoing proteins, and variants of any of the foregoing proteins. The "subunits or fragments of any of the foregoing proteins" include subunits or fragments of "partial domains of any of the foregoing proteins". The "variants of any of the foregoing proteins" include variants of "partial domains of any of the foregoing proteins, subunits or fragments of any of the foregoing proteins". The "variants of any of the foregoing proteins" include, but are not limited to, mutants of any of the foregoing proteins. In the present application, the meaning of "two or more" of the "foregoing" at other positions is similarly interpreted.

[0333] The structure of the foreign protein can be a complete structure, or can be selected from corresponding partial domains, subunits, fragments, dimers, multimers, fusion proteins, glycoproteins, etc.

[0334] For example, the exogenous protein that can be synthesized by the in vitro protein synthesis system of the present application can be selected from any one of the following proteins, a fusion protein in any combination of the foregoing, a mixture in any combination of the foregoing: 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, actin, variable region of an antibody, alpha-amylase, enterotoxin A, hepatitis C virus E2 glycoprotein, insulin precursor, interferon alpha A, interleukin-1 beta, lysozyme, serum albumin, single-chain antibody fragment (scFV), transthyretin, tyrosinase, xylanase, E. coli beta-galactosidase (LacZ), human lysine-tRNA synthetase, human leucine-tRNA synthetase, Arabidopsis thaliana glyceraldehyde-3-phosphate dehydrogenase, mouse catalase, and the like, a partial domain of any of the foregoing proteins, a subunit or fragment of any of the foregoing proteins, or a variant of any of the foregoing (as defined above, the variant includes a mutant, for example, a luciferase mutant, a mutant of eGFP). Reference can also be made to patent document CN109423496A. The mixture in any combination of the foregoing can include any of the foregoing proteins and any of the foregoing fusion proteins in any combination.

[0335] In one of the preferred embodiments, GFP, eGFP, or a mutant thereof is used as the exogenous protein to evaluate the protein synthesis capability of the in vitro protein synthesis system.

[0336] Exogenous nucleic acid template (including a nucleic acid template encoding an exogenous protein)

[0337] The exogenous nucleic acid template of the present application, unless otherwise specified, refers to a nucleic acid template encoding an exogenous protein. In addition, the exogenous nucleic acid template of the present application, when specified, can also include a nucleic acid template encoding a protein factor or protease required in the in vitro protein synthesis process, for example, an exogenous nucleic acid template encoding RNA polymerase, an exogenous nucleic acid template encoding DNA polymerase.

[0338] Without the nucleic acid template encoding an exogenous protein, the in vitro synthesis reaction of the exogenous protein cannot be performed.

[0339] The nucleic acid template encoding an exogenous protein in any of the embodiments of the present application can each independently be a DNA template, an mRNA template, or a combination thereof.

[0340] The nucleic acid template encoding the foreign protein in any embodiment of the present application can each independently be preferably a DNA template.

[0341] The nucleic acid template is a direct template (mRNA) or an indirect template (DNA) for synthesis of the foreign protein, or a combination thereof.

[0342] The nucleic acid template encoding the foreign protein can include a non-coding region. The expression product can be a polypeptide or a protein, or a fusion protein. One nucleic acid template molecule can be translated (or transcribed and translated) once, and the number of synthesized polypeptide or protein molecules can be one, two or more.

[0343] The protein synthesis process by transcription and translation uses a DNA template as an indirect template, and the protein synthesis process by translation only can use an mRNA template as a direct template.

[0344] Preferably, the in vitro protein synthesis system of the present application is an in vitro transcription and translation system, i.e. an IVTT system, which uses a DNA template as the nucleic acid template encoding the foreign protein.

[0345] The nucleic acid template encoding the foreign protein contains translation-related elements required for synthesis of the foreign protein.

[0346] In any embodiment of the present application, each independently preferably, the nucleic acid template encoding the foreign protein further contains a promoter element recognizable by the cell extract.

[0347] In one preferred mode, the nucleic acid template encoding the foreign protein contains a promoter element recognizable by the cell extract.

[0348] In one preferred mode, the transcription of the gene of the foreign protein is initiated by a T7 promoter on the nucleic acid template.

[0349] In one preferred mode, the T7 promoter is located upstream of the coding sequence of the foreign protein on the nucleic acid template, the transcription of the foreign protein is initiated by the T7 promoter, and the cell extract in the in vitro cell-free protein synthesis system contains endogenously expressed T7 RNA polymerase.

[0350] In one preferred mode, the nucleic acid template encoding the foreign protein includes a foreign protein translation system, a resistance gene translation system, and a Lac repressor translation system, each of which includes a corresponding promoter.

[0351] In one preferred mode, the nucleic acid template encoding the foreign protein further contains a gene for controlling the copy number of the plasmid.

[0352] In one preferred mode, the nucleic acid template encoding the foreign protein further contains a transcription enhancer element, such as a kozak sequence.

[0353] In one preferred mode, the nucleic acid template encoding the exogenous protein further comprises a translation enhancing element, such as a translation enhancer element, an IRES element, a kozak sequence, etc.

[0354] The exogenous DNA template (including the DNA template encoding the exogenous protein)

[0355] The exogenous DNA template of the present application, unless otherwise specified, refers to the DNA template encoding the exogenous protein.

[0356] The exogenous DNA template of the present application can be selected from DNA, cDNA, methylated DNA, or a combination thereof. Among them, the cDNA can be obtained by reverse transcription from RNA or miRNA. miRNA (MicroRNA) is a class of non-coding single-stranded RNA molecules with a length of about 20-25 nucleotides encoded by endogenous genes.

[0357] The DNA template encoding the exogenous protein contains the coding sequence of the exogenous protein.

[0358] Preferably, the DNA template encoding the exogenous protein contains the coding gene of the exogenous protein.

[0359] The DNA template encoding the exogenous protein is determined according to the exogenous protein.

[0360] The DNA template encoding the exogenous protein can further contain other functional elements selected from the group consisting of promoters, terminators, enhancers (for example, enhancer elements described in documents such as CN109423497A, CN109022478A, CN109837293A (CN201711194355.9), CN109971775A, and their cited documents, for example, omega sequence and its homologous sequence, combined enhancer element), kozak sequence (refer to documents such as CN109022478A, CN109837293A, CN109971775A, and their cited documents), IRES element (internal ribosome entry sequence, refer to documents such as CN109022478A, CN109423497A, and their cited documents), MCS (multiple cloning site), genes controlling plasmid copy number, etc. It can also contain coding sequences of other amino acid chains such as signal peptide (corresponding to signal sequence), leader peptide (corresponding to leader sequence), functional tag (such as purification tag), linker peptide, etc. It can also contain 5' untranslated sequence, 3' untranslated sequence.

[0361] The DNA template encoding the exogenous protein preferably contains a promoter element. The promoter element is required to be recognized by the cell extract used or other components of the in vitro protein synthesis system; it can be a promoter recognized by a wild-type cell extract, or the source strain of the cell extract can be modified to a strain that recognizes the promoter. The promoter in the DNA template encoding the exogenous protein can be selected from the following group: AOD1, MOX, AUG1, AOX1, GAP, FLD1, PEX8, YPT1, LAC4, PGK, ADH4, AMY1, GAM1, XYL1, XPR2, TEF, RPS7, T7, or a combination thereof. References include but are not limited to the following literature and its cited literature: "Cereghino G. Applications of yeast in biotechnology: protein production and genetic analysis. Current Opinion in Biotechnology, 1999, 10 (5), 422-427".

[0362] In Examples S3-S12, the exogenous DNA template uses a T7 promoter to initiate the transcription of the exogenous protein; the T7 promoter is a strong promoter that can specifically respond to T7 RNA polymerase.

[0363] Preferably, the gene transcription process of the foreign protein is initiated by the T7 promoter on the foreign DNA template.

[0364] The concentration of the exogenous DNA template is determined based on the amount of exogenous protein to be expressed in the experimental protocol. In one preferred embodiment, the concentration of the exogenous DNA template is 1 ng / μL to 400 ng / μL. In another preferred embodiment, the concentration of the exogenous DNA template is 1 ng / μL to 80 ng / μL. In another preferred embodiment, the concentration of the exogenous DNA template is 5 ng / μL to 50 ng / μL. In the present invention, unless otherwise specified, the concentration of the DNA template added is the final concentration, i.e., the starting concentration of the in vitro protein synthesis reaction.

[0365] The exogenous DNA template can be circular or linear; single-stranded or double-stranded. The gene encoding the exogenous protein can be selected from, including but not limited to, genomic sequences and cDNA sequences. The exogenous DNA template can also contain a promoter sequence, a 5' untranslated sequence, and a 3' untranslated sequence.

[0366] In one preferred embodiment, the exogenous DNA template further comprises any one element or combination thereof selected from the group consisting of a promoter, a terminator, a poly(A) element, a transport element, a gene targeting element, a selection marker gene, an enhancer, an IRES element, a kozak sequence, a resistance gene, a transposase-encoding gene, a signal sequence, a leading sequence (for example, the leading sequence described in CN109022478A and the leading sequence cited therein), a gene for controlling plasmid copy number (rop gene), a tag for enhancing translation level (for example, the polypeptide tag described in CN2019112066163), and the like. Reference can be made to US20060211083A1 and the like.

[0367] The exogenous DNA template can also be constructed in an expression vector. Those of ordinary skill in the art can use well-known methods to construct an expression vector containing a coding gene of an exogenous protein. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, and the like.

[0368] For example, a nucleic acid construct of the "Z1-Z2" structure is inserted into a cloning site of a plasmid vector as a plasmid DNA; wherein Z1 is a promoter, "-" is a covalent bond or a nucleotide fragment, and Z2 is a coding sequence of an exogenous protein. In one preferred embodiment of Z1, it is a T7 promoter.

