WO2018171747A1 - Système de synthèse in vitro d'adn en protéine (d2p), préparation, kit de réactif et procédé de préparation - Google Patents

Système de synthèse in vitro d'adn en protéine (d2p), préparation, kit de réactif et procédé de préparation Download PDF

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WO2018171747A1
WO2018171747A1 PCT/CN2018/080322 CN2018080322W WO2018171747A1 WO 2018171747 A1 WO2018171747 A1 WO 2018171747A1 CN 2018080322 W CN2018080322 W CN 2018080322W WO 2018171747 A1 WO2018171747 A1 WO 2018171747A1
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dna
protein
cell
synthesis
vitro
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Chinese (zh)
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郭敏
章小铃
王海鹏
王静
徐开
陈秋锦
于雪
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康码（上海）生物科技有限公司
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    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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

Definitions

  • the invention relates to the field of biotechnology, in particular to an in vitro DNA-to-Protein (D2P) synthesis system, a preparation, a kit and a preparation method.
  • D2P DNA-to-Protein
  • a traditional biosynthetic system refers to a molecular biology technique that expresses a foreign gene through a model organism, a fungus, a plant cell, or an animal cell.
  • cell-free expression systems also known as in vitro protein synthesis systems
  • In vitro protein synthesis system refers to the exogenous target mRNA or DNA as a template, and the synthesis of the target protein can be achieved by artificially controlling the substrate required for protein synthesis, transcription and translation related protein factors.
  • the in vitro protein synthesis system expresses proteins without a plasmid construction, transformation, cell culture, cell collection and fragmentation steps, and is a relatively fast, time-saving and convenient protein expression.
  • the present invention provides a theoretical model and design for simultaneous DNA, RNA, and protein biosynthesis in vitro.
  • the invention provides an in vitro synthesis system which is simple, convenient, high efficiency, high yield and low cost.
  • the invention provides a method for synthesizing DNA, RNA and protein in vitro by using a DNA template.
  • the invention provides a method for establishing and optimizing protein synthesis in vitro by using a trace DNA template to overcome the defects and deficiencies of the prior art.
  • the first object of the present invention is to theoretically construct an in vitro synthesis system for DNA replication, transcription and translation; the second object of the present invention is to establish and optimize an in vitro synthesis system for DNA replication, transcription and translation;
  • a third object of the invention is to provide a simple and feasible in vitro biosynthetic preparation for coupling DNA-to-Protein (D2P).
  • a fourth object of the present invention is to provide a simple, high-yield in vitro protein synthesis kit.
  • a first aspect of the invention provides for the establishment of a theory of a cell-free synthesis system in vitro, the cell-free synthesis system comprising:
  • a second aspect of the invention provides an in vitro synthesis system for coupling DNA replication, transcription and translation, the cell-free synthesis system comprising:
  • the cell-free protein synthesis system further comprises a component selected from the group consisting of:
  • the DNA polymerase comprises: phi29 DNA polymerase, T7 DNA polymerase, Bst DNA polymerase, Ecoli DNA polymerase, Klenow fragment of DNA polymerase I, etc. for isothermal amplification polymerization. Enzymes and mutants are not limited to this.
  • the helicase comprises: a helicase (4B), a UvrD helicase, and the like of the T7 bacteriophage replication system.
  • the DNA binding protein comprises: T4 phage gene 32 protein, RB49 phage gene 32 protein, single chain binding protein of T7 phage replication system, DNA binding protein 7 and the like.
  • the substrate for the synthetic DNA includes: deoxynucleoside monophosphate, deoxynucleoside triphosphate, or a combination thereof.
  • the substrate for the synthetic RNA includes: nucleoside monophosphate, nucleoside triphosphate, or a combination thereof.
  • the substrate of the synthetic protein comprises: 1-20 natural amino acids, and unnatural amino acids.
  • the cell-free protein synthesis system further comprises an exogenous DNA molecule for directing protein synthesis.
  • the DNA molecule is linear.
  • the DNA molecule is cyclic.
  • the DNA molecule contains a sequence encoding a foreign protein.
  • the sequence encoding the foreign protein comprises a genomic sequence, a cDNA sequence.
  • sequence encoding the foreign protein further comprises a promoter sequence, a 5' untranslated sequence, and a 3' untranslated sequence.
  • the cell-free protein synthesis system comprises a component selected from the group consisting of polyethylene glycol, sucrose, 4-hydroxyethylpiperazine ethanesulfonic acid, potassium acetate, magnesium acetate, and nucleoside III. Phosphoric acid, amino acids, creatine phosphate, dithiothreitol (DTT), phosphocreatine kinase, T7 RNA polymerase, or a combination thereof.
  • a third aspect of the invention provides a simple and feasible in vitro biosynthetic preparation coupled to DNA-to-Protein (D2P), comprising:
  • the DNA replication system can also be incubated with T1 and then combined with cell extracts and cell-free synthesis systems to synthesize DNA, RNA and protein.
  • the reaction temperature is 20-30 ° C and the reaction time is 2-12 h.
  • the reaction temperature is 25 to 65 °C.
  • the T1 reaction time is 2-6 h.
  • a fourth aspect of the invention provides a kit for in vitro cell-free synthesis of a protein comprising:
  • (k2) a second container, and a DNA polymerase, a helicase, a DNA binding protein located in the second container;
  • first container, the second container, and the third container are the same container or different containers.
  • the kit further comprises an optional one or more containers selected from the group consisting of:
  • a fifth aspect of the invention provides a cell-free synthesis system that couples or integrates DNA replication, RNA transcription and protein translation into a synthetic system.
  • the synthetic system comprises:
  • the cell-free synthesis system further comprises:
  • the cell-free synthesis system further comprises:
  • the cell-free synthesis system further comprises an optional template DNA.
  • the (i) DNA polymerization system comprises: (a) a DNA polymerase; (b) an optional helicase; (c) an optional DNA binding protein; and (d) A substrate for the synthesis of DNA.
  • the (ii) RNA transcription system comprises: (e) an RNA polymerase; and (f) a substrate for synthesizing RNA.
  • the (iii) protein translation system comprises: (g) a substrate for synthesizing a protein; and (h) a cell extract.
  • the cell extract of the cell extract is selected from the group consisting of one or more types of cells: prokaryotic cells and eukaryotic cells.
