In-vitro cell-free protein synthesis method based on hydrophobic interface, D2P kit and related application

Abstract

The invention provides an in-vitro cell-free synthesis method and kit of foreign protein, and belongs to the technical field of protein synthesis. According to the in-vitro cell-free synthesis method of the foreign protein, a water-phase in-vitro cell-free protein synthesis system and a nucleic acid template for coding the foreign protein are provided, a hydrophobic interface in surface contact with the water-phase in-vitro cell-free protein synthesis system is provided, incubation reaction is carried out, the foreign protein is synthesized, and the in-vitro cell-free synthesis method of the foreign protein is obtained. The protein synthesis efficiency and the protein expression quantity of the cell-free system are improved. The invention further provides an in-vitro protein synthesis kit which is more efficient and higher in throughput. The invention provides a simple and convenient hydrophobic interface optimization method which is simple and convenient to operate and saves cost.

Description

Technical Field

[0001] This invention relates to the field of protein synthesis technology, and particularly to the field of in vitro cell-free protein synthesis technology, specifically 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 Technology

[0002] Proteins are essential molecules in cells, participating in almost all cellular functions. Protein synthesis mainly includes traditional intracellular synthesis techniques and next-generation in vitro synthesis techniques. Traditional protein expression systems refer to molecular biology techniques that express exogenous genes using model organisms such as bacteria, fungi, plant cells, insect cells, or animal cells. In vitro protein synthesis systems, also known as cell-free expression systems, emerged in the 1960s. They use exogenous mRNA or DNA as templates for protein synthesis and achieve the synthesis of 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 systems generally refer to the rapid and efficient translation of exogenous proteins by adding nucleic acid templates (mRNA or DNA templates), RNA polymerase, amino acids, ATP, and other components to the lysates / extracts of bacterial, fungal, plant, insect, or animal cells. In vitro protein synthesis systems eliminate the need for plasmid construction, transformation, cell culture, cell collection, and disruption, offering a relatively rapid, time-saving, and convenient method for protein expression. They are an important tool in the field of protein research. (―Garcia RA, Riley MR. Applied biochemistry and biotechnology. Humana Press. 1981, 263-264”;―Fromm HJ, Hargrove M. Essentials of Biochemistry. 2012”;CN109988801A;―Assenberg R, Wan PT, Geisse S, Mayr LM. Advances in recombinant protein expression for use in pharmaceutical research[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").In vitro protein synthesis systems can also express specific proteins that are toxic to cells or contain non-natural amino acids (such as D-amino acids). They can simultaneously synthesize multiple proteins in parallel, facilitating high-throughput drug screening and proteomics research (Spirin AS, Swartz JR. Chapter 1. Cell-Free Protein Synthesis Systems: Historical Landmarks, Classification, and General Methods. Wiley-VCH Verlag GmbH & Co. KGaA, 2008: 1-34.). Protein products produced using in vitro synthesis systems can be widely used in pharmaceuticals, food, nutritional products, dietary supplements, cosmetics, and other fields, including but not limited to the applicant's PROTEINN™, Proton™, and Proton™ protein products.

[0003] Protein synthesis capacity is one of the key indicators determining whether an in vitro protein synthesis system can be industrialized, mainly including protein synthesis efficiency and protein synthesis amount (protein expression level). In order to improve the protein synthesis amount, many modifications have been made to cell extracts, energy systems, genetic templates (nucleic acid templates), reactors and operating methods (Zhang Xu. Key technologies and industrial applications of efficient synthesis of complex membrane proteins in cell-free systems [D]. Zhejiang University, 2014). Among them, regarding the modification of the reactor, for example, the continuous exchange cell-free reaction developed by Kim et al. (Biotechnol.Prog.,1996,12(5):645-649) uses a selectively permeable semipermeable membrane to divide the reaction system into two parts: a reaction zone and a supplement zone. The reaction zone is responsible for transcribing and reacting the template molecules to express the target protein molecules. The supplement zone is responsible for replenishing the consumed energy and substrate. The energy and substrate small molecules in the supplement zone can pass through the semipermeable membrane into the reaction zone (maintaining the reaction). The inorganic phosphorus and other small molecule byproducts in the reaction zone can also diffuse through the semipermeable membrane into the supplement zone (reducing the inhibitory effect). However, the expressed target protein, as a single molecule, cannot diffuse through the semipermeable membrane into the supplement zone. The duration of the cell-free reaction has been extended from tens of minutes or even several minutes to more than twenty hours. Sawasaki et al. also separated the reaction zone and the supplement zone by interfacial diffusion, which does not require a semipermeable membrane (FEBS Lett.,2002,514(1):102-105). Biyani et al. developed a solid-phase cell-free reaction system that increased local substrate concentration and template stability (Nucleic Acids Res., 2006, 34:e140). Park et al. also used DNA hydrogels for in vitro protein synthesis, significantly improving reaction efficiency (Nat. Mater., 2009, 8:432-437). The aforementioned methods for modifying reactors generally require specially designed or fabricated reaction equipment (selective semi-permeable membranes, hydrogels, solid-phase systems, etc.), which are either costly or cumbersome, and are not suitable for large-scale production.

[0004] In conclusion, there is still an urgent need to improve reaction vessels to achieve efficient and low-cost in vitro cell-free protein synthesis. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention discloses a simple, convenient, more efficient, and higher-throughput in vitro cell-free synthesis method for exogenous proteins. By providing a hydrophobic interface and utilizing the hydrophobic interface that makes surface contact with the aqueous in vitro cell-free protein synthesis system, a significant improvement in protein synthesis capacity can be achieved.

[0006] The first aspect of this invention provides a method for the in vitro cell-free synthesis of exogenous proteins, the method comprising:

[0007] Step 1: Add component ii to component i and mix.

[0008] Component i is an in vitro cell-free protein synthesis system; the in vitro cell-free protein synthesis system is an aqueous system, and is referred to as an aqueous synthesis system, aqueous system, aqueous synthesis system or synthesis system.

[0009] Component ii is a nucleic acid template encoding a foreign protein;

[0010] Components i and ii together provide the translation-related elements required for the synthesis of exogenous proteins;

[0011] Step 2: Incubate the exogenous protein with the assistance of a hydrophobic interface iii.

[0012] The hydrophobic interface iii is in surface contact with the aqueous in vitro cell-free protein synthesis system.

[0013] Optionally, step three may also be included: isolating and / or detecting the exogenous protein.

[0014] The hydrophobic interface is formed by the surface contact between the hydrophobic phase and the aqueous phase synthesis system.

[0015] Hydrophobic phase: In this invention, the hydrophobic phase refers to a surface or interface that is hydrophobic in contact with the aqueous synthesis system. There are no particular limitations on the hydrophilicity or hydrophobicity of the parts of the substance or device constituting the hydrophobic phase that are not in contact with the aqueous synthesis system.

[0016] Depending on 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, it is preferable that the entire liquid phase (including its internal and external surfaces / interfaces) is hydrophobic.

[0017] Preferably, the hydrophobic interface is provided by the following hydrophobic surfaces: the inner surface of the reactor, the hydrophobic outer surface of the hydrophobic embedded solid, the liquid interface of the oil phase system, or a combination thereof. The inner surface of the reactor may be, for example, the inner surface of the sidewall or the inner surface of the bottom of the reactor. The hydrophobic embedded solid refers to a solid with a hydrophobic outer surface, and its location is not particularly limited; it may be deposited below the aqueous synthesis system, suspended in the aqueous synthesis system, or float above the aqueous synthesis system. Preferably, the hydrophobic embedded solid is deposited below the aqueous synthesis system or floats above the aqueous synthesis system.

[0018] Preferably, the hydrophobic phase is provided by a hydrocarbon substance, the main component of which is 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 has a straight-chain structure, a branched-chain structure, a cyclic structure, or a combination thereof.

[0020] Preferably, the hydrocarbon substance has at least 6 carbon atoms, and more preferably, the hydrocarbon substance has 6 to 44 carbon atoms.

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

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

[0023] Preferably, the alkane has a straight-chain structure, a branched-chain structure, a cyclic structure, or a combination thereof.

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

[0025] Preferably, the alkane has at least 6 carbon atoms; more preferably, the alkane has 6 to 44 carbon atoms. Even more preferably, the alkane is selected from: cyclohexane, isooctane, decane, tetradecane, pentadecylcyclohexane, tetratetradecane, petrolatum, or combinations thereof.

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

[0027] The amount of alkane used to increase the synthesis of exogenous proteins is selected from the range of alkane usage in the YPRT(Chc) curve where the expression level of the exogenous protein is greater than Y0. This selected range can be continuous or discontinuous.

[0028] In this invention,

[0029] Qhc refers to the amount of alkane used in this invention that can increase the synthesis of exogenous proteins.

[0030] Chc refers to the amount of alkane used.

[0031] YPRT refers to the expression level of exogenous proteins;

[0032] The YPRT (Chc) curve refers to a curve with the amount of alkane as the independent variable, the expression of exogenous protein as the dependent variable, and other reaction parameters as determined. In this invention, it is also referred to as the YPRT-Chc curve. The "other reaction parameters" include, but are not limited to: other system components, the method of adding reaction raw materials, the reaction temperature program, the reaction time length, the properties of the reaction vessel, the volume of the reaction system, etc. One component i system can correspond to multiple YPRT-Chc curves. One component i system can react at different reaction temperature programs and for different time lengths, thereby generating several different YPRT-Chc curves.

[0033] Ymax refers to the highest expression level of the exogenous protein in the YPRT(Chc) curve.

[0034] Cmax refers to the amount of alkane used when the exogenous protein reaches its highest expression level in the YPRT(Chc) curve.

[0035] Ymin refers to the lowest expression level of exogenous protein in the range where YPRT>Y0 in the YPRT(Chc) concentration curve.

[0036] Y0 refers to the level of exogenous protein expression when Chc is 0.

[0037] YΔ is the difference between Ymax and Y0. Numerically, YΔ = Ymax - Y0.

[0038] Preferably, the Qhc is selected from the alkane dosage range when the expression level of the exogenous protein is at least Y0 + 50% YΔ;

[0039] More preferably, the Qhc is selected from the alkane dosage range when the expression level of the exogenous protein is at least Y0 + 60% YΔ;

[0040] More preferably, the Qhc is selected from the alkane dosage range when the expression level of the exogenous protein is at least Y0 + 70% YΔ;

[0041] More preferably, the Qhc is selected from the alkane dosage range when the expression level of the exogenous protein is at least Y0 + 80% YΔ;

[0042] More preferably, the Qhc is selected from the alkane dosage range when the expression level of the exogenous protein is at least Y0 + 90% YΔ;

[0043] More preferably, the Qhc is selected from the alkane dosage range when the expression level of the exogenous protein is at least Y0 + 95% YΔ;

[0044] More preferably, Qhc is the amount of alkane used, Cmax, at which the exogenous protein is expressed at its highest level.

[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 system components capable of recognizing promoter elements on nucleic acid templates, enabling the in vitro cell-free protein synthesis system to recognize promoter elements of nucleic acid templates encoding exogenous proteins. For example, the in vitro cell-free protein synthesis system contains RNA polymerase corresponding to the promoter element.

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

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

[0049] Preferably, the in vitro cell-free protein synthesis system further includes RNA polymerase. The sources of the RNA polymerase include, but are not limited to: cell extracts containing endogenously expressed RNA polymerase, exogenous RNA polymerase, translation products of exogenous nucleic acid templates encoding RNA polymerase, or combinations thereof. In each of the above technical solutions, the RNA polymerase is preferably T7 RNA polymerase. The exogenous nucleic acid template encoding RNA polymerase can be translated into RNA polymerase through the in vitro protein synthesis reaction of the system. Alternatively, the in vitro cell-free protein synthesis system further includes DNA polymerase. The sources of the DNA polymerase include, but are not limited to: cell extracts containing endogenously expressed DNA polymerase, exogenous DNA polymerase, translation products of exogenous nucleic acid templates encoding DNA polymerase, or combinations thereof. In each of the above technical solutions, the DNA polymerase is preferably phi29 DNA polymerase. The exogenous nucleic acid template encoding DNA polymerase can be translated into DNA polymerase through the in vitro protein synthesis reaction of the system. The RNA polymerase and DNA polymerase can be added directly from an exogenous source, or provided as reaction products or intermediates (e.g., by adding an exogenous nucleic acid template encoding the RNA polymerase or / and the DNA polymerase).

[0050] In one preferred embodiment, the cell extract contains endogenously expressed RNA polymerase. More preferably, the cell extract contains endogenously expressed T7 RNA polymerase.

[0051] In one preferred embodiment, the cell extract contains endogenously expressed RNA polymerase. More preferably, this is achieved by preparing the cell extract after endogenously modifying the source cells with a specific strain, including but not limited to the following endogenous strain modification methods: inserting the coding sequence of the RNA polymerase into a cell-free plasmid, integrating the coding gene of the RNA polymerase into the cell genome, or a combination of the aforementioned two methods. It should be noted that, in performing the above-mentioned endogenous strain modification, in addition to integrating the aforementioned coding sequence / coding gene, other nucleotide sequences are also allowed to be inserted, such as non-coding sequences, enhancer sequences, Kozak sequences, leader sequences, signal peptide sequences, tag sequences, codon sequences, etc. Through the above-mentioned endogenous strain modification, the modified strain can endogenously express RNA polymerase. The RNA polymerase is preferably T7 RNA polymerase.

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

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

[0054] The cell extract is selected from: prokaryotic cell extracts, eukaryotic cell extracts, or combinations thereof.

[0055] In one preferred embodiment, the cell extract is selected from any of the following sources: prokaryotic cells, yeast cells, mammalian cells, plant cells, insect cells, or a combination thereof. The prokaryotic cells are preferably *Escherichia coli*.

[0056] The yeast cells are preferably selected from Kluyveromyces, Saccharomyces cerevisiae, Pichia pastoris, or a combination thereof.

[0057] The Kluyveromyces is further preferably Kluyveromyces lactis, Kluyveromyces marx, Kluyveromyces dob, Kluyveromyces salina, Kluyveromyces wickheim, Kluyveromyces brittlewall, Kluyveromyces hubeiensis, Kluyveromyces multisporum, Kluyveromyces siamensis, Kluyveromyces yarbromentosus, or a combination thereof.

[0058] In one preferred manner, the cell extract is selected from any of the following sources: *Escherichia coli*, *Kluyveromyces lactis*, wheat germ cells, *Spodoptera frugiperda* cells (fall armyworm cells, an insect cell), *Leishmania tarentolae* cells (lizard leishmania cells), rabbit reticulocytes, Chinese hamster ovary cells (CHO cells), African green monkey kidney COS cells, African green monkey kidney VERO cells, young hamster kidney cells (BHK cells), human HeLa cells, human Hybridoma cells, human fibrosarcoma HT1080 cells, or combinations thereof.

[0059] Preferably, the nucleic acid template encoding the exogenous protein contains a promoter element that can be recognized by component i; the system component refers to the components of the aqueous in vitro cell-free protein synthesis system.

[0060] Preferably, 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;

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

[0062] In one preferred embodiment, 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, wherein the cell extract provides an endogenously expressed T7 RNA polymerase.

[0063] In one preferred embodiment, the in vitro cell-free protein synthesis system further includes an energy system; the energy system is preferably selected from: a sugar (e.g., monosaccharide, disaccharide, oligosaccharide, polysaccharide) and phosphate energy system, a sugar and phosphocreatine energy system, a phosphocreatine and phosphocreatine enzyme system, a phosphocreatine and phosphocreatine kinase system, a glycolysis pathway and its intermediate product energy system (e.g., a monosaccharide and its glycolysis intermediate product energy system, glycogen and its glycolysis intermediate product), or a combination thereof.

[0064] In one preferred embodiment, the in vitro cell-free protein synthesis system further includes a substrate for protein synthesis; the substrate for protein synthesis is preferably an amino acid mixture, including at least the amino acid mixture required for synthesizing exogenous proteins. 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 includes a substrate for synthesizing RNA; the substrate for synthesizing RNA is preferably a mixture of nucleotides selected from: nucleoside monophosphates, nucleoside triphosphates, or combinations thereof.

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

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

[0068] The crowding agent is preferably polyethylene glycol, polyvinyl alcohol, polypropylene, dextran, sucrose polymers (including Ficoll sucrose polymers, such as reagent, a nonionic synthetic sucrose polymer), polyethylene (vinylpyrrolidone), albumin, etc., or combinations thereof.

[0069] The magnesium ion source is preferably selected 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 combinations thereof;

[0070] Potassium ion sources are preferably selected from: potassium acetate, potassium glutamate, and chloride. Potassium chloride, potassium phosphate, potassium sulfate, potassium citrate, potassium hydrogen phosphate, potassium iodide, potassium lactate, potassium nitrate, potassium oxalate, or combinations thereof;

[0071] The antioxidant or reducing agent is preferably dithiothreitol, 2-mercaptoethanesulfonic acid, 2-mercaptoethanol, reduced glutathione, tricarboxymethyl phosphate, 3-mercapto-1,2-propanediol, or a combination thereof.

[0072] The reaction promoter is preferably aluminum oxide;

[0073] The buffer is preferably selected from: 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 can react with DNA templates or mRNA templates encoding exogenous proteins to synthesize exogenous proteins.

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

[0077] A second aspect of the present invention provides an in vitro protein synthesis kit, the in vitro protein synthesis kit comprising:

[0078] (i) The in vitro cell-free protein synthesis system in the in vitro cell-free synthesis method of the exogenous protein described in the first aspect; wherein 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 includes a nucleic acid template encoding a foreign protein;

[0080] (iii) Optionally includes a reaction vessel;

[0081] (iv) Hydrophobic phase; may include substances or devices that form hydrophobic interfaces;

[0082] The hydrophobic phase can form a hydrophobic interface by surface contact with the aqueous synthesis system.

[0083] The hydrophobic interface is selected from any hydrophobic interface in the in vitro cell-free synthesis method of exogenous protein provided in the first aspect.

[0084] (v) Label or instruction manual.

[0085] (i) and (ii) together provide the translation-related elements required for the synthesis of exogenous proteins.

[0086] The aforementioned in vitro protein synthesis kit can be used to perform in vitro protein synthesis reactions and synthesize exogenous proteins.

[0087] Each part of the kit corresponds to a component of the in vitro cell-free synthesis method for exogenous proteins described in the first aspect.

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

[0089] In one preferred embodiment, the nucleic acid template encoding the exogenous protein contains a promoter element that the cell extract can recognize. For example, the cell extract contains an endogenously expressed RNA polymerase that corresponds to the promoter element on the nucleic acid template.

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

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

[0092] Preferably, the transcription process of the exogenous protein is initiated by the T7 promoter on the nucleic acid template.

[0093] In one preferred manner, 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.

