WO2021237410A1

WO2021237410A1 - Co-production method for multiprotein system, co-production system for multiprotein system and use thereof

Info

Publication number: WO2021237410A1
Application number: PCT/CN2020/092110
Authority: WO
Inventors: 戴卓君; 李鹏程; 张曦
Original assignee: 深圳先进技术研究院
Priority date: 2020-05-25
Filing date: 2020-05-25
Publication date: 2021-12-02

Abstract

Provided are a co-production method for a multiprotein system, a co-production system for a multiprotein system and use thereof. Specifically, a vector for expressing a bacteriolytic protein and a plurality of vectors for expressing proteins are co-transferred into a protein expression strain, so that the obtained co-expression strain expresses corresponding proteins and bacteriolytic protein. As growth of the co-expression strain, the generated bacteriolytic protein acts on the co-expression strain, so that the co-expression strain is subject to autonomous lysis, and the expressed proteins are released, thereby forming a multi-protein system which can be used for in vitro synthesis of proteins and/or compounds.

Description

Multi-protein system symbiosis production method, multi-protein system co-production system and application technology field

[0001] This application relates to the technical field of protein synthesis in vitro, and specifically relates to a co-production method of a multi-protein system, a co-production system of a multi-protein system, and applications. Background technique

[0002] Cells are the basic structure and functional unit of life activities, and at the same time provide a place for biochemical reactions, and are called "cell factories". However, the intracellular biochemical reaction network is complex, and artificial modification is likely to have adverse or unpredictable effects on cells, and ultimately lead to reduced cell viability and even loss of function, which greatly limits the application of cell factories. Therefore, how to break through the limitations of cell factories is a huge challenge for biosynthesis. Aiming at the limitations of cell factories, cell-free protein synthesis (CFPS) provides a good solution. The cell-free protein synthesis system is a biological technology that does not rely on intact cells for protein synthesis in vitro. It uses DNA or mRNA as a template and uses protein synthesis elements, protein folding factors and other related enzyme systems in cell extracts. Add amino acids, tRNA and energy substances to complete protein synthesis in vitro, simulate the life phenomenon of biological cells, and reproduce the transcription and translation process of intracellular proteins. The resulting protein can be used in downstream experiments such as protein function testing and structural analysis. Compared with the living cell system, the modular cell-free protein synthesis system has clear and simple reaction components, easy control of the components, and short experiment period, providing an open and versatile reaction environment for many biochemical experiments.

[0003] Currently, cell-free protein synthesis systems are mainly divided into two types: one is directly derived from cell lysates (WCE: whole-cell extracts), and the other contains only the necessary for DNA transcription, protein translation and energy regeneration. Ingredients, namely the protein synthesis using recombinant elements PURE (protein synthesis using recombinant elements). Different from the WCE system, all additives in the F*URE system are fully known and the concentration is controllable, so it has many significant advantages: For example, the low-degrading enzyme environment greatly improves the stability of mRNA or protein; The protein or substrate components can be conveniently designed for the protein to be synthesized (such as the insertion of unnatural amino acids, etc.), making the system highly modular and capable Flexibility etc.

[0004] Although HJRE has been commercialized since 2002, its high price limits its application in most laboratories. To prepare the PURE system by traditional methods, it is necessary to express and purify more than 30 proteins related to transcription, translation, and energy regeneration. The workload is large, time-consuming, and the preparation cost is high. Generally, laboratories cannot prepare it by themselves. At present, large biologics companies represented by NEB (New England Biolab) still use this method to produce PURE systems. Summary of Invention Technical Problem

[0005] One of the objectives of the embodiments of the present application is to provide a method for co-production of a multi-protein system, a co-production system of a multi-protein system, and applications, aiming to solve the large workload and consumption in the synthesis of the existing PURE system. Issues such as time length and high cost. Problem solution technical solution

[0006] To solve the above technical problems, the technical solutions adopted in the embodiments of the present application are:

[0007] In the first aspect, a method for co-production of a multi-protein system is provided, which includes the following steps:

[0008] A vector for expressing a bacteriolytic protein, a vector for multiple expression proteins, and a protein expression strain are provided. Each protein expression vector expresses one protein in a multi-protein system, and different protein expression vectors express different proteins. same;

[0009] The vector expressing the bacteriolytic protein and the vector expressing each protein are co-transformed into the protein expression strain to obtain multiple co-expression strains;

[0010] All co-expression strains are co-cultured, and autonomous lysis occurs when the growth density of the co-expression strain reaches the autonomous lysis density, and the respective expressed proteins are released to obtain a polyprotein system;

[0011] Among them, the multi-protein system includes proteins used for protein translation, energy regeneration, and/or enzymes that catalyze the synthesis of compounds.