  • the cell extract is obtained from a cell source selected from the group consisting of one or more types of cells: Escherichia coli, bacteria, mammalian cells (eg, HF9, Hela, CHO, HEK293), plant cells , yeast cells, or a combination thereof.
  • a cell source selected from the group consisting of one or more types of cells: Escherichia coli, bacteria, mammalian cells (eg, HF9, Hela, CHO, HEK293), plant cells , yeast cells, or a combination thereof.
  • the cell extract comprises a yeast cell extract.
  • the yeast cell is selected from the group consisting of Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces, or a combination thereof; preferably, the yeast cell comprises: Kluyveromyces, preferably The ground is Kluyveromyces lactis.
  • the yeast cell extract is an aqueous extract of yeast cells.
  • the yeast cell extract is free of yeast endogenous long chain nucleic acid molecules.
  • the magnesium ion is derived from a source of magnesium ions selected from the group consisting of magnesium acetate, magnesium glutamate, or a combination thereof.
  • the potassium ion is derived from a source of potassium ions selected from the group consisting of potassium acetate, potassium glutamate, or a combination thereof.
  • the buffering agent is selected from the group consisting of 4-hydroxyethylpiperazineethanesulfonic acid, trishydroxymethylaminomethane, or a combination thereof.
  • the substrate for the synthetic RNA comprises: a nucleoside monophosphate, a nucleoside triphosphate, or a combination thereof.
  • the substrate of the synthetic protein comprises: 1-20 natural amino acids, and unnatural amino acids.
  • the synthetic system is a three-in-one synthetic system of DNA replication, RNA transcription, and protein translation.
  • the amount of the template DNA in the DNA polymerization system is ⁇ 1 ng, preferably ⁇ 0.1 ng, more preferably ⁇ 0.01 ng.
  • the template DNA is used in an amount of 0.0001 to 10 ng, preferably 0.001 to 1 ng, more preferably 0.001 to 0.1 ng, in the DNA polymerization system.
  • the template DNA is circular DNA.
  • the template DNA is plasmid DNA.
  • the plasmid DNA comprises tandem DNA elements capable of enhancing protein synthesis efficiency.
  • the plasmid DNA comprises the following elements: a yeast cell-derived IRES enhancer K1NCE102, an ⁇ sequence, and a yeast-specific Kozak sequence.
  • the synthetic system has a volume of from 10 to 50 microliters, preferably from 20 to 40 microliters.
  • the polymerase is selected from the group consisting of phi29 DNA polymerase, T7 DNA polymerase, Bst DNA polymerase, E. coli DNA polymerase, and DNA polymerase I. Klenow, or a combination thereof.
  • the polymerase in the DNA polymerization system, is phi29 DNA polymerase.
  • the concentration of the polymerase in the DNA polymerization system is 0.0005 to 0.5 mg/mL, preferably 0.01 to 0.2 mg/mL, more preferably 0.05 to 0.1 mg/mL. .
  • the DNA replication, RNA transcription, and protein translation do not include the step of removing unnecessary proteins from the synthetic system.
  • the method does not include the step of removing unnecessary proteins (such as DNase, RNA polymerase) from the synthetic system.
  • unnecessary proteins such as DNase, RNA polymerase
  • the polyethylene glycol is selected from the group consisting of PEG3000, PEG 8000, PEG 6000, PEG 3350, or a combination thereof.
  • the polyethylene glycol comprises polyethylene glycol having a molecular weight (Da) of from 200 to 10,000, preferably polyethylene glycol having a molecular weight of from 3,000 to 10,000.
  • the concentration of the polyethylene glycol (w/v, for example, g/ml) in the protein synthesis system is 0.1 to 8%, preferably 0.5 to 4%, more preferably, 1 -2%.
  • the energy regeneration system is selected from the group consisting of a phosphocreatine/phosphocreatase system, a glycolysis pathway and its intermediate energy system, or a combination thereof.
  • the saccharide is selected from the group consisting of glucose, starch, glycogen, sucrose, maltose, cyclodextrin, or a combination thereof.
  • the concentration of the saccharide is 10-100 mM, preferably 10-60 mM, preferably 20-50 mM, more preferably 20-30 mM.
  • the content of the saccharide (V/V) is from 1 to 10%, preferably from 3 to 8%, more preferably from 4 to 6%, based on the total amount of the cell-free synthesis system. Volume meter.
  • the phosphate compound is selected from the group consisting of potassium phosphate, magnesium phosphate, ammonium phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, or a combination thereof.
  • the concentration (v/v) of the phosphate compound is from 1 to 6%, preferably from 2 to 5%, more preferably from 2 to 3%, based on the total volume of the cell-free synthesis system. meter.
  • the concentration of the phosphate compound is 10-60 mM, preferably 20-50 mM, more preferably 20-30 mM.
  • a sixth aspect of the invention provides a method for cell-free synthesis of proteins in vitro, comprising the steps of:
  • a mixed system comprising the cell-free synthesis system of the fifth aspect of the invention and an exogenous template DNA for directing protein synthesis, the cell-free synthesis system comprising a DNA replication system and transcriptional, translational coupling a cell-free synthesis system, under suitable conditions, incubating the DNA replication system for a period of time T1, combining a transcriptionally, translationally coupled cell-free synthesis system with the DNA replication system, and incubated to synthesize the foreign DNA Encoded protein.
  • a seventh aspect of the invention provides a method for cell-free synthesis of proteins in vitro, comprising the steps of:
  • the template DNA is circular DNA.
  • the template DNA is plasmid DNA.
  • the method further comprises: (b) isolating or detecting the protein encoded by the template, optionally from the protein synthesis system.
  • the reaction temperature is 20 to 37 ° C, preferably 20 to 25 ° C.
  • the reaction time is 6-24 h, preferably 8-16 h.
  • the template DNA is used in an amount of ⁇ 1 ng, preferably ⁇ 0.1 ng, more preferably ⁇ 0.01 ng.
  • the template DNA is used in an amount of 0.0001 to 10 ng, preferably 0.001 to 1 ng, more preferably 0.001 to 0.1 ng.
  • An eighth aspect of the invention provides a formulation for cell-free synthesis in vitro comprising:
  • the preparation is a mixed solution or a lyophilized powder.