[0094] In one preferred embodiment, the T7 promoter is located upstream of the coding sequence of the exogenous protein in the nucleic acid template, and the transcription program of the exogenous protein is initiated by the T7 promoter. The in vitro cell-free protein synthesis system includes a cell extract, which provides 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 invention provides the use of alkane in the in vitro cell-free synthesis method of exogenous protein described in the first aspect, or in the in vitro protein synthesis kit described in the second aspect, or in the field of in vitro protein synthesis; wherein the alkane is used to construct a hydrophobic interface.

[0097] Preferably, the application in in vitro protein synthesis includes, but is not limited to, applications in protein manufacturing or applications in protein synthesis-based detection.

[0098] Beneficial effects:

[0099] The present invention provides a method for in vitro cell-free synthesis of exogenous proteins, which provides an aqueous in vitro cell-free protein synthesis system and a nucleic acid template encoding the exogenous protein, and introduces a hydrophobic interface that makes surface contact with the aqueous in vitro cell-free protein synthesis system for incubation reaction to synthesize the exogenous protein, thereby improving the protein synthesis capacity of the cell-free system (at least including protein synthesis efficiency and protein synthesis amount), with an improvement of up to 90%.

[0100] The hydrophobic interface utilizes hydrophilic-hydrophobic interactions to promote the enrichment of hydrophobic components at the interface, increase local concentration, improve reaction efficiency and raw material utilization, and enhance protein synthesis. On the other hand, for insoluble protein products, the hydrophobic interface also acts as an interface fixation mechanism, allowing the hydrophobic side of the protein product to adhere to the interface through hydrophilic-hydrophobic interactions, while the hydrophilic end dissolves in the aqueous phase of the in vitro cell-free protein synthesis system, thus achieving an interface solubilization effect.

[0101] This invention constructs a hydrophobic interface by simply adding alkanes. Compared to existing methods that require complex modifications to the reactor (such as constructing complex, costly, and cumbersome selective semi-permeable membranes), compared to methods that require prior polymerization to construct a gel crosslinking system, and compared to solid-phase reaction systems with covalent fixation, this invention is simple and convenient to operate, uses inexpensive and readily available raw materials, and is inexpensive.

[0102] The further provided in vitro protein synthesis kit has the advantages of higher efficiency and higher throughput. Attached Figure Description

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

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

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

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

[0107] Figure 5 The effect of isooctane on in vitro protein synthesis. Isooctane was in liquid form, used at a concentration of 0–5% (v / v). 3h and 18h ​​represent reaction times of 3 hours and 18 hours, respectively. A concentration of "0" corresponds to the blank control (BC group). Alkanes form an oily hydrophobic interface above the aqueous in vitro protein synthesis system. The reaction was conducted in 48-well cell culture plates.

[0108] Figure 6The effect of decane on in vitro protein synthesis reactions. Decane was n-decane, in a liquid state, and used in amounts ranging from 0% to 5% (v / v). 3h and 18h ​​represent reaction times of 3 hours and 18 hours, respectively. A concentration of "0" corresponds to the blank control (group BC). Alkanes form an oily hydrophobic interface above the aqueous in vitro protein synthesis system. The reaction was conducted in 48-well cell culture plates.

[0109] Figure 7 , ten The effect of tetradecane on in vitro protein synthesis reactions. Tetradecane is a straight-chain structure (n-tetradecane), physically liquid, and used in amounts ranging from 0% to 2% (v / v). 3h and 18h ​​represent reaction times of 3 hours and 18 hours, respectively. A concentration of "0" corresponds to the blank control (group BC). Alkanes form an oily hydrophobic interface above the aqueous in vitro protein synthesis system. The reaction was carried out in 48-well cell culture plates.

[0110] Figure 8 , ten The effect of tetradecane on in vitro protein synthesis reactions. Tetradecane is a straight-chain structure (n-tetradecane), physically liquid, and used in amounts ranging from 2% to 20% (v / v). 3h and 18h ​​represent reaction times of 3 hours and 18 hours, respectively. A concentration of "0" corresponds to the blank control (group BC). Alkanes form an oily hydrophobic interface above the aqueous in vitro protein synthesis system. The reaction was carried out in 48-well cell culture plates.

[0111] Figure 9 , ten The effect of pentaalkylcyclohexane on in vitro protein synthesis. Pentadecylcyclohexane was used in liquid form at concentrations ranging from 0% to 20% (v / v). 3h and 18h ​​represent reaction times of 3 hours and 18 hours, respectively. Concentration "0" corresponds to the blank control (BC group), and NC is the negative control. The alkane forms an oily hydrophobic interface above the aqueous in vitro protein synthesis system. The reaction was carried out in 48-well culture plates.

[0112] Figure 10 , Four The effect of tetradecane on in vitro protein synthesis. Tetradecane was used in solid form at concentrations ranging from 0 to 500 μg / μL. 3h and 18h ​​represent reaction times of 3 h and 18 h, respectively. Concentration "0" corresponds to the blank control (BC group), and NC is the negative control. During in vitro protein synthesis, the alkanes existed in an oily layer above the aqueous synthesis system. The reaction was conducted in 48-well cell culture plates.

[0113] Figure 11The effect of petrolatum on in vitro protein synthesis reactions. Petrolatum, a paste-like mixture of alkanes, was used at a rate of 0–0.09 g / well and applied to the sidewalls and inner walls of the wells to create a hydrophobic interface. The in vitro protein synthesis system consisted of a 300 μL volume and was conducted in a 48-well cell culture plate (Example S10). Group BC served as the blank control group.

[0114] Figure 12 The effect of petrolatum on in vitro protein synthesis reactions. The in vitro protein synthesis system consisted of a 300 μL volume and was carried out in a 48-well plate (Example S11). ZS01092 is the code for the modified strain used to produce the cell extract. This strain is a modified *Kluyveromyces lactis* that endogenously integrates the encoding gene for T7 RNA polymerase, enabling endogenous expression of T7 RNA polymerase. Vas indicates the application of petrolatum to the sidewalls and bottom walls of the wells to create a hydrophobic interface; AC indicates the addition of activated charcoal; AC+Vas indicates both petrolatum application and activated charcoal addition; BC group was the blank control group, which received neither petrolatum nor activated charcoal, but included the DNA template encoding the exogenous protein; NC was the negative control group, which received neither petrolatum nor activated charcoal, and did not include the DNA template encoding the exogenous protein. 3h, 6h, and 21h represent reaction times of 3h, 6h, and overnight (21h), respectively.

[0115] Figure 13 The effect of petroleum jelly on in vitro protein synthesis reactions. The in vitro protein synthesis system was 300 μL, and the reaction was carried out in 48-well plates (Example S12). In this context, Vas indicates that petroleum jelly was applied to the sidewalls and bottom walls of the wells to construct a hydrophobic interface; AC indicates the addition of activated charcoal; ssDNA indicates the addition of salmon sperm DNA to the in vitro protein synthesis system; and Amp indicates the addition of ampicillin to the in vitro protein synthesis system. AC+ssDNA+Amp represents the addition of activated charcoal, salmon sperm DNA, and ampicillin, serving as a blank control group without petroleum jelly; Vas+AC+ssDNA+Amp represents the addition of petroleum jelly to the AC+ssDNA+Amp blank control group to construct a hydrophobic interface; NC is the negative control group, where neither petroleum jelly nor DNA template encoding the exogenous protein was added. 3h, 6h, and 21h represent reaction times of 3h, 6h, and overnight (21h), respectively.

[0116] Nucleotide and / or amino acid sequence listing

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

[0118] SEQ ID No.:2 is the amino acid sequence of the exogenous protein mEGFP, consisting of 239 amino acids. Detailed Implementation

[0119] The meanings of terms, nouns, and phrases in this invention are as follows. The explanations in this section apply to the entire text of this invention, both below and above. Where references are cited in this invention, the definitions of relevant terms, nouns, and phrases in those references are also cited; however, in case of conflict with the definitions in this invention, the definitions in this invention shall prevail. In the event of a conflict between the definitions in the cited references and the definitions in this invention, the referenced components, substances, compositions, materials, systems, formulations, types, methods, equipment, etc., shall be taken as the definitive version.

[0120] In this invention, the aqueous synthesis system refers to an in vitro cell-free protein synthesis system in an aqueous phase.

[0121] In this invention, "synthetic system", "aqueous system", "aqueous synthetic system" and "aqueous synthetic system" have the same meaning and can be used interchangeably.

[0122] Interface: The boundary between two material phases.

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

[0124] Hydrophobic phase: In this invention, the hydrophobic phase refers to a surface or interface that is hydrophobic in contact with the aqueous synthesis system. The hydrophobicity of the hydrophobic phase refers to its hydrophobicity relative to the aqueous synthesis system. The hydrophobicity of the hydrophobic phase refers to the hydrophobicity of its outer surface or external interface. For the substance or device constituting the hydrophobic phase, there are no particular limitations on the hydrophilicity or hydrophobicity of the portion that is not in contact with the aqueous synthesis system. The hydrophobic phase can be a substance phase or device with a hydrophobic outer surface or external interface. Depending on 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, it is preferable that the entire liquid phase (including its internal and external surfaces / interfaces) is hydrophobic.

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

[0126] D2P, DNA-to-Protein, refers to the process of converting a DNA template into a protein product. Examples include D2P technology, D2P systems, D2P methods, and D2P kits.

[0127] IVTT, in vitro transcription translation.

[0128] RFU stands for Relative Fluorescence Unit.

[0129] eGFP: Enhanced green fluorescence protein.

[0130] mEGFP: an A206K mutant of eGFP.

[0131] wt% or %(wt): These are units of mass concentration, both representing a mass percentage.

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

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

[0134] g / well indicates grams per well.

[0135] Sucrose polymers: polymers containing at least two sucrose units. This includes, but is not limited to, polysucrose.

[0136] Ficoll sucrose polymer: unless otherwise specified, refers to... The reagent, a nonionic synthetic sucrose polymer, is a highly branched polymer copolymerized from sucrose and epichlorohydrin, and can be selected from commercially available products. Examples include Ficoll-400 (polysucrose 400, CAS: 26873-85-8) and Ficoll-70 (polysucrose 70, CAS: 72146-89-5). 400 (Sigma Aldrich) is a highly branched polymer copolymerized from sucrose and epichlorohydrin, with an average molecular weight of 400 kg / mol; Ficoll PM 70 (Sigma Aldrich) has an average molecular weight of 70 kg / mol.

[0137] Hydrocarbon substances are primarily composed of hydrocarbons, and impurities are generated during the preparation process, not introduced through addition. Examples include petrolatum and mineral oil.

[0138] Hydrocarbons are hydrocarbons consisting only of carbon and hydrogen atoms. Based on their degree of unsaturation, hydrocarbons are classified into saturated hydrocarbons and unsaturated hydrocarbons. Based on their aromaticity, hydrocarbons are classified into aromatic hydrocarbons and aliphatic hydrocarbons.

[0139] Aliphatic hydrocarbons, hydrocarbons that do not contain aromatic rings.

[0140] Alkanes, also known as saturated hydrocarbons, can be open-chain or contain cyclic structures. Open-chain structures include, but are not limited to, straight-chain and branched structures; branched structures include, but are not limited to, common branched structures with low branching degree, as well as comb-like and dendritic structures with special structures, and hyperbranched structures with high branching degree. Cyclic structures include, but are not limited to, cyclic structures and combinations of cyclic and open-chain structures. The alkanes used in this invention have at least 6 carbon atoms to form liquid, paste, solid (e.g., waxy, granular, powdered), or mixed forms, thereby constructing a hydrophobic interface. When containing cyclic structures, the number of cyclic structures can be one, two, or more.

[0141] Unsaturated hydrocarbons, including but not limited to alkenes, dienes, and alkynes, may each have an open-chain structure or contain a cyclic structure (for example, the alkenes include but are not limited to open-chain alkenes and cyclic alkenes). The alkenes contain only one carbon-carbon double bond. The dienes contain only two carbon-carbon double bonds and may be conjugated dienes or non-conjugated structures.

[0142] A cyclic structure in which all carbon atoms participate in forming the cyclic framework. This includes, but is not limited to, monocyclic, bicyclic, polycyclic, bridged, spirocyclic, and nested cyclic structures. In a bridged ring, adjacent rings share two carbon atoms; these shared carbon atoms are called bridgehead carbon atoms. The two bridgehead carbon atoms can be directly bonded by chemical bonds or separated by other carbon atoms without being directly connected. In a spirocyclic ring, adjacent rings share one carbon atom.

[0143] Open-chain hydrocarbons are hydrocarbons that do not contain cyclic structures, including straight-chain hydrocarbons and branched-chain hydrocarbons.

[0144] Straight-chain hydrocarbons are open-chain hydrocarbons in which all carbon atoms are located in the main chain, and they are linked together to form a chain structure, such as n-hexane, n-decane, and n-tetradecane.

[0145] Branched hydrocarbons are open-chain hydrocarbons in which at least one carbon atom is located in the side chain, wherein at least one carbon atom is bonded to at least three carbon atoms simultaneously. An example is isooctane.

[0146] Cyclic hydrocarbons are hydrocarbons containing a cyclic structure, such as cyclohexane (containing only a cyclic structure) and pentadecylcyclohexane (a combination of cyclic and open-chain structures). These cyclic hydrocarbons can be saturated or unsaturated.

[0147] The terms "expression system of the present invention," "in vitro expression system of the present invention," "in vitro cell-free expression system," and "in vitro cell-free expression system" are used interchangeably to refer to the in vitro protein expression system of the present invention. Other descriptive methods may also be used, such as: in vitro protein synthesis system, in vitro protein synthesis system, cell-free system, cell-free protein synthesis system, cell-free in vitro protein synthesis system, in vitro cell-free protein synthesis system, in vitro cell-free synthesis system, CFS system (cell-free system), CFPS system (cell-free protein synthesis system), etc. This includes in vitro translation systems and in vitro transcription-translation systems (IVTT systems). In this invention, the IVTT system is preferred. We also refer to the in vitro protein synthesis system as a "protein factory" (or "protein factory"). The components of the in vitro protein synthesis system provided by this invention are described in an open-ended manner.

[0148] "Cell-free system" refers to a method of in vitro protein synthesis that does not involve secretion and expression by intact cells. It should be noted that in the in vitro cell-free protein synthesis system of this invention, the addition of cellular components to promote the reaction is also permitted, but the added cells are not primarily intended for the secretion and expression of exogenous target proteins. Furthermore, in the cell-free CFPS system constructed under the guidance of this invention, the intentional addition of a small number of intact cells (e.g., whose protein content does not exceed 30 wt% compared to the protein content provided by cell extracts) is also included within the scope of protection of this invention.

[0149] In vitro protein synthesis refers to the synthesis of proteins in a cell-free in vitro synthesis system, including at least the translation process. This includes, but is not limited to, the IVTT reaction (in vitro transcription-translation reaction). In this invention, the IVTT reaction is preferred. The IVTT reaction, corresponding to the IVTT system, is the process of transcribing and translating DNA into protein in vitro. Therefore, we also refer to this type of in vitro protein synthesis system as a D2P system, D-to-P system, or DNA-to-Protein system; the corresponding in vitro protein synthesis methods are also referred to as D2P methods, D-to-P methods, or DNA-to-Protein methods.

[0150] Post-translational modification (PTM), also known as post-translational processing, plays a crucial role in the proper folding, activity, and stability of proteins.

[0151] In this invention, "translation-related elements" refers to the relevant functional elements required in the synthesis process from a nucleic acid template to a protein product, and is not limited to functional elements required for the translation process; when the nucleic acid template is DNA, it also broadly includes functional elements required for the transcription process. These translation-related elements can be provided through cell extracts (various endogenous factors), other exogenous components of in vitro protein synthesis systems (such as exogenous RNA polymerase, cofactors, etc.), and functional elements on the nucleic acid template (such as functional elements controlling exogenous protein transcription / translation, resistance gene translation systems, Lac repressor translation systems, plasmid copy number control translation systems, etc.). Examples of functional elements controlling exogenous protein transcription / translation include promoters, terminators, enhancers, IRES elements, Kozak sequences, other elements regulating translation levels, signal sequences, leader sequences, and functional tags (such as selection marker tags, tags enhancing translation levels).

[0152] Genes include coding and non-coding regions.

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

[0154] Nucleic acid sequence: The sequence of nucleic acid material, including DNA sequence and RNA sequence.

[0155] Coding sequence (CDS): A nucleotide sequence that perfectly corresponds to the codons of a protein, containing no other sequences that do not correspond to that protein (sequence changes during mRNA processing are not considered).

[0156] Encoding gene: A valid gene segment that encodes a protein; it can be continuous or discontinuous. A coding gene must contain a coding sequence.

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

[0158] "A nucleic acid template encoding protein X" refers to a nucleic acid template containing the coding sequence of protein X. Protein X can be synthesized based on this template through translation or transcription-translation. The template may contain non-coding regions and coding sequences for other polypeptides or proteins besides protein X. For example, "a nucleic acid template encoding RNA polymerase" must at least include the coding sequence of RNA polymerase, and may also include non-coding regions, fusion tags, and other nucleic acid sequences. The corresponding expression product must at least contain an RNA polymerase structure, and can be an RNA polymerase molecule or its fusion protein, or a mixture including an RNA polymerase molecule and / or its fusion protein.

[0159] Enhancer element: Unless otherwise specified, in this invention, it refers to a sequence in a nucleic acid sequence located between the promoter and the coding sequence of the target protein, which plays a role in promoting transcription and / or translation. Examples include Ω sequences, kozak sequences, and IRES sequences. This includes transcriptional enhancers and translational enhancers.

[0160] Endogenous / Endogenous: Dependent on the metabolic activity of living cells. Endogenously expressed proteins are secreted endogenously during cell culture and can be present in the cell extracts of this invention after processing.

[0161] Exogenous / Exogenous: Independent of active cellular metabolic activity. Exogenous components are added directly to the in vitro protein synthesis system, rather than through the addition of cells or cell extracts. For example, exogenous RNA polymerase can be added to the reaction system via the addition of precursors (e.g., inactive precursors that can be activated by enzymatic digestion or other methods to generate RNA polymerase), nucleic acid templates (which can be translated into proteins by the system), fusion proteins, pure substances, or mixtures. Similarly, exogenous DNA polymerase can also be added to the reaction system via the aforementioned exogenous methods.

[0162] Exogenous protein: The target expression product of the in vitro protein synthesis system of this invention is not synthesized by the secretion of host cells. It can be a protein, a fusion protein, or a mixture containing a protein or a fusion protein; it also broadly includes polypeptides. The product obtained after in vitro protein synthesis based on a nucleic acid template encoding an exogenous protein can be a single substance or a 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 RNA polymerase (or a nucleic acid template encoding DNA polymerase)" must include at least the coding sequence of RNA polymerase (or DNA polymerase), and may also include other nucleic acid sequences such as non-coding regions and fusion tags; correspondingly, the expression product must contain at least an RNA polymerase structure (or a DNA polymerase structure). Taking RNA polymerase as an example, it can be an RNA polymerase molecule or its fusion protein, or a mixture of RNA polymerase molecules and / or their fusion proteins.