[0369] In one preferred embodiment, the exogenous DNA template is a circular DNA, and is further preferably a plasmid DNA. The corresponding DNA plasmid is not particularly limited, as long as it can react with the cell extract of the system to synthesize the exogenous protein. Typically, the plasmid contains functional elements such as promoters, terminators, untranslated regions (UTRs), and the like. In one preferred embodiment of the plasmid, it contains a promoter that can be recognized by the components of the cell extract. For example, plasmids containing a T7 promoter can theoretically be used as the expression vector of the exogenous DNA template used in Examples S3-S12. For example, the pET series plasmids of Escherichia coli, the pGEM series plasmids, and the like can be used to replace the plasmid vector of the Kluyveromyces lactis extract in Examples S3-S12 to implement the present application. Another preferred embodiment of the plasmid is that it contains a promoter that can be recognized by an exogenously added component.

[0370] Taking the transcription of an exogenous protein using a T7 promoter as an example, the T7 promoter can be recognized and initiated by a T7 RNA polymerase endogenously expressed in the cell extract, or by a T7 RNA polymerase added exogenously.

[0371] The linear DNA can be obtained by in vitro nucleic acid amplification technology. The amplification technology that can be used is not particularly limited, including but not limited to PCR amplification technology, isothermal amplification technology, room temperature amplification technology, room temperature amplification technology, etc. Among them, the isothermal amplification technology is preferably the room temperature amplification technology. One of the preferred embodiments is that the exogenous DNA template is linear DNA, and is a PCR linear fragment. The PCR linear fragment can be obtained by the reported PCR technology.

[0372] Another preferred embodiment is that the exogenous DNA template is linear DNA, and is a double-stranded linear DNA obtained by an amplification system. The amplification system is not particularly limited, and can be selected from existing commercial kits, literature reported amplification systems, etc. as long as it can amplify the DNA template encoding the exogenous protein of the application. For example, the commercial DNA amplification system provided by enterprises including but not limited to Biocompare, Neta Scientific Inc, ABM Company, Thermo Fisher Scientific Company, Expedeon Company, Vivantis Company, etc.

[0373] Another preferred embodiment is to use double-stranded DNA as an exogenous DNA template, and construct it in a circular plasmid vector. The plasmid vector used typically contains functional elements such as T7 promoter, T7 terminator and / or 5' and 3' UTR.

[0374] As one of the preferred embodiments, in examples S3-S12, double-stranded DNA is used as an exogenous DNA template and is constructed in a circular plasmid vector; these plasmids contain a T7 promoter as a promoter for initiating the transcription and translation of the exogenous protein; in examples S2-S12, the modified Kluyveromyces lactis can endogenously express T7 RNA polymerase, and a cell extract is prepared from the modified strain to construct an in vitro cell-free protein synthesis system. The T7 promoter of the system can be used for in vitro cell-free expression of various proteins. The plasmid also contains functional elements such as T7 terminator, UTR, etc.

[0375] In an embodiment, the plasmid DNA includes the following functional elements: promoter, 5' non-coding region, coding sequence of exogenous protein, 3' non-coding region, terminator, replication initiation site (f1 ori), AmpR promoter, ampicillin resistance gene, high copy number replication initiation site (ori), gene controlling plasmid copy number (rop gene), LacI promoter, coding sequence of lacI.

[0376] In one embodiment, the plasmid DNA includes at least the structural elements indicated in Table 1. For example, Figure 1 the plasmid structure shown.

[0377] Table 1Figure 1 Explanation of structural elements of the indicated plasmid DNA (pD2P-mEGFP)

[0378]

[0379] In another embodiment, in addition to the indicated functional elements, there is a purification tag, such as a polyhistidine tag (His-tag), between the 5' UTR and the coding sequence of mEGFP. For example, as shown in Figure 1 Figure 2

[0380] In another embodiment, in addition to the indicated functional elements, there is a kozak sequence downstream of the 5' UTR for improved translation levels. For example, as shown in Figure 1 Figure 2

[0381] In another embodiment, in addition to the indicated functional elements, there is a coding sequence for a signal peptide (signal sequence) between the 5' UTR and the coding sequence of mEGFP, downstream of the 5' UTR. Figure 1

[0382] In another embodiment, the plasmid DNA includes the following functional elements: a promoter, a 5' non-coding region, a coding sequence for a signal peptide, a coding sequence for an exogenous protein, a 3' non-coding region, a terminator, a f1 ori, an AmpR promoter, an ampicillin resistance gene, an ori, a rop gene, a LacI promoter, a coding sequence for lacI. Specifically, for example, the plasmid DNA includes the following functional elements: a T7 promoter, a 5' non-coding region, a coding sequence for a signal peptide, a coding sequence for the exogenous protein mEGFP, a 3' non-coding region, a T7 terminator, a f1 ori, an AmpR promoter, an ampicillin resistance gene, an ori, a rop gene, a LacI promoter, a coding sequence for lacI. Figure 3

[0383] In another embodiment, the plasmid DNA includes the following functional elements: a promoter, a 5' non-coding region, a coding sequence for a signal peptide, a coding sequence for an exogenous protein, a 3' non-coding region, a terminator, a f1 ori, an AmpR promoter, an ampicillin resistance gene, an ori, a rop gene, a LacI promoter, a coding sequence for lacI. Specifically, for example, the plasmid DNA includes the following functional elements: a T7 promoter, a 5' non-coding region, a coding sequence for a signal peptide, a coding sequence for the exogenous protein mEGFP, a 3' non-coding region, a T7 terminator, a f1 ori, an AmpR promoter, an ampicillin resistance gene, an ori, a rop gene, a LacI promoter, a coding sequence for lacI.

[0384] ​​​​​​In another embodiment, the plasmid DNA comprises the following functional elements: a promoter, a 5' non-coding region, a coding sequence of a signal peptide, a coding sequence of a purification tag, a multiple cloning site (MCS), a coding sequence of a foreign protein, a 3' non-coding region, a terminator, a f1 ori, an AmpR promoter, an ampicillin resistance gene, an ori, a rop gene, a LacI promoter, a coding sequence of lacI. Specifically, for example, the plasmid DNA comprises the following functional elements: a T7 promoter, a 5' non-coding region, a coding sequence of a signal peptide, a coding sequence of a purification tag, an MCS, a coding sequence of a foreign protein mEGFP, a 3' non-coding region, a LAC4 terminator or a T7 terminator, a f1 ori, an AmpR promoter, an ampicillin resistance gene, an ori, a rop gene, a LacI promoter, a coding sequence of lacI.

[0385] The basic structure of the plasmid and the method of inserting the coding gene of the foreign protein into the plasmid vector can adopt conventional technical means in the art, which will not be described here. As an example, reference can be made to patent documents such as CN108690139A, CN107574179A, CN108949801A, etc. As an example, the basic structure of the plasmid can also be referred to in Chinese patent application document CN201910460987.8.

[0386] In the present application, the concentration of the DNA template encoding a non-foreign protein can refer to the dosage of the DNA template encoding a foreign protein described above, and is determined according to the required expression amount of the non-foreign protein. The non-foreign protein refers to a translation product synthesized for the purpose of promoting the reaction, rather than the target expression protein.

[0387] Foreign mRNA template

[0388] The present application can also use a foreign mRNA template instead of a foreign DNA template, or use a mixture of a foreign mRNA template and a foreign DNA template, to be added to the above-mentioned in vitro cell-free protein synthesis system to perform in vitro protein synthesis reaction to synthesize the foreign protein encoded by the mRNA template.

[0389] In vitro nucleic acid amplification (in vitro nucleic acid amplification technology, in vitro nucleic acid amplification method)

[0390] "In vitro nucleic acid amplification" is a process of replicating nucleic acids in vitro.

[0391] The nucleic acid template for the in vitro protein synthesis system of the present application includes a nucleic acid template encoding a foreign protein, and can also optionally include a nucleic acid template encoding other proteins, which can all be prepared by using in vitro nucleic acid amplification technology.

[0392] The in vitro nucleic acid amplification technology that can be used is not particularly limited, and can be non-isothermal amplification or isothermal amplification (also known as constant temperature amplification). It includes but is not limited to polymerase chain reaction technology (PCR amplification technology), isothermal amplification technology, room temperature amplification technology, room temperature amplification technology, etc. Among them, the isothermal amplification technology is preferably the room temperature amplification technology.

[0393] Among them, the isothermal amplification technology can refer to the isothermal DNA amplification technology disclosed in the documents such as “J Kim et al. Isothermal DNA amplification in bioanalysis: strategies and applications [J]. Bioanalysis, 2011, 3(2): 227-239”, “Gill P et al. Nucleic Acid Isothermal Amplification Technologies—A Review [J]. Nucleosides, Nucleotides, and Nucleic Acids, 2008, 27(3) 224-243”, “Yong-Joo Jeong, Kkothanah Reum Park and Dong-Eun Kim. Isothermal DNA amplification in vitro: the helicase-dependent amplification system [J]. Cell. Mol. Life Sci., 2009, 66: 3325-3336”, “Lü Bei, et al. Development and continuous innovation of in vitro nucleic acid rapid amplification technology [J]. Chinese Journal of Bioengineering, 2011, 31(3): 91-96”, “Wang Lin, et al. Research progress of nucleic acid isothermal amplification technology [J]. Biotechnology Communications, 2011, 22(2)”, and the cited documents thereof. Specifically, the nucleic acid isothermal amplification method that can be used in the technical means of the present application includes but is 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-based amplification method (NASBA), rolling circle amplification method (RCA), nicking enzyme nucleic acid isothermal amplification method (nick 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), etc. One of the preferred modes is the rolling circle amplification method.