  • the auxiliary agent is selected from the group consisting of:
  • the auxiliary reagent further comprises:
  • a ninth aspect of the invention provides a kit for in vitro cell-free synthesis comprising:
  • the kit further comprises an optional one or more containers selected from the group consisting of:
  • a tenth aspect of the invention provides a method of in vitro coupling of synthetic DNA, mRNA and protein, comprising the steps of:
  • an in vitro DNA replication system comprising a DNA polymerase, an exogenous DNA molecule for directing protein synthesis, a helicase, a DNA binding protein, a substrate for synthesizing DNA;
  • the incubation step (i) is carried out via T1 and then combined with a transcription- and translation-coupled cell-free synthesis system to synthesize DNA, mRNA and protein encoded by the foreign DNA;
  • An eleventh aspect of the present invention provides a method of in vitro coupling of synthetic DNA, mRNA and protein, comprising the steps of:
  • DNA molecule a DNA molecule, a helicase, a DNA binding protein for directing protein synthesis
  • the incubation step (i) is carried out via T1 and then combined with a transcription- and translation-coupled cell-free synthesis system to synthesize DNA, mRNA and protein encoded by the foreign DNA;
  • the foreign DNA can be directly encoded to synthesize the protein under suitable conditions without the need for an incubation step.
  • Figure 1 is a schematic diagram of a synthetic system of DNA-to-Protein (D2P) in vitro. The central principle of DNA replication, transcription, and translation.
  • D2P DNA-to-Protein
  • Figure 2 is a graphical representation of the effect of different volumes of DNA on the in vitro protein synthesis system in the phi29 DNA polymerase replication system.
  • the reaction temperature of phi29 DNA polymerase was 30 ° C, the reaction time was 6-16 h, and the plasmid containing the luciferase gene was 1 ng.
  • NC is a negative control with no in vitro synthesis of DNA.
  • PC is a positive control and DNA is derived from a PCR reaction of approximately 500 ng.
  • 0.5-3 microliters of phi29 DNA polymerase-replicated DNA can be used as a cell-free expression system for in vitro transcription and translation coupling.
  • Figure 3 is a graphical representation of the effect of DNA at different reaction times on the in vitro protein synthesis system in the phi29 DNA polymerase replication system.
  • the reaction temperature of phi29 DNA polymerase was 30 ° C
  • the reaction time was 1-28 days
  • the plasmid containing the luciferase gene was 1 ng.
  • NC is a negative control with no in vitro synthesis of DNA.
  • PC is a positive control and DNA is derived from a PCR reaction of approximately 500 ng.
  • 1-28 days of phi29 DNA polymerase-replicated DNA can still be used as a cell-free expression system for in vitro transcription and translation coupling.
  • Figure 4 is a graphical representation of the effect of different concentrations of phi29 DNA polymerase amplified DNA on in vitro protein synthesis systems in a replication system.
  • the reaction temperature of phi29 DNA polymerase was 30 ° C
  • the reaction time was 6-16 h
  • the plasmid containing eGFP gene was 1 ng
  • the reaction concentration of phi29 polymerase was 0.5 mg/mL-0.8 ug/mL.
  • NC is a negative control with no in vitro synthesis of DNA.
  • PC is a positive control and DNA is derived from a PCR reaction of approximately 500 ng.
  • the DNA replicated by phi29 DNA polymerase at 0.8 mg/mL to 0.4 ug/mL can be used as a cell-free expression system for in vitro transcription and translation coupling.
  • Figure 5 is a schematic diagram showing the effect of DNA template synthesized at different time points of phi29 DNA amplification system on in vitro protein synthesis system.
  • the reaction temperature of phi29 DNA polymerase was 20-30 ° C, the reaction time was 0-24 h, and the plasmid containing the eGFP gene was 1 ng.
  • NC is a negative control with no in vitro synthesis of DNA.
  • PC is a positive control and DNA is derived from a PCR reaction of approximately 500 ng. It can be seen from the figure that when the DNA amplification reaction proceeds to 6 h, the amplified DNA guides the amount of synthesized eGFP to the highest value and enters the platform.
  • Figure 6 is a graphical representation of the analysis of the amount of DNA synthesized at different time points in the phi29 DNA amplification system of Figure 5 using agarose gel. It can be seen from the figure that when the DNA amplification reaction is carried out until 6 hours, the amount of DNA amplified reaches a maximum value, and enters the platform with time, and there is no significant increase.
  • Figure 7 is a diagram showing the detection of eGFP synthesized by IVDTT using Western Blot.
  • Lane 1 is the IVDTT system to which the plasmid containing the eGFP coding sequence was added
  • Lane 2 is the IVDTT system (negative control) to which no DNA template was added.
  • Western Blot used the primary antibody of eGFP protein, and the molecular weight of the lane 1 band was very close to the theoretical molecular weight (26.7KDa), indicating that the synthetic target protein is the correct eGFP protein.
  • Figure 8 is a diagram showing the detection of IVDTT synthesized eGFP protein using a fluorescent SDS-PAGE imaging method.
  • Lane 1 is the IVDTT system to which the plasmid containing the eGFP coding sequence was added
  • Lane 2 is the IVDTT system (negative control) to which no DNA template was added.
  • the fluorophore in the eGFP protein is also capable of being excited and fluorescing in the event of incomplete denaturation.
  • the molecular weight of the fluorescent bands detected in lane 1 is very close to the theoretical molecular weight of eGFP (26.7 KDa), indicating that the synthesized eGFP protein is capable of being excited and fluorescing.
  • Figure 9 is the relative fluorescence value (RFU) of the fusion protein Ubiquitin-eGFP and eGFP alone synthesized using the IVDTT system.
  • the in vitro synthesis system was incubated for 3 h (blank histogram) and 20 h (shaded histogram).
  • the relative fluorescence value (RFU) emitted by eGFP and eGFP alone in the post-synthesized fusion protein, and the synthesized eGFP alone as a positive control for the IVDTT system.
  • the relative fluorescence value of the synthesized Ubiquitin-eGFP was significantly higher than that of the negative control (NC), indicating that the fusion protein Ubiquitin-eGFP was synthesized by the IVDTT system.
  • NC negative control
  • Figure 10 is the relative fluorescence of the fusion protein p53-eGFP and eGFP alone synthesized using the IVDTT system.
  • the in vitro synthesis system was incubated for 3 h (blank histogram) and 20 h (shaded histogram).