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

[0167] Polypeptides are peptides composed of 10 to 50 amino acids.

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

[0169] Derivatives of peptides and proteins: Unless otherwise specified (e.g., a specific sequence is designated), the present invention also includes derivatives of any peptide or protein. The peptide derivatives and protein derivatives include at least those containing a C-terminal tag, those containing an N-terminal tag, and those containing both C-terminal and N-terminal tags. Wherein, C-terminus refers to the COOH-terminus, and N-terminus refers to the NH2-terminus, the meaning of which is understood by those skilled in the art. The tags can be peptide tags or protein tags. Examples of tags include, but are not limited to, 6-histidine (6×-His, HHHHHH), Glu-Glu, c-myc epitope (EQKLISEEDL), octapeptide (DYKDDDDK), protein C (EDQVDPRLIDGK), Tag-100 (EETARFQPGYRS), V5 epitope (V5epitope, GKPIPNPLLGLDST), VSV-G (YTDIEMNRLGK), Xpress (DLYDDDDK), hemagglutinin (YPYDVPDYA), β-galactosidase, thioredoxin, histidine-patch thioredoxin, IgG-binding domain, intein-chitin binding domain, and T7 gene 10. 10) Glutathione S-transferase (GST), green fluorescent protein (GFP), maltose-binding protein (MBP), etc.

[0170] Homology, unless otherwise specified, refers to at least 50% homology; preferably at least 60% homology, more preferably at least 70% homology, more preferably at least 75% homology, more preferably at least 80% homology, more preferably at least 85% homology, more preferably at least 90% homology; and also, for example, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% homology. For example, homologous sequences of the Ω sequence mentioned in this invention.

[0171] A "variant" refers to a substance that has a different structure (including but not limited to minor variations) but still retains or substantially retains its original function or performance. Variants include, but are not limited to, nucleic acid variants, peptide variants, and protein variants. Methods for obtaining variants include, but are not limited to, recombination, deletion or omission, insertion, translocation, and substitution of structural units. Variants include, but are not limited to, modified products, genetically modified products, and fusion products. Methods for genetic modification to obtain genetically modified products include, but are not limited to, gene recombination (corresponding to gene recombination products), gene deletion or omission, insertion, frameshift, and base substitution. Gene mutation products, also known as gene mutants, are a type of genetically modified product.

[0172] Modified products include, but are not limited to, chemically modified products, amino acid modifiers, peptide modifiers, protein modifiers, etc. Chemically modified products refer to products modified using chemical synthesis methods such as organic chemistry, inorganic chemistry, and polymer chemistry. Examples of chemical modification methods include ionization, salting, desalination, complexation, decomplexation, chelation, dechelation, addition reactions, substitution reactions, and elimination reactions. Specific examples include oxidation, reduction, methylation, demethylation, amination, carboxylation, and sulfidation.

[0173] In this invention, unless otherwise specified, a "mutant" refers to a mutated product that retains or substantially retains its original function or performance, and there is no particular limitation on the number of mutation sites. Mutants include, but are not limited to, gene mutants, polypeptide mutants, and protein mutants. A mutant is a type of variant. Methods for obtaining relevant mutants include, but are not limited to, recombination, deletion or omission, insertion, shift, and substitution of structural units. The structural unit of a gene is a base, while the structural units of polypeptides and proteins are amino acids. Types of gene mutations include, but are not limited to, gene deletion or omission, insertion, frameshift, and base substitution.

[0174] In this invention, cell extract, cell extract solution, cell lysate, cell fragments, and cell lysate have the same meaning and can be used interchangeably. In English, they can be described as cell extract, cell lysate, etc.

[0175] In this invention, the terms "energy system," "energy system," and "energy supply system" have equivalent meanings and can be used interchangeably. Similarly, "energy regeneration system" and "energy regeneration system" have equivalent meanings and can be used interchangeably. An energy regeneration system is a preferred embodiment or component of an energy system.

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

[0177] In this invention, unless otherwise specified, the amino acids can be natural amino acids or non-natural amino acids, and can be L-amino acids, D-amino acids, or combinations thereof, as well as amino acids labeled with radioisotopes, modified amino acids, etc. Modified amino acids refer to amino acids with chemically modified groups attached, and their structures are not particularly limited, including but not limited to modifications via amino acid side groups. The definition of amino acids above covers any substance comprising an amino acid unit in this invention, including but not limited to: polypeptides and their derivatives, proteins and their derivatives, polypeptide tags, protein tags, polypeptide sequences, protein sequences, amino acid modifications, polypeptide modifications, protein modifications, partial domains of any of the aforementioned, subunits or fragments of any of the aforementioned, and variants of any of the aforementioned (including variants of the domains, subunits, and fragments of any of the aforementioned). "Variants of any of the aforementioned" include, but are not limited to, "mutants of any of the aforementioned." In this invention, the subscript and non-subscript forms of "L-" and "D-" indicating chirality have the same meaning.

[0178] Crowding agents are reagents used to simulate the crowded macromolecular environment within cells in vitro. References include “X Ge, D Luo and J Xu. Cell-free protein expression under macromolecular crowding conditions[J]. PLoS One, 2011, 6(12):e28707” and its cited references.

[0179] Phosphoric acid compounds include both organic and inorganic compounds.

[0180] Unless otherwise specified, phosphates refer to inorganic phosphates.

[0181] In this invention, "room temperature" is preferably room temperature to 37°C, specifically, preferably 20°C to 37°C, and more preferably 25°C to 37°C.

[0182] In this invention, preferred embodiments, better embodiments, more preferred embodiments, and most preferred embodiments are not intended to limit the implementation of this invention, but are only used to provide examples of embodiments with better technical effects.

[0183] In the description of this invention, the terms "preferred mode," "preferred embodiment," "preferred example," "preferred example," "in a preferred embodiment," "preferred," "preferred," "more preferred," "more preferably," "further preferred," "most preferred," etc., and the illustrative enumeration terms such as "one embodiment," "one mode," "example," "specific example," "for instance," "as an example," "for example," "like," etc., all describe specific features included in at least one specific embodiment of this invention. In this invention, the specific features described by each mode can be combined in any suitable manner in one or more specific embodiments. In this invention, the technical solutions corresponding to each preferred mode can also be combined in any suitable manner; for example, exogenous RNA polymerase and exogenous DNA polymerase can be added simultaneously, see patent document CN108642076A.

[0184] In this invention, "optionally" means that it may or may not be present.

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

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

[0187] In this invention, the terms "or / and" and "and / or" are used to indicate "either one or a combination thereof," or at least one of them. For example, "including substrates for synthesizing RNA and / or substrates for synthesizing proteins" means that it may include only substrates for synthesizing RNA, only substrates for synthesizing proteins, or both substrates for synthesizing RNA and substrates for synthesizing proteins.

[0188] The prior art methods described in this invention using terms such as "usually," "conventionally," "generally," "frequently," and "often" are also cited as references to the content of this invention, and unless otherwise specified, can be regarded as one of the preferred embodiments of this invention.

[0189] All documents mentioned in this invention, and those directly or indirectly cited by such documents, are incorporated herein by reference as if each document were cited individually.

[0190] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (including but not limited to the embodiments) can be combined with each other to form new or preferred technical solutions, as long as they can synthesize exogenous proteins in vitro or preferably synthesize exogenous proteins efficiently. Due to space limitations, they will not be described in detail here.

[0191] The first aspect of this invention provides a method for the in vitro cell-free synthesis of exogenous proteins, the method comprising:

[0192] Step 1: Add component ii to component i and mix.

[0193] 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] Component ii is a nucleic acid template encoding a foreign protein;

[0195] Step 2: Incubate the exogenous protein with the assistance of a hydrophobic interface iii.

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

[0197] Optionally, the procedure may also include the steps of isolating and / or detecting the exogenous protein.

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

[0199] By limiting the technical function to "expressing exogenous proteins," this invention only covers combinations of technical features that can achieve the aforementioned function; combinations of technical features that cannot achieve the aforementioned function are naturally excluded from the scope of this invention. That is, the in vitro cell-free protein synthesis system (component i system) must first be a working system, a system capable of expressing exogenous proteins.

[0200] Hydrophobic interface

[0201] The hydrophobic interface is formed by the surface contact between the hydrophobic phase and the aqueous phase synthesis system.

[0202] The hydrophobic interface provides local enrichment of raw materials through hydrophilic-hydrophobic interactions and also provides interfacial solubilization for insoluble protein products. Furthermore, the translation process from mRNA to protein occurs in ribosomes, and the endoplasmic reticulum (ER) membrane surface of the ribosome has a hydrophobic structure and properties; the hydrophobic interface provides a ribosome-like hydrophobic structure, suggesting that the hydrophobic interface constructed in this invention may also promote the folding of newly synthesized proteins.

[0203] Depending on 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, preferably, the entire liquid phase (including internal and external surfaces / interfaces) is hydrophobic. Preferably, the hydrophobic interface is provided by the following hydrophobic surfaces: the inner surface of the reactor, the hydrophobic outer surface of the hydrophobic embedded solid, the liquid interface of the oil phase system, or a combination thereof. The inner surface of the reactor may be, for example, the inner surface of the sidewall or the inner surface of the bottom of the reactor. The hydrophobic embedded solid refers to a solid with a hydrophobic outer surface, and its location is not particularly limited; it can be deposited below the aqueous synthesis system, suspended in the aqueous synthesis system, or float above the aqueous synthesis system; preferably, the hydrophobic embedded solid is deposited below the aqueous synthesis system or floats above the aqueous synthesis system.

[0204] Preferably, the hydrophobic phase is provided by a hydrocarbon substance. The main component of the hydrocarbon substance is hydrocarbon, and its physical state is paste, solid (e.g., waxy, granular, powder, etc.), liquid, or a mixture thereof. The hydrocarbon substance contains at least 6 carbon atoms, preferably 6 to 44. The hydrocarbon substance can contain saturated hydrocarbons or unsaturated hydrocarbons. Depending on the degree of unsaturation, the hydrocarbon substance contains, but is not limited to, alkanes, alkenes, dienes, and alkynes; depending on the structural type, the hydrocarbon substance contains, but is not limited to, open-chain hydrocarbons and cyclic hydrocarbons.

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

[0206] Preferably, the hydrocarbon is selected from alkanes.

[0207] Preferably, the hydrophobic phase is provided by an alkane.

[0208] Preferably, the alkane is in the form of paste, solid (such as wax, granules, powder, etc.), liquid, or a mixture thereof.

[0209] Preferably, the alkane has at least 6 carbon atoms.

[0210] Preferably, the alkane has a straight-chain structure, a branched structure, a cyclic structure, or a combination thereof. The general molecular formula for the straight-chain and branched structures is CnH2n+2, and the general molecular formula for the cyclic structure is typically CnH2n (monocyclic).

[0211] Preferably, the alkane has 6 to 44 carbon atoms.

[0212] More preferably, the alkane is selected from: cyclohexane, isooctane, decane, tetradecane, pentadecylcyclohexane, tetradecane, petrolatum, or combinations thereof.

[0213] Preferably, the hydrocarbon substance is selected from petrolatum, mineral oil, or a combination thereof.

[0214] The amount of alkane used to increase the synthesis of exogenous proteins (Qhc) is determined based on the expression level of exogenous proteins in the YPRT(Chc) curve, and is selected from the range where the expression level of exogenous proteins in the YPRT(Chc) curve is greater than Y0.

[0215] Preferably, the Qhc is selected from the alkane dosage range when the expression level of the exogenous protein is at least Y0 + 50% YΔ;

[0216] More preferably, the Qhc is selected from the alkane dosage range when the expression level of the exogenous protein is at least Y0 + 60% YΔ;

[0217] More preferably, the Qhc is selected from the alkane dosage range when the expression level of the exogenous protein is at least Y0 + 70% YΔ;

[0218] More preferably, the Qhc is selected from the alkane dosage range when the expression level of the exogenous protein is at least Y0 + 80% YΔ;

[0219] More preferably, the Qhc is selected from the alkane dosage range when the expression level of the exogenous protein is at least Y0 + 90% YΔ;

[0220] More preferably, the Qhc is selected from the alkane dosage range when the expression level of the exogenous protein is at least Y0 + 95% YΔ;

[0221] More preferably, Qhc is the amount of alkane used, Cmax, at which the exogenous protein is expressed at its highest level.

[0222] One preferred method is to determine the amount of alkane as follows: when the types and contents of the system components of the in vitro cell-free protein synthesis system are determined, the amount of alkane is adjusted within a wide concentration range. Under specified reaction conditions (reaction temperature, reaction time, etc.), the amount of alkane Cmax at which the expression level of the exogenous protein is highest is the most suitable amount of alkane under this technical solution.

[0223] The definitions of the YPRT(Chc) curve, Qhc, Chc, YPRT, Ymax, Cmax, Y0, and YΔ are consistent with those described above.

[0224] The scope of protection of this invention only covers the technical solutions corresponding to alkane dosages that can increase the expression level of exogenous proteins. In this invention, for any component i of the in vitro cell-free protein synthesis system, it is only required that at least one alkane dosage value can increase the expression level of exogenous proteins, and it is not required that all alkane dosage values ​​have an enhancing 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 invention is carried out in an aqueous in vitro cell-free protein synthesis system.

[0227] The aforementioned in vitro cell-free protein synthesis system can provide various factors required for the in vitro protein synthesis process. These factors can be provided integratedly via cell extracts or added separately.

[0228] The types and amounts of components in the in vitro cell-free protein synthesis system are not particularly limited, as long as the system can react with the nucleic acid template encoding the exogenous protein to synthesize the exogenous protein. Preferably, combinations that can efficiently express the exogenous protein are preferred. Combinations that cannot express the exogenous protein due to excessively low or high concentrations of certain components are naturally excluded from the scope of this invention.

[0229] There are no particular restrictions on the order in which the components of the in vitro cell-free protein synthesis system are added.

[0230] Preferably, the in vitro cell-free protein synthesis system contains system components capable of recognizing promoter elements on nucleic acid templates, such as RNA polymerases corresponding to promoter elements.

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

[0232] In one preferred embodiment, the in vitro cell-free protein synthesis system includes at least a cell extract. The cell extract is intended to provide structural or biological factors for protein transcription and translation. The selection criteria for the cell extract are: it enables the synthesis of a foreign protein using a nucleic acid template encoding a foreign protein as a basis through an in vitro protein synthesis reaction. The cell extract of this invention can be derived from wild-type or non-wild-type cells. Modification methods for non-wild-type cells include, but are not limited to, genetic modification. The cell extract of this invention is preferably derived from eukaryotic cells, more preferably from yeast cells, and even more preferably from Kluyveromyces lactis cells.

[0233] The components of the in vitro cell-free protein synthesis system preferably include cell extracts. More preferably, the cell extracts contain endogenously expressed RNA polymerases corresponding to promoter elements on the nucleic acid template. Specifically, for example, Kluyveromyces lactis cell extracts contain endogenously expressed T7 RNA polymerases capable of recognizing the T7 promoter on the nucleic acid template.

[0234] One preferred approach is to use the PURE system (Protein synthesis Using Recombinant Elements (PURE) system) developed by Japanese scientists to provide the various factors required for the in vitro protein synthesis process. Refer to the introduction of the PURE system in the literature and its cited references, such as "Lu, Y. Advances in Cell-Free Biosynthetic Technology. Current Developments in Biotechnology and Bioengineering, 2019, Chapter 2, 23-45." and "Y Shimizu, A Inoue, Y Tomari, et al. Cell-free translation reconstituted with purified components[J]. Nature Biotechnology, 2001, 19(8):751-755".

[0235] The process of synthesizing proteins in vitro includes at least translation and optionally transcription.

[0236] The transcription process, which converts DNA into mRNA, is inseparable from RNA polymerase. The in vitro cell-free protein synthesis system preferably also includes RNA polymerase. The RNA polymerase may be selected from: 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] To ensure that the cell extract contains endogenously expressed RNA polymerase, the coding sequence / gene of the RNA polymerase is preferably integrated into the host cell for which the cell extract is prepared. Specifically, this is preferably achieved through the following methods: inserting the coding sequence of the RNA polymerase into a cell-free plasmid, integrating the coding gene of the RNA polymerase into the cell genome, or modifying the bacterial strain using a combination of the above two methods, and then preparing the cell extract. The methods for integrating the coding sequence / gene of the RNA polymerase into the cell genome include, but are not limited to: insertion into the cell genome, in situ replacement of a portion of the genome, or combinations thereof.

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

[0240] The in vitro cell-free protein synthesis system preferably includes a DNA polymerase, which may be selected from: 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 may optionally include an exogenous RNA polymerase and / or a nucleic acid template encoding the RNA polymerase.

[0242] The in vitro cell-free protein synthesis system may optionally include an exogenous DNA polymerase and / or a nucleic acid template encoding the DNA polymerase.

[0243] In one preferred embodiment, the in vitro cell-free protein synthesis system includes exogenous RNA polymerase and exogenous DNA polymerase. References: CN108642076A, WO2018171747A1 (CN201710176691.4).

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

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

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

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

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

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

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

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

[0252] Cell extract

[0253] The cell extract should be able to express the nucleic acid template encoding the exogenous protein in the in vitro cell-free protein synthesis system, that is, be able to synthesize the exogenous protein encoded by the nucleic acid template.

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

[0255] Cell extracts can provide many of the key translation-related elements needed to synthesize exogenous proteins; this is an endogenous way of providing them.

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

[0257] The cell extract, in principle, does not contain intact cells because its preparation involves a cell disruption step (also known as cell lysis or lysis). Compared to traditional methods of protein synthesis from intact cells, this in vitro protein synthesis system is called a cell-free system. The cell extract may also contain other proteins derived from the cytoplasm of cells, especially soluble proteins.

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

[0259] The various protein factors provided by the aforementioned cell extracts, and the related coding genes, can be naturally present in the cell genome, or the related coding genes can be integrated into the cell genome (integrated into the chromosome), or the related genes, gene fragments, or coding sequences can be inserted into intracellular free plasmids. Taking RNA polymerase and DNA polymerase as examples, in one preferred embodiment, the cell extract contains endogenously expressed RNA polymerase and / or DNA polymerase.

[0260] For the source cells of cell extracts, the coding sequence or coding gene of a heterologous protein can be endogenously integrated to enable the endogenous expression of the heterologous protein. The heterologous protein may include, but is not limited to, RNA polymerase, DNA polymerase, etc. The method for endogenously integrating the coding sequence or coding gene of a heterologous protein can refer to the methods provided in existing literature and their cited literature, including but not limited to patent application documents CN109423496A, CN10697843A, CN2018116198190, "Molecular and Cellular Biology, 1990, 10(1):353-360", etc. Specifically, it includes, but is not limited to, inserting the coding sequence into a free cell plasmid, inserting the coding gene into the cell genome, replacing part of the gene in the cell genome with the coding gene in situ, or combinations thereof.