[0394] The in vitro nucleic acid amplification method, particularly the room temperature amplification method, that can be used in the present application is not particularly limited, and the room temperature amplification techniques that can be used in the existing art in vitro cell-free system are all incorporated into the scope of the present application by reference, including but not limited to rolling circle amplification (RCA), recombinase polymerase amplification (RPA), strand displacement amplification (SDA), helicase dependent amplification (HDA), 3SR technology (self-sustained sequence replication), etc. 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 Protoc. 2019, 2, 24, Y Lu. Advances in Cell-Free Biosynthetic Technology [J]. Current Developments in Biotechnology and Bioengineering, 2019, Chapter 2, 23-45, Y Lu. Cell-free synthetic biology: Engineering in an open world [J]. Synthetic and Systems Biotechnology, 2017, 2, 23-27, and other documents directly or indirectly cited by the documents, the disclosed in vitro nucleic acid amplification methods (particularly room temperature amplification methods) can be used as technical means of the present application, and are all incorporated into the present application by reference.

[0395] The in vitro nucleic acid amplification of the present application can also use SMART amplification (SMAP), single primer isothermal amplification (SPIA), exponential amplification reaction (EXPAR), heat-stable HDA (tHDA), multiple displacement amplification (MDA), restriction-assisted RCA, and other amplification techniques.

[0396] The in vitro nucleic acid amplification reaction of the present application can be continuously carried out at a specific temperature or temperature range that is conducive to the reaction. Any room temperature amplification technique of the present application also allows the reaction to be carried out under conditions with temperature fluctuations.

[0397] Incubation reaction (in vitro protein synthesis reaction)

[0398] The nucleic acid template (preferably a DNA template) encoding the exogenous protein is added to the in vitro cell-free protein synthesis system, and the reaction is incubated for a period of time to express and synthesize the exogenous protein.

[0399] The conditions for performing the in vitro protein synthesis reaction are determined according to the specific in vitro cell-free protein synthesis system, and can be referred to the reported reaction conditions, including but not limited to the reaction conditions reported in CN106978349A, CN108535489A, CN108642076A, etc. The in vitro protein synthesis reaction can be continuously performed at a specific temperature or temperature range that is conducive to the reaction. In one preferred manner, the change in the Celsius temperature of the mixture is less than 25% (for example, less than 20%, less than 15%, less than 10%, or less than 5%) throughout the reaction time, and / or the change in the temperature of the mixture is less than 15°C (for example, less than 10°C, less than 5°C, less than 2°C, or less than 1°C) throughout the reaction time. In one preferred manner, the in vitro protein synthesis is performed at room temperature. The room temperature is preferably room temperature to 37°C, and specifically, is preferably 20°C to 37°C. In one preferred manner, the temperature is 25°C to 37°C. In another preferred manner, the temperature is 20°C to 30°C. The reported room temperature protein synthesis method or isothermal protein synthesis method suitable for room temperature conditions can be used to implement the technical solutions of the present application.

[0400] The reaction time can be determined comprehensively according to the amount of raw materials (such as the amount of reaction substrates, the expected protein content, etc.), reaction efficiency, and other factors.

[0401] In one embodiment, the reaction time is 1h to 72h.

[0402] In another embodiment, the reaction time is 3h to 24h.

[0403] In another embodiment, the reaction time is 3h to 21h.

[0404] In another embodiment, the reaction time is 6h to 21h.

[0405] The reaction time can also be selected from any one of the following time lengths, or a time length between any two of the following time lengths (including both endpoints): 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, 24h, 36h, 48h.

[0406] Separation or / and detection step

[0407] The in vitro cell-free synthesis method of the exogenous protein can also optionally include a step of separating or / and detecting the exogenous protein. The separation or / and detection method can be achieved by using conventional techniques.

[0408] The second aspect of the present application provides an in vitro protein synthesis kit, comprising:

[0409] (i) the in vitro cell-free protein synthesis system in the method for in vitro cell-free synthesis of the exogenous protein according to the first aspect;

[0410] (ii) optionally, a nucleic acid template encoding the exogenous protein;

[0411] (iii) optionally, a reaction vessel;

[0412] (iv) a hydrophobic phase;

[0413] The hydrophobic phase is used to construct the hydrophobic interface in the method for in vitro cell-free synthesis of the exogenous protein according to the first aspect;

[0414] The component can be used to construct a hydrophobic interface capable of facing the in vitro cell-free protein synthesis system of the aqueous phase;

[0415] (v) a label or an instruction.

[0416] The (i) and (i) collectively provide the translation-related elements required for the synthesis of the exogenous protein.

[0417] The components of the in vitro cell-free protein synthesis system are placed in one or more containers in the form of dry powder, liquid, emulsion, suspension, or a combination thereof. One of the preferred forms of the dry powder is freeze-dried powder. The liquid includes pure substance, solution.

[0418] Using the in vitro protein synthesis kit, an in vitro protein synthesis reaction can be carried out to synthesize an exogenous protein.

[0419] The parts of the in vitro protein synthesis kit correspond to the components of the method for in vitro cell-free synthesis of the exogenous protein according to the first aspect.

[0420] The hydrophobic phase can be a substance phase or a device for constructing a hydrophobic interface.

[0421] The nucleic acid template encoding the exogenous protein is a DNA template, an mRNA template, or a combination thereof.

[0422] Preferably, the nucleic acid template encoding the exogenous protein is a DNA template.

[0423] The substrate of the reaction vessel is, for example, a cell culture plate, a centrifuge tube, etc. For example, a six-well plate, a twelve-well plate, a twenty-four-well plate, a forty-eight-well plate, a ninety-six-well plate, etc. It can be a flat-bottom plate or a sharp-bottom plate.

[0424] In one preferred embodiment, the components of the in vitro cell-free protein synthesis system together form an aqueous solution. The kit comprises a container holding the aqueous solution.

[0425] In one preferred embodiment, the kit comprises separate containers for each of the following components: (a) cell extract; (b) energy system; (c) optionally, nucleic acid template; (d) buffer; (e) optionally, pH adjusting component; (f) optionally, one or more additional solid components; (h) optionally, one or more additional liquid components. In this embodiment, components (a), (b), (c) are each independently either a dry powder or an aqueous solution. In this embodiment, components (c), (e), (f) are each independently either present or absent. The term "one or more" means one, two, or more.

[0426] In one preferred embodiment, the components are divided into two parts, a dry powder and a liquid reagent. The kit comprises two containers, one holding the dry powder components and the other holding the liquid reagent components. The liquid reagent includes any system that contains a liquid phase, either a homogeneous system or a mixed system, including but not limited to a pure substance, a solution, an emulsion, a suspension, combinations thereof.

[0427] In one preferred embodiment, the components are divided into a dry powder, a buffer, and one or more additional liquid reagents, optionally including aqueous solvent.

[0428] In one preferred embodiment, the following components are each contained in a separate container: cell extract (containing endogenously expressed RNA polymerase, optionally containing endogenously expressed DNA polymerase), energy system, substrate for RNA synthesis, substrate for protein synthesis, crowding agent, exogenous magnesium ions, exogenous potassium ions, buffer, and optionally one or more of the following: exogenously added RNA polymerase, exogenous DNA template encoding RNA polymerase, exogenously added DNA polymerase, exogenous DNA template encoding RNA polymerase, antioxidant or reducing agent, trehalose, reaction facilitator, aqueous solvent. The RNA polymerase is more preferably independently T7 RNA polymerase. The DNA polymerase is more preferably independently phi29 DNA polymerase. The cell extract contains transfer RNA (tRNA), ribosomes. The cell extract is preferably a eukaryotic cell extract, more preferably a Kluyveromyces cell extract, more preferably a Kluyveromyces lactis cell extract.

[0429] In one preferred mode, the following components are separately contained in different containers: cell extract (without endogenous integrated coding sequence / coding gene of RNA polymerase and without endogenous integrated coding sequence / coding gene of DNA polymerase), exogenously added RNA polymerase, energy system, substrate for synthesizing RNA, substrate for synthesizing protein, crowding agent, exogenously added magnesium ion, exogenously added potassium ion, buffer, and optionally including the following components in separate containers: exogenous DNA template for coding RNA polymerase, exogenously added DNA polymerase, exogenous DNA template for coding DNA polymerase, antioxidant or reducing agent, trehalose, reaction promoter, aqueous solvent. The RNA polymerase is more preferably independently T7 RNA polymerase. The DNA polymerase is more preferably independently phi29 DNA polymerase. The cell extract contains transport RNA and ribosome. The cell extract is preferably eukaryotic cell extract, more preferably Kluyveromyces cell extract, more preferably Kluyveromyces lactis cell extract.

[0430] The third aspect of the present application provides the use of alkanes in the in vitro cell-free synthesis method of the exogenous protein of the first aspect, or in the in vitro protein synthesis kit of the second aspect, or in the in vitro protein synthesis; wherein the alkanes are used to construct a hydrophobic interface.

[0431] Preferably, the use in the in vitro protein synthesis includes but is not limited to the use in protein manufacturing, or the use in detection based on protein synthesis, etc.

[0432] The application field of the in vitro cell-free synthesis method of the exogenous protein includes but is not limited to the fields of biological medicine, molecular biology, medicine, in vitro detection, medical diagnosis, regenerative medicine, biological engineering, tissue engineering, stem cell engineering, genetic engineering, polymer engineering, surface engineering, nano-engineering, cosmetics, food, food additives, nutritional agents, agriculture, feed, daily necessities, washing, environment, chemical dyeing, fluorescent labeling, etc.

[0433] The specific embodiments and drawings are described below. Figures 1-13to further illustrate the present application. It is to be understood that these examples are only used to illustrate the present application and not intended to limit the scope of the present application. The experimental methods in the following examples, if not otherwise specified, are generally carried out according to the conventional conditions, such as the experimental conditions described in the literatures, such as "Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989)", "Cell-free Protein Synthesis: Methods and Protocols" Edited by Alexander S. Spirin and James R. Swartz. 2008", or according to the conditions suggested by the manufacturers, or according to the conditions directed by or with reference to the above-mentioned detailed description. Unless otherwise specified, the percentages and parts mentioned in the present application are weight percentages and weight parts.

[0434] Unless otherwise specified, the materials and reagents used in the examples of the present application are commercially available products.

