An in vitro cell-free protein synthesis system (D2P system), its reagent kit and its applications

Abstract

This invention provides an in vitro cell-free protein synthesis system, its reagent kit, and its applications, belonging to the field of protein synthesis technology. This invention improves an in vitro cell-free protein synthesis system based on Kluyveromyces lactis by adding exogenous L-arabinose, significantly enhancing the system's protein synthesis capacity. Furthermore, it provides a more efficient and higher-throughput in vitro protein synthesis reagent kit and a method for synthesizing exogenous proteins. The improvements provided by this invention can be achieved without molecular modification, are simple and convenient to operate, and save costs.

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 L-arabinose-optimized in vitro cell-free protein synthesis system (DNA-to-Protein system, D2P system), its kit, and its applications. Background Technology

[0002] Proteins are essential molecules in cells, participating in almost all cellular functions. Protein synthesis mainly includes traditional intracellular synthesis techniques and next-generation in vitro synthesis techniques. Traditional protein expression systems refer to molecular biology techniques that express exogenous genes using model organisms such as bacteria, fungi, plant cells, 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. 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 (eukaryoticsystems. Chembiochem. 2015, 16:2420-2431”). Protein products produced using in vitro synthesis systems can be widely used in various fields such as medicine, food, nutritional products, dietary supplements, and cosmetics, including but not limited to PROTEINN. TM Proton TM Princeton TM Protein products, etc.

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

[0004] Kluyveromyces is an ascospore-forming yeast. Among them, 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 standards, and the ability to perform post-translational protein modifications.

[0005] Energy supply is one of the key issues affecting system efficiency and cost in order to synthesize large amounts of protein. Existing energy supply systems include carbohydrate and phosphate energy systems, phosphocreatine and phosphocreatinine enzyme systems, and monosaccharide or glycogen and their glycolysis intermediates energy systems. Monosaccharides, oligosaccharides, and polysaccharides all have energy systems for constructing or optimizing in vitro cell-free protein synthesis systems (Anderson MJ, Stark JC, Hodgman CE and Jewett M C. Energizing eukaryotic cell-free protein synthesis with glucose metabolism[J].FEBS Letters,2015,589(15):1723-1727).

[0006] In summary, there are still many technical gaps to be addressed in how to improve the in vitro cell-free protein synthesis system based on Kluyveromyces lactis. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention discloses a simple, convenient, more efficient, and higher-throughput in vitro cell-free protein synthesis system. This system is based on Kluyveromyces lactis cell extract and is optimized by adding L-arabinose, which can significantly improve protein synthesis capacity.

[0008] The first aspect of this invention provides an in vitro cell-free protein synthesis system, the in vitro cell-free protein synthesis system comprising Kluyveromyces lactis cell extract and exogenous L-arabinose; the in vitro cell-free protein synthesis system is capable of reacting with a nucleic acid template encoding an exogenous protein to synthesize the exogenous protein.

[0009] The exogenous L-arabinose can increase the expression level of exogenous proteins.

[0010] Preferably, the amount of exogenous L-arabinose added is based on C L-ara As the independent variable, Y PRT Y in the concentration curve when the dependent variable and other reaction parameters are determined PRT The value determines the concentration range.

[0011] In this invention,

[0012] C L-ara , refers to the concentration of exogenous L-arabinose.

[0013] Y PRT , refers to the expression level of exogenous proteins in the in vitro cell-free protein synthesis system of the present invention.

[0014] Unless otherwise specified, the "concentration curve of exogenous L-arabinose" in this invention refers to the concentration curve of exogenous L-arabinose in the in vitro cell-free protein synthesis system of this invention, with C L-ara As the independent variable, Y PRT The concentration curve when the dependent variable and other reaction parameters are determined is also denoted as "Y". PRT (C L-ara The concentration curve is also referred to as Y in this invention. PRT ~C L-ara Concentration curve.

[0015] The "other reaction parameters" include, but are not limited to: other system components (including type, concentration, etc.) besides L-arabinose, the method of adding reaction raw materials, reaction temperature program, reaction time length, the nature of the reaction vessel, reaction system volume, etc.

[0016] In this invention, the "in vitro cell-free protein synthesis system without added L-arabinose" is referred to as the CFS(ara-) basic system. One basic system can correspond to multiple Y... PRT ~CL-ara Concentration curves. A basic system can react at different temperature programs and for different durations, thereby producing several different Y atoms. PRT ~C L-ara Concentration curve.

[0017] Y max , refers to the highest expression level of the exogenous protein in the exogenous L-arabinose concentration curve.

[0018] C max The concentration curve of exogenous L-arabinose, Y max The corresponding concentration of exogenous L-arabinose.

[0019] Y min , refers to Y PRT (C L-ara In the concentration curve, Y PRT The lowest expression level of exogenous protein within the range of >Y0.

[0020] Y0 refers to Y PRT (C L-ara In the concentration curve, C L-ara =0 corresponds to the expression level of exogenous protein.

[0021] Y Δ , for Y max The difference between Y0 and Y0, numerically, Y Δ =Y max -Y0.

[0022] The C L-ara Selected from the concentration curves of exogenous L-arabinose, the concentration range of exogenous L-arabinose where the expression level of the exogenous protein is greater than Y0. In this invention, the C... L-ara Selected from, Y PRT (C L-ara The expression level of exogenous protein (Y) in the concentration curve PRT The concentration range where Y is greater than Y0. The above-mentioned selectable concentration range can be continuous or discontinuous.

[0023] Preferably, the C L-ara Selected from Y PRT ≥Y0+50%Y Δ The concentration range of exogenous L-arabinose (i.e., Y) min =Y0 + 50%Y Δ ).

[0024] More preferably, the C L-ara Selected from Y PRT ≥Y0+60%Y Δ The concentration range of exogenous L-arabinose (i.e., Y)min =Y0 + 60%Y Δ ).

[0025] More preferably, the C L-ara Selected from Y PRT ≥Y0+70%Y Δ The concentration range of exogenous L-arabinose (i.e., Y) min =Y0 + 70%Y Δ ).

[0026] More preferably, the C L-ara Selected from Y PRT ≥Y0+80%Y Δ The concentration range of exogenous L-arabinose (i.e., Y) min =Y0 + 80%Y Δ ).

[0027] More preferably, the C L-ara Selected from Y PRT ≥Y0+90%Y Δ The concentration range of exogenous L-arabinose (i.e., Y) min =Y0 + 90%Y Δ ).

[0028] More preferably, the C L-ara Selected from Y PRT ≥Y0+95%Y Δ The concentration range of exogenous L-arabinose (i.e., Y) min =Y0 + 95%Y Δ ).

[0029] Most preferably, the C L-ara C represents the concentration of exogenous L-arabinose corresponding to the highest expression level of the exogenous protein. max .

[0030] In one preferred embodiment, the concentration of the exogenous L-arabinose is 2 mM to 400 mM.

[0031] In one preferred embodiment, the concentration of the exogenous L-arabinose is 5 mM to 150 mM.

[0032] In one preferred embodiment, the concentration of the exogenous L-arabinose is selected from: 6mM to 110mM, 5mM to 50mM, or 4.8mM to 36mM.

[0033] In one preferred embodiment, the concentration of the exogenous L-arabinose is selected from: 7.56mM, 7.5mM, 7.6mM, 28.84mM, 29mM or 30mM.

[0034] 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 elements.

[0035] The system components (e.g., the corresponding RNA polymerase) capable of recognizing promoter elements on nucleic acid templates can be provided by the Kluyveromyces lactis cell extract, or by other exogenous components, or by a combination of two or more methods.

[0036] 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 the RNA polymerase can be translated into RNA polymerase through the in vitro protein synthesis reaction of the system.

[0037] In one preferred embodiment, the in vitro cell-free protein synthesis system further includes a DNA polymerase. The source of the DNA polymerase includes, but is not limited to: cell extracts containing endogenously expressed DNA polymerase, exogenous DNA polymerase, translation products of exogenous nucleic acid templates encoding DNA polymerase, or combinations thereof. In each of the above technical solutions, the DNA polymerase is preferably phi29 DNA polymerase. The exogenous nucleic acid template encoding the DNA polymerase can be translated into DNA polymerase through the in vitro protein synthesis reaction of the system.

[0038] 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).

[0039] In one preferred embodiment, the Kluyveromyces lactis cell extract contains endogenously expressed RNA polymerase. More preferably, the Kluyveromyces lactis cell extract contains endogenously expressed T7 RNA polymerase.

[0040] In one preferred embodiment, the *Kluyveromyces lactis* cell extract contains endogenously expressed RNA polymerase. More preferably, this is achieved by preparing the cell extract after endogenous strain modification of *Kluyveromyces lactis*, including but not limited to the following endogenous strain modification methods: inserting the coding sequence of RNA polymerase into an intracellular free plasmid, or integrating the coding gene of RNA polymerase into the cell genome, or a combination of the above two methods. It should be noted that, in performing the above endogenous strain modification, in addition to integrating the above coding sequence / coding gene, other nucleotide sequences are also allowed to be inserted, such as non-coding sequences, codon sequences, enhancer sequences, tag sequences, etc. Through the above endogenous strain modification, the modified strain can endogenously express RNA polymerase. The RNA polymerase is preferably T7 RNA polymerase.

[0041] In one preferred embodiment, the in vitro cell-free protein synthesis system includes exogenously added T7 RNA polymerase.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] The crowding agent is preferably polyethylene glycol, dextran, or Ficoll sucrose polymer (such as...). The reagent (a nonionic synthetic sucrose polymer), or a combination thereof;

[0048] 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;

[0049] The potassium ion source is preferably selected from: potassium acetate, potassium glutamate, potassium chloride, potassium phosphate, potassium sulfate, potassium citrate, potassium hydrogen phosphate, potassium iodide, potassium lactate, potassium nitrate, potassium oxalate, or combinations thereof;

[0050] The antioxidant or reducing agent is preferably dithiothreitol (DTT);

[0051] The reaction promoter is preferably aluminum oxide;

[0052] The buffer is preferably selected from: Tris-HCl, Tris base, HEPES, or combinations thereof;

[0053] The aqueous solvent is preferably a buffer.