[0012] In the second aspect, a multi-protein system co-production system is provided, which includes:

[0013] Co-expression strain preparation unit: used to prepare a variety of co-expression strains, each co-expression strain expresses a protein and bacteriolytic protein in a multi-protein system, and different co-expression strains express different proteins; [0014] Cell culture unit: used to cultivate co-expression strains in the co-expression strain unit;

[0015] Among them, the multi-protein system includes proteins used for protein translation, energy regeneration, and/or enzymes that catalyze the synthesis of compounds.

[0016] In the third aspect, the application of a multi-protein system co-production system in the synthesis of proteins and/or compounds in vitro is provided. The beneficial effects of the invention

[0017] The beneficial effects of the co-production method of the multi-protein system provided by the examples of the application are: by co-transferring the vector expressing the lysoprotein and the vector expressing multiple proteins into the protein expression strain, the obtained co-expression strain can be expressed The corresponding protein and bacteriolytic protein, as the co-expression strain grows, the produced bacteriolytic protein acts on the co-expression strain to cause autonomous lysis, release the expressed protein, and form a multi-protein system. The co-production method of the multi-protein system provided in the examples of the application can realize the co-cultivation of multiple co-expression strains and collect multiple proteins at one time, and does not require the steps of mechanically or non-mechanically disrupted protein expression strains, and is applicable The advantages of wide range, simple steps, high efficiency and low cost.

[0018] The beneficial effects of the multi-protein system co-production system provided in the examples of the application are: the multi-protein system co-production system includes a co-expression strain preparation unit and a cell culture unit. Among them, the co-expression strain preparation unit prepares multiple co-expression strains. Since each co-expression strain does not repeatedly express a protein and lysoprotein in the multi-protein system, when multiple co-expression strains are co-cultured in the cell culture unit The bacteriolytic protein will cause the co-expression strain to undergo autonomous lysis, which is beneficial to the stability of the coexistence of strains in the co-expression system (avoid some co-expression strains from robbing all resources due to excessive growth and uninhibited growth). At the same time, the autonomous lysis will Release various proteins in the multi-protein system, so as to realize the co-production of the multi-protein system. The multi-protein system co-production system provided in the examples of the present application can obtain multiple proteins at one time, and has the advantages of easy control of culture conditions, high production efficiency, and low cost.

[0019] The beneficial effects of the application of the multi-protein system co-production system in the in vitro synthesis of proteins and/or compounds provided by the examples of the application are: That is, the proteins necessary for the synthesis of proteins and/or compounds in vitro can be collected, and the synthesis of proteins and/or compounds can be performed in vitro without relying on intact cells. Compared with the existing method for preparing a cell-free protein synthesis system, the co-production system of the multi-protein system provided in the embodiments of the application is It's easier, faster, more efficient, and lower cost. Brief description of the drawings

[0020] FIG. 1 is a schematic diagram of the work flow of a method for co-production of a multi-protein system provided by one of the embodiments of the application;

[0021] FIG. 2 provides a specific example of this application, the synthetic red fluorescent protein mRFP content and fluorescence signal standard curve when excited at 580nm and emitted at 610nm;

[0022] FIG. 3 is a specific embodiment provided in this application, the real-time fluorescent signal monitoring result of the synthetic red fluorescent protein mRFP. Inventive embodiment of the present invention

[0023] In order to make the objectives, technical solutions, and advantages of this application clearer, the following embodiments will further describe this application in detail. It should be understood that the specific embodiments described here are only used to explain the present application, and are not used to limit the present application. In combination with the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative work shall fall within the protection scope of this application. If the specific conditions are not specified in the examples, proceed in accordance with the conventional conditions or the conditions recommended by the manufacturer; if the reagents or instruments used do not specify the manufacturer, they are all conventional products that can be purchased commercially.

[0024] In the description of this application, it should be understood that the weight of related components mentioned in the examples of this application can not only refer to the specific content of each component, but can also represent the weight ratio of each component. Therefore, as long as the content of the relevant components in the embodiments of the application is scaled up or down, it is within the scope of the disclosure of the application. Specifically, the weight described in the embodiments of the present application may be mass units known in the chemical industry, such as y g, mg, g, and kg.

[0025] In addition, unless the context clearly uses otherwise, the expression of the singular form of a word should be understood to include the plural form of the word. The term "including" or "having" is intended to specify the existence of features, quantities, steps, operations, elements, parts or combinations thereof, but is not used to exclude the existence or possible addition of one or more other features, quantities, steps, operations, elements , Part or a combination thereof.

