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WO2021237410A1 - Procédé de co-production de système multiprotéique, système de co-production pour système multiprotéique et son utilisation - Google Patents

Procédé de co-production de système multiprotéique, système de co-production pour système multiprotéique et son utilisation Download PDF

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Publication number
WO2021237410A1
WO2021237410A1 PCT/CN2020/092110 CN2020092110W WO2021237410A1 WO 2021237410 A1 WO2021237410 A1 WO 2021237410A1 CN 2020092110 W CN2020092110 W CN 2020092110W WO 2021237410 A1 WO2021237410 A1 WO 2021237410A1
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protein
translation
production
expression
factor
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Chinese (zh)
Inventor
戴卓君
李鹏程
张曦
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Shenzhen Institute of Advanced Technology of CAS
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Shenzhen Institute of Advanced Technology of CAS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • Multi-protein system symbiosis production method multi-protein system co-production system and application technology field
  • 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
  • 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".
  • 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.
  • CFPS cell-free protein synthesis
  • the cell-free protein synthesis system is a biological technology that does not rely on intact cells for protein synthesis in vitro.
  • DNA or mRNA 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.
  • 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.
  • 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).
  • PURE protein synthesis using recombinant elements
  • the concentration is controllable, so it has many significant advantages:
  • 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.
  • 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
  • a method for co-production of a multi-protein system which includes the following steps:
  • 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;
  • 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;
  • 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;
  • the multi-protein system includes proteins used for protein translation, energy regeneration, and/or enzymes that catalyze the synthesis of compounds.
  • a multi-protein system co-production system which includes:
  • 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;
  • Cell culture unit used to cultivate co-expression strains in the co-expression strain unit;
  • the multi-protein system includes proteins used for protein translation, energy regeneration, and/or enzymes that catalyze the synthesis of compounds.
  • the beneficial effects of the co-production method of the multi-protein system 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.
  • the multi-protein system co-production system includes a co-expression strain preparation unit and a cell culture unit.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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;
  • 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;
  • FIG. 3 is a specific embodiment provided in this application, the real-time fluorescent signal monitoring result of the synthetic red fluorescent protein mRFP.
  • 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.
  • 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.
  • vector includes plasmids, bacteriophages, viruses or other vectors.
  • the embodiments of the present application provide a co-production method of a multi-protein system, which includes the following steps:
  • 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
  • the multi-protein system includes proteins used for protein translation, energy regeneration, and/or enzymes that catalyze the synthesis of compounds.
  • the beneficial effects of the co-production method of the multi-protein system 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 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.
  • 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.
  • the co-expression strain 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.
  • the plasmid ePop expressing the phage is selected as the vector for expressing the lysoprotein.
  • 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.
  • the gene circuit is one This kind of adjustable circuit can adjust the corresponding module according to the function that needs to be performed.
  • the above example of selecting the plasmid ePop expressing the phage 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.
  • E. coli strains are selected as protein expression strains.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the timing of autonomous lysis of the co-expression strain can be controlled.
  • the vector expressing the bacteriolytic protein is the expression bacteriophage X174
  • 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.
  • the OD value can be determined at regular intervals.
  • 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.
  • 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%.
  • the multi-protein system includes proteins that synthesize in vitro and/or catalyze the synthesis of compounds.
  • a multi-protein system composed of proteins for protein translation and energy regeneration is the key to achieving protein synthesis in vitro.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the selection 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
  • translation release factor 1 translational release factor 1, RF1
  • translation release factor 2 translation release factor
  • 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
  • the present application provides a multi-protein system co-production system in some embodiments, which includes:
  • 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;
  • cell culture unit for culturing co-expression strains in the co-expression strain unit
  • the multi-protein system includes proteins used for protein translation, energy regeneration, and/or enzymes that catalyze the synthesis of compounds.
  • 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.
  • each co-expression strain does not repeatedly express one protein and bacteriolytic protein in the multi-protein system.
  • 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.
  • 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.
  • the co-expressing strain can further promote The copy number of the plasmid increases, which in turn promotes the expression of E protein.
  • the co-expression can also be adjusted The effect of the degree of spontaneous lysis of the strain.
  • 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.
  • 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.
  • translation initiation factor 1 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.
  • the DNA encoding the target protein when 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.
  • 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.
  • 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.
  • 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.
  • the glucose is in a culture environment (For example, in the culture medium), the final mass volume concentration is 0.5%-2%.
  • 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.
  • 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.
  • 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.
  • the beneficial effects of the application of the multi-protein system co-production system in the in vitro synthesis of proteins and/or compounds 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.
  • the co-production system of the multi-protein system is simpler, faster, more efficient and lower in cost.
  • step (3) 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.
  • 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.
  • 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/ ⁇ L after reacting at 37°C for 4 hours.

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Abstract

L'invention concerne un procédé de coproduction pour un système multiprotéique, un système de co-production pour un système multiprotéique et son utilisation. En particulier, un vecteur pour exprimer une protéine bactériolytique et une pluralité de vecteurs pour exprimer des protéines sont co-transférés dans une souche d'expression de protéine, de telle sorte que la souche de co-expression obtenue exprime des protéines correspondantes et une protéine bactériolytique. Lors de la croissance de la souche de co-expression, la protéine bactériolytique générée agit sur la souche de co-expression, de sorte que la souche de co-expression est soumise à une lyse autonome, et les protéines exprimées sont libérées, formant ainsi un système multiprotéique qui peut être utilisé pour la synthèse in vitro de protéines et/ou de composés.
PCT/CN2020/092110 2020-05-25 2020-05-25 Procédé de co-production de système multiprotéique, système de co-production pour système multiprotéique et son utilisation Ceased WO2021237410A1 (fr)

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