[0054] The in vitro cell-free protein synthesis system can react with DNA templates or mRNA templates encoding exogenous proteins to synthesize exogenous proteins.

[0055] The above-mentioned preferred methods can be combined in any suitable manner.

[0056] A second aspect of the present invention provides an in vitro protein synthesis kit, the kit comprising:

[0057] (i) The in vitro cell-free protein synthesis system provided in the first aspect;

[0058] (ii) Optionally includes a nucleic acid template encoding a foreign protein;

[0059] (iii) Labels or instructions.

[0060] This kit can be used to perform in vitro protein synthesis reactions and synthesize exogenous proteins.

[0061] A third aspect of this invention provides a method for synthesizing exogenous proteins, the method comprising the following steps:

[0062] (i) Provides the in vitro cell-free protein synthesis system provided in the first aspect of the present invention;

[0063] The methods of "providing" include, but are not limited to: obtaining, preparing;

[0064] (ii) Add a nucleic acid template encoding a foreign protein to the in vitro cell-free protein synthesis system described in step (i), incubate the reaction, and synthesize the foreign protein.

[0065] Optionally, step (iii) may also be included to isolate and / or detect the exogenous protein.

[0066] In the second and third aspects, the nucleic acid template encoding the foreign protein is a DNA template, an mRNA template, or a combination thereof; independently, the nucleic acid template encoding the foreign protein is preferably a DNA template.

[0067] For in vitro protein synthesis reactions to be carried out, the nucleic acid template encoding the exogenous protein preferably contains a promoter element that can be recognized by the system components.

[0068] In one preferred embodiment, the nucleic acid template encoding the exogenous protein contains a promoter element that can be recognized by the Kluyveromyces lactis cell extract. For example, the Kluyveromyces lactis cell extract contains an endogenously expressed RNA polymerase corresponding to the promoter element on the nucleic acid template.

[0069] 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.

[0070] In one preferred embodiment, the nucleic acid template encoding the exogenous protein contains a T7 promoter, and the Kluyveromyces lactis cell extract includes endogenously expressed T7 RNA polymerase.

[0071] Preferably, the transcription process of the exogenous protein is initiated by the T7 promoter on the nucleic acid template.

[0072] 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.

[0073] One preferred approach is that the T7 promoter is located upstream of the coding sequence of the exogenous protein in the nucleic acid template, initiating the transcription program of the exogenous protein. One of the components of the system, Kluyveromyces lactis cell extract, contains endogenously expressed T7 RNA polymerase.

[0074] A fourth aspect of this invention provides the application of the in vitro cell-free protein synthesis system described in the first aspect for protein synthesis. This application includes, but is not limited to, applications in protein manufacturing or protein synthesis-based detection.

[0075] The fifth aspect of the present invention provides the use of L-arabinose in the in vitro cell-free protein synthesis system described in the first aspect, or in the in vitro protein synthesis kit described in the second aspect, or in the method for synthesizing exogenous proteins described in the third aspect.

[0076] Beneficial effects:

[0077] The in vitro cell-free protein synthesis system employed in this invention utilizes cell extracts derived from Kluyveromyces lactis, optimized with the addition of L-arabinose. This unexpectedly and significantly increases the protein synthesis yield of the system (generally by more than 15%, and even by 100%). The improved method provided by this invention can be achieved without molecular modification, is simple to operate, and saves costs.

[0078] According to literature reports, hexoses such as glucose, mannose, galactose, and lactose, and pentoses such as xylose and arabinose can all serve as metabolic substrates in the Kluyveromyces system (Hou Shengbo, Feng Hualiang, Gao Jiaoqi, et al. Xylose and arabinose fermentation in Kluyveromyces [J]. Chinese Journal of Biotechnology, 2017, 33(6):923-935). However, in the Kluyveromyces lactis system, which is also a Kluyveromyces system, components such as L-arabinose, citrate, and D- or L-fucose cannot serve as carbon sources to provide energy (RC Dickson and JS Markin. Physiological studies of β-galactosidase induction in Kluyveromyces lactis [J]. Journal of Bacteriology, 1980, 142(3):777-785).

[0079] According to existing technology, L-arabinose is not suitable as a carbon source to improve the in vitro cell-free protein synthesis system of *Kluyveromyces lactis*. In fact, no existing reports have used L-arabinose as an additive in in vitro protein synthesis systems based on *Kluyveromyces lactis* cell extracts. However, in this invention, we accidentally discovered that L-arabinose can significantly improve the exogenous protein synthesis capacity of the in vitro cell-free protein synthesis system based on *Kluyveromyces lactis* cell extracts, particularly increasing the yield of exogenous proteins. It is speculated that L-arabinose may play a role in stabilizing the protein. Attached Figure Description

[0080] Figure 1A schematic diagram of the plasmid DNA template encoding the foreign protein mEGFP, denoted as D2P plasmid or pD2P. The mEGFP is a mutant of enhanced green fluorescent protein. This plasmid DNA template includes the following elements: a T7 promoter (recognizable by T7 RNA polymerase), a 5' untranslated region, a leader sequence (optional element), a purification tag (optional element), the coding sequence for the foreign protein mEGFP, a 3' untranslated region, a replication origin site, an AmpR promoter, an ampicillin resistance gene, a high copy number replication origin site, a gene controlling plasmid copy number (rop gene, not shown in the figure), the coding sequence for the Lac repressor (lacI), and the LacI promoter.

[0081] Figure 2 The effect of L-arabinose concentration on protein synthesis yield in an in vitro cell-free protein synthesis system (concentration curve of exogenous L-arabinose). Corresponding to Example 1. In the in vitro cell-free protein synthesis system, the concentration of L-arabinose was a variable, ranging from 6.05 mM to 110.01 mM. Group BC was the blank control, with no L-arabinose added but plasmid DNA added; group NC was the negative control, with neither L-arabinose nor plasmid DNA template added.

[0082] Figure 3 The effect of L-arabinose concentration on protein synthesis yield in an in vitro cell-free protein synthesis system (concentration curve of exogenous L-arabinose). See Example 2. In the in vitro cell-free protein synthesis system, the concentration of L-arabinose was a variable, ranging from 6.05 mM to 110.00 mM. Group BC served as a blank control, without L-arabinose but with added plasmid DNA; group NC served as a negative control, without L-arabinose or plasmid DNA template.

[0083] Figure 4 The effect of L-arabinose concentration on protein synthesis yield in an in vitro cell-free protein synthesis system (concentration curve of exogenous L-arabinose). See Example 3. In the in vitro cell-free protein synthesis system, the concentration of L-arabinose was a variable, ranging from 4.84 mM to 45.06 mM. Group BC served as a blank control, without L-arabinose but with added plasmid DNA; group NC served as a negative control, without L-arabinose or plasmid DNA template.

[0084] Figure 5Comparison of RFU values ​​for different in vitro cell-free protein synthesis systems. "L-ara-29mM" corresponds to the addition of 29mM L-arabinose (without glucose), "L-ara-15mM" corresponds to the addition of 15mM L-arabinose (without glucose), "Glu-15mM" corresponds to the addition of 15mM glucose (without L-arabinose), and NC is the negative control group (no L-arabinose, no glucose, and no exogenous DNA template added).

[0085] Nucleotide and / or amino acid sequence listing

[0086] SEQ ID No.:1 is the gene sequence of the exogenous protein mEGFP, which is 717 bases in length and contains the stop codon TAA.

[0087] SEQ ID No.:2 is the amino acid sequence of the exogenous protein mEGFP, consisting of 238 amino acids. Detailed Implementation

[0088] The meanings of the terms, nouns, and phrases used in this invention are as follows. The explanations in this section apply throughout the entire invention, both below and above.

[0089] D2P, DNA-to-Protein, refers to the process of converting a DNA template into a protein product. Examples include D2P technology, D2P systems, and D2P methods.

[0090] IVTT, in vitro transcription translation.

[0091] RFU stands for Relative Fluorescence Unit.

[0092] L-ara, L-arabinose, L-arabinose.

[0093] eGFP: Enhanced green fluorescence protein.

[0094] mEGFP: an A206K mutant of eGFP.

[0095] wt% or %(wt): These are units of mass concentration, both representing a mass percentage.

[0096] (v / v)% or %(v / v): both represent volume percentage.

[0097] % (w / v): a unit of mass-volume concentration, corresponding to g / 100mL.

[0098] Ficoll sucrose polymer: unless otherwise specified, refers to... The reagent, a nonionic synthetic sucrose polymer, is a highly branched polymer copolymerized from sucrose and epichlorohydrin, and can be selected from commercially available products. Examples include Ficoll-400 (polysucrose 400, CAS: 26873-85-8) and Ficoll-70 (polysucrose 70, CAS: 72146-89-5). PM 400 (Sigma Aldrich) is a highly branched polymer copolymerized from sucrose and epichlorohydrin, with an average molecular weight of 400 kg / mol. The average molecular weight of PM 70 (Sigma Aldrich) is 70 kg / mol.

[0099] 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." An in vitro protein synthesis reaction refers to a reaction that synthesizes proteins in an in vitro cell-free synthesis system, including at least a 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 a process of transcribing and translating DNA into protein in vitro. Therefore, we also refer to this type of in vitro protein synthesis system as the D2P system, D-to-P system, D_to_P system, or DNA-to-Protein system; the corresponding in vitro protein synthesis methods are also called the D2P method, D-to-P method, D_to_P method, or DNA-to-Protein method.

[0100] Post-translational modification (PTM), also known as post-translational processing, plays a crucial role in the proper folding, activity, and stability of proteins.

[0101] 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, signal sequences, leader sequences (such as selection tags, purification tags, tags that enhance translation levels), etc.

[0102] Genes include coding and non-coding regions.

[0103] Nucleotide sequence: A sequence composed of nucleotide units.

[0104] Nucleic acid sequence: The sequence of nucleic acid material, including DNA sequence and RNA sequence.

[0105] 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).

[0106] Encoding gene: A valid gene segment that encodes a protein; it can be continuous or discontinuous. A coding gene must contain a coding sequence.

[0107] Nucleic acid 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.

[0108] "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.

[0109] 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.