[0026] In the description of this application, the term "and/or" is regarded as a specific disclosure of each of the two specified features or components with or without the other. Therefore, as in the phrase "A and/or B" in this article The use of the term "and/or" is intended to include A and B; A or B; A (alone); and B (alone). Likewise, the term "and/or" as used in phrases such as "A, B and/or C" is intended to cover each of the following: A, B and C; A, BSC; A or C; A Or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0027] Unless otherwise defined in this application, the terms used in the content of this application shall have the meaning commonly understood by a person of ordinary skill in the art. For example, any terms and techniques related to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid in the examples of this application are well-known and commonly used in the art. In case of any potential ambiguity, the definition provided in this application takes precedence over any dictionary or external definition.

[0028] In the description of this application, the term "vector" includes plasmids, bacteriophages, viruses or other vectors.

[0029] In order to illustrate the technical solutions described in the present application, the following describes in detail with reference to specific embodiments.

[0030] The embodiments of the present application provide a co-production method of a multi-protein system, which includes the following steps:

[0031] S1. Provide vectors for expressing bacteriolytic proteins, vectors for multiple expression proteins, and protein expression strains. Each protein-expressing vector expresses one protein in a multi-protein system, and different protein-expressing vectors express proteins Different

[0032] S2. Co-transform the vector expressing the bacteriolytic protein and the vector expressing each protein into the protein expression strains to obtain multiple co-expression strains;

[0033] S3. All co-expression strains are co-cultured, and autonomous lysis occurs when the growth density of the co-expression strain reaches the autonomous lysis density, and the respective expressed proteins are released to obtain a polyprotein system;

[0034] Among them, the multi-protein system includes proteins used for protein translation, energy regeneration, and/or enzymes that catalyze the synthesis of compounds.

[0035] The beneficial effects of the co-production method of the multi-protein system provided by the examples of the application are: by co-transforming the vector expressing the lysoprotein and the vector expressing multiple proteins into the protein expression strain, the obtained co-expression strain can be expressed The corresponding protein and bacteriolytic protein, as the co-expression strain grows, the produced bacteriolytic protein acts on the co-expression strain to cause autonomous lysis, release the expressed protein, and form a multi-protein system. The co-production method of the multi-protein system provided in the examples of the application can realize the co-cultivation of multiple co-expression strains and collect multiple proteins at one time, and does not require the steps of mechanically or non-mechanically disrupted protein expression strains, and is applicable The advantages of wide range, simple steps, high efficiency and low cost. [0036] Specifically, in S1, the vector expressing the bacteriolytic protein can be used to express the bacteriolytic protein. The bacteriolytic protein is a type of protein that inhibits the cell wall synthesis of the host cell through different ways, or directly destroys the cell wall of the host cell. By transferring the vector expressing bacteriolytic protein into the protein expression strain, as the co-expression strain grows, the copy number of the vector expressing bacteriolytic protein increases and the expression of the bacteriolytic protein increases, thereby causing the co-expression strain to undergo autonomous lysis.

[0037] In some embodiments, the plasmid ePop expressing the phage (i>X174 E protein) is selected as the vector for expressing the lysoprotein. As an engineered gene circuit, ePop contains two main modules: a cell autonomous lysis module : This module is based on the gene from phage 4>X174 E protein. Phage (i>X174 contains only 10 genes, and its lysis mechanism is to produce a single E protein, E protein can effectively inhibit peptidoglycan synthase MraY, thereby inhibiting peptides The synthesis of glycans, and E protein can also inhibit the peptidoglycan precursor substance diaminoheptanoic acid from entering the cell wall, thereby causing the lysis of host cells. The other module is the cell density sensor module, which is based on the mutant luxR gene and ColEl-derived genes lacking Rom/Rop protein replication. Therefore, the use of plasmid ePop can achieve programmed autonomous lysis. By configuring the gene circuit as a cell density-dependent circuit coupled with E protein expression, when the protein expression strain is cultured and reproduced When the medium reaches a certain density, the copy number of the plasmid ePop expressing the phage (i>X174 E protein will increase, and the expression of the E protein will be higher at this time, which will lead to autonomous lysis of the co-expressing strain. It should be noted that the gene circuit is one This kind of adjustable circuit can adjust the corresponding module according to the function that needs to be performed. Therefore, the above example of selecting the plasmid ePop expressing the phage (i>X174 E protein) is only one of the examples of this application, even if the gene is not used The circuit, the use of other gene circuits that can make cells produce autonomous lysis or induce lysis; or the technical solutions that can make the protein-expressing strains undergo autonomous lysis when the growth density reaches a higher level should all fall within the protection scope of this application.

[0038] The protein expression strains are used in the examples of this application to co-express lysoproteins and proteins in a multi-protein system. Therefore, in principle, suitable for co-expression of lysins and other protein expression strains are applicable to this application. In some embodiments, in order to achieve stable protein production, E. coli strains are selected as protein expression strains. Specifically, E. coli strains include, but are not limited to, BL21 (DE3), MC4100, MG1655, NISSLE 1917 series of strains, and their mutant strains or derivative strains.