[0435] Examples 1-12 have the same meaning as Examples S1-S12, and can be used interchangeably.

[0436] In the present application, Kluyveromyces lactis (abbreviated as K. lactis or kl) is used as the source of cell extract in Examples S2-S12. In Examples S3-S12 of the present application, the cell extract is prepared from a modified strain of K. lactis. It should be noted that the same design and analysis, experimental methods are also applicable to other sources of cell extract described herein, including but not limited to, for example, prokaryotic cells (such as E. coli), for example, other yeast cells, for example, plant cells, insect cells, animal cells (such as mammalian cells, specifically, mouse, rabbit, monkey, human, etc.) and other eukaryotic cells.

[0437] The plasmid expression vectors used in the examples of the present application are only used to specifically illustrate the embodiments of the present application, and do not limit the scope of the present application; other plasmid vectors that can be used to implement the present application include but are not limited to common plasmid vectors that can be purchased through existing commercial channels, such as pET series plasmids, pGEM series plasmids, etc.

[0438] In the following examples, the alkanes used are linear chain structures (n-structures, straight chain structures) unless otherwise specified. For example, decane without specific limitation refers to n-decane. The physical parameters of various alkanes used in the following examples are shown in Table 2.

[0439] Table 2. Parameter description of hydrocarbon substances used in Examples S3-S12

[0440]

[0441] The reaction container is a 24-well plate or a 48-well plate, both of which are flat-bottomed cell culture plates. The bottom area of the 24-well plate is 1.77 cm 2 , and the bottom area of the 48-well plate is 0.8 cm 2 . 3

[0443] In the following examples, a negative control group (NC group) is set, without adding alkanes and without adding DNA templates encoding fluorescent proteins, and the other reaction conditions are consistent with those of the experimental groups in the examples. The RFU values of the reaction after 3 h and 18-22 h relative to the blank control group (BC group) can be ignored, and the RFU value is not more than 35. The experimental results of some NC groups are not shown in the charts.

[0444] Example S1: Preparation of nucleic acid template encoding exogenous protein mEGFP

[0445] A plasmid vector expressing mEGFP was constructed, and in vitro DNA amplification was performed to prepare the plasmid encoding the exogenous protein mEGFP.

[0446] The enhanced green fluorescent protein (mEGFP) was selected as the exogenous protein as the target expression product, and its amino acid sequence is shown in SEQ ID No. 2.

[0447] A plasmid vector was selected. An artificial plasmid vector designed for Kluyveromyces lactis cell extract was used, which contains functional elements such as T7 promoter, T7 terminator, 5' UTR and 3' UTR. This plasmid vector can be combined with Kluyveromyces lactis cell extract containing endogenous T7 RNA polymerase to construct an in vitro cell-free protein synthesis system to express various exogenous proteins in vitro.

[0448] Using PCR amplification and homologous fragment recombination methods, the DNA fragment containing the coding gene of mEGFP was inserted into the plasmid vector to construct a plasmid vector expressing mEGFP, denoted as plasmid D2P-mEGFP (abbreviated as pD2P-mEGFP), with a total length of 6384 bp. The plasmid was confirmed to be correct by gene sequencing. The gene sequence encoding mEGFP is shown in SEQ ID No. 1.

[0449] The map of the pD2P-mEGFP plasmid is shown in Figure 1 , and its structural elements are shown in Table 3.

[0450] Table 3: Structural element explanation of the plasmid (pD2P-mEGFP) encoding the exogenous protein mEGFP

[0451]

[0452] DNA amplification was performed. The amplification reaction system included the following components at the final concentrations: 1-5 μM random primers (primer sequence: NNNNNNN), 1.14 ng / μL of the above-mentioned plasmid (pD2P-mEGFP as a template), 0.5-1 mM dNTP mixture, 0.1-0.5 mg / mL BSA, 0.05-0.1 mg / mL phi29 DNA polymerase, and 1x phi29 reaction buffer (composition: 200 mM Tris-HCl, 20 mM MgCl2, 10 mM (NH4)2SO4, 10 mM KCl, pH 7.5). After mixing the above-mentioned reaction system, it was placed in an environment at 30°C for 2 h. The DNA template was obtained, and the nucleic acid concentration was determined by ultraviolet spectrophotometry. The reaction solution was refrigerated for standby, and used as a nucleic acid template in subsequent examples.

[0453] Example S2: Preparation of cell extract

[0454] In examples S3-S12 of the present application, a modified strain of Kluyveromyces lactis was used to prepare the cell extract.

[0455] The source of the cell extract was Kluyveromyces lactis (K. lactis). A modified strain based on Kluyveromyces lactis strain ATCC8585 was used, and the method described in CN109423496A was used to integrate the coding gene of T7 RNA polymerase into the genome of Kluyveromyces lactis, so that the modified strain can endogenously express T7 RNA polymerase. According to the comparison of the control experiment, without adding any exogenous RNA polymerase, Kluyveromyces lactis without endogenous integration of the coding gene of T7 RNA polymerase can hardly perform in vitro protein synthesis reaction; after the above-mentioned endogenous integration modification, without adding any exogenous RNA polymerase, it can achieve efficient expression of exogenous proteins; it can achieve the protein synthesis level of the traditional in vitro protein synthesis system (in the traditional in vitro protein synthesis system, a strain without T7 RNA polymerase endogenous modification is used to prepare the cell extract, and an exogenous T7 RNA polymerase is added in the synthesis system). The hydrophobic interface optimization method of examples S3-S12 of the present application is also applicable to the in vitro protein synthesis system of strains without T7 RNA polymerase endogenous modification (including but not limited to Kluyveromyces lactis strains), and can achieve the same or similar optimization effect.

[0456] The preparation process of the Kluyveromyces lactis cell extract adopts conventional technical means and refers to the method described in CN109593656A. In general, the preparation steps include: providing fermented and cultured Kluyveromyces lactis cell raw materials, rapidly freezing the cells with liquid nitrogen, crushing the cells, and centrifuging to collect the supernatant, which is the cell extract.

[0457] The protein concentration in the obtained Kluyveromyces lactis cell extract is 20-40 mg / mL.

[0458] Embodiments S2-S12 of the present application use the following Kluyveromyces lactis cell extract (lysate).

[0459] Table 4 Information of Kluyveromyces lactis (K. lactis) cell extract used in embodiments S2-S12

[0460]

[0461] Effect of cyclohexane (liquid) on protein synthesis capacity of in vitro protein synthesis system

[0462] 3.1 In vitro cell-free protein synthesis system (without adding exogenous RNA polymerase)

[0463] The volume of each system is 300 μL, and the reaction is carried out in a flat-bottom 48-well plate with a bottom area of 0.8 cm 2 Each sample is set up in triplicate, and the mean and standard deviation (error bar) are calculated.

[0464] In vitro cell-free protein synthesis system: the final concentrations of the components are as follows: 9.78 mM Tris-HCl (pH 8.0), 80 mM potassium acetate, 5.0 mM magnesium acetate, 1.5 mM nucleotide triphosphate mixture (adenine nucleotide triphosphate, guanine nucleotide triphosphate, cytosine nucleotide triphosphate, and uracil nucleotide triphosphate, each at a concentration of 1.5 mM), 0.7 mM amino acid mixture (glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine, each at a concentration of 0.7 mM), 0.44 mM dithiothreitol (DTT), 2% (w / v) polyethylene glycol 8000, 15 mM glucose, 52.48 mg / mL maltodextrin, 24 mM potassium phosphate, and 50% by volume of Kluyveromyces lactis cell extract. The water phase system is formed by mixing.

[0465] 3.2 Reaction interface (providing a hydrophobic interface)

[0466] Experimental group (concentration curve of cyclohexane): 0.1% (v / v) to 20% (v / v) cyclohexane was added to the system respectively; wherein, the cyclohexane is in liquid state. After mixing, an oil phase layer is formed on the water phase system, and a hydrophobic interface is provided by the reaction interface between the oil phase layer and the water phase.

[0467] Blank control group (BC group): relative to the above experimental group, no alkane (cyclohexane addition amount is 0) is added.

[0468] Negative control group (NC group): relative to the above experimental group, no alkane is added, and no DNA template is added subsequently.

[0469] 3.3 Perform in vitro protein synthesis reaction: the NC group does not add exogenous DNA template; 15 ng / μL of DNA template encoding mEGFP (obtained by in vitro RCA amplification in the above embodiment S1) is added to each independent in vitro cell-free protein synthesis system of the above experimental group and BC group respectively, and after mixing, all systems are placed in an environment of 20-30°C and shaken overnight. Sample at 3h, 18h for fluorescence protein activity test.

[0470] 3.4 Fluorescent protein activity determination: place the sample to be tested in Envision 2120 multifunctional enzyme label instrument (PerkinElmer), detect the strength of the fluorescence signal, and take the relative fluorescence unit value (Relative Fluorescence Unit, RFU) as the activity unit. The size of the RFU value can reflect the amount of mEGFP protein synthesis, and the conversion relationship between the mass volume concentration X (unit μg / mL) of mEGFP and the RFU value is: Within the test range of the present application, the linear relationship between X and RFU is basically met.

[0471] Sample the reaction system for fluorescence test respectively. Sample treatment: 4000 rpm, 25°C (room temperature) centrifugation for 1 minute. Place the sample to be tested in Envision 2120 multifunctional enzyme label instrument, and the detection wavelength used is excitation wavelength / emission wavelength (Ex / Em): 488nm / 507nm, and the relative fluorescence unit value (RFU) is obtained by detection.

[0472] 3.5 Experimental results: as shown in Figure 4 After 18h of reaction, when the addition amount of cyclohexane is 0.1% (v / v) to 5% (v / v), the RFU value of the experimental group is higher than that of the blank control group (BC group), which improves the protein synthesis capacity of the in vitro protein synthesis system. Especially when the addition amount of cyclohexane is 1.8% (v / v), the RFU value of the experimental group is increased by 25.64% relative to the BC group, and the synthesis amount of exogenous protein mEGFP is increased by 25.64%.