[0261] In one preferred embodiment, the cell extract originates from cells that endogenously integrate an RNA polymerase-encoding gene, enabling endogenous expression of RNA polymerase and allowing for in vitro cell-free protein synthesis without the addition of exogenous RNA polymerase. The methods for achieving endogenous integration of RNA polymerase include, but are not limited to: inserting the RNA polymerase-encoding gene into a cell plasmid or into the cell genome; replacing a portion of a gene or sequence in situ with the RNA polymerase-encoding gene (i.e., including the step of knocking out a portion of the original gene or sequence); knocking out a portion of the original gene and inserting the RNA polymerase-encoding gene; or combinations thereof. In Example S2, the T7 RNA polymerase-encoding gene was 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 no additional RNA polymerase is added to the in vitro cell-free protein synthesis system. In other specific embodiments, the RNA polymerase-encoding sequence is inserted into a free intracellular plasmid of Kluyveromyces lactis to prepare the cell extract. For specific preparation methods, please refer to CN109423496A.

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

[0263] The preparation method of the cell extract can employ reported techniques. In brief, it typically includes the following steps: flash freezing the cells with liquid nitrogen, breaking up the cells, centrifuging to collect the supernatant, thus obtaining the cell extract. Refer to documents CN106978349A, CN108535489A, CN108642076A, CN109593656A, CN109971783A, etc. Alternatively, seed cells can be fermented, centrifuged, the culture medium removed, and the cells collected to prepare the cell extract.

[0264] The cell extract prepared by the method provided in this invention enables in vitro protein synthesis reactions to proceed normally and contains essential components required for protein synthesis, such as tRNA with amino acid transport function and aminoacyl-tRNA synthetase. In some embodiments, the cell extract is a yeast cell extract, prepared by a method comprising the following steps: (i) providing source cells; (ii) washing the yeast cells to obtain washed yeast cells; (iii) performing cell disruption on the washed yeast cells to obtain a crude yeast extract; and (iv) performing solid-liquid separation on the crude yeast extract, with the collected supernatant being the cell extract. Preferably, the yeast cell extract is a Kluyveromyces lactis cell extract.

[0265] In this invention, the protein content of the cell extract is preferably 20 mg / mL to 100 mg / mL. Another preferred method is 20 mg / mL to 50 mg / mL. Yet another preferred method is 50 mg / mL to 100 mg / mL. Another preferred method is any one of the following concentrations: 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, and 90 mg / mL, or a concentration range (including both endpoints) between any two concentrations. The method for determining the protein content can be the Coomassie Brilliant Blue assay.

[0266] There is no particular limitation on the concentration of the cell extract in the in vitro protein synthesis system. In one preferred embodiment, the concentration of the cell extract is 20%-80% (v / v); in another preferred embodiment, the concentration is 20%-70% (v / v); in yet another preferred embodiment, the concentration is 30%-60% (v / v); in yet another preferred embodiment, the concentration is 40%-50% (v / v); and in yet another preferred embodiment, the concentration is 80% (v / v); all based on the total volume of the in vitro cell-free protein synthesis system. Examples of cell extract concentrations include, but are not limited to, any one of the following volume percentages, or a range between any two of the following volume percentages (the range may or may not include two endpoints): 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%.

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

[0268] One preferred method for obtaining the cell extract of the present invention is from prokaryotic cells, more preferably from Escherichia coli (E. coli cells) or Bacillus.

[0269] One preferred method for obtaining the cell extract of the present invention is to obtain it from eukaryotic cells.

[0270] Prokaryotic and eukaryotic cells differ fundamentally in their translation initiation and regulation mechanisms, and prokaryotic expression systems lack many post-translational protein processing mechanisms. Typically, cell-free systems based on E. coli lack the translation or post-translational modification capabilities that only eukaryotic cell-free systems can perform, making many eukaryotic proteins unsuitable for expression in this system; the synthesized proteins contain incomplete novel polypeptides. The synthetic mechanisms of cell-free protein synthesis systems based on prokaryotic systems differ significantly from those based on eukaryotic systems. References include, but are not limited to, the following literature: “Nicole E. Gregorio, Max Z. Levine and Javin P. Oza. Methods Protoc. 2019, 2, 24”, “Edited by Alexander S. Spirin and James R. Swartz. Cell-free protein synthesis: methods and protocols [M]. 2008, p. 5”, “Zhang Xu. Key technologies and industrial applications of efficient synthesis of complex membrane proteins in cell-free systems [D]. Zhejiang University, 2014.” etc. The in vitro cell-free protein synthesis system of this invention preferably uses eukaryotic cell extracts.

[0271] The cell source of the cell extract may be selected from, but is not limited to, eukaryotic cells from 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, rabbit, monkey, and human sources.

[0272] The cell source and preparation method of the cell extract can also refer to 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 literature, and the cell sources reported in the literature directly or indirectly cited are all included in this invention. For example, prokaryotic cell sources include, but are not limited to, Escherichia coli; 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 hamsterovary cell), human K562 cell, HEK293 cell, HeLa cell, mouse embryonic fibroblast, and Leishmania tarentolae cell (protozoan, a single-celled organism).

[0273] The yeast cells are preferably selected from *Saccharomyces cerevisiae*, *Pichia pastoris*, *Kluyveromyces*, or a combination thereof, in one preferred embodiment. The *Kluyveromyces* is further preferably *Kluyveromyces lactis* (K. lactis), *Kluyveromyces lactis* var. *drosophilarum*, *Kluyveromyces lactis* var. *lactis*, *Kluyveromyces marxianus*, *Kluyveromyces marxianus* var. *lactis*, *Kluyveromyces marxianus* var. *marxianus*, *Kluyveromyces marxianus* var. *vanudenii*, *Kluyveromyces dobzhanskii*, *Kluyveromyces aestuarii*, *Kluyveromyces nonfermentans*, or *Kluyveromyces* Wickheim. Kluyveromyces wickerhamii), Kluyveromyces thermomotolerans, Kluyveromyces fragilis, Kluyveromyces hubeiensis, Kluyveromyces polysporus, Kluyveromyces siamensis, Kluyveromyces yarrowii, or combinations thereof; references include, but are not limited to, the following: EP1197560A1, “Marc-André Lachance. The Yeasts (Fifth Edition), Chapter 35, Kluyveromyces van der Walt (1971). 2011, Pages 471-481”, “JL Souciet, BDujon, C Gaillardin, M Johnston et al. Comparative genomics of protoploid Saccharomycetaceae[J]. Genome Res. 2009, 19: 1696-1709.

[0274] Kluyveromyces is an ascospore-forming yeast, among which Kluyveromyces marxianus and Kluyveromyces lactis are widely used industrially. Compared with other yeasts, Kluyveromyces lactis has many advantages, such as superior secretion capacity, better large-scale fermentation characteristics, higher food safety levels, and the ability to perform post-translational protein modifications. The genome of wild-type Kluyveromyces lactis does not contain the gene encoding T7 RNA polymerase.

[0275] In one preferred embodiment, the cell extract is derived from Kluyveromyces lactis and endogenously integrates any one or a combination of the following gene sequences: the gene encoding RNA polymerase, or the gene encoding DNA polymerase. Preferably, the endogenous integration occurs through integration into a cell plasmid or into the cell genome.

[0276] In one preferred embodiment, the cell extract is derived from Kluyveromyces lactis, and endogenously integrates any one or a combination of the following: the encoding gene for T7 RNA polymerase and the encoding gene for phi29 DNA polymerase. Preferably, the endogenous integration is performed by integration into a cell plasmid or into the cell genome.

[0277] In one preferred embodiment of the cell extract of the present invention, the cell extract may be selected from any of the following sources: *Escherichia coli*, yeast cells, mammalian cells, plant cells, insect cells, or combinations thereof. The yeast cells are more preferably selected from *Kluyveromyces*, *Saccharomyces cerevisiae*, *Pichia pastoris*, or combinations thereof; the *Kluyveromyces* is further preferably *Kluyveromyces lactis* var. *drosophilarum*, *Kluyveromyces lactis* var. *lactis*, *Kluyveromyces marxianus* var. *lactis*, *Kluyveromyces marxianus* var. *marxianus*, *Kluyveromyces marxianus* var. *vanudenii*, *Kluyveromyces dobii*, *Kluyveromyces non-fermenting*, *Kluyveromyces var. *marxianus*, *Kluyveromyces wickheim*, *Kluyveromyces thermostable*, *Kluyveromyces brittle-walled*, *Kluyveromyces hubeiensis*, *Kluyveromyces multisporum*, *Kluyveromyces siamensis*, *Kluyveromyces yarrowensis*, or combinations thereof.

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

[0279] In another preferred embodiment, the cell extract may be selected from any of the following sources: Escherichia coli, Kluyveromyces lactis, wheat germ cells, Spodoptera frugiperda cells (sf cells, an insect cell), Leishmania lizard cells, rabbit reticulocytes, Chinese hamster ovary cells (CHO cells), African green monkey kidney COS cells, African green monkey kidney VERO cells, young hamster kidney cells (BHK cells), human HeLa cells, human Hybridoma cells (human hybridoma cells), human fibrosarcoma HT1080 cells, or combinations thereof.

[0280] Exogenous RNA polymerase, exogenous DNA polymerase

[0281] When the genome of the cell from which the cell extract is derived does not contain the gene for RNA polymerase, nor does it have the coding sequence / gene for endogenous integrative RNA polymerase, it is usually necessary to add exogenous RNA polymerase to promote the reaction. For example, when using cell extracts from wild-type Kluyveromyces lactis.

[0282] Adding exogenous RNA polymerase to in vitro protein synthesis systems is a traditional technique. Existing in vitro protein synthesis systems with added exogenous RNA polymerases reported in the art are all included in this invention as optional components of system i. For example, the *Kluyveromyces lactis* in vitro protein synthesis system with added exogenous RNA polymerase (such as T7 RNA polymerase) described in CN108535489A is also included in this invention as an optional component of system i.

[0283] The in vitro cell-free protein synthesis system may also include 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, and nucleic acid template encoding exogenous DNA polymerase.

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

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

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

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

[0288] The polymerase (exogenous RNA polymerase, exogenous DNA polymerase) is preferably a polymerase capable of room-temperature amplification, wherein room temperature is preferably room temperature to 37°C, specifically, preferably 20°C to 37°C, more preferably 25°C to 37°C. The room-temperature amplification polymerase can be selected based on the exogenous nucleic acid template. Polymerases capable of room-temperature amplification in cell-free in vitro systems are included in the scope of this invention, including but not limited to phi29 DNA polymerase, T4 DNA polymerase, T7 DNA polymerase, exo-klenow DNA polymerase, Bsu DNA polymerase, Pol III DNA polymerase, T7 RNA polymerase, T3 RNA polymerase, T4 RNA polymerase, T5 RNA polymerase, etc., any partial domain of any of the above polymerases, any subunit or fragment of any of the above polymerases, any variant of any of the above polymerases, and any combination of the above polymerases and their partial domains, subunits, fragments, variants (including but not limited to mutants). This invention may also employ other DNA polymerases such as Taq DNA polymerase, Pfu DNA polymerase, Pol I DNA polymerase, and Pol II DNA polymerase.

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

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

[0291] The amplification techniques that can be used in this invention, especially room temperature amplification methods, are not particularly limited. Room temperature amplification techniques that can be used in cell-free in vitro systems are all included in the scope of this invention for reference.

[0292] Energy System / Energy Regeneration System

[0293] Energy systems / energy regeneration systems are used to provide the energy required for protein synthesis.

[0294] The energy systems / energy regeneration systems previously reported for cell-free in vitro protein synthesis systems can all provide energy for the in vitro protein synthesis system of the present invention. 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 withglucosemetabolism[J].FEBS Letters,2015,589(15):1723-1727","Y Lu.Advances in Cell-FreeBiosynthetic Technology[J].Current Developments in BiotechnologyandBioengineering,2019,Chapter 2,23-45","P Shrestha,MT Smith and BCBundy.Cell-free unnatural amino acid incorporation with alternative energysystems and linear expression templates[J].New Energy systems / energy regeneration systems reported in literature such as Biotechnology, 2014, 31(1):28-34, and other directly or indirectly cited literature are all incorporated herein by reference.

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

[0296] There are no particular restrictions on the concentration of each component in the energy system, including but not limited to the use of existing reported technical solutions and their equivalents. Example S3 uses an energy system of monosaccharide (glucose), polysaccharide (maltodextrin), and phosphate; Examples S11 and S12 use a polysaccharide and phosphate energy system, wherein the polysaccharide is maltodextrin, and the phosphate is tripotassium phosphate (potassium phosphate is equivalent to tripotassium phosphate).

[0297] substrates for RNA synthesis

[0298] The substrate for the synthesized RNA is a mixture of nucleotides, selected from: nucleoside monophosphates, nucleoside triphosphates, or combinations thereof, in one embodiment. A mixture of nucleoside triphosphates (NTPs) is preferred. The nucleoside triphosphate mixture is preferably a mixture of adenine nucleoside triphosphate, guanine nucleoside triphosphate, cytosine nucleoside triphosphate, and uracil nucleoside triphosphate. In this invention, the concentration of each mononucleotide is not particularly limited, and is measured according to the nucleotides required for protein synthesis. In one preferred embodiment, the concentration of each mononucleotide is 0.5 mM to 5 mM; in another preferred embodiment, it is 1.0 mM to 2.0 mM. Each mononucleotide concentration is independently exemplified by any of the following concentrations, or a concentration range between any two of the following concentration values ​​(the 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] substrates for DNA synthesis

[0300] When DNA amplification or in vitro protein synthesis reactions involve steps involving DNA replication, a substrate for DNA synthesis needs to be added. The substrate for DNA synthesis is preferably a mixture of deoxynucleotides, and more preferably a mixture of deoxynucleoside triphosphates (dNTPs).

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

[0302] substrates for protein synthesis

[0303] The substrate for the synthesized protein is a mixture of amino acids. The concentration is measured in nucleotides required for protein synthesis. One preferred embodiment typically uses a concentration of 0.01 mM to 5 mM for each amino acid, while another preferred embodiment uses a concentration of 0.1 mM to 1 mM. Examples of the concentrations of each amino acid independently include any of the following concentrations, or a concentration range (including both endpoints) between any two of the following concentration values: 0.1 mM, 0.2 mM, 0.4 mM, 0.5 mM, 1.0 mM, 1.2 mM, 1.5 mM, 1.8 mM, 2.0 mM, 2.5 mM, 3.0 mM, 3.5 mM, 4.0 mM, 4.5 mM, 5.0 mM, 5.5 mM, and 6.0 mM.

[0304] The amino acid mixture includes at least the amino acid mixture required for the synthesis of the exogenous protein, selected from, but not limited to: glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine. The amino acid mixture required for the synthesis of the exogenous protein includes not only the amino acids constituting the primary sequence of the exogenous protein but may also include other amino acids involved in the synthesis process.

[0305] The amino acid mixture may include natural amino acids and non-natural amino acids.

[0306] The amino acid mixture may include L-amino acids, D-amino acids, or combinations thereof.

[0307] In addition to natural amino acids, the amino acid mixture may also include non-natural amino acids, D-amino acids, radioisotope-labeled amino acids, modified amino acids, and other components. The non-natural amino acids are not particularly limited and may be selected from, but are not limited to, those reported or cited in literature such as “Y Lu. Cell-free synthetic biology: Engineering in an open world [J]. Synthetic and Systems Biotechnology, 2017, 2, 23-27” and “W Gao, E Cho, Y Liu and Y Lu. Advances and challenges in cell-free incorporation of unnatural aminoacids into proteins [J]. Frontiers in pharmacology, 2019, 10: 611” and other directly or indirectly cited literature. The radioisotope-labeled amino acids are not particularly limited and include, but are not limited to, isotope labeling used in the field of protein synthesis as reported. The modified amino acids are not particularly limited and include, but are not limited to, modifications via amino acid side groups.

[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 may also 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, and aqueous solvents. References may be made to WO2016005982A1, US20060211083A1, “L Kai, VR Kaldenhoff and F Bernhard. Artificial environments for the co-translational stabilization of cell-free expressed proteins[J]. PloS one,2013,8(2):e56637”, US20030119091A1, US20180245087A1, US5665563, WO2019033095A1, US9410170B2, US9528137B2, and other references directly or indirectly cited.

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

[0312] In some preferred embodiments, the molecular weight of the congesting agent does not exceed 400 kDa. In some preferred embodiments, the molecular weight of the congesting agent does not exceed 200 kDa. Typically, the preferred molecular weight distribution is ±10% or narrower. In one preferred manner, the amount of the crowding agent, expressed as a weight percentage (wt%), volume percentage (%(v / v)), or mass-volume concentration (%(w / v)) in the in vitro protein synthesis reaction mixture, is selected from 0.5% to 15%, more preferably from 1% to 12%; for example, any of the following concentration values, or a concentration range between any two of the following concentration values: 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 contains polyethylene glycol and / or its analogues, used as a congesting agent and / or to stabilize mRNA (Reference: 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.). Polyethylene glycol, for example, can also adjust the viscosity of the system. Polyethylene glycol, having repeating units of CH2CH2O (EO units), is commonly abbreviated as PEG (polyethylene glycol), PEO (poly(ethylene oxide)), or POE (polyoxyethylene). The polyethylene glycol analogues include, but are not limited to, copolymers rich in EO units, polyethylene glycol derivatives, other polyoxyolefins (e.g., polyoxypropylene, POP) that can act as congestors, derivatives of the other polyoxyolefins, etc.; the derivatives, taking polyethylene glycol derivatives as an example, include, but are not limited to, chemical modifiers (e.g., methoxy polyethylene glycol, amino modifiers, carboxyl modifiers, etc.), amino acid modifiers, peptide modifiers, protein modifiers, block polymers containing polyethylene glycol blocks, polymers containing polyethylene glycol side chains, etc. There is no particular limitation on the concentration of polyethylene glycol or its analogues. Typically, the concentration of polyethylene glycol or its analogues is 0.1%-10%, preferably 0.1%-8%, more preferably 0.5%-4%, and even more preferably 1%-2%, expressed as the mass-volume concentration (%(w / v)) of the protein synthesis system or as the total weight (wt%). Unless otherwise specified, all values ​​in this invention refer to mass-volume concentration, expressed in %(w / v), such as 2%, which means 2%(w / v), corresponding to 2g / 100mL or 20mg / mL. In some preferred embodiments, the molecular weight of polyethylene glycol and / or its analogues does not exceed 40,000 Da, with representative molecular weights including, for example: 200, 400, 500, 600, 800, 1000, 1200, 1400, 1450, 1500, 1600, 1800, 2000, 2500, 3000, 3350, 4000, 4500, 5000, and 5500. The molecular weights listed are: 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 12000, 14000, 15000, 16000, 18000, 20000, 25000, 30000, 35000, 40000, and combinations thereof; the unit is Da; each of the above numbers is numerically equal to the weight-average molecular weight or the number-average molecular weight. Typically, a molecular weight distribution of ±10% or narrower is preferred.The preferred molecular weight of the polyethylene glycol and / or its analogues is 200 Da to 10000 Da, more preferably 3000 Da to 10000 Da. Another preferred molecular weight is 200 Da to 8000 Da. In this invention, unless otherwise specified, the molecular weight of polyethylene glycol or its analogues refers to the weight-average molecular weight (Mw). Representative PEGs are selected from the following group: PEG200, PEG400, PEG1000, PEG1500, PEG2000, PEG3000, PEG3350, PEG5000, PEG6000, PEG8000, PEG10000, etc., and combinations thereof; wherein, numbers such as 3350 are numerically equal to the weight-average molecular weight.