[0039] A variety of protein expression vectors are used for protein expression. Among them, these protein-expressing vectors do not repeatedly express a protein in the multi-protein system, so the number of types of protein-expressing vectors is different from that of the multi-protein system. The number of types of protein is equal. After multiple protein expression vectors are respectively transferred to the protein expression strain, the protein expression strain can express the corresponding protein.

[0040] In S2, the co-expression strain can be obtained by co-transferring the vector expressing the bacteriolytic protein and the vector expressing the multi-protein system protein into the protein expression strain, and the co-expression strain expresses the bacteriolytic protein and the multi-protein system protein at the same time. In this step, the number of types of co-expression strains obtained is equal to the number of types of protein-expressing vectors, and is also equal to the number of types of proteins in the multi-protein system. Therefore, it is understandable that each co-expression strain is not repeated separately. Express a protein in a multi-protein system.

[0041] In S3, by co-cultivating all co-expression strains obtained from S2, when the strain grows to a comparable density, the concentration of the lysoprotein expressed by it reaches a concentration that destroys the cell wall of the co-expression strain, the co-expression strain occurs Autonomous cleavage releases the expressed protein. Among them, each co-expression strain separately expresses a protein in the polyprotein system, and collects all the proteins released by the autonomous cleavage of all co-expression strains to form a polyprotein system.

[0042] In some embodiments, the timing of autonomous lysis of the co-expression strain can be controlled. Specifically, when the vector expressing the bacteriolytic protein is the expression bacteriophage X174

In the case of the plasmid ePop of the E protein, you can set the cell autonomous lysis module in the gene circuit of the ePop, so that when the growth density of the co-expression strain reaches a certain value, the phage (i>X174 E protein plasmid ePop can be set. The copy number will increase significantly, and more lysoproteins will be expressed, which will promote autonomous lysis of co-expressing strains.

[0043] In some embodiments, during the process of culturing the co-expression strain, the OD value can be determined at regular intervals. When the growth density of the co-expression strain reaches the autonomous lysis density, that is, 0D600 is greater than or equal to 0.05, the co-expression strain begins to undergo autonomous lysis.

[0044] In some embodiments, before co-cultivating multiple co-expression strains, each co-expression strain is cultured separately. By culturing each co-expression strain separately, the co-expression strain can be activated to achieve a better state. At this time, glucose is added to the environment where each co-expression strain is cultured separately to inhibit the spontaneous lysis of the strain, so that the strain can reproduce smoothly to a certain density; then the co-expression strain is inoculated into a co-culture system without adding Glucose makes the autonomous lysis circuit open, and autonomous lysis occurs when the co-cultured strains reach the density of autonomous lysis. Specifically, when glucose is added to inhibit autonomous lysis of co-cultured strains, the final mass of glucose in the culture environment (such as in the culture medium) The product concentration is 0.5%-2%.

[0045] There can be many types of multi-protein systems, which are a general term for a series of proteins with catalytic functions included in each metabolic pathway. In the examples of the present application, the multi-protein system includes proteins that synthesize in vitro and/or catalyze the synthesis of compounds. Among them, a multi-protein system composed of proteins for protein translation and energy regeneration is the key to achieving protein synthesis in vitro. In the examples of this application, the proteins necessary for energy regeneration include creatine kinase, myokinase, and nucleoside diphosphate kinase. And pyrophosphatase; The multi-enzyme system composed of enzymes that catalyze the synthesis of compounds can realize the efficient synthesis of multiple target compounds, and is currently used in the fields of biology, chemical engineering, and medical treatment.

[0046] In some embodiments, a polyketide synthase system and/or a non-ribosomal polypeptide synthetase system are selected as the target multi-protein system of the multi-protein system co-production method. Among them, polyketides are a large class of natural products produced by bacteria, actinomycetes, fungi or plants, including macrolides, tetracyclines, anthracyclines, polyethers and other compounds. Since these natural products have various activities such as anti-infection, anti-fungal, anti-tumor, and immunosuppressive activities, it is of great significance in the medical field to provide a method for efficiently producing a polyketide synthase system. Enzymatic Synthesis of polyketides is the polyketide synthase ^{(polyket idesynthase,} PKS), specifically includes PKS I, PKS II, PKS III . Non-ribosomal peptide synthase is a special type of enzyme that can utilize amino acids and other compounds (such as salicylic acid and pyridine carboxylic acid) without ribosomes, mRNA as a template, and tRNA as a carrier. Etc.) Synthesis of special peptides. Bacteria and fungi can synthesize penicillin, vancomycin, actinomycin D, bacitracin, cyclosporin A, etc. through the non-ribosomal polypeptide synthetase system in vivo. The efficient production of non-ribosomal polypeptide synthetase system will effectively synthesize the above Pharmaceutical compounds provide more convenient conditions.