[0110] 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, or single substances or mixtures. Similarly, exogenous DNA polymerase can also be added to the reaction system via the aforementioned exogenous methods.

[0111] 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.

[0112] "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.

[0113] 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.

[0114] Polypeptides are peptides composed of 10 to 50 amino acids.

[0115] Proteins are peptides composed of 50 or more amino acids. Fusion proteins are also a type of protein.

[0116] Exogenous RNA polymerase: has the same meaning as exogenous RNA polymerase.

[0117] Exogenous DNA polymerase: has the same meaning as exogenous DNA polymerase.

[0118] In this invention, cell extract, cell extract solution, cell lysate, cell fragments, and cell lysate have the same meaning, and can be described in English as cell extract, cell lysate, etc.

[0119] 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.

[0120] An amino acid mixture refers to a mixture containing at least two or more amino acids.

[0121] In this invention, unless otherwise specified, the amino acids can be natural amino acids or non-natural amino acids. L -amino acids, D - Amino acids or combinations thereof, and may also be radioisotope-labeled amino acids, modified amino acids, etc. The 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.

[0122] Crowding agents are reagents used to mimic the crowded macromolecular environment within cells. See references such as "XGe,D Luo and J Xu. Cell-free protein expression under macromolecular crowding conditions[J].PLoS One,2011,6(12):e28707" and its cited references.

[0123] Phosphoric acid compounds include both organic and inorganic compounds.

[0124] Unless otherwise specified, phosphates refer to inorganic phosphates.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] In this invention, "optionally" means that it may or may not be present.

[0129] In this invention, the descriptions of "one or more", "one or more", etc., have the same meaning as "at least one", "at least one", etc., indicating a quantity equal to "1" or "greater than 1".

[0130] 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.

[0131] 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.

[0132] 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.

[0133] This invention provides an in vitro cell-free protein synthesis system based on Kluyveromyces lactis cell extract and containing exogenous L-arabinose; the system can react with a nucleic acid template encoding an exogenous protein to synthesize the exogenous protein.

[0134] The exogenous L-arabinose can increase the expression level of exogenous proteins.

[0135] 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 must first be a working system, a system capable of expressing exogenous proteins.

[0136] The scope of protection of this invention only covers technical solutions corresponding to L-arabinose concentrations that can increase the expression level of exogenous proteins. In this invention, for any CFS(ara-) base system of the aforementioned in vitro cell-free protein synthesis system, it is only required that at least one L-arabinose concentration can increase the expression level of exogenous proteins, and it is not required that all L-arabinose concentrations have an enhancing effect.

[0137] The in vitro cell-free protein synthesis system can provide the translation-related elements required for the synthesis of exogenous proteins.

[0138] 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.

[0139] The system components (e.g., the corresponding RNA polymerase) capable of recognizing promoter elements on nucleic acid templates can be provided by the Kluyveromyces lactis cell extract, or by exogenous addition, or by a combination of the two methods.

[0140] For example, Kluyveromyces lactis cell extract contains endogenously expressed RNA polymerases that correspond to promoter elements on nucleic acid templates. Specifically, for example, Kluyveromyces lactis cell extract contains endogenously expressed T7 RNA polymerases that can recognize the T7 promoter on nucleic acid templates.

[0141] In vitro cell-free protein synthesis system (L-arabinose optimization)

[0142] The in vitro protein synthesis reaction of the present invention is carried out in an in vitro cell-free protein synthesis system based on Kluyveromyces lactis cell extract.

[0143] 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.

[0144] There are no particular restrictions on the order in which the components of the in vitro cell-free protein synthesis system are added.

[0145] 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 is derived from Kluyveromyces lactis cells, which can be wild-type or non-wild-type. Non-wild-type Kluyveromyces lactis includes, but is not limited to, genetically modified types.

[0146] 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.

[0147] The endogenously expressed RNA polymerase is not added independently but is present in the cell extract.

[0148] To ensure that the cell extract contains endogenously expressed RNA polymerase, it 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 yeast cell genome, or modifying the strain using a combination of the above two methods, and then preparing the cell extract. The methods for integrating the coding 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 gene, or combinations thereof.

[0149] The exogenously added or endogenously expressed RNA polymerase is preferably T7 RNA polymerase.

[0150] The in vitro cell-free protein synthesis system preferably further 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.

[0151] 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.

[0152] 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.

[0153] 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).

[0154] In one preferred embodiment, the in vitro cell-free protein synthesis system further includes an energy system.

[0155] In one preferred embodiment, the in vitro cell-free protein synthesis system further includes a substrate for synthesizing RNA.

[0156] In one preferred embodiment, the in vitro cell-free protein synthesis system further includes a substrate for the synthesized protein.

[0157] In one preferred embodiment, the in vitro cell-free protein synthesis system includes Kluyveromyces lactis cell extract (containing endogenously expressed RNA polymerase), exogenous L-arabinose, an energy system, a substrate for synthesizing RNA, and a substrate for synthesizing protein.

[0158] 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.

[0159] Exogenous L-arabinose

[0160] L-arabinose (L-Ara) is a plant-specific monosaccharide found in 10%–20% of the non-cellulose cell wall polysaccharides in Arabidopsis thaliana. L-Ara is present in arabinogalactan and type I arabinogalactan, which are major components of pectin walls. Besides polysaccharides, many glycoproteins, such as extensins and leucine-rich repetitive extensins, as well as some secreted CLE peptides and ubiquitous arabinogalactan proteins (AGP), are also arabinoglycosylated (Marzol E, Borassi C, Bringas M, Sede A, Rosa Rodríguez Garcia D, Capece L, Estevez JM, Garcia RD. Filling the gaps to solve the extensin puzzle. Molecular Plant. 2018, 11:645-658). L-arabinose contains multiple hydroxyl groups in its structure.

[0161] In one preferred embodiment, the selectable concentration range of the exogenous L-arabinose is determined according to Y. PRT (C L-ara The expression level of exogenous protein in the concentration curve was determined. The Y... PRT (C L-ara Concentration curve, which is also referred to as Y in this invention. PRT ~C L-ara Concentration curve, referring to the concentration of exogenous L-arabinose (C) in the in vitro cell-free protein synthesis system of the present invention. L-ara ) is the independent variable, and the expression level of exogenous protein (Y) is the independent variable. PRT () represents the concentration curve when the dependent variable and other reaction parameters are determined.

[0162] Preferably, the C L-ara Selected from Y PRT At least Y0 + 50%Y Δ The concentration range of exogenous L-arabinose.

[0163] More preferably, the C L-ara Selected from Y PRT At least Y0 + 60%Y Δ The concentration range of exogenous L-arabinose.

[0164] More preferably, the C L-ara Selected from Y PRT At least Y0 + 70%Y Δ The concentration range of exogenous L-arabinose.

[0165] More preferably, the C L-ara Selected from Y PRT At least Y0 + 80%Y ΔThe concentration range of exogenous L-arabinose.

[0166] More preferably, the C L-ara Selected from Y PRT At least Y0 + 90%Y Δ The concentration range of exogenous L-arabinose.

[0167] More preferably, the C L-ara Selected from Y PRT At least Y0 + 95%Y Δ The concentration range of exogenous L-arabinose.

[0168] One preferred method is that the C L-ara For Y PRT (C L-ara Y in the concentration curve max The corresponding concentration of exogenous L-arabinose.

[0169] One preferred method is to determine the concentration of exogenous L-arabinose as follows: when the types and contents of other components are determined, the concentration of L-arabinose is adjusted within a relatively wide range, such as 0–200 mM. Under specified reaction conditions (reaction temperature, reaction time, etc.), the concentration C of exogenous L-arabinose at which the expression level of the exogenous protein is highest is determined. max This is the optimal concentration (most preferred concentration) of L-arabinose under this technical solution.

[0170] The C L-ara Y PRT Y max C max Y0, Y Δ The definition is the same as above.

[0171] In one preferred embodiment, the concentration of the exogenous L-arabinose is 2 mM to 400 mM.

[0172] In one preferred embodiment, the concentration of the exogenous L-arabinose is 5 mM to 150 mM.

[0173] In one preferred embodiment, the concentration of the exogenous L-arabinose is 6 mM to 110 mM.

[0174] In one preferred embodiment, the concentration of the exogenous L-arabinose is 5 mM to 50 mM.

[0175] In one preferred embodiment, the concentration of the exogenous L-arabinose is 4.8 mM to 36 mM.

[0176] In one preferred embodiment, the concentration of the exogenous L-arabinose is 7.56 mM, 7.5 mM, or 7.6 mM.

[0177] In one preferred embodiment, the concentration of the exogenous L-arabinose is 28.84 mM, 29 mM, or 30 mM.

[0178] Cell extract (Kluyveromyces lactis cell extract)

[0179] 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.

[0180] The cell source of the cell extract described in this invention is Kluyveromyces lactis (K. lactis).

[0181] The cell extract is intended to provide structural and / or biological factors for protein transcription and translation.

[0182] Cell extracts can provide many of the key translation-related elements needed to synthesize exogenous proteins; this is an endogenous way of providing them.

[0183] 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.

[0184] The cell extract preferably does not contain intact cells.

[0185] Cell extracts may also contain other proteins derived from the cytoplasm of cells, especially soluble proteins.

[0186] Preferably, the cell extract contains various factors required for protein synthesis.

[0187] The various protein factors provided by the aforementioned cell extracts, and the corresponding encoding genes, can be naturally present in the cell genome, or the relevant encoding genes can be integrated into the cell genome (integrated into chromosomes), or the relevant genes, gene fragments, or coding sequences can be inserted into intracellular free plasmids. RNA polymerase and DNA polymerase are used as examples. In one preferred embodiment, the cell extract contains endogenously expressed RNA polymerase and / or DNA polymerase.

[0188] 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, or a combination thereof.

[0189] The genome of the wild-type Kluyveromyces lactis strain of this invention does not contain the gene encoding T7 RNA polymerase. Previous studies have shown that when constructing an in vitro cell-free protein synthesis system using cell extracts of wild-type Kluyveromyces lactis and using the T7 promoter to control the transcription and translation of exogenous proteins, exogenous proteins cannot be synthesized during in vitro protein synthesis reactions without the addition of exogenous T7 RNA polymerase.