  • the relative fluorescence value of the synthesized p53-eGFP was significantly higher than that of the negative control, indicating that the fusion protein p53-eGFP was synthesized by the IVDTT system.
  • Figure 11 is a relative fluorescence value of the fusion protein ⁇ 2AR-eGFP synthesized by the IVDTT system and eGFP alone, and the synthesis of the in vitro synthesis system was measured after incubation for 3 h (blank histogram) and 20 h (shaded histogram).
  • the relative fluorescence value of the synthesized ⁇ 2AR-eGFP was significantly higher than that of the negative control, indicating that the fusion protein ⁇ 2AR-eGFP was synthesized by the IVDTT system.
  • Figure 12 is the relative fluorescence of the fusion protein AQP1-eGFP and eGFP alone synthesized using the IVDTT system.
  • the in vitro synthesis system was incubated for 3 h (blank histogram) and 20 h (shaded histogram).
  • the relative fluorescence value of the synthesized AQP1-eGFP was significantly higher than that of the negative control, indicating that the fusion protein AQP1-eGFP was synthesized by the IVDTT system.
  • Figure 13 is a relative fluorescence value of the fusion protein IFN- ⁇ 2A-eGFP synthesized by the IVDTT system and eGFP alone, and the in vitro synthesis system was incubated for 3 h (blank histogram) and 20 h (shaded histogram).
  • the relative fluorescence value of the synthesized IFN- ⁇ 2A-eGFP was significantly higher than that of the negative control, indicating that the fusion protein IFN- ⁇ 2A-eGFP was synthesized by the IVDTT system.
  • Figure 14 and Figure 15 show the relative fluorescence values of the fusion proteins ate-H-eGFP, ate-L-eGFP and eGFP alone synthesized using the IVDTT system.
  • the in vitro synthesis system was incubated for 3 h (blank histogram).
  • the relative fluorescence values of the synthesized ate-H-eGFP and ate-L-eGFP were significantly higher than those of the negative control, indicating that the fusion proteins ate-H-eGFP and ate-L-eGFP were synthesized by the IVDTT system.
  • Figure 16 is a diagram showing the advantages of the in vitro DNA-to-Protein (D2P) synthesis system.
  • D2P DNA-to-Protein
  • the D2P system can use a very small amount of template (the amount can be reduced by 2-4 orders of magnitude or more), which can effectively reduce the cost, simultaneously and efficiently synthesize DNA, RNA and protein at the same time, greatly reducing the complexity of the use of cell-free synthesis systems. Sex and cost.
  • the D2P in vitro cell-free expression system provided by the present invention can synthesize specific proteins continuously, simply, and efficiently using a very small amount (nock-microgram) of DNA.
  • the relative light unit value of the synthesized luciferase activity can be as high as about 60 times that of the current commercial system (such as the rabbit reticulocyte in vitro expression system), saving the user.
  • D2P DNA-to-Protein
  • the “Center Law” is the basic principle of the occurrence and development of living things on the earth. It is the process by which genetic information is transmitted from DNA to DNA, that is, the process of DNA replication, and from DNA to RNA, and from RNA to protein, that is, the transcription and translation of genetic information. This is the core rule followed by all organisms with cellular structures. The advancement of modern biology is largely based on the understanding and application of this law. Such as nucleic acid amplification technology (PCR), molecular cloning, genomic engineering, cell signal regulation, neural networks, disease principles and treatment, and expression of recombinant proteins, and so on. In the past 20 years, with the development of gene sequencing, omics, computer and Internet technology, biological research has also made a lot of revolutionary progress.
  • PCR nucleic acid amplification technology
  • protein synthesis methods can be divided into two types: traditional cellular protein synthesis and in vitro cell-free protein synthesis.
  • Traditional protein synthesis methods originated in the 1970s and refer to a molecular biology technique that expresses foreign genes through model organisms such as bacteria, fungi, plant cells or animal cells [1-2].
  • cell-free expression systems also known as in vitro protein synthesis systems, emerged in the 1990s [3-5], which is an exogenous target mRNA or DNA as a protein synthesis template, supplemented by artificial control of protein synthesis.
  • the desired substrate and transcription and translation related protein factors can achieve the synthesis of the target protein.
  • Protein expression in an in vitro translation system requires a plasmid construction, transformation, cell culture, cell collection and fragmentation steps, and is a relatively fast, time-saving, and convenient way to express proteins.
  • RNA DNA
  • RNA protein
  • the rate of biosynthesis of protein 0.20 pg was: 34 ⁇ g/mL/hour DNA, 200 ⁇ g/mL/hour RNA, 400 ⁇ g/mL/hour protein [6-8].
  • Eukaryotic yeast cells (Saccharomyces cerevisiae, Sc), according to their 90-fold replication rate, the rate of biosynthesis is 73 ml (cell volume, 79 pg, dry weight 40 pg, DNA 0.06 pg, RNA 4 pg, protein 20 pg). : 0.55 ⁇ g/mL/hour DNA, 36 ⁇ g/mL/hour RNA, 182 ⁇ g/mL/hour protein [9-12]. No matter the speed of synthetic protein, or the amplification ratio from DNA to RNA to Protein, it is much larger than the existing in vitro protein synthesis system.
  • the core foundation of these manufacturing processes is the "central rule" of DNA-RNA-Protein. Each of these steps is required and cannot be missed.
  • the most basic for achieving high-efficiency biosynthesis is the self-replication of DNA in the first step.
  • RNA: Protein 1:64:330
  • D2P DNA-to-Protein
  • Implementations of D2P technology include: nucleic acid amplification technology, RNA polymerization technology, and in vitro protein translation technology.
  • Nucleic acid amplification techniques include non-isothermal amplification techniques and isothermal amplification techniques.
  • Polymerase chain reaction is a typical representative of nucleic acid amplification technology.
  • PCR technology is dependent on temperature cycling, and often requires higher temperature denaturation of DNA template and amplification and extension of newly synthesized DNA molecules. High temperature can cause denaturation of protein factors in in vitro synthesis systems, so it is not suitable for use. In vitro synthesis system.
  • isothermal amplification of nucleic acids is characterized by amplification of nucleic acids under specific, relatively mild temperature conditions, thereby allowing DNA replication, mRNA transcription, and protein synthesis to be coupled in vitro.
  • DNA polymerases for isothermal amplification of nucleic acids including phi29 DNA polymerase and T7 DNA polymerase, have great advantages in temperature and amplification efficiency, so that it is not necessary to prepare a large amount of DNA molecules in advance, and only a small amount is needed.