[0047] In some embodiments, a recombinant element protein synthesis system is selected as the target multi-protein system for the co-production method of the multi-protein system. The recombination element protein synthesis system is also a kind of multi-protein system, which includes a series of proteins that can be used to synthesize the target protein under in vitro conditions. Different from non-ribosomal polypeptide synthetase, in the process of synthesizing the target protein, the protein synthesis system of the recombination element requires the participation of ribosomes and amino acids. At the same time, it also needs to use mRNA as a template and tRNA as a tool for carrying amino acids.

[0048] In some embodiments, when mRNA is used as a template, the selection includes translation initiation factor 1 (translati onal initiation factor 1, IF1), translational initiation factor 2 (translational initiation factor 2, IF2), and fanci Translational initiation factor 3 factor 3, IF3), translational elongation factor G (EF-G), translational elongation factor Tu (EF_Tu), translational elongation factor Ts (EF_Ts), translation elongation factor 4 (translational elongation factor

4. EF-4), translation release factor 1 (translational release factor 1, RF1), translation release factor 2 (translational release factor

2, RF2), translation release factor 3 (translational release factor 3, RF3), ribosome recycling factor (RRF), methionyl-tRNA formyl transferase (Met-tRNA formyl transferase) _> creatine kinase (creatine kinase, CK), myokinase and diphosphonucleotide kinase (NDK), pyrophosphatase (Pyrophosphatase) and 22 aminoacyl-tRNA synthetase as a combination of recombination element protein system, through multiple The co-production method of protein system is produced, which can quickly and efficiently realize the synthesis of target protein in vitro. Among them, 22 kinds of aminoacyl-tRNA synthetase specifically include: methionyl-tRNA synthetase (Met-tRNA-synthetase), threonyl-tRNA-synthetase (Thr-tRNA-synthetase)

, Glu-tRNA-synthetase, Ala-tRNA-synthetase, Aspartyl-tRNA-synthetase, Asparagus Asn-tRNA-synthetase, Cys-tRNA-s ynthetase, Pro-tRNA-synthetase, Tyrosyl- tRNA synthetase (Tyr-tRNA-synthetase), glutaminyl-tRNA synthetase (Gln-tRNA-syntheta se), histidyl-tRNA synthetase (His-tRNA-synthetase), glycyl-tRNA synthetase A (G ly-tRNA-synthetase -A), Gan aminoacyl -tRNA synthetase B (Gly-tRNA-synthetase- B), Jiao ^_ aminoacyl tRNA synthetase (Val-tRNA-synthetase), lysyl -tRNA Synthetase (Lys-tRNA-synthetase), Leu-tRNA-synthetase, tryptophan-tRNA-synthetase, phenylalanyl-tRNA synthetase B ( Phe-tRNA-B synthetase), seryl-tRNA synthetase (Ser-tRNA-synthetase), phenylalanyl-tRNA synthetase A (Phe-tRNA- A synthetase), arginyl-tRNA synthetase (Arg -tRNA synthetase), Ile-tRNA synthetase (Ile-tRNA synthetase) _D [0049] In some embodiments, when the DNA encoding the target protein is used as a template, the above-mentioned polyprotein body 17 RNA polymerase is added to the system; when the target protein is some protein that is difficult to synthesize in vitro, the addition of disulfide isomerase and/or molecular chaperone protein in the above-mentioned multi-protein system can help to improve this purpose The efficiency of protein synthesis in vitro.

[0050] In the second aspect, the present application provides a multi-protein system co-production system in some embodiments, which includes:

[0051] The co-expression strain preparation unit is used to prepare a variety of co-expression strains, each co-expression strain expresses one protein and bacteriolytic protein in the multi-protein system, and different co-expression strains express different proteins;

[0052] cell culture unit for culturing co-expression strains in the co-expression strain unit;

[0053] Among them, the multi-protein system includes proteins used for protein translation, energy regeneration, and/or enzymes that catalyze the synthesis of compounds.

[0054] The beneficial effects of the multi-protein system co-production system provided in the examples of the application are: the multi-protein system co-production system includes a co-expression strain preparation unit and a cell culture unit, wherein the co-expression strain preparation unit prepares multiple co-production systems Expression strains, since each co-expression strain does not repeatedly express a protein and lysoprotein in the multi-protein system, therefore, when multiple co-expression strains are co-cultured in the cell culture unit, the lysoprotein will cause the co-expression strain to undergo autonomous lysis ，Release a variety of proteins and realize the co-production of multi-protein systems. The co-production system of the multi-protein system realizes autonomous lysis by editing strains, simplifies the subsequent protein purification steps, and maintains the stability of the flora during the co-cultivation process (avoid some co-expression strains that rob all of them because they grow too fast and are not inhibited. Resources), a variety of proteins can be obtained at one time, with the advantages of easy control of culture conditions, high production efficiency, and low cost.