[0473] Example S4 Effect of isooctane (liquid) on protein synthesis ability of in vitro protein synthesis system

[0474] 4.1 In vitro cell-free protein synthesis system (without adding exogenous RNA polymerase)

[0475] The same in vitro cell-free protein synthesis system in water phase as 3.1 in Example 3 was used. Each system had a volume of 300 μL, and the reaction was carried out in a flat-bottom 48-well plate.

[0476] 4.2 Reaction interface (providing hydrophobic interface)

[0477] Experimental group (concentration curve of isooctane): 0.5% (v / v) to 5% (v / v) isooctane was added to the system, respectively; the isooctane was in liquid state. After mixing, an oil phase layer was formed on the water phase system, and the hydrophobic interface was provided by the reaction interface between the oil phase and the water phase.

[0478] Blank control group (BC group): relative to the above experimental group, no alkane was added (the amount of isooctane added was 0).

[0479] Negative control group (NC group): relative to the above experimental group, no alkane was added, and no DNA template was added subsequently.

[0480] 4.3 In vitro protein synthesis reaction: the NC group did not add exogenous DNA template; 15 ng / μL of DNA template encoding mEGFP (obtained by in vitro RCA amplification in Example S1) was added to each independent in vitro cell-free protein synthesis system of the above experimental group and BC group, respectively, and after mixing, all systems were placed in an environment of 20-30°C and shaken overnight. The samples were taken at 3h and 18h for fluorescence protein activity test.

[0481] 4.4 Fluorescent protein activity determination: the method of 3.4 in Example 3 was used to determine the RFU value of the synthesized exogenous fluorescent protein mEGFP in the sample.

[0482] 4.5 Experimental results: as shown in Table 2 Figure 5The addition amount of isooctane is 0.5% (v / v) to 5% (v / v) after 18 hours of reaction, and the RFU value of the experimental group is higher than that of the blank control group (BC group), which improves the protein synthesis capacity of the in vitro protein synthesis system. Especially when the addition amount of isooctane is 0.5% (v / v), 0.8% (v / v), 1% (v / v), 1.2% (v / v), 1.4% (v / v), 4% (v / v), and 5% (v / v), the RFU value of the experimental group is increased by 89.16%, 88.58%, 75.29%, 77.15%, 70.04%, 73.66%, and 79.49% respectively relative to the BC group, which significantly increases the synthesis amount of exogenous protein mEGFP.

[0483] Example S5 Influence of decane (liquid) on the protein synthesis capacity of the in vitro protein synthesis system

[0484] 5.1 In vitro cell-free protein synthesis system (without adding exogenous RNA polymerase)

[0485] The same aqueous phase in vitro cell-free protein synthesis system as in 3.1 of Example 3 was used. The volume of each system was 300 μL, and the reaction was carried out in a flat-bottom 48-well plate.

[0486] 5.2 Reaction interface (providing a hydrophobic interface)

[0487] Experimental group (concentration curve of n-decane): 0.1% (v / v) to 5% (v / v) n-decane (n-decane) was added to the system, respectively; wherein the n-decane was in liquid state. After mixing, an oil phase layer was formed above the aqueous phase system, and a hydrophobic interface was provided through the reaction interface between the oil phase and the aqueous phase.

[0488] Blank control group (BC group): relative to the above experimental group, no alkane (n-decane addition amount is 0) was added.

[0489] Negative control group (NC group): relative to the above experimental group, no alkane was added, and no DNA template was added subsequently.

[0490] 5.3 Perform in vitro protein synthesis reaction: NC group does not add exogenous DNA template; 15 ng / μL of DNA template encoding mEGFP (obtained by in vitro RCA amplification in the above Example S1) was added to each independent in vitro cell-free protein synthesis system of the above experimental group and BC group, respectively, and mixed well. All systems were placed in an environment of 20-30°C and shaken overnight. Sampling was performed at 3h and 18h for fluorescence protein activity test.

[0491] 5.4 Fluorescent protein activity determination: the method of 3.4 in Example 3 was used to determine the RFU value of the synthesized exogenous fluorescent protein mEGFP in the sample.

[0492] 5.5 Experimental results: as shown in Figure 6 Table 5.5. After 18h of reaction, the RFU values of the experimental groups were higher than that of the blank control group (BC group) when the addition amount of decane was 0.1% (v / v)~1.4% (v / v), 2% (v / v), and 5% (v / v), which improved the protein synthesis capacity of the in vitro protein synthesis system. Especially when the addition amount of decane was 1.2% (v / v), 1.4% (v / v), and 5% (v / v), the RFU values of the experimental groups were increased by 28.66%, 23.93%, and 22.18% respectively relative to the BC group, which significantly improved the synthesis amount of the exogenous protein mEGFP.

[0493] Example S6 Tetradecane (liquid) effect on the protein synthesis capacity of the in vitro protein synthesis system

[0494] 6.1 In vitro cell-free protein synthesis system (without adding exogenous RNA polymerase)

[0495] The same aqueous phase in vitro cell-free protein synthesis system as in 3.1 of Example 3 was used. The volume of each system was 300 μL, and the reaction was carried out in a flat-bottom 48-well plate.

[0496] 6.2 Reaction interface (providing a hydrophobic interface)

[0497] Experimental group (tetradecane concentration curve): 0.1% (v / v)~2% (v / v) tetradecane was added to the system; the tetradecane was in a liquid state. After mixing, an oil phase layer was formed above the aqueous phase system, and a hydrophobic interface was provided through the reaction interface between the oil phase and the aqueous phase.

[0498] Blank control group (BC group): relative to the above experimental group, no alkane was added (the addition amount of tetradecane was 0).

[0499] Negative control group (NC group): relative to the above experimental group, no alkane was added, and no DNA template was added subsequently.

[0500] 6.3 In vitro protein synthesis reaction: the NC group did not add exogenous DNA template; 15 ng / μL of DNA template encoding mEGFP (obtained by in vitro RCA amplification in Example S1) was added to each independent in vitro cell-free protein synthesis system of the above experimental groups and BC group, respectively, and mixed well. All systems were placed in an environment of 20-30°C and shaken overnight. Samples were taken at 3h and 18h for fluorescence protein activity testing.

[0501] 6.4 Fluorescent protein activity determination: the method of 3.4 in Example 3 was used to determine the RFU value of the synthesized exogenous fluorescent protein mEGFP in the sample.

[0502] 6.5 Experimental results: as shown inFigure 7 The RFU values of the experimental groups were higher than that of the blank control group (BC group) when the addition amount of tetradecane was 0.1% (v / v) to 2% (v / v) after 3h and 18h of reaction. The protein synthesis amount of the in vitro protein synthesis system was increased by 10.24% to 26.61% at 3h and by 11.73% to 47.09% at 18h. See Table 5 below for details. Both the reaction efficiency and the protein synthesis amount were improved.

[0503] Table 5 Experimental results of optimizing the in vitro protein synthesis system by providing a hydrophobic interface with tetradecane (see Example S6) Figure 7 )

[0504]

[0505]

[0506] Example S7 Effect of tetradecane (liquid) on the protein synthesis capacity of the in vitro protein synthesis system

[0507] 7.1 In vitro cell-free protein synthesis system (without adding exogenous RNA polymerase)

[0508] The same aqueous phase in vitro cell-free protein synthesis system as in 3.1 of Example 3 was used. The volume of each system was 300 μL, and the reaction was carried out in a flat-bottom 48-well plate.

[0509] 7.2 Reaction interface (providing a hydrophobic interface)

[0510] Experimental group (tetradecane concentration curve): 2% (v / v) to 20% (v / v) of tetradecane was added to the system, respectively; the tetradecane was in a liquid state. After mixing, an oil phase layer was formed above the aqueous phase system, and a hydrophobic interface was provided through the reaction interface between the oil phase and the aqueous phase.

[0511] Blank control group (BC group): relative to the above experimental group, no alkane was added (the addition amount of tetradecane was 0).

[0512] Negative control group (NC group): relative to the above experimental group, no alkane was added, and no DNA template was added subsequently.

[0513] 7.3 Perform in vitro protein synthesis reaction: the NC group does not add exogenous DNA template; 15 ng / μL of DNA template encoding mEGFP (obtained by in vitro RCA amplification in Example S1) was added to each independent in vitro cell-free protein synthesis system of the above experimental group and BC group, respectively, and mixed well. All systems were placed in an environment of 20-30°C and shaken overnight. Sampling was performed at 3h and 18h for fluorescence protein activity testing.

[0514] 7.4 Fluorescent protein activity assay: The RFU value of the synthesized exogenous fluorescent protein mEGFP in the sample was determined by the method of 3.4 in Example 3.

[0515] 7.5 Experimental results: As shown in Figure 8 After 3h of reaction, the RFU value of the experimental group was higher than that of the blank control group (BC group) when the addition amount of tetradecane was 2% (v / v) to 20% (v / v), which was 1.65% to 34.12% higher, respectively, thereby improving the reaction efficiency. After 18h of reaction, the RFU value of the experimental group was higher than that of the blank control group (BC group) when the addition amount of tetradecane was 2% (v / v) and 5% (v / v), which increased the protein synthesis amount of the in vitro protein synthesis system by 21.10% and 11.77%, respectively, thereby increasing the protein synthesis amount.

[0516] Example S8 Influence of pentadecylcyclohexane (liquid) on the protein synthesis capacity of in vitro protein synthesis system

[0517] 8.1 In vitro cell-free protein synthesis system (without adding exogenous RNA polymerase)

[0518] The same aqueous phase in vitro cell-free protein synthesis system as in 3.1 of Example 3 was used. The volume of each system was 300 μL, and the reaction was carried out in a flat-bottom 48-well plate.