[0314] The magnesium ions are derived from a magnesium ion source, which is not particularly limited. The magnesium ion source may be selected from the group consisting of: magnesium acetate, magnesium glutamate (preferably L-glutamate magnesium), magnesium aspartate (preferably L-aspartate magnesium), magnesium chloride, magnesium phosphate, magnesium sulfate, magnesium citrate, magnesium hydrogen phosphate, magnesium iodide, magnesium lactate, magnesium nitrate, magnesium oxalate, and combinations thereof. One preferred embodiment has a concentration range of 0.1 mM to 50 mM. Another preferred embodiment has a concentration range of 0.5 mM to 20 mM. Yet another preferred embodiment has a concentration range of 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 glutamate.

[0315] The potassium ions are derived from a potassium ion source, which is not particularly limited. The potassium ion source can be selected from the group consisting of: potassium acetate, potassium glutamate (preferably L-glutamate), potassium chloride, potassium phosphate, potassium sulfate, potassium citrate, potassium hydrogen phosphate, potassium iodide, potassium lactate, potassium nitrate, potassium oxalate, and combinations thereof. One preferred embodiment has a concentration range of 0–500 mM. Another preferred embodiment has a concentration range of 1 mM–250 mM. Another preferred embodiment has a concentration range of 5 mM–200 mM. Another preferred embodiment has a concentration range of 10 mM–100 mM.

[0316] The optimization effects and preferred methods of polyethylene glycol, magnesium ions, and potassium ions reported in patent document WO2016005982A1 can also be incorporated into this invention as a reference.

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

[0318] The reaction promoters include, but are not limited to, the reaction promoters provided in CN109971783A (such as aluminum salts).

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

[0320] The aqueous solvent is preferably a buffer.

[0321] Examples of specific implementation methods for in vitro protein synthesis systems

[0322] One preferred embodiment is that the in vitro protein synthesis system contains cell extracts, endogenously expressed RNA polymerase (included in the aforementioned cell extracts) or exogenously added RNA polymerase, an energy system, a substrate for RNA synthesis, a substrate for protein synthesis, a congestant, magnesium ions, potassium ions, and a buffer solution. Optionally, it may also include any of the following components: an exogenous nucleic acid template encoding the RNA polymerase (independently preferably a DNA template), an endogenously expressed DNA polymerase or exogenously added DNA polymerase, an exogenous nucleic acid template encoding the DNA polymerase (independently preferably a DNA template), a substrate for DNA synthesis, an antioxidant or reducing agent, trehalose, a reaction promoter, and an aqueous solvent. The cell extract is preferably a eukaryotic cell extract, more preferably a yeast cell extract, and one of the more preferred methods is Kluyveromyces lactis cell extract.

[0323] One preferred embodiment is that the in vitro protein synthesis system contains a cell extract (by integrating the RNA polymerase encoding gene into the cell genome or inserting it into an intracellular free plasmid for strain modification), and also contains one or more components selected from the group consisting of: potassium 4-hydroxyethylpiperazine ethanesulfonate (HEPES-K) or tris(hydroxymethyl)aminomethane (Tris), potassium acetate, magnesium acetate, magnesium glutamate, magnesium aspartate, a mixture of nucleoside triphosphates (NTPs), a mixture of amino acids, creatine phosphate, creatine phosphokinase, creatine phosphokinase, dithiothreitol (DTT), an RNase inhibitor, L-arabinose, glucose, sucrose, starch, dextrin, zeadonine, maltodextrin, and phosphates (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 methods is Kluyveromyces lactis cell extract.

[0324] One preferred embodiment is that the in vitro protein synthesis system contains a cell extract and also contains one or more components selected from the group consisting of: HEPES-K or Tris, potassium acetate, magnesium acetate, magnesium glutamate, magnesium aspartate, a mixture of nucleoside triphosphates, a mixture of amino acids, creatine phosphate, creatine phosphokinase, creatine phosphokinase, DTT, RNase inhibitors, L-arabinose, glucose, sucrose, starch, dextrin, zeadonine, maltodextrin, phosphates (such as potassium phosphate), exogenous T7 RNA polymerase, exogenous phi29 DNA polymerase, and a mixture of deoxynucleoside triphosphates. The cell extract is preferably a eukaryotic cell extract, more preferably a yeast cell extract, and one of the more preferred methods is Kluyveromyces lactis cell extract.

[0325] One preferred embodiment is that the in vitro protein synthesis system contains cell extracts (optionally, the gene encoding RNA polymerase is integrated into the cell genome or inserted into an intracellular free plasmid for strain modification), and also contains one or more components selected from the group consisting of: HEPES-K or Tris·HCl, potassium acetate, magnesium acetate, magnesium glutamate (preferably L-glutamate magnesium), magnesium aspartate (preferably L-aspartate magnesium), a mixture of nucleoside triphosphates, a mixture of amino acids, creatine phosphate, creatine phosphokinase, creatine phosphokinase, DTT, RNase inhibitors, L-arabinose, glucose, sucrose, starch, dextrin, corn dextrin, maltodextrin, 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, and a mixture of deoxynucleoside triphosphates. The cell extract is preferably a eukaryotic cell extract, more preferably a yeast cell extract, and one of the more preferred methods is Kluyveromyces lactis cell extract.

[0326] Another preferred embodiment is that the in vitro protein synthesis system contains a cell extract and also contains one or more components selected from the group consisting of: Tris-HCl (pH 8.0), potassium acetate, magnesium acetate, magnesium glutamate, magnesium aspartate (preferably L-aspartate magnesium), dithiothreitol, polyethylene glycol, dextran, sucrose polymer, glucose, L-arabinose, a mixture of nucleoside triphosphates (a mixture of four nucleoside triphosphates, with each nucleoside triphosphate having the same concentration), a mixture of amino acids (glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine; wherein each amino acid has the same concentration), potassium phosphate, exogenous T7 RNA polymerase, exogenous phi29 DNA polymerase, and a mixture of deoxynucleoside triphosphates. The cell extract is preferably a eukaryotic cell extract, more preferably a yeast cell extract, and one of the more preferred methods is Kluyveromyces lactis cell extract.

[0327] Specifically, one preferred embodiment contains 50%–80% (v / v) cell extract, and further contains one or more or all of the following components: 9.78 mM Tris-HCl pH 8.0, 20 mM–80 mM potassium acetate, 2 mM–10 mM magnesium acetate, 1.5 mM–6 mM... Magnesium L-aspartate, 0.4mM–5mM dithiothreitol (e.g., 0.44mM), 0.5%–5% (w / v) polyethylene glycol (e.g., 2% (w / v)), 0.5mM–5mM four nucleoside triphosphates (each nucleoside triphosphate has the same concentration, e.g., 1.8mM), 0.1mM–1mM 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, each amino acid has the same concentration, e.g., 0.5mM), 5mM–110mM L-arabinose, 200mM–400mM maltodextrin (measured as glucose monomer, e.g., 320mM corresponds to approximately 52mg / mL), and 10mM–40mM potassium phosphate. The cell extract is preferably a eukaryotic cell extract, more preferably a yeast cell extract, and one of the more preferred methods is Kluyveromyces lactis cell extract.

[0328] One specific implementation of the in vitro protein synthesis system includes, but is not limited to, the cell-free protein synthesis system based on *E. coli* described in WO2016005982A1. Other in vitro cell-free protein synthesis systems based on wheat germ cells, rabbit reticulocytes, *Saccharomyces cerevisiae*, *Pichia pastoris*, and *Kluyveromyces martensii*, as described in other cited literature of this invention, including but not limited to those based on wheat germ cells, rabbit reticulocytes, *Saccharomyces cerevisiae*, *Pichia pastoris*, and *Kluyveromyces martensii*, are also included as implementation methods in this invention. For example, the in vitro cell-free protein synthesis system 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 literature cited in section "2.1 Systems and Advantages" on pages 27-28, can all be used as the in vitro cell-free protein synthesis system for implementing this invention. For example, documents CN106978349A, CN108535489A, CN108690139A, CN108949801A, CN108642076A, CN109022478A, CN109423496A, CN109423497A, CN109837293A, CN109971783A, CN109988801A, CN110551700A, CN109971775A, CN110551745A, CN110551700A, CN2018116083534, CN2018116198186, CN The in vitro cell-free protein synthesis systems described in CN2018116198190, CN2019102128619, CN2019102355148, CN2019107298813, CN2019112066163, CN2018108881848, CN2018109550734, CN2018111131300, CN2018111423277, CN2018112862093, CN201911418151.8, CN202010069383.3 and their cited references can all be used as the in vitro cell-free protein synthesis systems for implementing the present invention.

[0329] exogenous proteins

[0330] The exogenous proteins applicable to the in vitro protein synthesis system of the present invention are not particularly limited, as long as they can be synthesized in vitro based on cell extracts (including prokaryotic cell extracts, eukaryotic cell extracts; particularly eukaryotic cell extracts, more particularly yeast cell extracts, and even more particularly Kluyveromyces lactis cell extracts). Exogenous proteins of existing in vitro protein synthesis systems applicable to prokaryotic cell extracts, eukaryotic cell extracts (preferably yeast cell extracts, more preferably Kluyveromyces lactis), or endogenous proteins applicable to intracellular synthesis in prokaryotic or eukaryotic cell systems (preferably yeast cell systems, more preferably Kluyveromyces lactis systems) can also be synthesized using the system of the present invention, or attempts can be made to synthesize them using the in vitro protein synthesis system provided by the present invention.

[0331] The application fields of the exogenous protein include, but are not limited to, biomedicine, molecular biology, medicine, in vitro detection, medical diagnosis, regenerative medicine, bioengineering, tissue engineering, stem cell engineering, genetic engineering, polymer engineering, surface engineering, nanoengineering, cosmetics, food, food additives, nutrients, agriculture, feed, daily necessities, detergents, environment, chemical staining, and fluorescent labeling.

[0332] The types of exogenous proteins include, but are not limited to, polypeptides (in this invention, "exogenous protein" broadly includes polypeptides), fluorescent proteins, enzymes and corresponding zymogens, antibodies and their fragments, antigens, immunoglobulins, hormones, collagen, polyamino acids, vaccines, etc., as well as partial domains of any of the aforementioned proteins, subunits or fragments of any of the aforementioned proteins, and variants of any of the aforementioned proteins. The phrase "subunits or fragments of any of the aforementioned proteins" includes subunits or fragments of "partial domains of any of the aforementioned proteins." The phrase "variants of any of the aforementioned proteins" includes variants of "partial domains of any of the aforementioned proteins, subunits or fragments of any of the aforementioned proteins." The phrase "variants of any of the aforementioned proteins" includes, but is not limited to, mutants of any of the aforementioned proteins. In this invention, the meaning of two or more consecutive instances of "as described above" in other positions is interpreted similarly.

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

[0334] For example, the exogenous proteins synthesized by the in vitro protein synthesis system of the present invention can be selected from, but are not limited to, any one of the following proteins, fusion proteins in any combination, or mixtures in any combination: luciferase (such as 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 regions of antibodies, α-amylase, enterotoxin A, hepatitis C virus E2 glycoprotein, insulin precursor, interferon αA, interleukin-1β, lysozyme, serum albumin, single-chain antibody fragment (scFV), thyroxine transporter protein, tyrosinase, xylanase, *Escherichia coli* β-galactosidase (LacZ), human lysine-tRNA synthetase, and human leucine-tRNA synthetase. Synthetase, Arabidopsis thaliana glyceraldehyde-3-phosphate dehydrogenase, catalase, etc., partial domains of any of the aforementioned proteins, subunits or fragments of any of the aforementioned proteins, or variants of any of the aforementioned proteins (as defined above, variants include mutants, such as luciferase mutants or eGFP mutants). See also patent document CN109423496A. Mixtures of any of the aforementioned combinations may include any of the aforementioned proteins, or may include fusion proteins of any of the aforementioned combinations.

[0335] One preferred embodiment involves using GFP, eGFP, or a mutant thereof as exogenous proteins to evaluate the protein synthesis capability of an in vitro protein synthesis system.

[0336] Exogenous nucleic acid templates (including nucleic acid templates encoding exogenous proteins)

[0337] Unless otherwise specified, the exogenous nucleic acid template of the present invention specifically refers to a nucleic acid template encoding an exogenous protein. Furthermore, when specified, the exogenous nucleic acid template of the present invention may also include a nucleic acid template encoding a protein factor or protease required for in vitro protein synthesis, such as an exogenous nucleic acid template encoding RNA polymerase or an exogenous nucleic acid template encoding DNA polymerase.

[0338] Without the nucleic acid template encoding the foreign protein, the in vitro synthesis reaction of the foreign protein cannot proceed.

[0339] In any embodiment of the present invention, the nucleic acid template encoding the foreign protein can be independently a DNA template, an mRNA template, or a combination thereof.

[0340] In any embodiment of the present invention, the nucleic acid template encoding the foreign protein can be independently preferred to be a DNA template.

[0341] Nucleic acid templates serve as direct templates (mRNA), indirect templates (DNA), or combinations thereof for the synthesis of exogenous proteins.

[0342] The nucleic acid template encoding the foreign protein may include non-coding regions. The expression product may be a polypeptide or a protein, or a fusion protein. One translation (or transcription-translation) process is completed for one nucleic acid template molecule, and the number of polypeptide or protein molecules that can be synthesized may be one, two, or more.

[0343] Transcriptional protein synthesis uses DNA templates as indirect templates, while translational protein synthesis can use mRNA templates as direct templates.

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

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

[0346] In any embodiment of the present invention, preferably independently, the nucleic acid template encoding the exogenous protein further contains a promoter element that can be recognized by the cell extract.

[0347] In one preferred embodiment, the nucleic acid template encoding the exogenous protein contains a promoter element that the cell extract can recognize.

[0348] One preferred approach is that the transcription of exogenous proteins is initiated by the T7 promoter on the nucleic acid template.

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

[0350] In one preferred embodiment, the nucleic acid template encoding the exogenous protein includes an exogenous protein translation system, an resistance gene translation system, and a Lac repressor translation system; each of the above translation systems includes a corresponding promoter.

[0351] In one preferred embodiment, the nucleic acid template encoding the foreign protein further contains a gene that controls the copy number of the plasmid.

[0352] In one preferred embodiment, the nucleic acid template encoding the foreign protein further contains a transcriptional enhancement element, such as a kozak sequence.

[0353] In one preferred embodiment, the nucleic acid template encoding the exogenous protein further contains translation enhancement elements, such as translation enhancer elements, IRES elements, kozak sequences, etc.

[0354] Exogenous DNA template (including DNA templates encoding exogenous proteins)

[0355] Unless otherwise specified, the exogenous DNA template of this invention refers specifically to the DNA template encoding the exogenous protein.

[0356] The exogenous DNA template of this invention can be selected from DNA, cDNA, methylated DNA, or a combination thereof. cDNA can be obtained by reverse transcription of RNA or miRNA. miRNA (microRNA) is a class of non-coding single-stranded RNA molecules approximately 20-25 nucleotides in length encoded by endogenous genes.

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

[0358] Preferably, the DNA template encoding the foreign protein contains the gene encoding the foreign protein.

[0359] The DNA template encoding the foreign protein is determined based on the foreign protein.

[0360] The DNA template encoding the exogenous protein may also contain other functional elements selected from promoters, terminators, enhancers (e.g., enhancer elements described in CN109423497A, CN109022478A, CN109837293A (CN201711194355.9), CN109971775A, etc., such as Ω sequences and their homologous sequences, combined enhancer elements), kozak sequences (refer to CN109022478A, CN109837293A, CN109971775A, etc., and their cited references), IRES elements (internal ribosome entry sequences, refer to CN109022478A, CN109423497A, etc., and their cited references), multiple cloning sites (MCS), genes controlling plasmid copy number, etc. It may also contain coding sequences for other amino acid chains, such as signal peptides, leader sequences, functional tags (e.g., purification tags), and linker peptides. It may also contain 5' untranslated sequences and 3' untranslated sequences.

[0361] The DNA template encoding the foreign protein preferably contains a promoter element. The promoter element is required to be recognizable by the cell extract or other components of the in vitro protein synthesis system used; it can be a promoter recognizable by the wild-type cell extract, or the source strain of the cell extract can be modified to recognize the promoter. The promoter in the DNA template encoding the foreign 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 combinations thereof. References include, but are not limited to, the following literature and its cited references: “Cereghino G. Applications of yeast in biotechnology: protein production and genetic analysis. Current Opinion in Biotechnology, 1999, 10(5), 422-427.”

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

[0363] Preferably, the 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 selected based on the amount of exogenous protein to be expressed in the experimental protocol. One preferred embodiment is a concentration of 1 ng / μL to 400 ng / μL for the exogenous DNA template. Another preferred embodiment is a concentration of 1 ng / μL to 80 ng / μL for the exogenous DNA template. Yet another preferred embodiment is a concentration of 5 ng / μL to 50 ng / μL for the exogenous DNA template. Unless otherwise specified, the concentration of DNA template added in this invention is the final concentration, i.e., the initial concentration for the in vitro protein synthesis reaction.

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

[0366] In one preferred embodiment, the exogenous DNA template further includes any element or combination thereof selected from the group consisting of: promoters, terminators, poly(A) elements, transport elements, gene targeting elements, selection marker genes, enhancers, IRES elements, kozak sequences, resistance genes, transposase-encoding genes, signal sequences, leading sequences (e.g., the leading sequences described and cited in CN109022478A), genes controlling plasmid copy number (rop genes), tags enhancing translation levels (e.g., polypeptide tags described in CN2019112066163), etc. See also US20060211083A1, etc.

[0367] The exogenous DNA template can also be constructed in an expression vector. Those skilled in the art can construct expression vectors containing genes encoding exogenous proteins using well-known methods. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology.