  • the DNA template can be used to synthesize proteins in vitro.
  • T7 DNA polymerase is not pure wild type or mutant type, and will carry out the fidelity, synthesis efficiency and extension ability of DNA polymerase according to the needs of the in vitro synthesis system.
  • the T7 RNA polymerase used in the D2P in vitro synthesis system has high specificity and high transcription efficiency, and can rapidly and efficiently transcribe a large amount of mRNA molecules from a DNA template. T7 RNA polymerase combined with a highly efficient in vitro protein translation system further reduces the amount of DNA molecules required.
  • D2P DNA-to-Protein
  • the in vitro synthesis system comprises: a cell extract, 4-hydroxyethylpiperazineethanesulfonic acid, potassium acetate, magnesium acetate, adenosine triphosphate (ATP), and guanosine tris Phosphoric acid (GTP), cytosine triphosphate (CTP), thymidine triphosphate (TTP), amino acid mixture, creatine phosphate, dithiothreitol (DTT), phosphocreatine kinase, RNase inhibitor , RNA polymerase, spermidine, heme, DNA polymerase, helicase, DNA binding protein, and the like.
  • the RNA polymerase is not particularly limited and may be selected from one or more RNA polymerases, and a typical RNA polymerase is T7 RNA polymerase.
  • the DNA polymerase is not particularly limited, and can be used for a thermostatically amplified polymerase, and can be selected from one or more DNA polymerases, and a typical DNA polymerase is phi29 DNA polymerase, T7 DNA polymerase, Bst DNA polymerase or the like is not limited thereto.
  • the helicase may be selected from one or more, and a typical helicase is a helicase (4B), a UvrD helicase or the like of the T7 phage replication system, and is not limited thereto.
  • the DNA binding protein may be selected from one or more of: T4 phage gene 32 protein, RB49 phage gene 32 protein, T7 phage replication system single-stranded binding protein, DNA binding protein 7, etc., not limited thereto this.
  • D2P DNA-to-Protein
  • the present invention provides a cell-free synthetic system preparation for in vitro coupled DNA replication, transcription and translation, the formulation comprising:
  • Cell-free synthesis systems include: 4-hydroxyethylpiperazineethanesulfonic acid, potassium acetate, magnesium acetate, amino acid mixtures, creatine phosphate, dithiothreitol (DTT), phosphocreatine kinase, RNase inhibitors, poly A reactant such as ethylene glycol.
  • the DNA replication system can also be combined with cell extracts, DNA transcription systems and cell-free synthesis systems for an incubation time T1 to synthesize the DNA, RNA and protein.
  • the temperature of the DNA replication reaction system is 25-65 ° C, and the reaction time of T1 is 2-6 h.
  • D2P DNA-to-Protein
  • the invention provides a DNA-to-Protein (D2P) coupled kit for in vitro cell-free synthesis, comprising:
  • the first container, the second container and the third container are the same container or different containers.
  • a particularly preferred kit for in vitro protein synthesis comprises an in vitro synthesis system comprising: a cell extract, 4-hydroxyethylpiperazineethanesulfonic acid, potassium acetate, magnesium acetate, adenosine triphosphate (ATP), Guanine nucleoside triphosphate (GTP), cytosine triphosphate (CTP), thymidine triphosphate (TTP), amino acid mixture, creatine phosphate, dithiothreitol (DTT), phosphocreatine kinase , RNase inhibitor, T7 RNA polymerase, spermidine, heme, DNA polymerase, RNA polymerase, helicase, DNA binding protein.
  • ATP adenosine triphosphate
  • GTP Guanine nucleoside triphosphate
  • CTP cytosine triphosphate
  • TTP thymidine triphosphate
  • amino acid mixture amino acid mixture
  • creatine phosphate dithiothreitol (DTT
  • Yeast combines the advantages of simple, efficient protein folding, and post-translational modification. Among them, Saccharomyces cerevisiae and Pichia pastoris are model organisms that express complex eukaryotic proteins and membrane proteins. Yeast can also be used as a raw material for the preparation of in vitro translation systems.
  • Kluyveromyces is an ascomycete, in which Kluyveromyces marxianus and Kluyveromyces lactis are industrially widely used yeasts.
  • Kluyveromyces cerevisiae has many advantages over other yeasts, such as superior secretion capacity, better large-scale fermentation characteristics, food safety levels, and the ability to simultaneously modify post-translational proteins.
  • the yeast in vitro expression system is not particularly limited, and a preferred yeast in vitro expression system is the Kluyveromyces expression system (more preferably, the K. lactis expression system).
  • the in vitro cell-free synthesis system of the invention comprises a yeast in vitro synthesis system.
  • Yeast combines the advantages of simple, efficient protein folding, and post-translational modification. Among them, Saccharomyces cerevisiae and Pichia pastoris are model organisms that express complex eukaryotic proteins and membrane proteins. Yeast can also be used as a raw material for the preparation of in vitro translation systems.
  • Kluyveromyces is an ascomycete, in which Kluyveromyces marxianus and Kluyveromyces lactis are industrially widely used yeasts.
  • Kluyveromyces cerevisiae has many advantages over other yeasts, such as superior secretion capacity, better large-scale fermentation characteristics, food safety levels, and the ability to simultaneously modify post-translational proteins.
  • the yeast in vitro synthesis system is not particularly limited, and a preferred yeast in vitro synthesis system is the Kluyveromyces yeast expression system (more preferably, the K. lactis expression system).
  • Kluyveromyces cerevisiae e.g., Kluyveromyces lactis
  • Kluyveromyces lactis is not particularly limited, and includes any Kluvi (e.g., Kluyveromyces lactis) strain capable of improving the efficiency of synthetic proteins.
  • the cell-free in vitro synthesis system comprises:
  • the cell-free synthesis system further comprises:
  • the saccharide is selected from the group consisting of glucose, starch, glycogen, sucrose, maltose, cyclodextrin, or a combination thereof.
  • the concentration of the saccharide is 10-100 mM, preferably 10-60 mM, preferably 20-50 mM, more preferably 20-30 mM.
  • the content of the saccharide (V/V) is from 1 to 10%, preferably from 3 to 8%, more preferably from 4 to 6%, based on the total amount of the cell-free synthesis system. Volume meter.