[0055] There are multiple co-expression strains in the co-expression strain preparation unit, and each co-expression strain does not repeatedly express one protein and bacteriolytic protein in the multi-protein system. In some embodiments, multiple expression proteins can be constructed first, and each protein expression vector does not repeatedly express one protein in the above-mentioned multi-protein system; by co-transforming these expression vectors and the vectors expressing lysoproteins. Into the protein expression strain, a variety of co-expression strains are thus obtained, and each co-expression strain does not repeatedly express one protein and bacteriolytic protein in the multi-protein system. The vector expressing lysoprotein can specifically select the plasmid ePop expressing phage 4) X174 E protein. This plasmid also has the function of sensing cell density on the basis of expressing E protein. When the growth of the co-expressing strain reaches a certain density, it can further promote The copy number of the plasmid increases, which in turn promotes the expression of E protein. At the same time, by adding different concentrations of glucose, the co-expression can also be adjusted The effect of the degree of spontaneous lysis of the strain.

[0056] In some embodiments, a polyketide synthase system and/or a non-ribosomal polypeptide synthetase system are selected as the target polyprotein system of the co-production system. By synthesizing these two polyprotein systems, it is helpful to realize the efficient synthesis of a variety of pharmaceutical compounds.

[0057] In some embodiments, the recombinant element protein synthesis system is selected as the target multi-protein system of the co-production system. By synthesizing recombination element proteins, it helps to achieve efficient synthesis of target proteins in vitro.

[0058] In some embodiments, when mRNA is used as a template, translation initiation factor 1, translation initiation factor 2, translation initiation factor 3, translation elongation factor G, translation elongation factor Tu, translation elongation factor Ts, Translation elongation factor 4, translation release factor 1, translation release factor 2, translation release factor 3, ribosomal cycle factor, methionyl-tRNA formyl transferase, creatine kinase, myokinase, nucleoside diphosphate kinase, pyrophosphate The combination of enzyme and 22 kinds of aminoacyl-tRNA synthetase is the target multi-protein system of the co-production system of multi-protein system. Through the co-production and synthesis of the above-mentioned protein, the in vitro synthesis of the target protein can be quickly and efficiently achieved.

[0059] In some embodiments, when the DNA encoding the target protein is used as a template, it is also necessary to add 17 RNA polymerase to the above-mentioned polyprotein system; when the target protein is some protein that is difficult to synthesize in vitro, it is The addition of disulfide bond isomerase, molecular chaperone proteins, etc. to the multi-protein system can help improve the in vitro synthesis efficiency of such target proteins.

[0060] The conditions for co-cultivating co-expression strains in the cell culture unit involve various aspects such as culture medium, culture temperature, culture humidity, culture time, auxiliary additives, light conditions and so on. In some embodiments, the method of co-cultivating co-expression strains in a cell culture unit is as follows: first pick a single clone of each co-expression strain in an LB medium containing dual resistance to kanamycin and chloramphenicol, And add glucose with a final mass and volume concentration of 2% to inhibit cell lysis, ^{culture at 37°} C, 220rpm for 14h-18h, then inoculate and transfer to M9 medium with dual resistance to kanamycin and chloramphenicol After co-cultivation at 37 ^° C and 220 rpm for 5-6 hours, IPTG with a final concentration of 0.1 mM was added to induce 3-5 hours. Centrifuge the co-culture, collect the supernatant, and purify to obtain a multi-protein system.

[0061] In some embodiments, glucose is added before co-cultivation of the co-expression strains in the cell culture unit to inhibit autonomous lysis of the co-cultured strains; glucose is not added during the expansion of the culture, so that the autonomous lysis circuit is opened, At this time, when the co-cultured strains reach the density of autonomous lysis, autonomous lysis will occur. Specifically, when glucose is added to inhibit autonomous lysis of co-cultured strains, the glucose is in a culture environment (For example, in the culture medium), the final mass volume concentration is 0.5%-2%.

[0062] In some embodiments, the co-production system of the multi-protein system may also include a protein purification unit for purifying the multi-protein system released and collected after the co-expression strain is lysed autonomously to remove excess cell debris and impurities protein. The purification method can use common protein purification methods in the art, for example, recombinant protein purification tags such as His tags can be fused during the construction of protein expression vectors to make the subsequent protein purification process faster and more convenient. As a purification tag for metal chelation affinity chromatography, the His tag is a commonly used tag for protein purification. The protein purification method in the embodiments of the present application can also use any tag suitable for protein purification other than the His tag.

[0063] In the third aspect, the present application provides in some embodiments the application of a multi-protein system co-production system in the synthesis of proteins and/or compounds in vitro.