[0368] For example, a nucleic acid construct with a "Z1-Z2" structure can be inserted into the cloning site of a plasmid vector to serve as plasmid DNA; where Z1 is the promoter, "-" represents a covalent bond or nucleotide fragment, and Z2 is the coding sequence of the foreign protein. One preferred method for Z1 is the T7 promoter.

[0369] In one preferred embodiment, the exogenous DNA template is circular DNA, more preferably 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 exogenous proteins. Typically, the plasmid contains functional elements such as a promoter, terminator, and untranslated region (UTR). One preferred embodiment of the plasmid is one containing a promoter that can be recognized by the cell extract components. For example, plasmids containing the T7 promoter can theoretically be used as expression vectors for the exogenous DNA template used in Examples S3-S12. For example, pET series plasmids and pGEM series plasmids of *E. coli* can be used to replace the plasmid vectors of *Kluyveromyces lactis* extract in Examples S3-S12 to implement the present invention. Another preferred embodiment of the plasmid is one containing a promoter that can be recognized by the exogenously added components.

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

[0371] Linear DNA can be obtained through in vitro nucleic acid amplification techniques. There are no particular limitations on the amplification techniques that can be used, including but not limited to PCR amplification, isothermal amplification, room temperature amplification, and room temperature amplification. Room temperature amplification is preferred over isothermal amplification. In one preferred embodiment, the exogenous DNA template is linear DNA, specifically a PCR linear fragment. This PCR linear fragment can be obtained using reported PCR techniques.

[0372] Another preferred embodiment is that the exogenous DNA template is linear DNA, specifically double-stranded linear DNA obtained through an amplification system. The amplification system is not particularly limited and can be selected from, but is not limited to, existing commercial kits and amplification systems reported in the literature, as long as they can amplify the DNA template encoding the exogenous protein of this invention. Examples include, but are not limited to, commercial DNA amplification systems provided by companies such as Biocompare, Neta Scientific Inc., ABM, Thermo Fisher Scientific, Expedeon, and Vivantis.

[0373] Another preferred embodiment uses double-stranded DNA as the exogenous DNA template and constructs it in a circular plasmid vector. The plasmid vector typically contains functional elements such as a T7 promoter, a T7 terminator, and / or 5' and 3' UTRs.

[0374] As one of the preferred embodiments, in Examples S3-S12, double-stranded DNA was used as the exogenous DNA template and constructed into a circular plasmid vector. These plasmids contain a T7 promoter, which serves as a promoter for initiating the transcription and translation of exogenous proteins. In Examples S2-S12, the modified Kluyveromyces lactis can endogenously express T7 RNA polymerase. Cell extracts were prepared from the modified strain to construct an in vitro cell-free protein synthesis system. The T7 promoter in this system is suitable for the in vitro cell-free expression of various proteins. The plasmid also contains functional elements such as a T7 terminator and a UTR.

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

[0376] In one embodiment, the plasmid DNA includes at least the structural elements shown in Table 1. Specific examples include... Figure 1 The plasmid structure is shown.

[0377] Table 1 Figure 1 Description of structural elements of the plasmid DNA (pD2P-mEGFP) indicated in the sample.

[0378]

[0379] In another embodiment, except Figure 1 The labeled functional elements also possess purification tags, such as polyhistidine tags (His-tags), between the 5' UTR and the coding sequence of mEGFP. For example... Figure 2 As shown.

[0380] In another embodiment, except Figure 1 The indicated functional element contains a kozak sequence downstream of the 5' UTR, which is used to improve translation accuracy. For example... Figure 2 As shown.

[0381] In another embodiment, except Figure 1 The indicated functional element has a signal peptide coding sequence (signal sequence) between the 5'UTR and the coding sequence of mEGFP, and downstream of the 5'UTR.

[0382] In another embodiment, the plasmid DNA includes the following functional elements: promoter, 5' untranslated region, leader sequence, coding sequence of foreign protein, 3' untranslated region, terminator, replication origin site (f1 ori), AmpR promoter, ampicillin resistance gene, high copy number replication origin site (ori), gene controlling plasmid copy number (rop gene), LacI promoter, and coding sequence of lacI. For example... Figure 3 The plasmid structure is shown. The rop gene is not labeled. The exogenous protein translation system uses the T7 promoter and LAC4 terminator.

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

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

[0385] The basic structure of the plasmid and the method for inserting the coding gene of the exogenous protein into the plasmid vector can be achieved using conventional techniques in the field, and will not be elaborated here. For example, refer to patent documents such as CN108690139A, CN107574179A, and CN108949801A. For example, the basic structure of the plasmid can also be seen in the figure of Chinese patent application document CN201910460987.8.

[0386] In this invention, the concentration of the DNA template encoding the non-exogenous protein can be determined based on the amount of DNA template encoding the exogenous protein used, as described above, and according to the desired expression level of the non-exogenous protein. The non-exogenous protein refers to a translation product synthesized to promote the reaction, rather than the intended expression protein.

[0387] exogenous mRNA template

[0388] The present invention can also use an exogenous mRNA template instead of an exogenous DNA template, or use a mixture of an exogenous mRNA template and an exogenous DNA template, add it to the above-mentioned in vitro cell-free protein synthesis system to carry out an in vitro protein synthesis reaction and synthesize the exogenous protein encoded by the mRNA template.

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

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

[0391] The nucleic acid templates used in the in vitro protein synthesis system of the present invention include nucleic acid templates encoding exogenous proteins, and optionally also include nucleic acid templates encoding other proteins, all of which can be prepared using in vitro nucleic acid amplification technology.

[0392] There are no particular restrictions on the in vitro nucleic acid amplification techniques that can be used; they can be non-isothermal amplification or isothermal amplification (also known as isothermal amplification). These include, but are not limited to, polymerase chain reaction (PCR) amplification, isothermal amplification, room temperature amplification, and room temperature amplification. Among these, room temperature amplification is preferred.

[0393] Among them, for isothermal amplification technology, please refer to "J Kim et al. Acids,2008,27(3)224-243","Yong-JooJeong,Kkothanahreum Park and Dong-Eun Kim.Isothermal DNA amplificationinvitro: the helicase-dependent amplification system[J].Cell.Mol.Life “Sci., 2009, 66: 3325–3336”, “Lv Bei et al. Development and continuous innovation of rapid in vitro nucleic acid amplification technology [J]. China Biotechnology Journal, 2011, 31(3): 91-96”, “Wang Lin et al. Research progress of nucleic acid isothermal amplification technology [J]. Biotechnology Communications, 2011, 22(2)” and other literature and their cited literature disclose isothermal amplification technology. Specifically, the nucleic acid isothermal amplification methods that can be used with the technical means of this invention include, but are not limited to: loop-mediated isothermal amplification (LAMP), chain substitution amplification (SDA), sequence-dependent amplification (NASBA), rolling circle amplification (RCA), nicking enzyme nucleic acid isothermal amplification (nicking enzyme amplification reaction, NEAR), helicase-dependent isothermal amplification (HDA), transcription-dependent amplification, hybridization capture, transcription-mediated amplification (TMA), recombinase-mediated amplification (RAA), recombinase polymerase amplification (RPA), etc., with rolling circle amplification being one of the preferred methods.

[0394] The in vitro nucleic acid amplification methods used in this invention, especially room temperature amplification methods, are not particularly limited. All room temperature amplification techniques that can be used in cell-free in vitro systems in the prior art are included in the scope of this invention, including but not limited to rolling circle amplification (RCA), recombinase polymerase amplification (RPA), chain displacement amplification (SDA), helicase-dependent amplification (HDA), 3SR (self-sustained sequence replication), etc. 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 directly or indirectly cited references, and the in vitro nucleic acid amplification methods disclosed therein (especially room temperature amplification methods) can all be used as technical means of this invention and are all included in this invention by reference.

[0395] The in vitro nucleic acid amplification of the present invention can also employ amplification techniques such as SMART amplification (SMAP), single primer isothermal amplification (SPIA), exponential amplification reaction (EXPAR), thermally stable HDA (tHDA), multiple substitution amplification (MDA), and restriction-assisted RCA.

[0396] The in vitro nucleic acid amplification reaction of the present invention can be carried out continuously at a specific temperature or temperature range that is favorable for the reaction. Any of the room-temperature amplification techniques of the present invention also allows for operation under fluctuating temperature conditions.

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

[0398] A nucleic acid template (preferably a DNA template) encoding a foreign 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 foreign protein.

[0399] The conditions for in vitro protein synthesis reactions are determined based on the specific in vitro cell-free protein synthesis system. Reported reaction conditions can be referenced, including but not limited to those described in documents such as CN106978349A, CN108535489A, and CN108642076A. The in vitro protein synthesis reaction can be carried out continuously at a specific temperature or temperature range favorable to the reaction. Preferably, the temperature change of the mixture in Celsius is less than 25% (e.g., less than 20%, less than 15%, less than 10%, less than 5%) and / or the temperature change of the mixture is less than 15°C (e.g., less than 10°C, less than 5°C, less than 2°C, or less than 1°C) throughout the reaction. In vitro protein synthesis is preferably carried out at room temperature. The room temperature is preferably from room temperature to 37°C, specifically, preferably from 20°C to 37°C. One preferred method is 25°C to 37°C. Another preferred method is 20°C to 30°C. All reported methods for synthesizing proteins at room temperature or isothermal conditions can be used to implement the technical solution of this invention.

[0400] The reaction time can be determined comprehensively based on factors such as the amount of raw materials used (e.g., the amount of reaction substrate, the expected protein content, etc.) and reaction efficiency.

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

[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 may also be selected from any of the following time lengths, or the time length between any two time lengths (including the two 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 and / or detection steps

[0407] The in vitro cell-free synthesis method for the exogenous protein may optionally include the steps of isolating and / or detecting the exogenous protein. The isolation and / or detection methods can be implemented using conventional techniques.

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

[0409] (i) The in vitro cell-free protein synthesis system in the in vitro cell-free synthesis method of exogenous proteins described in the first aspect;

[0410] (ii) Optionally includes a nucleic acid template encoding a foreign protein;

[0411] (iii) Optionally includes a reaction vessel;

[0412] (iv) Hydrophobic phase;

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

[0414] This component can be used to construct a hydrophobic interface that can make surface contact with an aqueous in vitro cell-free protein synthesis system;

[0415] (v) Label or instruction manual.

[0416] (i) and (i) together provide the translation-related elements required for the synthesis of exogenous proteins.

[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 preferred form of the dry powder is lyophilized powder. The liquid includes pure substances and solutions.

[0418] The aforementioned in vitro protein synthesis kit can be used to perform in vitro protein synthesis reactions and synthesize exogenous proteins.

[0419] Each part of the in vitro protein synthesis kit corresponds to each component of the in vitro cell-free synthesis method for exogenous proteins described in the first aspect.

[0420] The hydrophobic phase can be a material phase or device that forms a hydrophobic interface.

[0421] The nucleic acid template encoding the foreign 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 can be, for example, a cell culture plate or centrifuge tube. Examples include six-well plates, twelve-well plates, twenty-four-well plates, forty-eight-well plates, and ninety-six-well plates. It can be a flat-bottomed plate or a pointed-bottomed plate.

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

[0425] In one preferred embodiment, the kit includes dispensing containers comprising the following components: (a) cell extract; (b) energy system; (c) optionally, nucleic acid template; (d) buffer; (e) optionally, pH adjustment component; (f) optionally, several other solid components; and (h) optionally, several other liquid components. Components (a), (b), and (c) are each independently a dry powder or an aqueous solution. Components (c), (e), and (f) are independently present or absent. "Several" here means one, two, or more.

[0426] One preferred method is to package each component into two parts: a dry powder and a liquid reagent. The kit includes two containers, one for the dry powder component and the other for the liquid reagent component. The liquid reagent includes all systems containing a liquid phase, which can be homogeneous or mixed systems, including but not limited to pure substances, solutions, emulsions, suspensions, and combinations thereof.

[0427] One preferred method is to dispense the components as dry powder, buffer solution, or other liquid reagents, optionally including water as a solvent.

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

[0429] In one preferred embodiment, the following components are packaged in separate containers: cell extract (from cells lacking the coding sequence / gene for both endogenous and endogenous integrated RNA polymerase and DNA polymerase), exogenously added RNA polymerase, an energy system, a substrate for RNA synthesis, a substrate for protein synthesis, a congestant, exogenous magnesium ions, exogenous potassium ions, and a buffer solution. Optionally, the container may also include any of the following components: an exogenous DNA template encoding the RNA polymerase, exogenously added DNA polymerase, an exogenous DNA template encoding the DNA polymerase, an antioxidant or reducing agent, trehalose, a reaction accelerator, and an aqueous solvent. The RNA polymerase is independently and more preferably T7 RNA polymerase. The DNA polymerase is independently and more preferably phi29 DNA polymerase. The cell extract contains transfer RNA and ribosomes. The cell extract is preferably a eukaryotic cell extract, more preferably a Kluyveromyces cell extract, and even more preferably a Kluyveromyces lactis cell extract.

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

[0431] Preferably, the application in in vitro protein synthesis includes, but is not limited to, applications in protein manufacturing or applications in protein synthesis-based detection.

[0432] The application fields of the in vitro cell-free synthesis method of the exogenous protein include, but are not limited to, biomedicine, molecular biology, medicine, in vitro detection, medical diagnosis, regenerative medicine, bioengineering, tissue engineering, stem cell engineering, genetic engineering, polymer engineering, surface engineering, nanoengineering, cosmetics, food, food additives, nutrients, agriculture, feed, daily necessities, detergents, environment, chemical staining, fluorescent labeling and other fields.

[0433] The following describes specific embodiments and appendices. Figure 1-13The present invention is further illustrated below. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specifically described in the following embodiments are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), Cell-Free Protein Synthesis Manual (Edited by Alexander S. Spirin and James R. Swartz, Cell-free protein synthesis: methods and protocols [M]. 2008), or as recommended by the manufacturer, or as indicated in the specific implementation guidelines above. Unless otherwise stated, percentages and parts mentioned in this invention are weight percentages and parts by weight.

[0434] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available products.

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

[0436] This invention uses Kluyveromyces lactis (K. lactis or kl) as the source of cell extracts in Examples S2-S12. Examples S3-S12 of this invention all use modified strains of Kluyveromyces lactis to prepare cell extracts. It should be noted that the same design, analysis, and experimental methods are also applicable to other cell extract sources described herein, including but not limited to, prokaryotic cells (such as Escherichia coli), other yeast cells, plant cells, insect cells, animal cells (such as mammalian cells, specifically mouse, rabbit, monkey, and human cells), and other eukaryotic cells.

[0437] The plasmid expression vectors used in the embodiments of this invention are only used to illustrate the implementation of the invention and are not intended to limit the scope of the invention. Other plasmid vectors that can be used to implement the invention include, but are not limited to, common plasmid vectors that are available through existing commercial channels, such as pET series plasmids, pGEM series plasmids, etc.

[0438] Unless otherwise specified, the alkanes used in the following examples refer to linear chain structures (n-structure, straight chain structure). For example, decane, unless otherwise specified, refers to n-decane. The physical parameters of the various alkanes used in the following examples are shown in Table 2 below.

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

[0440]

[0441] The reaction vessels used were either 24-well or 48-well plates, both flat-bottomed cell culture plates. The 24-well plate had a bottom area of ​​1.77 cm². 2 The bottom area of ​​the 48-well plate is 0.8 cm². 2 .

[0442] In the following examples, a negative control group (NC group) was set up. No alkanes or DNA templates encoding fluorescent proteins were added, and other reaction conditions were the same as those of the experimental groups in the examples. After 3 hours and 18-22 hours of reaction, the RFU values ​​were negligible compared to the blank control group (BC group), and the RFU values ​​did not exceed 35. Some experimental results of the NC group are not shown in the figures.

[0443] Example S1: Preparation of nucleic acid template encoding the exogenous protein mEGFP

[0444] A plasmid vector expressing mEGFP was constructed, and in vitro DNA amplification was performed to prepare a plasmid encoding the exogenous protein mEGFP.

[0445] Enhanced green fluorescent protein (mEGFP) was selected as the exogenous protein and used as the target expression product. Its amino acid sequence is shown in SEQ ID No. 2.

[0446] Plasmid vectors were selected. Artificially constructed plasmid vectors targeting Kluyveromyces lactis cell extract were used. These vectors contained functional elements such as the T7 promoter, T7 terminator, 5'UTR, and 3'UTR. This plasmid vector, combined with Kluyveromyces lactis cell extract containing endogenously expressed T7 RNA polymerase, could be used to construct an in vitro cell-free protein synthesis system for expressing various exogenous proteins in vitro.

[0447] Using PCR amplification and homologous fragment recombination, a DNA fragment containing the encoding gene of mEGFP was inserted into a plasmid vector to construct a plasmid vector expressing mEGFP, denoted as plasmid D2P-mEGFP (abbreviated as pD2P-mEGFP), with a total length of 6384 bp. The plasmid was confirmed to be correct by gene sequencing. The gene sequence encoding mEGFP is shown in SEQ ID No. 1.

[0448] The map of the pD2P-mEGFP plasmid is as follows: Figure 1 As shown in Table 3, its structural components are composed of the following elements.

[0449] Table 3. Structural elements of the plasmid (pD2P-mEGFP) encoding the exogenous protein mEGFP.

[0450]

[0451] DNA amplification was performed. The amplification reaction system included the following components at final concentrations: 1 μM–5 μM random primers (primer sequence: NNNNNNN), 1.14 ng / μL of the above plasmid (pD2P-mEGFP, as template), 0.5 mM–1 mM deoxyribonucleoside triphosphate mixture (dNTPs), 0.1 mg / mL–0.5 mg / mL BSA, 0.05 mg / mL–0.1 mg / mL phi29 DNA polymerase, and 1×phi29 reaction buffer (composed of 200 mM Tris-HCl, 20 mM MgCl2, 10 mM (NH4)2SO4, 10 mM KCl, pH 7.5). The reaction system was thoroughly mixed and incubated at 30°C for 2 h. The DNA template was obtained, and its nucleic acid concentration was determined using a UV spectrophotometer. The reaction solution was refrigerated for later use as the nucleic acid template in subsequent examples.

[0452] Example S2: Preparation of Cell Extracts

[0453] In Examples S3-S12 of the present invention, cell extracts were prepared using modified strains of Kluyveromyces lactis.