  • the phosphate compound is selected from the group consisting of potassium phosphate, magnesium phosphate, ammonium phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, or a combination thereof.
  • the concentration (v/v) of the phosphate compound is from 1 to 6%, preferably from 2 to 5%, more preferably from 2 to 3%, based on the total volume of the cell-free synthesis system. meter.
  • the ratio of the yeast cell extract in the in vitro synthesis system is not particularly limited, and usually the content (wt%) of the yeast cell extract is 10% to 95%, preferably 20%- 80%, more preferably 40% to 60%, based on the total weight of the synthetic system.
  • the yeast cell extract does not contain intact cells, and typical yeast cell extracts include ribosomes for protein translation, transfer RNA, aminoacyl tRNA synthetase, initiation factors required for protein synthesis, and The elongation factor and the termination release factor.
  • the yeast extract contains some other proteins in the cytoplasm derived from yeast cells, especially soluble proteins.
  • the yeast cell extract contains a protein content of 20 to 100 mg/mL, preferably 50 to 100 mg/mL.
  • the method for determining protein content is a Coomassie Brilliant Blue assay.
  • the preparation method of the yeast cell extract is not limited, and a preferred preparation method comprises the following steps:
  • the solid-liquid separation method is not particularly limited, and a preferred mode is centrifugation.
  • the centrifugation is carried out in a liquid state.
  • the centrifugation conditions are not particularly limited, and a preferred centrifugation condition is 5,000 to 100,000 g, preferably 8,000 to 30,000 g.
  • the centrifugation time is not particularly limited, and a preferred centrifugation time is from 0.5 min to 2 h, preferably from 20 min to 50 min.
  • the temperature of the centrifugation is not particularly limited.
  • the centrifugation is carried out at 1-10 ° C, preferably at 2-6 ° C.
  • the washing treatment method is not particularly limited, and a preferred washing treatment method is treatment with a washing liquid at a pH of 7-8 (preferably, 7.4), and the washing liquid is not particularly Typically, the wash liquor is typically selected from the group consisting of potassium 4-hydroxyethylpiperazine ethanesulfonate, potassium acetate, magnesium acetate, or combinations thereof.
  • the manner of the cell disruption treatment is not particularly limited, and a preferred cell disruption treatment includes high pressure disruption, freeze-thaw (e.g., liquid nitrogen low temperature) disruption.
  • the mixture of nucleoside triphosphates in the in vitro protein synthesis system is adenine nucleoside triphosphate, guanosine triphosphate, cytidine triphosphate, and uridine nucleoside triphosphate.
  • the concentration of each of the single nucleotides is not particularly limited, and usually the concentration of each single nucleotide is from 0.5 to 5 mM, preferably from 1.0 to 2.0 mM.
  • the mixture of amino acids in the in vitro synthesis system can include natural or unnatural amino acids, and can include D-form or L-form amino acids.
  • Representative amino acids include, but are not limited to, 20 natural amino acids: glycine, alanine, valine, leucine, isoleucine, phenylalanine, valine, tryptophan, serine, Tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine.
  • the concentration of each amino acid is usually from 0.01 to 0.5 mM, preferably from 0.02 to 0.2 mM, such as 0.05, 0.06, 0.07, 0.08 mM.
  • the in vitro synthesis system further comprises a polyethylene glycol analog.
  • Representative PEG examples in the present invention include, but are not limited to, PEG 3000, PEG 8000, PEG 6000, and PEG 3350. It should be understood that the system of the present invention may also include other various molecular weight polyethylene glycols (e.g., PEG 200, 400, 1500, 2000, 4000, 6000, 8000, 10000, etc.).
  • the in vitro synthesis system further comprises sucrose.
  • concentration of sucrose is not particularly limited, and usually, the concentration (w/v) of sucrose is 0.2 to 4%, preferably 0.5 to 4%, more preferably 0.5 to 1%, based on the total volume of the synthetic system. .
  • the in vitro synthesis system further contains heme.
  • concentration of hemoglobin is not particularly limited, and usually, the concentration of heme is 0.01 to 0.1 mM, preferably 0.02 to 0.08 mM, more preferably 0.03 to 0.05 mM, most preferably 0.04 mM.
  • the in vitro synthesis system further comprises spermidine.
  • concentration of spermidine is not particularly limited, and usually, the concentration of spermidine is 0.05 to 1 mM, preferably 0.1 to 0.8 mM, more preferably, more preferably 0.2 to 0.5 mM, still more preferably 0.3 to 0.4. mM, optimally, 0.4 mM.
  • the in vitro synthesis system further contains a buffer, the composition of which is not particularly limited, and a preferred buffer contains 4-hydroxyethylpiperazineethanesulfonic acid, and/or Tris buffer. .
  • the buffer may further contain other buffer components such as potassium acetate or magnesium acetate to form a reaction solution or a reaction buffer having a pH of 6.5 to 8.5 (preferably 7.0 to 8.0).
  • the type and content of the buffer are not particularly limited.
  • the buffer is present at a concentration of 1-200 mM or 1-100 mM, preferably 5-50 mM.
  • a particularly preferred in vitro synthesis system in addition to the yeast extract, further comprises one or more or all of the components selected from the group consisting of 0.05-0.1 mg/mL phi29 DNA polymerase, 0.01-0.05 mg/mL RNA polymerase, 22 mM, 4-hydroxyethylpiperazineethanesulfonic acid, pH 7.4, 30-150 mM potassium acetate, 1.0-5.0 mM magnesium acetate, 1.5-4 mM nucleoside triphosphate mixture, 0.08-0.24 mM amino acid mixture, 25 mM phosphate muscle Acid, 1.7 mM dithiothreitol, 0.27 mg/mL phosphocreatine kinase, 0.5%-2% sucrose, 0.027-0.054 mg/mL T7 RNA polymerase, 0.03-0.04 mM heme, 0.3-0.4 mM subspermine, 1%-10% polyethylene glycol, 10-100 mM glucose, 10-60 mM potassium phosphate.
  • the term "coding sequence of a foreign protein” is used interchangeably with “exogenous template DNA”, “template DNA”, and refers to a foreign DNA molecule for directing protein synthesis.
  • the DNA molecule is circular or plasmid DNA.
  • the DNA molecule contains a sequence encoding a foreign protein.