[0064] The beneficial effects of the application of the multi-protein system co-production system in the in vitro synthesis of proteins and/or compounds provided by the examples of the application are: The protein and/or compound necessary for synthesizing proteins and/or compounds in vitro can be collected in one step, and the synthesis of proteins and/or compounds can be performed in vitro without relying on intact cells. Compared with the existing preparation method of a cell-free protein synthesis system, the co-production system of the multi-protein system is simpler, faster, more efficient and lower in cost.

[0065] The following describes in conjunction with specific embodiments.

Embodiment 1

[0067] (1) 39 kinds of plasmids expressing proteins fused with His tags and plasmid ePop expressing bacteriophage i>X174 E protein were respectively transformed into protein expression strain BL21 (DE3) to obtain 39 kinds of co-expression strains; wherein , 39 protein-expressing plasmids separately express translation initiation factor 1, translation initiation factor 2, translation initiation factor 3, translation elongation factor G, translation elongation factor Tu, translation elongation factor Ts, translation elongation factor 4, translation release Factor 1, translation release factor 2, translation release factor 3, ribosomal cycle factor, methionyl-tRNA formyl transferase, creatine kinase, myokinase and nucleoside diphosphate kinase, pyrophosphatase, T7 RNA polymerase, and 22 kinds of aminoacyl-tRNA synthetase;

(2) Pick single clones from 39 co-expression strains in 4ml LB medium (dual resistance to kanamycin and chloramphenicol), and add 200 μl, 40wt% glucose to inhibit the cells For autonomous lysis, ^{incubate for 14-18 hours in a shaker at 37 °} C and 220 rpm for 14-18 hours; [0069] (3) The co-expression strain obtained in step (2) was inoculated with M9 medium (dual resistance to kanamycin and chloramphenicol) at a volume ratio of 1:100, at 37 ^° C in a shaker at 220 rpm Expand the culture to 0D600=0. 6-0.8, add IPTG with a final concentration of 0.1 mM for induction, continue the culture for 3-5 hours, ^{centrifuge at 4°} C, collect the supernatant, and purify and collect on a nickel column 39 mixed proteins;

[0070] (4) The 39 mixed proteins obtained in step (3) were dialyzed with a 3.5kDa dialysis bag. After the dialyzed protein was concentrated, ribosomes, energy buffer and a DNA linear template expressing the red fluorescent protein mRFP were added Then, react in a metal bath ^{at 37°C for 4 hours.} After the reaction is completed, the reaction system is transferred to Corning Fluorescence Detection 384-well plate, and the fluorescence signal is detected by a microplate reader. Among them, the energy buffer consists of potassium L-glutamate, a mixture of 20 amino acids, HEPES-KOH buffer, spermidine, magnesium acetate, creatine phosphate, dithiothreitol, formyl folate, NTPs, IPTG, tRNA , RNase inhibitor, and sterile water.

[0071] It can be seen from FIG. 2 that under the conditions of excitation at 580 nm and emission at 610 nm, the content of mRFP has a linear relationship with the detected fluorescence signal. The transcription and translation elements, energy regeneration system, substrates, inorganic salts, ribosomes, etc. in the protein synthesis system of the recombination elements were prepared into the reaction mixture. The mRFP DNA linear template was added to the two repeated reactions, and the mRFP DNA was not added to the negative control. Linear template, add the reaction system to the Corning Fluorescence Detection 384-well plate, set the incubation temperature to 37 ^° C, and monitor the fluorescence signal under 580nm excitation and 610nm emission in real time. The fluorescence signal continues to increase during the reaction, indicating 2 repeated reactions In vitro synthesis of mRFP is occurring (Figure 3). The reaction system was added to a 0.2mL PCR tube, one tube was added with mRFP DNA linear template, and the other tube without mRFP DNA linear template was used as a negative control, ^{and reacted in a 37°} C metal bath for 4 hours. After the reaction is complete, The reaction system was transferred to the Corning Fluorescence Detection 384-well plate and detected by a microplate reader. The fluorescence signal was detected as shown in Table 1: (mRFP excitation: 580nm, emission: 610nm) According to the linear relationship between the fluorescence signal and the content of mRFP, It can be calculated that the in vitro synthesis system can synthesize mRFP protein 1.4ng/μL after reacting ^{at 37°C for 4 hours.}

[0072] Table 1 End-point fluorescence detection results of synthetic red fluorescent protein mRFP [] [Table 1]

[0073] The above are only optional embodiments of the application, and are not used to limit the application. For those skilled in the art, this application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of this application shall be included in the scope of the claims of this application.