[0519] 8.2 Reaction interface (providing a hydrophobic interface)

[0520] Experimental group (concentration curve of pentadecylcyclohexane): 0.1% (v / v) to 20% (v / v) of pentadecylcyclohexane was added to the system, respectively; wherein the pentadecylcyclohexane was in a liquid state. After mixing, an oil phase layer was formed above the aqueous phase system, and a hydrophobic interface was provided through the reaction interface between the oil phase and the aqueous phase.

[0521] Blank control group (BC group): relative to the above experimental group, no alkane (pentadecylcyclohexane addition amount was 0) was added.

[0522] Negative control group (NC group): relative to the above experimental group, no alkane (pentadecylcyclohexane addition amount was 0) was added, and no DNA template encoding exogenous protein was added subsequently.

[0523] 8.3 In vitro protein synthesis reaction: the NC group did not add exogenous DNA template; 15 ng / μL of DNA template encoding mEGFP (obtained by in vitro RCA amplification in the above Example S1) was added to each independent in vitro cell-free protein synthesis system of the above experimental group and BC group, respectively, and mixed. After that, all systems were placed in an environment of 20-30°C and shaken overnight. Fluorescent protein activity test was carried out at 3h and 18h, respectively.

[0524] 8.4 Fluorescent protein activity assay: The RFU value of the synthesized exogenous fluorescent protein mEGFP in the sample was determined using the method of 3.4 in Example 3.

[0525] 8.5 Experimental results: As shown in Figure 9 After 3h of reaction, the RFU value of the experimental group was higher than that of the blank control group (BC group) when the addition amount of pentadecylcyclohexane was 0.1% (v / v) to 15% (v / v), which was 0.03% to 14.97% higher, and the protein synthesis efficiency was improved. After 18h of reaction, the RFU value of the experimental group was higher than that of the blank control group (BC group) when the addition amount of pentadecylcyclohexane was 12% (v / v) to 20% (v / v), and the protein synthesis amount of the in vitro protein synthesis system was increased by 20.35% to 36.60%. When the addition amount of pentadecylcyclohexane was 12% (v / v), 15% (v / v), 18% (v / v), and 20% (v / v), respectively, the RFU value of the experimental group was increased by 20.35%, 29.50%, 36.60%, and 23.00% compared with the BC group, which significantly improved the synthesis amount of the exogenous protein mEGFP.

[0526] Example S9 Effect of tetradecane (solid powder) on protein synthesis capacity of in vitro protein synthesis system

[0527] 9.1 In vitro cell-free protein synthesis system (without adding exogenous RNA polymerase)

[0528] The same aqueous in vitro cell-free protein synthesis system as in 3.1 of Example 3 was used. The volume of each system was 300 μL, and the reaction was carried out in a flat-bottom 48-well plate.

[0529] 9.2 Reaction interface (providing a hydrophobic interface)

[0530] Experimental group (concentration curve of tetradecane): 0-500 μg / μL of tetradecane was added to the system, respectively; wherein the tetradecane was in solid state. After mixing, a suspension was formed, and after complete dissolution, an oil phase layer floating on the reaction solution was formed.

[0531] Blank control group (BC group): relative to the above experimental group, no alkane (tetradecane addition amount is 0) was added.

[0532] Negative control group (NC group): relative to the above experimental group, no alkane (tetradecane addition amount is 0) was added, and the subsequent DNA template encoding the exogenous protein was not added.

[0533] 9.3 Perform in vitro protein synthesis reaction: NC group without adding exogenous DNA template; add 15 ng / μL of DNA template encoding mEGFP (obtained by in vitro RCA amplification in Example S1 above) into each independent in vitro cell-free protein synthesis system of the above experimental groups and BC group, mix well, and then place all systems in an environment of 20-30°C and shake overnight. During the reaction, tetradecane exists in the oil layer on the top. Sample at 3h and 18h for fluorescence protein activity test.

[0534] 9.4 Fluorescent protein activity determination: use the method of 3.4 in Example 3 to determine the RFU value of the synthesized exogenous fluorescent protein mEGFP in the sample.

[0535] 9.5 Experimental results: as shown in Figure 10 After 18h of reaction, the added amount of tetradecane is 0-500 μg / μL, and the RFU value of the experimental group is higher than that of the blank control group (BC group), and the protein synthesis amount of the in vitro protein synthesis system is increased by 20.35%-36.60%. When the added amount of tetradecane is 0.02% (v / v), 0.035% (v / v), 0.04% (v / v), and 0.05% (v / v), the RFU value of the experimental group is increased by 26.47%, 30.90%, 27.97%, and 26.86% respectively relative to the BC group, significantly increasing the synthesis amount of the exogenous protein mEGFP.

[0536] Example S10 Influence of vaseline (paste) on the protein synthesis capacity of in vitro protein synthesis system

[0537] 10.1 In vitro cell-free protein synthesis system (without adding exogenous RNA polymerase)

[0538] Each system has a volume of 300 μL, and the reaction is carried out in a flat-bottom 48-well plate with a bottom area of 0.8 cm 2 for each well. Three parallel samples are set for each sample, and the mean value and standard deviation (error bar) are calculated.

[0539] In vitro cell-free protein synthesis system: the final concentrations of each component are as follows: 9.78 mM Tris-HCl (pH 8.0), 80 mM potassium acetate, 5.0 mM magnesium acetate, 1.5 mM nucleotide triphosphate mixture (adenine nucleotide triphosphate, guanine nucleotide triphosphate, cytosine nucleotide triphosphate and uracil nucleotide triphosphate, each at a concentration of 1.5 mM), 0.7 mM amino acid mixture (glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine, each at a concentration of 0.7 mM), 0.44 mM dithiothreitol (DTT), 2% (w / v) polyethylene glycol 8000, 15 mM glucose, 52.48 mg / mL maltodextrin, 24 mM potassium phosphate, 50% by volume of Kluyveromyces lactis cell extract. Among them, the cell extract is numbered ZS12181, and the protein concentration is 25.16 mg / mL.

[0540] 10.2 Reaction interface (providing a hydrophobic interface)

[0541] Experimental group (concentration curve of vaseline): 5 μg / μL to 450 μg / μL of vaseline (corresponding to 1 mg to 90 mg of solid alkane added to each 200 μL of reaction system) was added to the system; wherein the vaseline is a paste-like alkane. It is applied to the inner wall and side wall of the bottom of the hole, and the amount added is measured by the weight difference before and after application. The hydrophobic interface is provided by the interface between the vaseline layer and the aqueous phase.

[0542] Blank control group (BC group): relative to the above experimental group, no alkane is added (the amount of vaseline applied is 0).

[0543] Negative control group (NC group): relative to the above experimental group, no alkane is added (the amount of vaseline applied is 0), and no DNA template encoding an exogenous protein is added subsequently.

[0544] 10.3 Perform in vitro protein synthesis reaction: the NC group does not add an exogenous DNA template; 15 ng / μL of DNA template encoding mEGFP (obtained by in vitro RCA amplification according to the above embodiment S1) is added to each independent in vitro cell-free protein synthesis system of the above experimental group and BC group, respectively, and mixed well. All systems are placed in an environment of 20-30°C and shaken overnight. Sample is taken at 18h for fluorescence protein activity test.

[0545] 10.4 Fluorescent protein activity determination: the method of 3.4 in embodiment 3 is used to determine the RFU value of the synthesized exogenous fluorescent protein mEGFP in the sample.

[0546] 10.5 Experimental results: as shown in Figure 11 After 18h of reaction, the RFU values of the experimental groups were higher than that of the blank control group (BC group) when the amount of vaseline added was 1mg-3.5mg / well, and the amount of protein synthesis in the in vitro protein synthesis system was increased by 9.96%-39.95%. When the amount of vaseline added in each well was 1mg, 1.5mg, 2.5mg and 3.5mg, respectively, the RFU values of the experimental groups were increased by 39.95%, 21.42%, 21.91% and 18.99% relative to the BC group, significantly increasing the amount of exogenous protein mEGFP synthesis.

[0547] Example S11 Optimization of the combination of vaseline and activated carbon for in vitro protein synthesis system

[0548] 11.1 In vitro cell-free protein synthesis system (without adding exogenous RNA polymerase)

[0549] The volume of each system was 300μL, and the reaction was carried out in a flat-bottom 48-well plate with a bottom area of 0.8cm 2 Each sample was set up in triplicate, and the mean and standard deviation (error bar) were calculated.

[0550] In vitro cell-free protein synthesis system: the final concentrations of the components were as follows: 9.78mM Tris (pH 8.0, adjusted with hydrochloric acid), 80mM potassium acetate, 5.0mM magnesium acetate, 1.5mM nucleotide triphosphate mixture (adenine nucleotide triphosphate, guanine nucleotide triphosphate, cytosine nucleotide triphosphate and uracil nucleotide triphosphate, each at a concentration of 1.5mM), 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), 0.44mM dithiothreitol, 2% (w / v) polyethylene glycol 8000, 15mM glucose, 52.48mg / mL maltodextrin, 24mM potassium phosphate, 50% (by volume) of Kluyveromyces lactis cell extract. The cell extract was numbered ZS01092, and the protein concentration was 23mg / mL.

[0551] 11.2 Reaction interface (providing a hydrophobic interface)

[0552] BC group (blank control group): no alkane (vaseline application amount was 0) and no activated carbon were added.

[0553] Vas group (vaseline group): 0.005 g / mL vaseline was applied to the inner wall of the reactor's side wall and bottom (1 mg per well). The hydrophobic interface was provided by the interface between the vaseline layer and the aqueous phase.

[0554] AC group (activated carbon group): 0.04 g / mL activated carbon particles were added to the system (12 mg per well), which were deposited on the inner wall of the bottom.

[0555] AC+Vas group (activated carbon + vaseline combination optimization group): 0.005 g / mL vaseline was added to the system (1 mg per well), and 0.04 g / mL activated carbon particles were added (12 mg per well).

[0556] NC group (negative control group): no alkanes were added (the amount of vaseline applied was 0), no activated carbon was added, and no DNA template encoding an exogenous protein was subsequently added.