  • examples of the sequence encoding the foreign protein include, but are not limited to, a genomic sequence, a cDNA sequence.
  • the sequence encoding the foreign protein further comprises a promoter sequence, a 5' untranslated sequence, and a 3' untranslated sequence.
  • the selection of the exogenous DNA is not particularly limited.
  • the exogenous DNA is selected from the group consisting of a luciferin protein, or a luciferase (such as firefly luciferase), a green fluorescent protein, and a yellow fluorescent protein. , aminoacyl tRNA synthetase, glyceraldehyde-3-phosphate dehydrogenase, catalase, actin, exogenous DNA of a variable region of an antibody, DNA of a luciferase mutant, or a combination thereof.
  • the exogenous DNA may also be selected from the group consisting of alpha-amylase, enteromycin A, hepatitis C virus E2 glycoprotein, insulin precursor, interferon alpha A, interleukin-1 beta, lysozyme, serum white. Protein, single-chain antibody fragment (scFV), thyroxine transporter, tyrosinase, exogenous DNA of xylanase, or a combination thereof.
  • alpha-amylase enteromycin A
  • hepatitis C virus E2 glycoprotein insulin precursor
  • interferon alpha A interleukin-1 beta
  • lysozyme serum white.
  • Protein single-chain antibody fragment (scFV), thyroxine transporter, tyrosinase, exogenous DNA of xylanase, or a combination thereof.
  • the exogenous DNA encodes a protein selected from the group consisting of: green fluorescent protein (eGFP), yellow fluorescent protein (YFP), and Escherichia coli beta-galactosidase ( ⁇ -galactosidase, LacZ), human lysine-tRNA synthetase, human leucine-tRNA synthetase, Arabidopsis glyceraldehyde 3-phosphate dehydrogenase (Glyceraldehyde-3-phosphate) Dehydrogenase), murine catalase (Catalase), or a combination thereof.
  • eGFP green fluorescent protein
  • YFP yellow fluorescent protein
  • Escherichia coli beta-galactosidase ⁇ -galactosidase, LacZ
  • human lysine-tRNA synthetase human leucine-tRNA synthetase
  • valuable template DNA may be included in the cell-free synthesis system of the present invention, or the corresponding exogenous template DNA may be added to the cell-free synthesis system of the present invention depending on the foreign protein of interest.
  • the system of the present invention can be used to simultaneously synthesize DNA, RNA, and protein in vitro.
  • the system of the present invention can be used to rapidly generate a target protein directly from a very small amount of DNA template.
  • the preparation of the present invention can directly perform in vitro protein synthesis from a trace amount of DNA as a template, and is simpler, faster, and more efficient than in vitro protein expression using RNA or DNA as a template.
  • the preparation of the present invention can be used for the synthesis of a large amount of a target protein while being easy to store, easy to use, and requiring no additional additives.
  • the kit of the present invention can be used for in vitro protein synthesis of trace DNA or plasmid as a template, and is simpler, faster, and more efficient than in vitro protein expression using RNA or DNA as a template.
  • the D2P in vitro expression system of the present invention can be used to express a variety of complex proteins and to obtain a higher protein content.
  • the D2P in vitro expression system of the invention is simple, rapid, and efficient to express a variety of proteins, and is convenient for rapid and efficient synthesis of high-throughput proteins, far superior to existing in vitro synthesis kits and traditional intracellular protein synthesis systems. .
  • the D2P in vitro cell-free biosynthesis system of the present invention omits time-consuming and labor-intensive mass cloning, transformation, and cell culture processes as compared to conventional cell protein expression systems.
  • the D2P in vitro cell-free biosynthesis system of the present invention omits the preparation and concentration process of a large number of DNA samples, omits the mRNA preparation process, greatly improves the work efficiency, improves the synthesis efficiency, and synthesizes.
  • the protein is easier to purify, saving users a lot of time and cost, and enabling large-scale, high-throughput protein manufacturing.
  • Example 1 Amplification of a plasmid template using phi29 DNA polymerase
  • DNA amplification system random primer with a final concentration of 20-30 ⁇ M, plasmid template of 0.05-0.15 ⁇ g/mL, 0.5-1 mM dNTP, 2 ⁇ BSA, 0.05-0.1 mg/mL phi29 DNA polymerase, 1 x phi29 reaction buffer (component: 50 mM Tris-HCl, 10 mM MgCl 2 , 10 mM (NH 4 ) 2 SO 4 , 4 mM DTT, pH 7.5).
  • Example 2 In vitro protein synthesis using amplified template DNA
  • Firefly luciferase (Fluc) activity assay After the reaction is completed, add an equal volume of substrate luciferine to a 96-well white plate or a 384-well white plate, and immediately place it on the Envision 2120 multi-function microplate reader. (Perkin Elmer), reading, detecting firefly luciferase activity, relative light unit (RLU) as the unit of activity, as shown in Figures 2 and 3.
  • Fluc activity assay After the reaction is completed, add an equal volume of substrate luciferine to a 96-well white plate or a 384-well white plate, and immediately place it on the Envision 2120 multi-function microplate reader. (Perkin Elmer), reading, detecting firefly luciferase activity, relative light unit (RLU) as the unit of activity, as shown in Figures 2 and 3.
  • RLU relative light unit
  • Example 3 Effect of different volumes of phi29 amplification system on in vitro protein synthesis efficiency
  • Example 4 Effect of phi29 DNA amplification system for different days of reaction on protein synthesis in vitro
  • the DNA amplification system was placed in an environment of 20-30 ° C for 1, 7, 14, 21 and 28 days, and heated at 65 ° C for 10 min to terminate the reaction, and the terminated reaction system was stored at -20 ° C;
  • Example 5 Effect of different concentrations of phi29 DNA polymerase amplified DNA template on in vitro protein synthesis system
  • Example 6 Effect of phi29 DNA amplification system at different reaction time points on in vitro protein synthesis system
  • the phi29 DNA amplification system using the plasmid containing the eGFP-encoding DNA sequence as a template was terminated at different reaction time points and stored at -20 °C until use.