[0190] One preferred method is that the cell extract is derived from *Kluyveromyces lactis*, whose genome integrates the coding gene for RNA polymerase. This includes, but is not limited to, insertion into the cell genome, in situ replacement of a portion of the genome (i.e., including the step of knocking out a portion of the original gene), and knocking out a portion of the original gene and inserting the coding gene for RNA polymerase. In Examples 1-4, the coding gene for T7 RNA polymerase was integrated into the genome of *Kluyveromyces lactis* cells, which endogenously express T7 RNA polymerase. The cell extract prepared in this way contains endogenously expressed T7 RNA polymerase; therefore, in vitro cell-free protein synthesis can be performed without adding exogenous RNA polymerase. Furthermore, the coding sequence for RNA polymerase can also be inserted into an intracellular free plasmid, such as an intracellular free plasmid of *Kluyveromyces lactis*, to prepare the cell extract. Refer to the preparation method in CN109423496A for details.

[0191] In one preferred embodiment, the cell extract is derived from Kluyveromyces lactis, and its genome integrates any one or a combination of the following gene sequences: an RNA polymerase encoding gene and a DNA polymerase encoding gene.

[0192] 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.

[0193] 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.

[0194] The cell extract prepared by the method provided in this invention can enable in vitro protein synthesis reactions to proceed normally and contains the necessary components required for protein synthesis, such as tRNA with amino acid transport function and aminoacyl-tRNA synthetase.

[0195] In one embodiment, the cell extract is prepared by a method comprising the following steps: (i) providing source cells, Kluyveromyces lactis cells; (ii) washing the Kluyveromyces lactis cells to obtain washed Kluyveromyces lactis cells; (iii) performing cell-breaking treatment on the washed Kluyveromyces lactis cells to obtain crude Kluyveromyces lactis extract; and (iv) performing solid-liquid separation on the crude Kluyveromyces lactis extract, the collected supernatant being the cell extract (Kluyveromyces lactis cell extract).

[0196] 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. Still another preferred method is 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, or 45 mg / mL. The method for determining the protein content can be the Coomassie Brilliant Blue assay.

[0197] 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.

[0198] Exogenous RNA polymerase, exogenous DNA polymerase

[0199] When the genome of the cell from which the cell extract is derived does not contain the gene for RNA polymerase, nor does it contain the coding sequence / gene for endogenous integrative RNA polymerase, it is usually necessary to add exogenous RNA polymerase to promote the reaction.

[0200] Adding exogenous RNA polymerase to in vitro protein synthesis systems is a traditional technique. Existing Kluyveromyces lactis in vitro protein synthesis systems with added exogenous RNA polymerases are all included in this invention as optional methods for the CFS(ara-) basic system of this invention. For example, the Kluyveromyces lactis in vitro protein synthesis system with added exogenous RNA polymerase (such as T7 RNA polymerase) described in CN108535489A is also included in this invention as an optional method for the CFS(ara-) basic system.

[0201] 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.

[0202] You can add exogenous RNA polymerase directly, or you can add an exogenous nucleic acid template encoding RNA polymerase, or a combination thereof. The coding sequence of 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.

[0203] 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.

[0204] When the nucleic acid template encoding the exogenous protein is a DNA template, the process may or may not include DNA amplification. 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 Examples 1-4 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.

[0205] 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 all 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, fragments of any of the above polymerases, and any combination of the above polymerases and their fragments. This invention may also use other DNA polymerases such as Taq DNA polymerase, Pfu DNA polymerase, Pol I DNA polymerase, and Pol II DNA polymerase.

[0206] 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.

[0207] Energy System / Energy Regeneration System

[0208] Energy systems / energy regeneration systems are used to provide the energy required for protein synthesis.

[0209] 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 with glucose metabolism[J].FEBS Letters,2015,589(15):1723-1727","Y Lu.Advances inCell-Free Biosynthetic Technology[J].Current Developments in Biotechnologyand Bioengineering,2019,Chapter 2,23-45","P Shrestha,MT Smith and BCBundy.Cell-free unnatural amino acid incorporation with alternative energysystems and linear expression templates[J].New Energy systems / energy regeneration systems reported in literature such as Biotechnology, 2014, 31(1):28-34, and other directly or indirectly cited literature are all incorporated herein by reference.

[0210] 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.

[0211] 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. The energy system used in Examples 1-4 is a polysaccharide and phosphate energy system, wherein the polysaccharide is maltodextrin and the phosphate is tripotassium phosphate (potassium phosphate is equivalent to tripotassium phosphate).

[0212] substrates for RNA synthesis

[0213] The substrate for synthesizing RNA is a mixture of nucleotides, selected from: nucleoside monophosphates, nucleoside triphosphates, or combinations thereof, in one embodiment. Preferably, it is a mixture of nucleoside triphosphates (dNTPs). 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.

[0214] substrates for protein synthesis

[0215] The substrate for the synthesized protein is a mixture of amino acids. The concentration is measured using the 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 0.1 mM to 1 mM.

[0216] 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.

[0217] The amino acid mixture may include natural amino acids and non-natural amino acids.

[0218] The amino acid mixture may include L -amino acids, D - Amino acids, or combinations thereof.

[0219] In addition to natural amino acids, the amino acid mixture may also include non-natural amino acids. D- Components including amino acids, radioisotope-labeled amino acids, and modified amino acids. The non-natural amino acids are not particularly limited and can be selected from, but are not limited to, those reported or cited in 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 their 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.

[0220] Preferably, the amino acid mixture is a mixture of natural amino acids.

[0221] Other additive components

[0222] The in vitro cell-free protein synthesis system may further include at least one of the following components: polyethylene glycol and / or its analogues, magnesium ions, potassium ions, antioxidants or reducing agents, trehalose, reaction promoters, buffers, and aqueous solvents. See references WO2016005982A1, US20060211083A1, and "L Kai, V R Kaldenhoff and F. Bernhard. Artificial environments for the co-translational stabilization of cell-free expressed proteins[J]. PloS one, 2013, 8(2):e56637”, US20030119091A1, US20180245087A1, US5665563, WO2019033095A1, US9410170B2, US9528137B2 and other literature and their direct or indirect citations.

[0223] The in vitro cell-free protein synthesis system may also contain polyethylene glycol and / or its analogues to simulate the crowded macromolecular environment within cells, acting as crowding agents. Polyethylene glycol, for example, can also adjust the viscosity of the system. Polyethylene glycol, having repeating units of CH2CH2O, is commonly abbreviated as PEG (polyethylene glycol), PEO (poly(ethylene oxide)), or POE (polyoxyethylene). The concentration of polyethylene glycol or its analogues is not particularly limited; typically, the concentration is 0.1%-10%, preferably 0.1%-8%, more preferably 0.5%-4%, and even more preferably 1%-2%, expressed as a mass-volume concentration (%(w / v)) or a total weight (wt%) of the protein synthesis system. Unless otherwise specified, all concentrations in this invention refer to mass-volume concentrations, expressed in %(w / v), such as 2%, which means 2%(w / v), corresponding to 2 g / 100 mL or 20 mg / mL. The preferred molecular weight of the polyethylene glycol 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 M. w Representative PEGs are selected from the following group: PEG3000, PEG8000, PEG6000, PEG3350, and combinations thereof; where 3350, etc., are numerically equal to the weight-average molecular weight. The molecular weight of polyethylene glycol can also be, for example, 200 Da, 400 Da, 1500 Da, 2000 Da, 4000 Da, 6000 Da, 8000 Da, 10000 Da, etc. Typically, the molecular weight specification is preferably ±10% or less than ±10%. Other macromolecules that can act as crowding agents include, for example, polyvinyl alcohol (PVA) and dextran. Reagents (such as Ficoll-400), and also refer to 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 the crowding agents disclosed in the cited literature.

[0224] 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 to 20 mM. Yet another preferred embodiment has a concentration range of 1 mM to 10 mM.

[0225] 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.

[0226] 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.

[0227] The antioxidant, also known as a reducing agent, can be selected from, but is not limited to, dithiothreitol (DTT), 2-mercaptoethanesulfonic acid, 2-mercaptoethanol, 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.

[0228] The reaction promoters include, but are not limited to, the reaction promoters provided in CN109971783A (such as aluminum salts).

[0229] 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).

[0230] The aqueous solvent is preferably a buffer.

[0231] Examples of specific implementation methods for in vitro protein synthesis systems

[0232] One preferred embodiment is that the in vitro protein synthesis system contains Kluyveromyces lactis cell extract, L-arabinose, endogenously expressed RNA polymerase (included in the aforementioned Kluyveromyces lactis cell extract) or exogenously added RNA polymerase, an energy system, a substrate for RNA synthesis, a substrate for protein synthesis, a congesting agent, 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), an antioxidant or reducing agent, trehalose, a reaction promoter, and an aqueous solvent.

[0233] One preferred embodiment is that the in vitro protein synthesis system contains Kluyveromyces lactis cell extract (by integrating the RNA polymerase encoding gene into the cell genome or inserting it into an intracellular free plasmid to modify the strain), L-arabinose, and 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 (dNTPs), a mixture of amino acids, creatine phosphate, creatine phosphokinase, creatine phosphokinase, dithiothreitol (DTT), an RNase inhibitor, sucrose, glucose, starch, dextrin, zeadrin, maltodextrin, and phosphates (such as potassium phosphate).

[0234] One preferred embodiment is that the in vitro protein synthesis system contains Kluyveromyces lactis cell extract, L-arabinose, and one or more components selected from the group consisting of: HEPES-K or Tris, potassium acetate, magnesium acetate, magnesium glutamate, magnesium aspartate, dNTPs, amino acid mixture, creatine phosphate, creatine phosphokinase, creatine phosphokinase, DTT, RNase inhibitor, sucrose, glucose, starch, dextrin, zeadrin, maltodextrin, phosphate (such as potassium phosphate), exogenous T7 RNA polymerase, and exogenous phi29 DNA polymerase.