[0454] The cell extract was obtained from *Kluyveromyces lactis* (K. lactis) cell extract. A modified strain based on *Kluyveromyces lactis* strain ATCC8585 was used. Following the method described in CN109423496A, the coding gene for T7 RNA polymerase was integrated into the genome of *Kluyveromyces lactis* to obtain a modified strain capable of endogenously expressing T7 RNA polymerase. According to control experiments, without the addition of any exogenous RNA polymerase, *Kluyveromyces lactis* without endogenously integrated T7 RNA polymerase coding gene could hardly perform in vitro protein synthesis reactions. After the above endogenous integration modification, high-efficiency expression of exogenous proteins could be achieved without the addition of any exogenous RNA polymerase, reaching the protein synthesis level of traditional in vitro protein synthesis systems (in traditional in vitro protein synthesis systems, cell extracts are prepared using strains without endogenous T7 RNA polymerase modification, and exogenous T7 RNA polymerase is added to the synthesis system). The hydrophobic interface optimization method in embodiments S3-S12 of this invention is also applicable to the in vitro protein synthesis system of strains (including but not limited to Kluyveromyces lactis strain) that have not undergone endogenous modification of T7 RNA polymerase, and can achieve the same or similar optimization effect.

[0455] The preparation process of Kluyveromyces lactis cell extract adopts conventional technical methods, referring to the method described in CN109593656A. In summary, the preparation steps include: providing fermented Kluyveromyces lactis cells as raw material; flash-freezing the cells with liquid nitrogen; breaking up the cells; and collecting the supernatant by centrifugation, which is the cell extract.

[0456] The protein concentration in the obtained Kluyveromyces lactis cell extract was 20–40 mg / mL.

[0457] Examples S2-S12 of the present invention use the following Kluyveromyces lactis cell extract (lysate).

[0458] Table 4. Information on Kluyveromyces lactis cell extracts used in Examples S2-S12

[0459]

[0460] Example S3: Effect of cyclohexane (liquid) on the protein synthesis capacity of an in vitro protein synthesis system

[0461] 3.1 In vitro cell-free protein synthesis system (without added exogenous RNA polymerase)

[0462] Each system consisted of 300 μL and was reacted in a flat-bottomed 48-well plate with a bottom area of ​​0.8 cm² per well. Three replicates were prepared for each sample, and the mean and standard deviation (error bar) were calculated.

[0463] 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 nucleoside triphosphate mixture (adenine nucleoside triphosphate, guanine nucleoside triphosphate, cytosine nucleoside triphosphate and uracil nucleoside triphosphate, each nucleoside triphosphate 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 tripotassium phosphate, 50% volume of Kluyveromyces lactis cell extract. They mix to form an aqueous system.

[0464] 3.2 Reaction Interface (Providing a Hydrophobic Interface)

[0465] Experimental group (cyclohexane concentration curve): 0.1% (v / v) to 20% (v / v) of cyclohexane were added to the system; the cyclohexane 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 layer and the aqueous phase.

[0466] Blank control group (BC group): Compared with the above experimental groups, no alkanes were added (cyclohexane addition was 0).

[0467] Negative control group (NC group): Compared with the above experimental groups, no alkane was added, and no DNA template was added subsequently.

[0468] 3.3 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 above) was added to each independent in vitro cell-free protein synthesis system of the above experimental groups and BC groups. After mixing, all systems were placed in an environment of 20-30℃ and reacted overnight on a shaker. Samples were taken at 3h and 18h ​​for fluorescent protein activity testing.

[0469] 3.4 Fluorescent Protein Activity Assay: The sample to be tested was placed in an Envision 2120 multi-functional microplate reader (PerkinElmer), and the intensity of the fluorescence signal was detected. The relative fluorescence unit (RFU) value was used as the activity unit. The RFU value reflects the amount of mEGFP protein synthesized. The conversion relationship between the mEGFP mass-volume concentration X (unit μg / mL) and the RFU value is as follows: Within the testing scope of this invention, X and RFU are essentially linearly related.

[0470] Fluorescence testing was performed on samples from each reaction system. Sample preparation: Centrifuge at 4000 rpm, 25℃ (room temperature) for 1 minute. Place the samples to be tested in an Envision 2120 multi-mode microplate reader, using excitation / emission wavelengths (Ex / Em): 488 nm / 507 nm, and obtain the relative fluorescence unit (RFU) value.

[0471] 3.5 Experimental Results: (e.g.) Figure 4 As shown in the figure, after 18 hours of reaction, when the amount of cyclohexane added was 0.1% (v / v) to 5% (v / v), the RFU values ​​of the experimental groups were all higher than those of the blank control group (BC group), indicating an improvement in the protein synthesis capacity of the in vitro protein synthesis system. In particular, when the amount of cyclohexane added was 1.8% (v / v), the RFU value of the experimental group was 25.64% higher than that of the BC group, and the synthesis of exogenous protein mEGFP was also 25.64% higher.

[0472] Example S4: Effect of isooctane (liquid) on the protein synthesis capacity of an in vitro protein synthesis system

[0473] 4.1 In vitro cell-free protein synthesis system (without added exogenous RNA polymerase)

[0474] The same aqueous in vitro cell-free protein synthesis system as described in Example 3.1 was used. Each system consisted of 300 μL and the reaction was carried out in a flat-bottomed 48-well plate.

[0475] 4.2 Reaction Interface (Providing a Hydrophobic Interface)

[0476] Experimental group (concentration curve of isooctane): 0.5% (v / v) to 5% (v / v) of isooctane were added to the system; the isooctane 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 layer and the aqueous phase.

[0477] Blank control group (BC group): Compared with the above experimental groups, no alkanes were added (the amount of isooctane added was 0).

[0478] Negative control group (NC group): Compared with the above experimental groups, no alkane was added, and no DNA template was added subsequently.

[0479] 4.3 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 above) was added to each independent in vitro cell-free protein synthesis system of the above experimental groups and BC groups. After mixing, all systems were placed in an environment of 20-30℃ and reacted overnight on a shaker. Samples were taken at 3h and 18h ​​for fluorescent protein activity testing.

[0480] 4.4 Fluorescent protein activity assay: The RFU value of the synthesized exogenous fluorescent protein mEGFP in the sample was determined using the method in 3.4 of Example 3.

[0481] 4.5 Experimental Results: (e.g.) Figure 5As shown in the figure, after 18 hours of reaction, when the amount of isooctane added was 0.5% (v / v) to 5% (v / v), the RFU values ​​of the experimental groups were all higher than those of the blank control group (BC group), indicating an improvement in the protein synthesis capacity of the in vitro protein synthesis system. In particular, when the amount of isooctane added was 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 values ​​of the experimental groups were increased by 89.16%, 88.58%, 75.29%, 77.15%, 70.04%, 73.66%, and 79.49% respectively compared with the BC group, significantly increasing the synthesis of the exogenous protein mEGFP.

[0482] Example S5: Effect of decane (liquid) on the protein synthesis capacity of an in vitro protein synthesis system

[0483] 5.1 In vitro cell-free protein synthesis system (without added exogenous RNA polymerase)

[0484] The same aqueous in vitro cell-free protein synthesis system as described in Example 3.1 was used. Each system consisted of 300 μL and the reaction was carried out in a flat-bottomed 48-well plate.

[0485] 5.2 Reaction Interface (Providing a Hydrophobic Interface)

[0486] Experimental group (concentration curve of n-decane): 0.1% (v / v) to 5% (v / v) decane (n-decane) were added to the system; 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 layer and the aqueous phase.

[0487] Blank control group (BC group): Compared with the above experimental groups, no alkanes were added (decane addition was 0).

[0488] Negative control group (NC group): Compared with the above experimental groups, no alkane was added, and no DNA template was added subsequently.

[0489] 5.3 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 above) was added to each independent in vitro cell-free protein synthesis system of the above experimental groups and BC groups. After mixing, all systems were placed in an environment of 20-30℃ and reacted overnight on a shaker. Samples were taken at 3h and 18h ​​for fluorescent protein activity testing.

[0490] 5.4 Fluorescent protein activity assay: The RFU value of the synthesized exogenous fluorescent protein mEGFP in the sample was determined using the method in 3.4 of Example 3.

[0491] 5.5 Experimental Results: (e.g.) Figure 6 As shown in the figure, after 18 hours of reaction, when the amount of decane added was 0.1% (v / v) to 1.4% (v / v), 2% (v / v), and 5% (v / v), the RFU values ​​of the experimental groups were all higher than those of the blank control group (BC group), indicating an improvement in the protein synthesis capacity of the in vitro protein synthesis system. In particular, when the amount of decane added was 1.2% (v / v), 1.4% (v / v), and 5% (v / v), the RFU values ​​of the experimental groups were 28.66%, 23.93%, and 22.18% higher than those of the BC group, respectively, significantly increasing the synthesis of the exogenous protein mEGFP.

[0492] Example S6 Effect of tetradecane (liquid) on the protein synthesis capacity of an in vitro protein synthesis system

[0493] 6.1 In vitro cell-free protein synthesis system (without added exogenous RNA polymerase)

[0494] The same aqueous in vitro cell-free protein synthesis system as described in Example 3.1 was used. Each system consisted of 300 μL and the reaction was carried out in a flat-bottomed 48-well plate.

[0495] 6.2 Reaction Interface (Providing a Hydrophobic Interface)

[0496] Experimental group (concentration curve of tetradecane): 0.1% (v / v) to 2% (v / v) tetradecane was added to the system; the tetradecane 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 layer and the aqueous phase.

[0497] Blank control group (BC group): Compared with the above experimental groups, no alkanes were added (tetradecane addition was 0).

[0498] Negative control group (NC group): Compared with the above experimental groups, no alkane was added, and no DNA template was added subsequently.

[0499] 6.3 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 above) was added to each independent in vitro cell-free protein synthesis system of the above experimental groups and BC groups. After mixing, all systems were placed in an environment of 20-30℃ and reacted overnight on a shaker. Samples were taken at 3h and 18h ​​for fluorescent protein activity testing.

[0500] 6.4 Fluorescent protein activity assay: The RFU value of the synthesized exogenous fluorescent protein mEGFP in the sample was determined using the method in 3.4 of Example 3.

[0501] 6.5 Experimental Results: (e.g.) Figure 7 As shown in Table 5, after 3 and 18 hours of reaction, when the amount of tetradecane added was 0.1% (v / v) to 2% (v / v), the RFU values ​​of the experimental groups were higher than those of the blank control group (BC group). At 3 hours, the protein synthesis amount in the in vitro protein synthesis system increased by 10.24% to 26.61%, and at 18 hours, it increased by 11.73% to 47.09%, as detailed in Table 5 below. Both reaction efficiency and protein synthesis amount were improved.

[0502] Table 5. Experimental results of optimizing the in vitro protein synthesis system using tetradecane to provide a hydrophobic interface (see Table 5). Figure 7 )

[0503]

[0505] Example S7 Effect of tetradecane (liquid) on the protein synthesis capacity of an in vitro protein synthesis system

[0506] 7.1 In vitro cell-free protein synthesis system (without added exogenous RNA polymerase)

[0507] The same aqueous in vitro cell-free protein synthesis system as described in Example 3.1 was used. Each system consisted of 300 μL and the reaction was carried out in a flat-bottomed 48-well plate.

[0508] 7.2 Reaction Interface (Provides a hydrophobic interface)

[0509] Experimental group (concentration curve of tetradecane): 2% (v / v) to 20% (v / v) tetradecane was added to the system; the tetradecane 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 layer and the aqueous phase.

[0510] Blank control group (BC group): Compared with the above experimental groups, no alkanes were added (tetradecane addition was 0).

[0511] Negative control group (NC group): Compared with the above experimental groups, no alkane was added, and no DNA template was added subsequently.

[0512] 7.3 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 above) was added to each independent in vitro cell-free protein synthesis system of the above experimental groups and BC groups. After mixing, all systems were placed in an environment of 20-30℃ and reacted overnight on a shaker. Samples were taken at 3h and 18h ​​for fluorescent protein activity testing.

[0513] 7.4 Fluorescent protein activity assay: The RFU value of the synthesized exogenous fluorescent protein mEGFP in the sample was determined using the method in 3.4 of Example 3.

[0514] 7.5 Experimental Results: (e.g.) Figure 8 As shown in the figure. After 3 hours of reaction, when tetradecane was added at concentrations of 2% (v / v) to 20% (v / v), the RFU values ​​of the experimental groups were all higher than those of the blank control group (BC group), exceeding them by 1.65% to 34.12%, respectively, thus improving the reaction efficiency. After 18 hours of reaction, when tetradecane was added at concentrations of 2% (v / v) and 5% (v / v), the RFU values ​​of the experimental groups were all higher than those of the blank control group (BC group), and the protein synthesis amount in the in vitro protein synthesis system increased by 21.10% and 11.77%, respectively, thus increasing the protein synthesis amount.

[0515] Example S8 Effect of pentadecylcyclohexane (liquid) on the protein synthesis capacity of an in vitro protein synthesis system

[0516] 8.1 In vitro cell-free protein synthesis system (without added exogenous RNA polymerase)

[0517] The same aqueous in vitro cell-free protein synthesis system as described in Example 3.1 was used. Each system consisted of 300 μL and the reaction was carried out in a flat-bottomed 48-well plate.

[0518] 8.2 Reaction Interface (Provides a hydrophobic interface)

[0519] Experimental group (concentration curve of pentadecylcyclohexane): 0.1% (v / v) to 20% (v / v) of pentadecylcyclohexane were added to the system; the pentadecylcyclohexane 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 layer and the aqueous phase.

[0520] Blank control group (BC group): Compared with the above experimental groups, no alkanes were added (the amount of pentadecylcyclohexane added was 0).

[0521] Negative control group (NC group): Compared with the above experimental groups, no alkanes were added (the amount of pentadecylcyclohexane added was 0), and no DNA template encoding exogenous proteins was added subsequently.

[0522] 8.3 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 above) was added to each independent in vitro cell-free protein synthesis system of the above experimental groups and BC groups. After mixing, all systems were placed in an environment of 20-30℃ and reacted overnight on a shaker. Samples were taken at 3h and 18h ​​for fluorescent protein activity testing.

[0523] 8.4 Fluorescent protein activity assay: The RFU value of the synthesized exogenous fluorescent protein mEGFP in the sample was determined using the method in 3.4 of Example 3.

[0524] 8.5 Experimental Results: (e.g.) Figure 9 As shown in the figure. After 3 hours of reaction, when the amount of pentadecylcyclohexane added was 0.1% (v / v) to 15% (v / v), the RFU values ​​of the experimental groups were all higher than those of the blank control group (BC group), exceeding them by 0.03% to 14.97%, respectively, thus improving the protein synthesis efficiency. After 18 hours of reaction, when the amount of pentadecylcyclohexane added was 12% (v / v) to 20% (v / v), the RFU values ​​of the experimental groups were all higher than those of the blank control group (BC group), and the protein synthesis amount of the in vitro protein synthesis system increased by 20.35% to 36.60%. When the amount of pentadecylcyclohexane added was 12% (v / v), 15% (v / v), 18% (v / v), and 20% (v / v), the RFU values ​​of the experimental groups were 20.35%, 29.50%, 36.60%, and 23.00% higher than those of the BC group, respectively, significantly increasing the synthesis amount of the exogenous protein mEGFP.

[0525] Example S9 Effect of tetradecane (solid powder) on the protein synthesis ability of an in vitro protein synthesis system

[0526] 9.1 In vitro cell-free protein synthesis system (without added exogenous RNA polymerase)

[0527] The same aqueous in vitro cell-free protein synthesis system as described in Example 3.1 was used. Each system consisted of 300 μL and the reaction was carried out in a flat-bottomed 48-well plate.

[0528] 9.2 Reaction Interface (Provides a hydrophobic interface)

[0529] Experimental group (concentration curve of tetradecane): 0–500 μg / μL tetradecane was added to the system; the tetradecane was in solid form. After adding tetradecane to the reaction system and mixing, a suspension was formed. After complete dissolution, an oil phase layer floated on top of the reaction liquid.

[0530] Blank control group (BC group): Compared with the above experimental groups, no alkanes were added (tetradecane addition was 0).

[0531] Negative control group (NC group): Compared with the above experimental group, no alkanes were added (tetradecane addition was 0), and no DNA template encoding exogenous proteins was added subsequently.

[0532] 9.3 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 above) was added to each independent in vitro cell-free protein synthesis system of the above experimental groups and BC groups. After mixing, all systems were placed in an environment of 20-30℃ and reacted overnight on a shaker. During the reaction, tetrachetane was present in the oily layer on top. Samples were taken at 3 h and 18 h for fluorescent protein activity testing.

[0533] 9.4 Fluorescent protein activity assay: The RFU value of the synthesized exogenous fluorescent protein mEGFP in the sample was determined using the method in 3.4 of Example 3.

[0534] 9.5 Experimental Results: (e.g.) Figure 10 As shown in the figure. After 18 hours of reaction, the RFU values ​​of the experimental groups were higher than those of the blank control group (BC group) when the amount of tetratetradecane added ranged from 0 to 500 μg / μL, and the protein synthesis amount of the in vitro protein synthesis system increased by 20.35% to 36.60%. When the amount of tetratetradecane added was 0.02% (v / v), 0.035% (v / v), 0.04% (v / v), and 0.05% (v / v), the RFU values ​​of the experimental groups were 26.47%, 30.90%, 27.97%, and 26.86% higher than those of the BC group, respectively, which significantly increased the synthesis amount of the exogenous protein mEGFP.

[0535] Example S10: Effect of Vaseline (paste) on the protein synthesis capacity of an in vitro protein synthesis system

[0536] 10.1 In vitro cell-free protein synthesis system (without added exogenous RNA polymerase)

[0537] Each system consisted of 300 μL and was reacted in a flat-bottomed 48-well plate with a bottom area of ​​0.8 cm² per well. Three replicates were prepared for each sample, and the mean and standard deviation (error bar) were calculated.

[0538] In vitro cell-free protein synthesis system: The final concentrations of each component were: 9.78 mM Tris-HCl (pH 10). 8.0), 80 mM potassium acetate, 5.0 mM magnesium acetate, 1.5 mM nucleoside triphosphate mixture (adenine nucleoside triphosphate, guanine nucleoside triphosphate, cytosine nucleoside triphosphate and uracil nucleoside triphosphate, each nucleoside triphosphate 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 tripotassium phosphate, 50% volume of Kluyveromyces lactis cell extract. The cell extract prepared was numbered ZS12181, with a protein concentration of 25.16 mg / mL.

[0539] 10.2 Reaction Interface (Provides a hydrophobic interface)

[0540] Experimental group (concentration curve of petrolatum): 5 μg / μL to 450 μg / μL petrolatum was added to the system (corresponding to 1 mg to 90 mg of solid alkane per 200 μL of reaction system); the petrolatum was in paste form. It was applied to the bottom inner wall and sidewalls of the wells, with the amount added measured as the weight difference before and after application. A hydrophobic interface was provided between the petrolatum layer and the aqueous phase.

[0541] Blank control group (BC group): Compared with the above experimental groups, no alkane was added (the amount of petroleum jelly applied was 0).

[0542] Negative control group (NC group): Compared with the above experimental group, no alkanes were added (the amount of petroleum jelly applied was 0), and no DNA template encoding exogenous proteins was added subsequently.