[0557] 11.3 Perform in vitro protein synthesis reactions: the NC group does not add an exogenous DNA template; 15 ng / μL of DNA template encoding mEGFP (obtained by in vitro RCA amplification in Example S1) is added to each independent in vitro cell-free protein synthesis system of the above-mentioned BC group, Vas group, AC group, and AC+Vas group, respectively, and mixed well. All systems are placed in an environment of 20-30°C and shaken overnight. Samples are taken at 3h, 6h, and 21h for fluorescence protein activity testing.

[0558] 11.4 Fluorescent protein activity determination: the method of 3.4 in Example 3 is used to determine the RFU value of the synthesized exogenous fluorescent protein mEGFP in the sample.

[0559] 11.5 Experimental results: as shown in Table 1. Figure 12 After 21h of reaction, the RFU value of the Vas group with vaseline applied to the reactor was increased by 40.38% compared to the BC group without vaseline applied; the RFU value of the AC+Vas group with vaseline applied to the reactor was increased by 57.06% compared to the AC group without vaseline applied, and by 72.64% compared to the BC group; the combination of vaseline and activated carbon was optimized, and the RFU value was increased by 171.13% compared to the blank control group (BC group), showing a synergistic optimization effect. In addition, compared with the blank control group (BC group), the Vas group, AC group, and AC+Vas group were increased by 61.79%, 56.61%, and 103.98% at 3h, and by 48.28%, 81.91%, and 144.56% at 6h, respectively.

[0560] Example S12 Combination optimization of vaseline on in vitro protein synthesis system

[0561] 12.1 In vitro cell-free protein synthesis system (without adding exogenous RNA polymerase)

[0562] Each system volume was 300 μL, and the reaction was carried out in a flat-bottom 48-well plate with a bottom area of 0.8 cm 2 Three parallel samples were set for each sample, and the mean value and standard deviation (error bar) were calculated.

[0563] In vitro cell-free protein synthesis system: the final concentrations of each component were as follows: 9.78 mM Tris-HCl (pH 8.0), 80 mM potassium acetate, 5.0 mM magnesium acetate, 1.5 mM nucleotide triphosphate mixture (adenine nucleotide triphosphate, guanine nucleotide triphosphate, cytosine nucleotide triphosphate, and uracil nucleotide triphosphate, each at a concentration of 1.5 mM), 0.7 mM amino acid mixture (glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine, each at a concentration of 0.7 mM), 0.44 mM DTT, 2% (w / v) polyethylene glycol 8000, 15 mM glucose, 52.48 mg / mL maltodextrin, 24 mM potassium phosphate, 0.3 mg / mL ampicillin (Amp), 16.6 μg / mL salmon sperm DNA (ssDNA), and 50% by volume of Kluyveromyces lactis cell extract. The cell extract was numbered ZS12181, and the protein concentration was 25.16 mg / mL.

[0564] 12.2 Reaction interface (providing a hydrophobic interface)

[0565] AC+ssDNA+Amp group (BC group, blank control group): the amount of vaseline applied was 0, and 12 mg of activated carbon was added to each well.

[0566] Vas+AC+ssDNA+Amp group (vaseline group): 0.05 g / mL of vaseline was applied to the side wall and inner wall of the bottom of the reactor (1 mg was applied to each well), and 12 mg of activated carbon was added to each well. The hydrophobic interface was provided through the interface between the vaseline layer and the aqueous phase.

[0567] NC group (negative control group): no alkane (the amount of vaseline applied was 0) and no activated carbon were added, and no DNA template encoding an exogenous protein was subsequently added.

[0568] 12.3 In vitro protein synthesis reaction: No exogenous DNA template was added to the NC group; 15 ng / μL of DNA template encoding mEGFP (obtained by in vitro RCA amplification in Example S1) was added to each independent in vitro cell-free protein synthesis system in the BC and vaseline groups. After mixing, all systems were placed at 20°C to 30°C and shaken overnight. Samples were collected at 3, 6, and 21 hours for fluorescent protein activity testing.

[0569] 12.4 Fluorescent protein activity determination: The RFU value of the exogenous fluorescent protein mEGFP synthesized in the sample was determined using the method described in 3.4 of Example 3.

[0570] 12.5 Experimental results: Figure 13 After 3h, 6h, and 21h of reaction, the RFU values ​​of the Vas+AC+ssDNA+Amp group (Vaseline group) in which Vaseline was applied to the reactor increased by 9.06%, 6.14%, and 20.27%, respectively, compared with the BC group without Vaseline.

[0571] The above are only some preferred embodiments of the present invention, and the present invention is not limited to the contents of the above embodiments. For those skilled in the art, various changes and modifications may be made within the scope of the concept of the technical solution of the present invention or under the guidance and inspiration of the technical solution of the present invention. Any changes and modifications made with equivalent technical effects are within the scope of protection of the present invention. Sequence Listing <110> Kangma (Shanghai) Biotechnology Co., Ltd. <120> An in vitro cell-free protein synthesis method based on hydrophobic interface, D2P kit and related applications <130> 2020 <141> 2020-03-16 <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 717 <212> DNA <213> artificial sequence <400> 1 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60 ggcgacgtaa acggccacaa gttcagcgtg cgcggcgagg gcgagggcga tgccaccaac 120 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatctcc 300 ttcaaggacg acggcaccta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420 aagctggagt acaacttcaa cagccacaac gtctatatca cggccgacaa gcagaagaac 480 ggcatcaagg cgaacttcaa gatccgccac aacgtcgagg acggcagcgt gcagctcgcc 540 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600 tacctgagca cccagtccaa gctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaag 717 <210> 2 <211> 239 <212> PRT <213> Artificial sequence <400> 2 Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15 Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Arg Gly 20 25 30 Glu Gly Glu Gly Asp Ala Thr Asn Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45 Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60 Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys 65 70 75 80 Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95 Arg Thr Ile Ser Phe Lys Asp Asp Gly Thr Tyr Lys Thr Arg Ala Glu 100 105 110 Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125 Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140 Asn Phe Asn Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn 145 150 155 160 Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp Gly Ser 165 170 175 Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190 Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Lys Leu 195 200 205 Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210 215 220 Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys 225 230 235

Claims

1. A method for in vitro cell-free synthesis of exogenous proteins, characterized by: The in vitro cell-free synthesis method of the exogenous protein comprises: Step 1: Add component ii to component i and mix; Wherein, component i is an in vitro cell-free protein synthesis system; the in vitro cell-free protein synthesis system is an aqueous phase synthesis system; Wherein, component ii is a nucleic acid template encoding an exogenous protein; Component i and component ii together provide translation-related elements required for synthesizing the exogenous protein; Step 2: Under the assistance of the hydrophobic interface iii, an incubation reaction is performed to synthesize the exogenous protein; wherein the hydrophobic interface iii is in surface contact with the aqueous in vitro cell-free protein synthesis system; The hydrophobic interface iii is an interface formed by surface contact between the hydrophobic phase and the synthetic system of the aqueous phase; The hydrophobic phase is provided by a hydrocarbon substance; the hydrocarbon substance is in the form of paste, wax, granules, powder, liquid, or a mixture thereof; The hydrocarbon substance has a carbon number of 6 to 44; The hydrocarbon contained in the hydrocarbon substance is a linear structure, a branched structure, a cyclic structure, or a combination thereof; Also including or excluding step three: isolating and / or detecting the exogenous protein; The in vitro cell-free protein synthesis system includes cell extracts; The cell extract is selected from Kluyveromyces lactis; The cell extract contains endogenously expressed RNA polymerase.

2. The method for in vitro cell-free synthesis of exogenous proteins according to claim 1, characterized in that: The hydrophobic interface iii is provided by the following hydrophobic surfaces: the inner surface of the reactor, the hydrophobic outer surface of the hydrophobic built-in solid object, the liquid interface of the oil phase system, or a combination thereof.

3. The method for in vitro cell-free synthesis of exogenous proteins according to claim 1, characterized in that: The hydrocarbon material is selected from aliphatic hydrocarbons.

4. The method for in vitro cell-free synthesis of exogenous proteins according to claim 3, characterized in that: The hydrocarbon material is selected from alkanes.

5. The method for in vitro cell-free synthesis of exogenous proteins according to claim 1, characterized in that: The hydrocarbon material is selected from vaseline, mineral oil, or a combination thereof.

6. The method for in vitro cell-free synthesis of exogenous proteins according to claim 4, characterized in that: The alkane is selected from cyclohexane, isooctane, decane, tetradecane, pentadecylcyclohexane, tetratetradecane, vaseline, or a combination thereof.

7. The method for in vitro cell-free synthesis of exogenous proteins according to claim 1, characterized in that: The amount of alkane that can increase the synthesis of exogenous protein is recorded as Qhc; The Qhc is selected from the alkane dosage interval when YPRT is greater than Y0 in the YPRT (Chc) curve; wherein Chc refers to the alkane dosage, YPRT refers to the exogenous protein expression level, and the YPRT (Chc) curve refers to a curve with the alkane dosage as the independent variable, the exogenous protein expression as the dependent variable, and other reaction parameters determined; Y0 refers to the exogenous protein expression level corresponding to Chc being 0; The Qhc is selected from the alkane dosage interval when the exogenous protein expression level is at least Y0+50%YΔ; Wherein, YΔ=Ymax-Y0; wherein, Ymax refers to the highest expression level of the exogenous protein in the YPRT (Chc) curve.

8. The method for in vitro cell-free synthesis of exogenous protein according to any one of claims 1 to 7, characterized in that: The cell extract includes an endogenously expressed RNA polymerase that recognizes a promoter on a nucleic acid template and initiates transcription of a gene for an exogenous protein.

9. The method for in vitro cell-free synthesis of exogenous protein according to any one of claim 8, characterized in that: The RNA polymerase is T7 RNA polymerase.