[0235] One preferred embodiment is that the in vitro protein synthesis system contains Kluyveromyces lactis cell extract (optionally, the gene encoding RNA polymerase is integrated into the cell genome or inserted into an intracellular free plasmid for strain modification), L-arabinose, and 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), dNTPs, an amino acid mixture, creatine phosphate, creatine phosphokinase, creatine phosphokinase, DTT, RNase inhibitor, sucrose, glucose, starch, dextrin, corn dextrin, maltodextrin, potassium phosphate, polyethylene glycol, alumina promoter, exogenous T7 RNA polymerase, exogenous phi29 DNA polymerase, DNA template encoding T7 RNA polymerase, and DNA template encoding phi29 DNA polymerase.

[0236] Another preferred embodiment is that the in vitro protein synthesis system contains Kluyveromyces lactis cell extract, L-arabinose, and one or more components selected from the group consisting of: Tris-HCl (pH 8.0), potassium acetate, magnesium acetate, magnesium glutamate, magnesium aspartate, dithiothreitol, polyethylene glycol, glucose, dNTPs (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, and exogenous phi29 DNA polymerase.

[0237] Specifically, one preferred embodiment contains 50%–80% (v / v) Kluyveromyces lactis cell extract, 5 mM–110 mM L-arabinose, and also 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... L-Aspartate magnesium, 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), 200mM–400mM maltodextrin (measured as glucose monomer, e.g., 320mM, corresponding to approximately 52mg / mL), and 10mM–40mM potassium phosphate.

[0238] The in vitro cell-free protein synthesis system of *Kluyveromyces lactis* described in other cited documents, as well as their direct and indirect citations, are also incorporated into this invention as implementation methods of the CFS(ara-) basic system. For example, documents CN106978349A, CN108535489A, CN108690139A, CN108949801A, CN108642076A, CN109022478A, CN109423496A, CN109423497A, CN109837293A, CN109971783A, CN109988801A, CN110551700A, CN109971775A, CN1105517 The in vitro cell-free protein synthesis systems described in CN110551700A, CN2018116083534, CN2018116198186, CN2018116198190, CN2019102128619, CN2019102355148, CN2019107298813, CN2019112066163, CN2018108881848, CN2018109550734, CN2018111131300, CN2018111423277, CN2018112862093 and their cited references can all be included in this invention as implementation methods of the CFS(ara-) basic system.

[0239] A second aspect of the present invention provides an in vitro protein synthesis kit, the kit comprising:

[0240] (i) The in vitro cell-free protein synthesis system described in the first aspect;

[0241] (ii) Optionally includes a nucleic acid template encoding a foreign protein;

[0242] (iii) Labels or instructions.

[0243] Preferably, the nucleic acid template encoding the exogenous protein is a DNA template.

[0244] In one preferred embodiment, the components together constitute an aqueous solution. The kit includes a container holding the aqueous solution.

[0245] 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 lyophilized powder or an aqueous solution. Components (c), (e), and (f) are independently present or absent. "Several" here means one, two, or more.

[0246] One preferred method is to package each component into two parts: a lyophilized powder and a liquid reagent. The kit includes two containers, one for the lyophilized powder component and the other for the liquid reagent component.

[0247] One preferred method is to dispense the components as lyophilized powder, buffer solution, or other liquid reagents, optionally including water as a solvent.

[0248] In one preferred embodiment, the following components are packaged in separate containers: Kluyveromyces lactis cell extract (containing endogenously expressed RNA polymerase, optionally containing endogenously expressed DNA polymerase), L-arabinose, 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: 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.

[0249] In one preferred embodiment, the following components are packaged in separate containers: Kluyveromyces lactis cell extract (the source cells do not contain the coding sequence / gene for endogenous integrated RNA polymerase or the coding sequence / gene for endogenous integrated DNA polymerase), L-arabinose, 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 one of the following components: an exogenous DNA template encoding the RNA polymerase, an exogenous 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.

[0250] A third aspect of this invention provides a method for synthesizing exogenous proteins, the method comprising the following steps:

[0251] (i) Providing the in vitro cell-free protein synthesis system described in the first aspect of the present invention;

[0252] (ii) Add a nucleic acid template encoding a foreign protein to the in vitro cell-free protein synthesis system described in step (i), incubate the reaction, and synthesize the foreign protein;

[0253] Optionally, step (iii) may also be included to isolate and / or detect the exogenous protein.

[0254] Preferably, the nucleic acid template encoding the exogenous protein is a DNA template.

[0255] The incubation reaction refers to an in vitro protein synthesis reaction, which includes at least the translation process (in which case the nucleic acid template may only include the mRNA template), and optionally includes the transcription process.

[0256] One preferred approach is to use a DNA template encoding a foreign protein, and the incubation reaction includes transcription and translation processes.

[0257] The separation and / or detection methods can be implemented using conventional techniques.

[0258] A fourth aspect of this invention provides the application of the in vitro cell-free protein synthesis system described in the first aspect for protein synthesis. This application includes, but is not limited to, applications in protein manufacturing and detection of synthesized proteins.

[0259] 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.

[0260] In any embodiment of the present invention, the nucleic acid template encoding the foreign protein can be independently preferred to be a DNA template.

[0261] In any embodiment of the present invention, preferably independently, the nucleic acid template encoding the foreign protein further contains a promoter element that can be recognized by the Kluyveromyces lactis cell extract. Specifically, for example, the T7 promoter is located upstream of the coding sequence of the foreign protein in the nucleic acid template, initiating the transcription program of the foreign protein, and the Kluyveromyces lactis cell extract, one of the components of the system, contains endogenously expressed T7 RNA polymerase.

[0262] exogenous proteins

[0263] The exogenous proteins applicable to the in vitro protein synthesis system of this invention are not particularly limited, as long as they can be synthesized in vitro based on Kluyveromyces lactis cell extract. Proteins disclosed in the prior art that are applicable to in vitro protein synthesis systems derived from Kluyveromyces lactis can all be synthesized using the system of this invention. Exogenous proteins disclosed that are applicable to in vitro protein synthesis systems derived from other yeasts, or endogenous proteins applicable to Kluyveromyces lactis systems or other yeast systems for intracellular synthesis, can also be synthesized using the system of this invention, or attempts can be made to synthesize them using the in vitro protein synthesis system provided by this invention.

[0264] 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, luciferase mutants, 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, mouse catalase, or mutants of any of the foregoing (e.g., luciferase mutants, eGFP mutants). See also patent document CN109423496A. The mixture of any of the aforementioned combinations may include any of the foregoing proteins, or may include fusion proteins of any of the aforementioned combinations.

[0265] 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.

[0266] Exogenous nucleic acid templates (including nucleic acid templates encoding exogenous proteins)

[0267] 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.

[0268] Without the nucleic acid template encoding the foreign protein, the in vitro synthesis reaction of the foreign protein cannot proceed.

[0269] The nucleic acid template encoding the exogenous protein is a DNA template, an mRNA template, or a combination thereof, preferably a DNA template.

[0270] Nucleic acid templates serve as direct templates (mRNA), indirect templates (DNA), or combinations thereof for the synthesis of exogenous proteins.

[0271] 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.

[0272] Transcriptional protein synthesis uses DNA templates as indirect templates, while translational protein synthesis can use mRNA templates as direct templates.

[0273] 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.

[0274] The nucleic acid template encoding the foreign protein contains translation-related elements required for the synthesis of the foreign protein.

[0275] For in vitro protein synthesis reactions to be carried out, the nucleic acid template encoding the exogenous protein preferably contains a promoter element that can be recognized by the system components.

[0276] In one preferred embodiment, the nucleic acid template encoding the exogenous protein contains a promoter element that can be recognized by the Kluyveromyces lactis cell extract.

[0277] One preferred approach is that the transcription of exogenous proteins is initiated by the T7 promoter on the nucleic acid template.

[0278] 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.

[0279] In one preferred embodiment, the nucleic acid template encoding the foreign protein further contains a gene that controls the copy number of the plasmid.

[0280] In one preferred embodiment, the nucleic acid template encoding the exogenous protein further contains translation enhancement elements, such as enhancer elements, IRES elements, etc.

[0281] Exogenous DNA template (including DNA templates encoding exogenous proteins)

[0282] Unless otherwise specified, the exogenous DNA template of this invention refers specifically to the DNA template encoding the exogenous protein.

[0283] The DNA template encoding the foreign protein contains the coding sequence of the foreign protein.

[0284] Preferably, the DNA template encoding the foreign protein contains the gene encoding the foreign protein.

[0285] The DNA template encoding the foreign protein is determined based on the foreign protein.

[0286] The DNA template encoding the foreign protein may also contain other functional elements selected from promoters, terminators, enhancers (e.g., enhancer elements described in CN109423497A, CN109022478A, CN109837293A (CN201711194355.9) and their cited references, such as Ω sequences and their homologous sequences, combined enhancer elements), IRES elements (internal ribosome entry sequences, see CN109022478A, CN109423497A and their cited references), multiple cloning sites (MCS), genes controlling plasmid copy number, etc. It may also contain sequences encoding signal peptides (corresponding to signal sequences), leader peptides (corresponding to leader sequences), functional tags (such as purification tags), linker peptides, and other amino acid chains. It may also contain 5' untranslated sequences and 3' untranslated sequences.

[0287] The DNA template encoding the exogenous protein preferably contains a promoter element. The promoter element is required to be recognizable by the cell extract used or other components of the in vitro protein synthesis system; it can be a promoter recognizable by the wild-type cell extract, or the source cells of the cell extract can be modified to recognize the promoter. The promoter in the DNA template encoding the exogenous protein can be selected from the following group: AOD1, MOX, AUG1, AOX1, GAP, FLD1, PEX8, YPT1, LAC4, PGK, ADH4, AMY1, GAM1, XYL1, XPR2, TEF, RPS7, T7, or 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.”

[0288] In Examples 1-4, the transcription process of the exogenous protein was initiated by the T7 promoter using the exogenous DNA template; the T7 promoter is a strong promoter that can specifically respond to T7 RNA polymerase.

[0289] Preferably, the transcription process of the exogenous protein gene is initiated by the T7 promoter on the exogenous DNA template. The concentration of the exogenous DNA template is determined 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. In this invention, unless otherwise specified, the concentration of the added DNA template is the final concentration, i.e., the initial concentration of the in vitro protein synthesis reaction.