[0543] 10.3 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 above) was added to each independent in vitro cell-free protein synthesis system of the above experimental groups and BC groups. After mixing, all systems were placed in an environment of 20-30℃ and reacted overnight on a shaker. Samples were taken at 18 h for fluorescent protein activity testing.

[0544] 10.4 Fluorescent protein activity assay: The RFU value of the synthesized exogenous fluorescent protein mEGFP in the sample was determined using the method in 3.4 of Example 3.

[0545] 10.5 Experimental Results: (e.g.) Figure 11 As shown in the figure. After 18 hours of reaction, when the amount of petroleum jelly added was 1 mg to 3.5 mg / well, the RFU values ​​of the experimental groups were all higher than those of the blank control group (BC group), and the protein synthesis amount of the in vitro protein synthesis system increased by 9.96% to 39.95%. When the amount of petroleum jelly added to each well was 1 mg, 1.5 mg, 2.5 mg, and 3.5 mg, respectively, the RFU values ​​of the experimental groups increased by 39.95%, 21.42%, 21.91%, and 18.99% compared with the BC group, respectively, which significantly increased the synthesis amount of exogenous protein mEGFP.

[0546] Example S11 Optimization of the combination of petrolatum and activated carbon in an in vitro protein synthesis system

[0547] 11.1 In vitro cell-free protein synthesis system (without added exogenous RNA polymerase)

[0548] Each system consisted of 300 μL and was reacted in a flat-bottomed 48-well plate with a bottom area of ​​0.8 cm² per well. Three replicates were prepared for each sample, and the mean and standard deviation (error bar) were calculated.

[0549] In vitro cell-free protein synthesis system: The final concentrations of each component were as follows: 9.78 mM tris(hydroxymethyl)aminomethane (Tris, pH 10). 8.0 (pH adjusted with hydrochloric acid solution), 80 mM potassium acetate, 5.0 mM magnesium acetate, 1.5 mM nucleoside triphosphate mixture (adenine nucleoside triphosphate, guanine nucleoside triphosphate, cytosine nucleoside triphosphate and uracil nucleoside triphosphate, each nucleoside triphosphate 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 final concentration of 0.7 mM), 0.44 mM dithiothreitol, 2% (w / v) polyethylene glycol 8000, 15 mM glucose, 52.48 mg / mL maltodextrin, 24 mM tripotassium phosphate, 50% volume of Kluyveromyces lactis cell extract. The cell extract prepared was numbered ZS01092, and the protein concentration was 23 mg / mL.

[0550] 11.2 Reaction Interface (Providing a Hydrophobic Interface)

[0551] Group BC (blank control group): No alkanes were added (the amount of petroleum jelly applied was 0), and no activated charcoal was added.

[0552] Vas group (Vaseline group): 0.005 g / mL Vaseline was applied to the sidewalls and bottom inner walls of the reactor (1 mg per well). A hydrophobic interface was provided between the Vaseline layer and the aqueous phase.

[0553] AC group (activated carbon group): 0.04 g / mL activated carbon particles (12 mg per well) were added to the system and deposited on the bottom inner wall.

[0554] AC+Vas group (optimized combination of activated carbon and petrolatum): 0.005 g / mL petrolatum (1 mg per well) and 0.04 g / mL activated carbon particles (12 mg per well) were added to the system.

[0555] NC group (negative control group): No alkanes were added (the amount of petroleum jelly applied was 0), no activated charcoal was added, and no DNA template encoding exogenous proteins was added subsequently.

[0556] 11.3 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 above) was added to each independent in vitro cell-free protein synthesis system of the BC group, Vas group, AC group, and AC+Vas group. After mixing, all systems were placed in an environment of 20℃~30℃ and reacted overnight on a shaker. Samples were taken at 3h, 6h, and 21h for fluorescent protein activity testing.

[0557] 11.4 Fluorescent protein activity assay: The RFU value of the synthesized exogenous fluorescent protein mEGFP in the sample was determined using the method in 3.4 of Example 3.

[0558] 11.5 Experimental Results: (e.g.) Figure 12 As shown in the figure, after 21 hours of reaction, the RFU value of the Vas group (coated with Vaseline) increased by 40.38% compared to the BC group (without Vaseline); the RFU value of the AC+Vas group (coated with Vaseline) increased by 57.06% compared to the AC group (without Vaseline) and by 72.64% compared to the BC group; the optimized combination of Vaseline and activated carbon increased the RFU value by 171.13% compared to the blank control group (BC group), demonstrating a synergistic optimization effect. Furthermore, compared to the blank control group (BC group), at 3 hours, the RFU values ​​of the Vas group, AC group, and AC+Vas group increased by 61.79%, 56.61%, and 103.98%, respectively; and at 6 hours, the RFU values ​​of the Vas group, AC group, and AC+Vas group increased by 48.28%, 81.91%, and 144.56%, respectively.

[0559] Example S12: Combination optimization of Vaseline for in vitro protein synthesis systems

[0560] 12.1 In vitro cell-free protein synthesis system (without added exogenous RNA polymerase)

[0561] Each system consisted of 300 μL and was reacted in a flat-bottomed 48-well plate with a bottom area of ​​0.8 cm² per well. Three replicates were prepared for each sample, and the mean and standard deviation (error bar) were calculated.

[0562] 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 nucleoside triphosphate mixture (adenine triphosphate, guanine triphosphate, cytosine triphosphate, and uracil 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), and 0.44 mM... DTT, 2% (w / v) polyethylene glycol 8000, 15 mM glucose, 52.48 mg / mL maltodextrin, 24 mM tripotassium phosphate, 0.3 mg / mL ampicillin (Amp), 16.6 μg / mL salmon sperm DNA (ssDNA), and 50% volume of Kluyveromyces lactis cell extract. The cell extract prepared was designated ZS12181, with a protein concentration of 25.16 mg / mL.

[0563] 12.2 Reaction Interface (Providing a Hydrophobic Interface)

[0564] AC+ssDNA+Amp group (BC group, blank control group): Vaseline application amount was 0, and activated charcoal was added to each well.

[0565] Vas+AC+ssDNA+Amp group (Vaseline group): 0.05 g / mL Vaseline was applied to the sidewalls and bottom inner walls of the reactor (1 mg per well), and 12 mg of activated carbon was added to each well. A hydrophobic interface was provided between the Vaseline layer and the aqueous phase.

[0566] NC group (negative control group): No alkanes were added (the amount of petroleum jelly applied was 0), no activated charcoal was added, and no DNA template encoding exogenous proteins was added subsequently.

[0567] 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 above) was added to each independent in vitro cell-free protein synthesis system of the BC group and the Vaseline group. After mixing, all systems were placed in an environment of 20℃~30℃ and reacted overnight on a shaker. Samples were taken at 3h, 6h, and 21h for fluorescent protein activity testing.

[0568] 12.4 Fluorescent protein activity assay: The RFU value of the synthesized exogenous fluorescent protein mEGFP in the sample was determined using the method in 3.4 of Example 3.

[0569] 12.5 Experimental Results: (e.g.) Figure 13 As shown. After 3h, 6h, and 21h of reaction, the RFU values ​​of the Vas+AC+ssDNA+Amp group (Vaseline group) coated with Vaseline increased by 9.06%, 6.14%, and 20.27% respectively compared with the BC group without Vaseline coating.

[0570] The above are merely some preferred embodiments of the present invention, and the present invention is not limited to the contents of the above embodiments. For those skilled in the art, various changes and modifications can be made within the scope of the concept of the technical solution of the present invention or under its guidance or inspiration. Any changes and modifications with equivalent technical effects are within the protection scope of the present invention.

[0571] sequence list

[0572] <110> Kangma (Shanghai) Biotechnology Co., Ltd.

[0573] <120> A method for in vitro cell-free protein synthesis based on hydrophobic interfaces, a D2P reagent kit, and related applications.

[0574] <130> 2020

[0575] <141> 2020-03-16

[0576] <160> 2

[0577] <170> SIPOSequenceListing 1.0

[0578] <210> 1

[0579] <211> 717

[0580] <212> DNA

[0581] <213> Artificial sequence

[0582] <400>1

[0583] atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60

[0584] ggcgacgtaa acggccacaa gttcagcgtg cgcggcgagg gcgagggcga tgccaccaac 120

[0585] ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180

[0586] ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240

[0587] cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatctcc 300

[0588] ttcaaggacg acggcaccta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

[0589] gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

[0590] aagctggagt acaacttcaa cagccacaac gtctatatca cggccgacaa gcagaagaac 480

[0591] ggcatcaagg cgaacttcaa gatccgccac aacgtcgagg acggcagcgt gcagctcgcc 540

[0592] gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600

[0593] tacctgagca cccagtccaa gctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

[0594] ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaag 717

[0595] <210>2

[0596] <211>239

[0597] <212>PRT

[0598] <213>Artificial sequence

[0599] <400>2

[0600] Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu 1 5 10 15

[0601] Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Arg Gly 20 25 30

[0602] Glu Gly Glu Gly Asp Ala Thr Asn Gly Lys Leu Thr Leu Lys Phe Ile 35 40 45

[0603] Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr 50 55 60

[0604] Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys 65 70 75 80

[0605] Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu 85 90 95

[0606] Arg Thr Ile Ser Phe Lys Asp Asp Gly Thr Tyr Lys Thr Arg Ala Glu 100 105 110

[0607] Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly 115 120 125

[0608] Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr 130 135 140

[0609] Asn Phe Asn Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn 145 150 155 160

[0610] Gly Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp Gly Ser 165 170 175

[0611] Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly 180 185 190

[0612] Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Lys Leu 195 200 205

[0613] Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe 210 215 220

[0614] 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 in that: The in vitro cell-free synthesis method of the exogenous protein includes: Step 1: Add component ii to component i and mix. Component i is an in vitro cell-free protein synthesis system; the in vitro cell-free protein synthesis system is an aqueous synthesis system. Component ii is a nucleic acid template encoding a foreign protein; Components i and ii together provide the translation-related elements required for the synthesis of the exogenous protein; Step 2: Incubate the exogenous protein with the assistance of a hydrophobic interface iii. 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 aqueous phase in a synthetic system. The hydrophobic phase is provided by a hydrocarbon substance; The hydrocarbon substance is selected from aliphatic hydrocarbons.

2. The method for in vitro cell-free synthesis of exogenous proteins according to claim 1, characterized in that: Depending on the physical state of the hydrophobic phase in surface contact with the aqueous phase of the synthetic system, the hydrophobic interface iii is selected from: a solid-phase hydrophobic interface, a liquid-phase hydrophobic interface, or a combination thereof. Preferably, the hydrophobic interface iii is provided by the following hydrophobic surfaces: the inner surface of the reactor, the hydrophobic outer surface of a hydrophobic embedded solid, the liquid interface of an oil phase system, or a combination thereof; Preferably, the hydrocarbon substance is in the form of paste, wax, granules, powder, liquid, or a mixture thereof; Preferably, the hydrocarbon contained in the hydrocarbon substance has a straight-chain structure, a branched-chain structure, a cyclic structure, or a combination thereof; Preferably, the hydrocarbon substance has at least 6 carbon atoms; more preferably, the hydrocarbon substance has 6 to 44 carbon atoms. Preferably, the hydrocarbon is selected from alkanes; Preferably, the hydrocarbon substance is selected from petrolatum, mineral oil, or a combination thereof; More preferably, the alkane is selected from: cyclohexane, isooctane, decane, tetradecane, pentadecylcyclohexane, tetradecane, petrolatum, or combinations thereof; The amount of alkane that can increase the synthesis of exogenous proteins is denoted as Qhc; The Qhc is selected from the alkane dosage range in the YPRT(Chc) curve where YPRT is greater than Y0; where Chc refers to the alkane dosage, YPRT refers to the exogenous protein expression level, the YPRT(Chc) curve refers to the curve with alkane dosage as the independent variable, exogenous protein expression as the dependent variable, and other reaction parameters as the determination; Y0 refers to the exogenous protein expression level corresponding to Chc being 0. Preferably, the Qhc is selected from the alkane dosage range when the expression level of the exogenous protein is at least Y0 + 50% YΔ; More preferably, the Qhc is selected from the alkane dosage range when the expression level of the exogenous protein is at least Y0 + 60% YΔ; More preferably, the Qhc is selected from the alkane dosage range when the expression level of the exogenous protein is at least Y0 + 70% YΔ; More preferably, the Qhc is selected from the alkane dosage range when the expression level of the exogenous protein is at least Y0 + 80% YΔ; More preferably, the Qhc is selected from the alkane dosage range when the expression level of the exogenous protein is at least Y0 + 90% YΔ; More preferably, the Qhc is selected from the alkane dosage range when the expression level of the exogenous protein is at least Y0 + 95% YΔ; More preferably, Qhc is the amount of alkane used, Cmax, at which the exogenous protein is expressed at its highest level; Wherein, YΔ = Ymax - Y0; where, Ymax refers to the highest expression level of the exogenous protein in the YPRT(Chc) curve.

3. The method for in vitro cell-free synthesis of exogenous proteins according to any one of claims 1-2, characterized in that, The in vitro cell-free protein synthesis system includes any one or more of the following: (1) Cell extracts; (2) RNA polymerase; the RNA polymerase is selected from any of the following: cell extracts containing endogenously expressed RNA polymerase, exogenous RNA polymerase, translation products of exogenous nucleic acid templates encoding RNA polymerase, or combinations thereof; Preferably, the RNA polymerase is T7 RNA polymerase; (3) DNA polymerase; the source of said DNA polymerase is selected from any of the following: cell extract containing endogenously expressed DNA polymerase, exogenous DNA polymerase, translation product of exogenous nucleic acid template encoding DNA polymerase, or a combination thereof; Preferably, the DNA polymerase is phi 29 DNA polymerase.

4. The method for in vitro cell-free synthesis of exogenous proteins according to any one of claims 3, characterized in that: The cell extract is selected from: prokaryotic cell extracts, eukaryotic cell extracts, or combinations thereof; Preferably, the cell extract is selected from any of the following sources: Escherichia coli, yeast cells, mammalian cells, plant cells, insect cells, or a combination thereof; More preferably, the yeast cells are selected from Kluyveromyces, Saccharomyces cerevisiae, Pichia pastoris, or a combination thereof; More preferably, the Kluyveromyces is selected from any one of the following: Kluyveromyces lactis, Kluyveromyces marx, Kluyveromyces dob, Kluyveromyces salina, Kluyveromyces wickheim, Kluyveromyces brittlewall, Kluyveromyces hubeiensis, Kluyveromyces multisporum, Kluyveromyces siamensis, Kluyveromyces yarbromentosus, or a combination thereof.

5. The method for in vitro cell-free synthesis of exogenous proteins according to any one of claims 1-4, characterized in that: The in vitro cell-free protein synthesis system also includes substrates for synthesizing RNA and / or substrates for synthesizing proteins; Preferably, the substrate for synthesizing RNA is a mixture of nucleotides, more preferably selected from: nucleoside monophosphates, nucleoside triphosphates, or combinations thereof; Preferably, the substrate for the synthesized protein is a mixture of amino acids, including at least the mixture of amino acids required for the synthesis of exogenous proteins; more preferably, the mixture of amino acids is a mixture of natural amino acids. The in vitro cell-free protein synthesis system may optionally include a substrate for synthesizing DNA; the substrate for synthesizing DNA is preferably a mixture of deoxynucleotides, more preferably a mixture of deoxynucleotide triphosphates.

6. The method for in vitro cell-free synthesis of exogenous proteins according to any one of claims 1-5, characterized in that: The in vitro cell-free protein synthesis system also includes at least one of the following components: crowding agent, magnesium ions, potassium ions, antioxidant or reducing agent, trehalose, reaction promoter, buffer, and aqueous solvent; The crowding agent is preferably polyethylene glycol, polyvinyl alcohol, polypropylene, dextran, sucrose polymer, polyethylene (vinylpyrrolidone), albumin, or a combination thereof; The magnesium ions are preferably derived 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 combinations thereof. The potassium ions are preferably derived from: potassium acetate, potassium glutamate, potassium chloride, potassium phosphate, potassium sulfate, potassium citrate, potassium hydrogen phosphate, potassium iodide, potassium lactate, potassium nitrate, potassium oxalate, or combinations thereof; The antioxidant or reducing agent is preferably dithiothreitol, 2-mercaptoethanesulfonic acid, 2-mercaptoethanol, reduced glutathione, tricarboxymethyl phosphate, 3-mercapto-1,2-propanediol, or a combination thereof. The buffer is preferably selected from any one of the following: Tris-HCl, Tris base, HEPES, or a combination thereof; The aqueous solvent is preferably a buffer.

7. The method for in vitro cell-free synthesis of exogenous proteins according to any one of claims 1-6, characterized in that, The exogenous protein is selected from any one of the following proteins, fusion proteins in any combination, or mixtures in any combination: luciferase, green fluorescent protein, enhanced green fluorescent protein, yellow fluorescent protein, aminoacyl-tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, variable regions of antibodies, α-amylase, enterotoxin A, hepatitis C virus E2 glycoprotein, insulin precursor, interferon αA, interleukin-1β, lysozyme, serum albumin, single-chain antibody fragments, thyroxine transporter protein, tyrosinase, xylanase, Escherichia coli β-galactosidase, human lysine-tRNA synthetase, human leucine-tRNA synthetase, Arabidopsis thaliana glyceraldehyde-3-phosphate dehydrogenase, mouse catalase, partial domains of any of the foregoing, subunits or fragments of any of the foregoing, or variants of any of the foregoing. Preferably, the variant is a mutant.

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

9. An in vitro protein synthesis kit, characterized in that, The in vitro protein synthesis kit includes: (i) The in vitro cell-free protein synthesis system in the in vitro cell-free synthesis method of exogenous protein according to any one of claims 1-8; Preferably, 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) Optionally includes a nucleic acid template encoding a foreign protein; (iii) Optionally includes a reaction vessel; (iv) Hydrophobic phase: capable of forming a hydrophobic interface with the aqueous phase of the in vitro cell-free protein synthesis system; The hydrophobic interface is selected from any of the hydrophobic interfaces described in claim 1 or 2; (i) and (ii) together provide the translation-related elements required for the synthesis of exogenous proteins; Preferably, the nucleic acid template encoding the foreign protein contains a promoter element that can be recognized by (i); Preferably, 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; In one preferred embodiment, the transcription process of the exogenous protein gene is initiated by the T7 promoter on the nucleic acid template, and the in vitro cell-free protein synthesis system includes T7 RNA polymerase; The nucleic acid template encoding the foreign protein is a DNA template, an mRNA template, or a combination thereof.

10. The use of alkanes in the in vitro cell-free synthesis method of exogenous proteins according to any one of claims 1-8, or in the in vitro protein synthesis kit according to claim 9, characterized in that, The alkane is used to construct a hydrophobic interface.

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