Claims

[Claim 1] A method for co-production of a multi-protein system, characterized in that it comprises the following steps: providing a vector for expressing lysoprotein, a vector for expressing multiple proteins, and a protein expression strain, each of the vectors for expressing the protein is provided separately Expressing a protein in the polyprotein system, and the proteins expressed by different vectors expressing the protein are different; the vectors expressing the lysoprotein and the vectors expressing each of the proteins are co-transformed into Among the protein expression strains, multiple co-expression strains are obtained; all of the co-expression strains are co-cultured, and autonomous lysis occurs when the growth density of the co-expression strain reaches the autonomous lysis density, and the respective expressed proteins are released To obtain the multi-protein system; wherein, the multi-protein system includes proteins used for protein translation, energy regeneration, and/or enzymes that catalyze the synthesis of compounds.

[Claim 2] The method for co-production of a multi-protein system according to claim 1, wherein the multi-protein system is selected from the group consisting of polyketide synthetase system, non-ribosomal polypeptide synthetase system, and recombinant element protein synthesis system At least one of them.

[Claim 3] The method for co-production of a multi-protein system according to claim 2, wherein the recombination element protein system includes translation initiation factor 1, translation initiation factor 2, translation initiation factor 3, and translation extension Factor G, Translation Elongation Factor Tu, Translation Elongation Factor Ts, Translation Elongation Factor 4, Translation Release Factor 1, Translation Release Factor 2, Translation Release Factor 3, Ribosome Circulation Factor, Methionyl-tRNA Formyltransferase, Creatine Kinase, myokinase, nucleoside diphosphate kinase, pyrophosphatase and 22 aminoacyl-tRNA synthetases.

[Claim 4] The method for co-production of a multi-protein system according to claim 3, wherein the recombination element protein synthesis system further comprises at least 17 RNA polymerase, disulfide isomerase, and molecular chaperone protein A sort of.

[Claim 5] The method for co-production of a multi-protein system according to claim 1, wherein the vector expressing the lysoprotein is a plasmid ePop that ^{expresses the bacteriophage 4) X174 E protein.}

[Claim 6] The method for co-production of a multi-protein system according to any one of claims 1 to 5, wherein the protein expression strain is Escherichia coli, Escherichia coli mutant strain or Escherichia coli-derived Bacteria.

[Claim 7] The method for co-production of a multi-protein system according to any one of claims 1-5, characterized in that the growth density of the co-expression strain undergoing autonomous lysis is an OD value greater than or equal to 0.05.

[Claim 8] The method for co-production of a multi-protein system according to any one of claims 1-5, characterized in that, before co-cultivating the multiple co-expression strains, the co-expression strains are separately A separate culture is performed to activate the co-expression strain.

[Claim 9] The method for co-production of a multi-protein system according to any one of claim 8, characterized in that, when the co-expression strains are separately cultured, a final mass volume concentration of 0.5%- is added. 2% glucose.

[Claim 10] A co-production system for a multi-protein system, characterized in that it comprises: a co-expression strain preparation unit for preparing multiple co-expression strains, each of the co-expression strains expressing in the multi-protein system The protein and bacteriolytic protein expressed by different co-expression strains are different from each other; a cell culture unit for cultivating co-expression strains in the co-expression strain unit; wherein, the multi-protein system includes Proteins used for protein translation, energy regeneration, and/or enzymes that catalyze the synthesis of compounds.

[Claim 11] The co-production system of the multi-protein system according to claim 10, wherein the multi-protein system is selected from the group consisting of a polyketide synthase system, a non-ribosomal polypeptide synthetase system, and a recombinant element protein system At least one of.

[Claim 12] The multi-protein system co-production system according to claim 11, wherein the recombination element protein system includes translation initiation factor 1, translation initiation factor 2, translation initiation factor 3, and translation extension Factor G, Translation Elongation Factor Tu, Translation Elongation Factor Ts, Translation Elongation Factor 4, Translation Release Factor 1, Translation Release Factor 2, Translation Release Factor 3, Ribosome Circulation Factor, Methionyl-tRNA Formyltransferase, Creatine Kinase, myokinase, nucleoside diphosphate kinase, pyrophosphatase and 22 aminoacyl-tRNA synthetases.

[Claim 13] The co-production system of the multi-protein system according to claim 12, wherein the recombination element protein system further comprises at least one of RNA polymerase, disulfide isomerase, and molecular chaperone protein. kind. [Claim 14] The co-production system of a multi-protein system according to any one of claims 10-13, wherein the bacteriolytic protein is bacteriophage 4 ⁾ X174 E protein.

[Claim 15] The co-production system for a multi-protein system according to any one of claims 10-13, wherein the co-expression strain is Escherichia coli, Escherichia coli mutant or Escherichia coli-derived bacteria.

[Claim 16] The application of the multi-protein system co-production system of any one of claims 10-13 in the synthesis of proteins and/or compounds in vitro.