[0290] 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.

[0291] 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, resistance genes, transposase-encoding genes, signal sequences, leading sequences (e.g., the leading sequences described and cited in CN109022478A), genes controlling plasmid copy number (rop genes), tags that enhance translation levels (e.g., polypeptide tags described in CN2019112066163), etc. See also US20060211083A1, etc.

[0292] 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.

[0293] For example, a nucleic acid construct with a "Z1-Z2" structure can be inserted into the cloning site of a plasmid vector as a plasmid DNA template; where Z1 is the promoter, "-" represents a covalent bond or nucleotide fragment, and Z2 is the coding sequence of a foreign protein. One preferred method for Z1 is the T7 promoter.

[0294] 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 templates used in Examples 1-4. For example, pET series plasmids and pGEM series plasmids of *E. coli* can be used to replace the plasmid vectors of *Kluyveromyces lactis* extract in Examples 1-4 to implement this invention. Another preferred embodiment of the plasmid is one containing a promoter that can be recognized by the exogenously added components.

[0295] 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 the T7 RNA polymerase expressed endogenously in Kluyveromyces lactis cell extract, or by the T7 RNA polymerase added exogenously.

[0296] Linear DNA can be obtained through 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. Among these, isothermal amplification is preferred.

[0297] One preferred embodiment is that the exogenous DNA template is linear DNA, and specifically a PCR linear fragment. The PCR linear fragment can be obtained using reported PCR techniques.

[0298] 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.

[0299] 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, an LAC4 terminator (or a T7 terminator), and / or 5' and 3' UTRs.

[0300] As one of the preferred embodiments, in Examples 1-4, double-stranded DNA was used as the exogenous DNA template to construct a circular plasmid vector. These plasmids contain the T7 promoter, which serves as a promoter for initiating the transcription and translation of exogenous proteins. In Examples 1-4, 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 the UTR.

[0301] 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.

[0302] In another embodiment, the plasmid structure includes at least the structural elements 1-2 and 3-12 listed in Table 1 of Example 1.

[0303] In another embodiment, in addition to at least including Figure 1 In addition to the labeled functional elements, there are purification tags, such as polyhistidine tags (His-tag), between the 5'UTR and the coding sequence of mEGFP.

[0304] In another embodiment, in addition to at least including Figure 1 In addition to the indicated functional elements, there is a signal peptide coding sequence (signal sequence) between the 5'UTR and the coding sequence of mEGFP, and downstream of the 5'UTR.

[0305] 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.

[0306] 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, a replication initiation site (f1 ori), an AmpR promoter, an ampicillin resistance gene, a high copy number replication initiation site (ori), a rop gene, a LacI promoter, and a coding sequence for lacI. Specifically, for example, the plasmid DNA includes the following functional elements: a T7 promoter, a 5' untranslated region, a coding sequence for a signal peptide, a coding sequence for a purification tag, an MCS, a coding sequence for the foreign protein mEGFP, a 3' untranslated region, a LAC4 terminator or a T7 terminator, f1 ori, an AmpR promoter, an ampicillin resistance gene, ori, a rop gene, a LacI promoter, and a coding sequence for lacI.

[0307] 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.

[0308] 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.

[0309] exogenous mRNA template

[0310] 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.

[0311] In vitro nucleic acid amplification (in vitro nucleic acid amplification technology, in vitro nucleic acid amplification methods)

[0312] "In vitro nucleic acid amplification" is the process of replicating nucleic acids in vitro.

[0313] 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.

[0314] There are no particular restrictions on the in vitro nucleic acid amplification techniques that can be used, including but not limited to polymerase chain reaction (PCR) amplification, isothermal amplification, room temperature amplification, and room temperature amplification. Among these, isothermal amplification is preferred.

[0315] 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 amplification invitro: 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):296-302” and other literature and their cited literature disclose isothermal amplification techniques. Specifically, the nucleic acid isothermal amplification methods that can be used in the technical means of this invention include, but are not limited to: loop-mediated isothermal amplification (LAMP), chain substitution amplification (SDA), sequence-dependent amplification (NASBA), rolling circle amplification (RCA), nicking enzyme nucleic acid isothermal amplification, helicase-dependent isothermal amplification (HDA), transcription-dependent amplification, hybridization capture, transcription-mediated amplification (TMA), recombinase-mediated amplification (RAA), recombinase polymerase amplification (RPA), etc., with loop-mediated isothermal amplification being preferred.

[0316] The in vitro nucleic acid amplification methods 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, 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.

[0317] Incubation reaction (in vitro protein synthesis reaction)

[0318] 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.

[0319] 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. Room temperature conditions are preferred for in vitro protein synthesis. The room temperature conditions are preferably room temperature to 37°C, specifically, 20°C to 37°C. One preferred method is 25°C to 37°C. Another preferred method is 20°C to 30°C. Any reported room-temperature amplification methods or isothermal amplification methods suitable for room-temperature conditions can be used to implement the technical solution of this invention.

[0320] 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.

[0321] In one embodiment, the reaction time is 1 hour to 72 hours.

[0322] In another embodiment, the reaction time is 3h to 24h.

[0323] In another embodiment, the reaction time is 3h to 21h.

[0324] In another embodiment, the reaction time is 6h to 21h.

[0325] The reaction time can also be selected from: 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, 24h, 36h, and 48h.

[0326] The following describes specific embodiments and appendices. Figure 1-5The 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.

[0327] Unless otherwise specified, all materials and reagents used in the embodiments of this invention are commercially available products.

[0328] This invention uses Kluyveromyces lactis (K. lactis or Kl) as the source of cell extracts in Examples 1-4, and the protein concentration in the prepared cell extracts is 30 mg / mL to 50 mg / mL. It should be noted that the plasmid expression vectors used in the embodiments of this invention are only used to specifically 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 this invention include, but are not limited to, common plasmid vectors that are commercially available, such as pET series plasmids and pGEM series plasmids.

[0329] Example 1: Effect of L-arabinose concentration on the protein synthesis capacity of an in vitro protein synthesis system

[0330] 1.1 Preparation of nucleic acid template: Construct a plasmid vector expressing mEGFP, perform in vitro DNA amplification, and prepare a DNA template (plasmid DNA template) encoding the exogenous protein mEGFP.

[0331] 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.

[0332] Plasmid vectors were selected. Artificially constructed plasmid vectors targeting Kluyveromyces lactis cell extract were used. These artificially constructed plasmid vectors contained functional elements such as the T7 promoter, 5'UTR, and 3'UTR. This plasmid vector, together with Kluyveromyces lactis cell extract containing endogenously expressed T7 RNA polymerase, can be used to construct an in vitro cell-free protein synthesis system for expressing various exogenous proteins in vitro.

[0333] A DNA fragment containing the encoding gene of mEGFP was inserted into a plasmid vector using PCR amplification and homologous fragment recombination to construct a plasmid vector expressing mEGFP, denoted as D2P plasmid or pD2P. The plasmid was confirmed to be correct by gene sequencing. The gene sequence encoding mEGFP is shown in SEQ ID No. 1.

[0334] The spectrum of the D2P plasmid is as follows: Figure 1 As shown in Table 1, its structural components are composed of the following elements.

[0335] Table 1 Figure 1 Explanation of the structural elements of the plasmid shown.

[0336]

[0337] DNA amplification was performed. The amplification reaction system consisted of the following components at final concentrations: 1 μM-5 μM random primers (primer sequence: NNNNNNN), 1.14 ng / μL of the plasmid template described above, 0.5 mM-1 mM dNTPs, 0.1 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 hours. The plasmid DNA template (double-stranded DNA structure) was obtained, and its nucleic acid concentration was determined using a UV spectrophotometer. The reaction solution was refrigerated for later use.

[0338] 1.2 Preparation of cell extract: Preparation of cell extract (Kluyveromyces lactis cell extract)

[0339] The cell extract was obtained from *Kluyveromyces lactis* (K. lactis). 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*, resulting in a modified strain capable of endogenously expressing T7 RNA polymerase. The resulting cell extract was designated CM1122. Comparative experiments showed that, 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, efficient expression of exogenous proteins could be achieved without the addition of any exogenous RNA polymerase.

[0340] 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.

[0341] 1.3 In vitro cell-free protein synthesis system (without added exogenous RNA polymerase)

[0342] Each system consisted of 200 μL and the reaction was carried out in a flat-bottomed 48-well plate. Three replicates were prepared for each sample, and the mean and standard deviation (error bar) were calculated.

[0343] The final concentrations of each component in the experimental group (concentration curve of L-arabinose, denoted as L-ara group) were: 9.78 mM Tris-HCl (pH 8.0), 80 mM potassium acetate, and 5.6 mM... Magnesium L-aspartate, 1.8 mM nucleoside triphosphate mixture (adenine triphosphate, guanine triphosphate, cytosine triphosphate and uracil triphosphate, each at 1.5 mM), 0.5 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 0.5 mM), 6% (w / v) trehalose, 2% (w / v) polyethylene glycol 8000, 6.05 mM–110 mM L-arabinose (L-ara), 5.3 wt% maltodextrin, 18 mM tripotassium phosphate, 50% v volume of Kluyveromyces lactis cell extract.

[0344] Blank control group (BC group): Compared with the above experimental group, L-arabinose was not added.

[0345] Negative control group (NC group): Compared with the above experimental group, no L-arabinose (concentration of 0mM) was added, and no exogenous DNA template was added subsequently.

[0346] 1.4 In vitro protein synthesis reaction: No exogenous DNA template was added to the NC group; a DNA template encoding mEGFP (obtained by in vitro RCA amplification in step 1.1) was added to each independent in vitro cell-free protein synthesis system of the L-ara group and BC group at a final concentration of 0.33 μg / μL. After mixing, all systems were placed in a shaking environment at 30°C and reacted overnight. Samples were taken at 3h and 20h for fluorescent protein activity testing.

[0347] 1.5 Fluorescent Protein Activity Assay: After the reaction, each reaction system was immediately placed in an Envision 2120 multi-functional microplate reader (Perkin Elmer) to detect the fluorescence signal intensity. The relative fluorescence unit (RFU) value was used as the activity unit. The RFU value reflects the amount of mEGFP protein synthesized.