10. The method for in vitro cell-free synthesis of exogenous proteins according to claim 8, characterized in that: The in vitro cell-free protein synthesis system further comprises a DNA polymerase; the source of the DNA polymerase is selected from any one of the following: a cell extract containing endogenously expressed DNA polymerase, an exogenous DNA polymerase, a translation product of an exogenous nucleic acid template encoding a DNA polymerase, or a combination thereof.

11. The method for in vitro cell-free synthesis of exogenous proteins according to claim 10, characterized in that: The DNA polymerase is phi 29 DNA polymerase.

12. The method for in vitro cell-free synthesis of exogenous proteins according to claim 1, characterized in that: This is achieved by preparing a cell extract after modifying an endogenous strain of Kluyveromyces lactis. The modification method is selected from: inserting the coding sequence of RNA polymerase into a free plasmid in the cell, or integrating the coding gene of RNA polymerase into the cell genome, or using a combination of the above two methods.

13. The method for in vitro cell-free synthesis of exogenous proteins according to claim 1, characterized in that: The endogenously expressed RNA polymerase is endogenously expressed T7 RNA polymerase.

14. The method for in vitro cell-free synthesis of exogenous protein according to any one of claims 1 to 7, characterized in that: The nucleic acid template encoding the foreign protein contains a promoter element that can be recognized by component i.

15. The method for in vitro cell-free synthesis of exogenous protein according to any one of claims 1 to 7, characterized in that: The nucleic acid template encoding the exogenous protein includes an exogenous protein translation system, a resistance gene translation system, and a Lac repressor translation system; each of the above translation systems includes a corresponding promoter.

16. The in vitro cell-free synthesis method according to any one of claims 1 to 7, characterized in that: The nucleic acid template encoding the foreign protein also contains a gene for controlling the copy number of the plasmid; And / or, the nucleic acid template encoding the exogenous protein further contains a transcription enhancing element and / or a translation enhancing element.

17. The method for in vitro cell-free synthesis of exogenous protein according to any one of claims 1 to 7, characterized in that: The nucleic acid template encoding the exogenous protein contains a promoter element that can be recognized by component i; The nucleic acid template encoding the exogenous protein contains a T7 promoter, and the in vitro cell-free protein synthesis system includes T7 RNA polymerase.

18. The in vitro cell-free synthesis method of exogenous protein according to any one of claims 1 to 7, characterized in that: The gene transcription process of the exogenous protein is initiated by the T7 promoter on the nucleic acid template, and the in vitro cell-free protein synthesis system includes T7 RNA polymerase.

19. The method for in vitro cell-free synthesis of exogenous protein according to any one of claims 1 to 7, characterized in that: The nucleic acid template encoding the exogenous protein contains a T7 promoter, and the in vitro cell-free protein synthesis system includes a cell extract, which provides endogenously expressed T7 RNA polymerase.

20. The method for in vitro cell-free synthesis of exogenous protein according to any one of claims 1 to 7, characterized in that: The in vitro cell-free protein synthesis system also includes an energy system.

21. The method for in vitro cell-free synthesis of exogenous protein according to any one of claims 1 to 7, characterized in that: The energy system is selected from any one of the following: a sugar and phosphate energy system, a sugar and creatine phosphate energy system, a creatine phosphate and creatine phosphate enzyme system, a creatine phosphate and creatine phosphate kinase system, monosaccharides and their glycolysis intermediates, glycogen and their glycolysis intermediates, or a combination thereof.

22. The method for in vitro cell-free synthesis of exogenous protein according to any one of claims 1 to 7, characterized in that: The in vitro cell-free protein synthesis system further comprises a substrate for synthesizing RNA and / or a substrate for synthesizing protein; 23. The method for in vitro cell-free synthesis of exogenous proteins according to claim 22, characterized in that: The substrate for synthesizing RNA is a nucleotide mixture.

24. The method for in vitro cell-free synthesis of exogenous proteins according to claim 23, characterized in that: The substrate for synthesizing RNA is selected from: nucleoside monophosphate, nucleoside triphosphate, or a combination thereof.

25. The method for in vitro cell-free synthesis of exogenous proteins according to claim 22, characterized in that: The substrate for synthesizing protein is an amino acid mixture, which at least includes the amino acid mixture required for the process of synthesizing exogenous protein.

26. The method for in vitro cell-free synthesis of exogenous proteins according to claim 25, characterized in that: The amino acid mixture is a mixture of natural amino acids.

27. The method for in vitro cell-free synthesis of exogenous protein according to any one of claims 1 to 7, characterized in that: The in vitro cell-free protein synthesis system also includes a substrate for synthesizing DNA.

28. The method for in vitro cell-free synthesis of exogenous protein according to claim 27, characterized in that: The substrate for synthesizing DNA is a deoxynucleotide mixture.

29. The method for in vitro cell-free synthesis of exogenous protein according to claim 28, characterized in that: The substrate for synthesizing DNA is a deoxynucleoside triphosphate mixture.

30. The method for in vitro cell-free synthesis of exogenous protein according to any one of claims 1 to 7, characterized in that: The in vitro cell-free protein synthesis system further comprises at least one of the following components: a crowding agent, magnesium ions, potassium ions, an antioxidant or a reducing agent, trehalose, a reaction accelerator, a buffer, and an aqueous solvent.

31. The method for in vitro cell-free synthesis of exogenous protein according to claim 30, characterized in that: The crowding agent is selected from polyethylene glycol, polyvinyl alcohol, polypropylene, dextran, sucrose polymer, polyvinyl pyrrolidone, albumin, or a combination thereof.

32. The method for in vitro cell-free synthesis of exogenous proteins according to claim 30, characterized in that: The magnesium ion comes from: magnesium aspartate, magnesium acetate, magnesium glutamate, magnesium chloride, magnesium phosphate, magnesium sulfate, magnesium citrate, magnesium hydrogen phosphate, magnesium iodide, magnesium lactate, magnesium nitrate, magnesium oxalate, or a combination thereof.

33. The method for in vitro cell-free synthesis of exogenous protein according to claim 30, characterized in that: The potassium ions are selected from potassium acetate, potassium glutamate, potassium chloride, potassium phosphate, potassium sulfate, potassium citrate, potassium hydrogen phosphate, potassium iodide, potassium lactate, potassium nitrate, potassium oxalate, or a combination thereof.

34. The method for in vitro cell-free synthesis of exogenous proteins according to claim 30, characterized in that: The antioxidant or reducing agent is dithiothreitol, 2-mercaptoethanesulfonic acid, 2-mercaptoethanol, reduced glutathione, triscarboxymethylphosphonic acid, 3-mercapto-1,2-propanediol, or a combination thereof.

35. The method for in vitro cell-free synthesis of exogenous proteins according to claim 30, characterized in that: The buffer is selected from any one of the following: Tris-HCl, Tris base, HEPES, or a combination thereof.

36. The method for in vitro cell-free synthesis of exogenous protein according to any one of claims 1 to 7, characterized in that: The exogenous protein is selected from the following: any one protein, any combination of fusion proteins, or any combination of mixtures: luciferase, green fluorescent protein, yellow fluorescent protein, aminoacyl-tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, variable region of an antibody, α-amylase, enterocin A, hepatitis C virus E2 glycoprotein, insulin precursor, interferon αA, interleukin-1β, lysozyme, serum albumin, single-chain antibody fragment, transthyretin, tyrosinase, xylanase, Escherichia coli β-galactosidase, human lysine-tRNA synthetase, human leucine-tRNA synthetase, Arabidopsis glyceraldehyde-3-phosphate dehydrogenase, mouse catalase, a partial domain of any of the foregoing, a subunit or fragment of any of the foregoing, or a variant of any of the foregoing.

37. The method for in vitro cell-free synthesis of exogenous protein according to claim 36, characterized in that: The variants are mutants.

38. The method for in vitro cell-free synthesis of exogenous protein according to any one of claims 1 to 7, characterized in that: The in vitro cell-free protein synthesis system can react with a DNA template or an mRNA template encoding an exogenous protein to synthesize the exogenous protein.

39. An in vitro protein synthesis kit, characterized in that The in vitro protein synthesis kit comprises: (i) an in vitro cell-free protein synthesis system in the in vitro cell-free synthesis method of an exogenous protein according to any one of claims 1 to 38; The components of the in vitro cell-free protein synthesis system are placed in one or more containers in the form of dry powder, liquid, emulsion, suspension, or a combination thereof; (ii) a nucleic acid template encoding a foreign protein; (iii) a reaction vessel; (iv) a hydrophobic phase capable of making surface contact with the in vitro cell-free protein synthesis system of the aqueous phase to construct a hydrophobic interface; The hydrophobic interface is selected from any one of claims 1 to 7; (v) labels or instructions; The aforementioned (i) and (ii) together provide translation-related elements required for synthesizing foreign proteins.

40. The method for in vitro cell-free synthesis of exogenous proteins according to claim 39, characterized in that: The nucleic acid template encoding the foreign protein contains a promoter element that can be recognized by component i.

41. The method for in vitro cell-free synthesis of exogenous protein according to claim 39, characterized in that: The nucleic acid template encoding the exogenous protein contains a T7 promoter, and the in vitro cell-free protein synthesis system includes T7 RNA polymerase.

42. The in vitro protein synthesis kit according to claim 39, characterized in that: The gene transcription process of the exogenous protein is initiated by the T7 promoter on the nucleic acid template, and the in vitro cell-free protein synthesis system includes T7 RNA polymerase.

43. The in vitro protein synthesis kit according to claim 39, characterized in that: The nucleic acid template encoding the exogenous protein contains a T7 promoter, and the in vitro cell-free protein synthesis system includes a cell extract, which provides endogenously expressed T7 RNA polymerase; The nucleic acid template encoding the exogenous protein is a DNA template, an mRNA template, or a combination thereof.

44. Use of an alkane in the in vitro cell-free synthesis method of an exogenous protein according to any one of claims 1 to 38, or in the in vitro protein synthesis kit according to any one of claims 39 to 43, characterized in that: The alkanes are used to construct a hydrophobic interface.

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