[0348] Fluorescence tests were performed on each reaction system. Test parameters: 4000 rpm, centrifugation for 1 minute, then the sample was placed in an Envision 2120 multi-microplate reader to obtain the relative fluorescence unit (RFU) value.

[0349] 1.6 Experimental Results: (e.g.) Figure 2 As shown, in the L-arabinose group (the experimental group with added L-arabinose), within the concentration range of 7.56 mM to 110 mM of added L-arabinose, it showed a positive effect compared to the blank control group (BC group) without added L-arabinose, with protein synthesis efficiency increasing by 10.77% to 73.96%. Specifically, when the concentrations of added L-arabinose were 28.84 mM and 18.46 mM, protein synthesis yield increased by 73.96% and 64.74%, respectively, significantly improving the protein synthesis capacity of the in vitro protein synthesis system.

[0350] Example 2: Effect of L-arabinose concentration on the protein synthesis capacity of an in vitro protein synthesis system

[0351] 2.1 Preparation of nucleic acid template: Using the method in 1.1 of Example 1, a plasmid vector expressing mEGFP was constructed and in vitro DNA amplification was performed to prepare a plasmid DNA template encoding the exogenous protein mEGFP.

[0352] 2.2 Following the method described in 1.2 of Example 1, based on the Kluyveromyces lactis strain ATCC8585, 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. After fermenting and culturing this modified strain, a Kluyveromyces lactis cell extract was prepared, designated YY09161.

[0353] 2.3 In vitro cell-free protein synthesis reaction system (without adding exogenous RNA polymerase)

[0354] All reaction systems were 300 μL in volume and carried out in 48-well flat-bottom plates. Three replicates were prepared for each sample, and the mean and standard deviation were calculated.

[0355] The final concentrations of each component in the experimental group (concentration curve of L-arabinose, denoted as L-ara group) were as follows: 9.78 mM Tris-HCl at pH 8.0, 80 mM potassium acetate, and 5.6 mM... Magnesium L-aspartate, 1.8 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), 0.6 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), 6% (w / v) trehalose, 2% (w / v) polyethylene glycol 8000, 6.05 mM–110 mM L-arabinose, 5.3 wt% maltodextrin, 24 mM tripotassium phosphate, 50% v volume of Kluyveromyces lactis cell extract.

[0356] Blank control group (BC group): Compared with the above L-ara group, L-arabinose was not added.

[0357] Negative control group (NC group): Compared with the L-arabose experimental group above, no L-arabinose (concentration of 0mM) was added, and no exogenous DNA template was added subsequently.

[0358] 2.4 In vitro protein synthesis reaction: No exogenous DNA template was added to the NC group; DNA template encoding mEGFP (obtained by DNA amplification in step 2.1) was added to each independent in vitro cell-free protein synthesis system of the L-ara group and BC group at a final concentration of 0.33 μg / μL. After mixing, all systems were placed in an environment of 30°C and reacted on a shaker for 20 h.

[0359] 2.5 Fluorescent protein activity assay: The RFU value of mEGFP fluorescent protein was determined using the method described in 1.5 of Example 1.

[0360] 2.6 Experimental Results: (e.g.) Figure 3 As shown, in the L-arabinose group (the experimental group with added L-arabinose), within the concentration range of 6.05 mM to 110 mM of added L-arabinose, it showed a positive effect compared to the blank control group (BC group) without added L-arabinose, with protein synthesis efficiency increasing by 7.22% to 32%. Specifically, when the concentration of added L-arabinose was 7.56 mM, protein synthesis yield increased by 32.0%.

[0361] Example 3: Effect of L-arabinose concentration on the protein synthesis capacity of an in vitro protein synthesis system

[0362] 3.1 Preparation of nucleic acid template: Using the method in 1.1 of Example 1, a plasmid vector expressing mEGFP was constructed and in vitro DNA amplification was performed to prepare a plasmid DNA template encoding the exogenous protein mEGFP.

[0363] 3.2 Following the method described in 1.2 of Example 1, based on the Kluyveromyces lactis strain ATCC8585, the encoding 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. After fermenting and culturing this modified strain, a Kluyveromyces lactis cell extract was prepared, designated YY102211.

[0364] 3.3 In vitro cell-free protein synthesis reaction system (without adding exogenous RNA polymerase).

[0365] The reaction system had a volume of 200 μL and was carried out in a flat-bottomed 48-well plate. Three parallel samples were prepared for each sample, and the mean and standard deviation were calculated.

[0366] The final concentrations of each component in the experimental group (for the concentration curve of L-arabinose, denoted as the L-ara group) were as follows: 9.78 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl, pH 8.0), 80 mM potassium acetate, and 5.6 mM... Magnesium L-aspartate, 1.8 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), 0.5 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 0.5 mM), 6% (w / v) trehalose, 2% (w / v) polyethylene glycol 8000, 4.84 mM–45.06 mM L-arabinose, 5.3 wt% maltodextrin, 18 mM tripotassium phosphate, 50% v volume of Kluyveromyces lactis cell extract.

[0367] Blank control group (BC group): Compared with the above L-ara group, L-arabinose was not added.

[0368] Negative control group (NC group): Compared with the L-ara group above, no L-arabinose (concentration of 0mM) was added, and no exogenous DNA template was added subsequently.

[0369] 3.4 In vitro protein synthesis reaction: No exogenous DNA template was added to the NC group; DNA template encoding mEGFP (obtained by DNA amplification in step 3.1) was added to each independent in vitro cell-free protein synthesis system of the L-ara group and BC group to a final concentration of 0.33 μg / μL. After mixing, all systems were placed in an environment of 30°C and reacted on a shaker for 20 h.

[0370] 3.5 Fluorescent protein activity assay: The RFU value of mEGFP fluorescent protein was determined using the method described in 1.5 of Example 1.

[0371] 3.6 Experimental Results: (e.g.) Figure 4 As shown in the figure, in the L-arabinose group (the experimental group with added L-arabinose), within the concentration range of 4.84 mM to 45 mM of added L-arabinose, it showed a positive effect compared to the blank control group (BC group) without added L-arabinose, with protein synthesis efficiency increasing by 15.13% to 100.20%. Specifically, when the concentrations of added L-arabinose were 9.45 mM, 28.84 mM, and 7.56 mM, the protein synthesis yield increased by 100.2%, 86.25%, and 71.71%, respectively, significantly improving the protein synthesis capacity of the in vitro protein synthesis system.

[0372] The experimental results of Examples 1-3 above show that adding L-arabinose to the in vitro cell-free protein synthesis system based on Kluyveromyces lactis cell extract can improve the in vitro protein synthesis capacity, with the protein synthesis amount generally increasing by more than 15%; under appropriate concentration, it can even increase by 100% (double).

[0373] Example 4: Comparison of the effects of L-arabinose and glucose on the protein synthesis capacity of in vitro protein synthesis systems.

[0374] 4.1 Preparation of nucleic acid template: Using the method in 1.1 of Example 1, a plasmid vector expressing mEGFP was constructed and in vitro DNA amplification was performed to prepare a plasmid DNA template encoding the exogenous protein mEGFP.

[0375] 4.2 Using the method described in 2.2 of Example 2, based on the Kluyveromyces lactis strain ATCC8585, 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. After fermenting and culturing this modified strain, Kluyveromyces lactis cell extract was prepared, designated YY09161.

[0376] 4.3 In vitro cell-free protein synthesis reaction system (without adding exogenous RNA polymerase)

[0377] All reaction systems were 300 μL in volume and carried out in 48-well flat-bottom plates. Three replicates were prepared for each sample, and the mean and standard deviation were calculated.

[0378] The final concentrations of each component in the experimental group (with added L-arabinose or glucose) 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), 1.7 mM dithiothreitol, 2% (w / v) polyethylene glycol 8000, and 29 mM... L-arabinose (L-ara-29mM) or 15mM L-arabinose (L-ara-15mM) or 15mM glucose (Glu-15mM), 5.3wt% maltodextrin, 24mM tripotassium phosphate, and 50% volume of yeast cell extract. The group with added L-arabinose was designated as the L-ara group (without added glucose), and the group with added glucose was designated as the GC group (without added L-arabinose).

[0379] Negative control group (NC group): Compared with the experimental group, no L-arabinose (concentration of 0mM) was added, and no exogenous DNA template was added subsequently.

[0380] 4.4 In vitro protein synthesis reaction: No exogenous DNA template was added to the NC group. DNA template encoding mEGFP (obtained by DNA amplification in step 4.1 above) was added to each independent in vitro cell-free protein synthesis system of the L-ara group and GC group at a final concentration of 0.33 μg / μL. After mixing, all systems were placed in an environment of 30°C and reacted on a shaker for 20 h.

[0381] 4.5 Fluorescent protein activity assay: The RFU value of mEGFP fluorescent protein was determined using the method described in 1.5 of Example 1.

[0382] 4.6 Experimental results are as follows Figure 5 As shown, compared with the protein yield at 15 mM glucose (mean RFU of 1288), the exogenous protein synthesis yield (mean RFU of 1897 and 1644, respectively) increased by 47.3% and 27.6% when L-arabinose concentrations were 29 mM and 15 mM, respectively.

[0383] The above are merely some preferred embodiments of the present invention, and the present invention is not limited to the contents of the above embodiments. For those skilled in the art, various changes and modifications can be made within the scope of the concept of the technical solution of the present invention or under its guidance or inspiration. Any changes and modifications with equivalent technical effects are within the protection scope of the present invention. sequence list <110> Kangma (Shanghai) Biotechnology Co., Ltd. <120> An in vitro cell-free protein synthesis system (D2P system), its reagent kit and its applications <130> 2020 <141> 2020-01-21 <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 717 <212> DNA <213> Artificial sequence <400> 1 gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc 60 gacgtaaacg gccacaagtt cagcgtgcgc ggcgagggcg agggcgatgc caccaacggc 120 aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 180 gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag 240 cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catctccttc 300 aaggacgacg gcacctacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg 360 aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag 420 ctggagtaca acttcaacag ccacaacgtc tatatcacgg ccgacaagca gaagaacggc 480 atcaaggcga acttcaagat ccgccacaac gtcgaggacg gcagcgtgca gctcgccgac 540 cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac 600 ctgagcaccc agtccaagct gagcaaagac cccaacgaga agcgcgatca catggtcctg 660 ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaa 717 <210> 2 <211> 238 <212> PRT <213> Artificial Sequence <400> 2 Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val 1 5 10 15 Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Arg Gly Glu 20 25 30 Gly Glu Gly Asp Ala Thr Asn Gly Lys Leu Thr Leu Lys Phe Ile Cys 35 40 45 Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu 50 55 60 Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln 65 70 75 80 His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg 85 90 95 Thr Ile Ser Phe Lys Asp Asp Gly Thr Tyr Lys Thr Arg Ala Glu Val 100 105 110 Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile 115 120 125 Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn 130 135 140 Phe Asn Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly 145 150 155 160 Ile Lys Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp Gly Ser Val 165 170 175 Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro 180 185 190 Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Lys Leu Ser 195 200 205 Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val 210 215 220 Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys 225 230 235

Claims

1. An in vitro cell-free protein synthesis system, characterized by: The in-vitro cell-free protein synthesis system comprises a Kluyveromyces lactis cell extract, exogenous L-arabinose and an RNA polymerase; the in-vitro cell-free protein synthesis system can react with a nucleic acid template encoding an exogenous protein to synthesize the exogenous protein; and the exogenous L-arabinose can increase the expression amount of the exogenous protein.

2. The in vitro cell-free protein synthesis system according to claim 1, characterized in that: The concentration of the exogenous L-arabinose is in the range of 2 mM to 400 mM.

3. The in vitro cell-free protein synthesis system according to claim 2, characterized in that: The concentration of the exogenous L-arabinose is in the range of 5 mM to 150 mM.

4. The in vitro cell-free protein synthesis system of claim 2, wherein: The concentration of the exogenous L-arabinose is in the range of 6 mM to 110 mM.

5. The in vitro cell-free protein synthesis system of claim 2, wherein: The concentration of the exogenous L-arabinose is in the range of 5 mM to 50 mM.

6. The in vitro cell-free protein synthesis system of claim 2, wherein: The concentration of the exogenous L-arabinose is in the range of 4.8 mM to 36 mM.

7. The in vitro cell-free protein synthesis system of claim 2, wherein: The concentration of the exogenous L-arabinose is 7.56 mM, 7.5 mM, 7.6 mM, 28.84 mM, 29 mM or 30 mM.

8. The in vitro cell-free protein synthesis system according to any one of claims 1 to 7, characterized in that: The source of the RNA polymerase is selected from any one of the following: a cell extract containing endogenously expressed RNA polymerase, an exogenous RNA polymerase, a translation product of an exogenous nucleic acid template encoding the RNA polymerase, or a combination thereof, and the RNA polymerase is a T7 RNA polymerase.

9. The in vitro cell-free protein synthesis system of claim 1, wherein: The in-vitro cell-free protein synthesis system further comprises a DNA polymerase; the source of the DNA polymerase is selected from any one of the following: a cell extract containing endogenously expressed DNA polymerase, an exogenous DNA polymerase, a translation product of an exogenous nucleic acid template encoding the DNA polymerase, or a combination thereof; and the DNA polymerase is a phi 29 DNA polymerase.

10. The in vitro cell-free protein synthesis system of claim 1, wherein: The Kluyveromyces lactis cell extract contains endogenously expressed RNA polymerase; the cell extract is prepared by endogenous strain modification of Kluyveromyces lactis, and the modification mode is selected from the following: inserting the coding sequence of the RNA polymerase into a free plasmid in the cell, or integrating the coding gene of the RNA polymerase into the genome of the cell, or a combination of the above two modes. The endogenously expressed RNA polymerase is an endogenously expressed T7 RNA polymerase.

11. The in vitro cell-free protein synthesis system of claim 1, wherein: The in-vitro cell-free protein synthesis system further comprises an energy system; the energy system is selected from any one of the following: a sugar and phosphate energy system, a sugar and phosphagen energy system, a phosphagen and phosphagen kinase system, a phosphagen and phosphagen kinase system, a monosaccharide and its catabolic intermediate, a glycogen and its catabolic intermediate, or a combination thereof.

12. The in vitro cell-free protein synthesis system of claim 1, wherein: The in-vitro cell-free protein synthesis system further comprises a substrate for synthesizing RNA and / or a substrate for synthesizing protein; the substrate for synthesizing RNA is a nucleotide mixture selected from the following: nucleoside monophosphate, nucleoside triphosphate, or a combination thereof; the substrate for synthesizing protein is an amino acid mixture, which at least includes an amino acid mixture required in the process of synthesizing the exogenous protein; and the amino acid mixture is a mixture of natural amino acids.

13. The in vitro cell-free protein synthesis system of claim 1, wherein: The in-vitro cell-free protein synthesis system further comprises at least one of the following components: a crowding agent, magnesium ions, potassium ions, an antioxidant or reducing agent, trehalose, a reaction promoter, a buffer, and an aqueous solvent.

14. The in vitro cell-free protein synthesis system of claim 13, wherein the crowding agent is selected from the group consisting of polyethylene glycol, dextran, Ficoll sucrose polymer, or a combination thereof. the magnesium ion is selected from the group consisting of magnesium aspartate, magnesium acetate, magnesium glutamate, magnesium chloride, magnesium phosphate, magnesium sulfate, magnesium citrate, magnesium hydrogen phosphate, magnesium iodide, magnesium lactate, magnesium nitrate, magnesium oxalate, or a combination thereof; the potassium ion is selected from the group consisting of potassium acetate, potassium glutamate, potassium chloride, potassium phosphate, potassium sulfate, potassium citrate, potassium hydrogen phosphate, potassium iodide, potassium lactate, potassium nitrate, potassium oxalate, or a combination thereof; the anti-oxidant or reducing agent is dithiothreitol; the buffer is selected from the group consisting of Tris-HCl, Tris base, HEPES, or a combination thereof. the exogenous protein is selected from the group consisting of luciferase, green fluorescent protein, enhanced green fluorescent protein, yellow fluorescent protein, aminoacyl tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, variable region of an antibody, luciferase mutant, alpha-amylase, enterotoxin A, hepatitis C virus E2 glycoprotein, insulin precursor, interferon alpha A, interleukin-1 beta, lysozyme, serum albumin, single chain antibody fragment, transthyretin, tyrosinase, xylanase, E. coli beta-galactosidase, human lysine-tRNA synthetase, human leucine-tRNA synthetase, Arabidopsis glyceraldehyde 3-phosphate dehydrogenase, murine catalase, mutant of any of the foregoing, in any combination.

15. The in vitro cell-free protein synthesis system of claim 1, wherein the in vitro cell-free protein synthesis system is capable of reacting with a DNA template or an mRNA template encoding an exogenous protein to synthesize the exogenous protein.

16. The in vitro cell-free protein synthesis system of claim 1, wherein: the in vitro protein synthesis kit comprises:

17. An in vitro protein synthesis kit, characterized in that, (i) the in vitro cell-free protein synthesis system of any one of claims 1-16; (ii) a label or an instruction. the in vitro protein synthesis kit further comprises a nucleic acid template encoding an exogenous protein, which comprises a promoter element recognizable by the components of the system in (i); 18. The in vitro protein synthesis kit according to claim 17, characterized in that, the nucleic acid template encoding an exogenous protein comprises a T7 promoter, and the in vitro cell-free protein synthesis system comprises a T7 RNA polymerase; the nucleic acid template encoding an exogenous protein comprises a T7 promoter, and the K. lactis cell extract comprises an endogenously expressed T7 RNA polymerase; the nucleic acid template encoding an exogenous protein is a DNA template, an mRNA template, or a combination thereof; the nucleic acid template encoding an exogenous protein further comprises other translation-related elements; the nucleic acid template encoding an exogenous protein comprises an exogenous protein translation system, a resistance gene translation system, a Lac repressor translation system, each of which comprises a corresponding promoter; the nucleic acid template encoding an exogenous protein further comprises a gene that controls the copy number of the plasmid; the nucleic acid template encoding an exogenous protein further comprises a translation enhancer element. the method for synthesizing an exogenous protein comprises the following steps:

19. A method of synthesizing an exogenous protein, comprising: (i) providing the in vitro cell-free protein synthesis system of any one of claims 1-16; ​ (ii) adding a nucleic acid template encoding an exogenous protein into the in vitro cell-free protein synthesis system of step (i), incubating the reaction, and synthesizing the exogenous protein; the nucleic acid template encoding the exogenous protein contains a promoter element recognizable by the components of the system in (i); the transcription of the gene of the exogenous protein is initiated by a T7 promoter on the nucleic acid template, and the in vitro cell-free protein synthesis system comprises a T7 RNA polymerase; the transcription of the gene of the exogenous protein is initiated by a T7 promoter on the nucleic acid template, and the Kluyveromyces lactis cell extract comprises an endogenously expressed T7 RNA polymerase; the nucleic acid template encoding the exogenous protein is a DNA template, an mRNA template, or a combination thereof; the nucleic acid template encoding the exogenous protein further contains other translation-related elements; the nucleic acid template encoding the exogenous protein comprises an exogenous protein translation system, a resistance gene translation system, a Lac repressor translation system; each of the above translation systems comprises a corresponding promoter; the nucleic acid template encoding the exogenous protein further contains a gene for controlling the copy number of plasmid; the nucleic acid template encoding the exogenous protein further contains a translation enhancer element.

20. A method for synthesizing an exogenous protein according to claim 19, characterized in that, Further comprising a step of: (iii) isolating or / and detecting the exogenous protein.

21. Use of the in vitro cell-free protein synthesis system according to any one of claims 1 to 16, characterized in that For protein synthesis, including protein production and protein synthesis-based detection.

22. Use of L-arabinose in the in vitro cell-free protein synthesis system of any one of claims 1-16, or in the in vitro protein synthesis kit of claims 17-18, or in the method for synthesizing an exogenous protein of claims 19-20.

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