  • the different reaction time points were 0 min, 5 min, 10 min, 30 min. , 1h, 2h, 4h, 6h, 8h, 12h, 16h and 24h;
  • Example 7 Detection of target protein synthesized by IVDTT using Western Blot
  • the primary antibody and the secondary antibody are incubated, and then subjected to tablet exposure treatment, wherein the primary antibody used is an antibody that specifically recognizes the eGFP protein;
  • Example 8 Detection of target protein synthesized by IVDTT using fluorescent SDS-PAGE
  • Ubiquitin protein which contains 76 amino acid residues and is covalently linked to other proteins, constitutes an important post-translational modification that can mediate a variety of cellular processes including protein degradation.
  • Ubiquitin in human cells is encoded by four genes, in which UBA52 and RPS27A contain a single copy of the Ubiquitin coding sequence, while the other two genes, UBB and UBC, contain multiple copies of the Ubiquitin coding sequence.
  • the IVDTT expression plasmid we constructed a coding sequence of eGFP located at the 3' end of the coding sequence of the target protein, so that the expressed protein is a fusion protein of the target protein and eGFP, and a peptide containing 9 amino acid residues is used in the middle.
  • the segments are connected and the amount of expression of the target protein can be quickly determined by detecting the amount of synthetic eGFP.
  • the fusion protein is a fusion protein of Ubiquitin and eGFP, which we named Ubiquitin-eGFP.
  • a plasmid containing the coding sequence of Ubiquitin-eGFP and eGFP was added to the amplification system containing phi29 DNA polymerase, and the reaction system was placed in an environment of 20-30 ° C overnight.
  • Example 10 Synthesis of the core domain of protein p53 using the IVDTT system
  • Protein p53 is a tumor suppressor that interacts with a wide variety of proteins and plays a very important role in cells. It also has a relatively complex structure.
  • the P53 core domain contains 221 amino acid residues. In many types of cancer cells, almost all mutations that inactivate p53 are located in the core domain, so the study of this domain has implications for understanding cancer occurrence. Important role.
  • the coding sequence of the p53 core domain was constructed into the expression plasmid of IVDTT, encoding the fusion protein p53-eGFP containing eGFP.
  • a plasmid containing the coding sequence of p53-eGFP and eGFP was added to an amplification system containing phi29 DNA polymerase, and the reaction system was placed in an environment of 20-30 ° C overnight.
  • G protein coupled receptor is a type of membrane protein containing seven transmembrane regions that can accept external signals and conduct them into cells, causing a downstream G protein complex.
  • a series of different cellular processes is a very important membrane protein molecule and a target protein for many drugs. It is crucial to study these proteins.
  • Beta2-adrenergic receptor ( ⁇ 2AR) is a GPCR protein that has been studied and contains 413 amino acid residues. Robert J. Lefkowitz and Brian K. Kobilka were awarded 2012 for their research. Bell Chemistry Prize.
  • the coding sequence of ⁇ 2AR was constructed into the expression plasmid of IVDTT, encoding the fusion protein ⁇ 2AR-eGFP containing eGFP.
  • a plasmid containing the coding sequence of ⁇ 2AR-eGFP and eGFP was added to an amplification system containing phi29 DNA polymerase, and the reaction system was placed in an environment of 20-30 ° C overnight.
  • Example 12 Synthesis of membrane protein aquaporin AQP1 using the IVDTT system
  • Aquaporin 1 is a membrane protein containing six transmembrane helices containing 269 amino acid residues, which generally form tetramers, but each individual AQP1 forms an independent water channel. .
  • the water channel allows rapid transfer of water molecules along the osmotic pressure gradient and is important for maintaining cell osmotic pressure. Peter Agre was awarded the 2003 Nobel Prize in Chemistry for his research on AQP1.
  • the coding sequence of AQP1 was constructed into the expression plasmid of IVDTT, encoding the fusion protein AQP1-eGFP containing eGFP.
  • a plasmid containing the AQP1-eGFP and eGFP coding sequences was added to an amplification system containing phi29 DNA polymerase, and the reaction system was placed in an environment of 20-30 ° C overnight.
  • Interferon is an important cytokine in the human body.
  • certain cells in the body can synthesize and secrete interferon, and pathogens that cause interferon secretion include viruses, bacteria, parasites and Cancer cells, etc.
  • interferon In addition to resisting pathogen invasion, interferon has other functions, such as activating some immune cells and improving the efficiency of pathogen presentation.
  • Interferon can be used clinically for the treatment of antiviral and advanced cancer, so the research and production of interferon plays a very important role in scientific research and clinical applications.
  • the coding sequence of IFN- ⁇ 2A was constructed into the expression plasmid of IVDTT, encoding the fusion protein IFN- ⁇ 2A-eGFP containing eGFP.
  • a plasmid containing the IFN- ⁇ 2A-eGFP and eGFP coding sequences was added to an amplification system containing phi29 DNA polymerase, and the reaction system was placed in an environment of 20-30 ° C overnight.
  • PD1 programmed death 1
  • its two substrates, PD-L1 and PD-L2 are co-inhibitory molecules of T cell-mediated immune responses.
  • the FDA approved several monoclonal antibodies against PD-L1 for the treatment of several cancers, such as atezolizumab, durvalumab and avelumab, and more and more for PD1 and PD-L1.
  • Monoclonal antibodies enter clinical trials. Protein drugs such as antibodies have become more and more widely used in clinical practice. Research on the production of protein drugs has also received more and more attention. How to produce protein drugs with high efficiency and low cost has become a hot research direction.
  • we used the IVDTT system to express the heavy chain (Healy chain, ate-H) and light chain (ate-L) of Atezolizumab, respectively.
  • the coding sequences of ate-H and ate-L were constructed into the expression plasmid of IVDTT, encoding the fusion proteins ate-H-eGFP and ate-L-eGFP containing eGFP.
  • a plasmid containing the coding sequences of ate-H-eGFP, ate-L-eGFP and eGFP was added to an amplification system containing phi29 DNA polymerase, and the reaction system was placed in an environment of 20-30 ° C overnight.
  • DNA replication, mRNA transcription and protein translation are the main genetic information transmission pathways of the central law of nature, and are the core theory of this patent.
  • 0.5-3 ⁇ L of Phi29 DNA polymerase amplified DNA can be used as an in vitro protein synthesis system coupled with in vitro transcription and translation.
  • the results of the protein amplification system of 0.5 ⁇ L or 3 ⁇ L for in vitro protein synthesis were not significantly different from those of the positive control, and the relative light unit (RLU) reached 1 ⁇ 10e9.
  • the phi29 DNA polymerase reaction time was 1-28 days, and the luciferase gene-containing plasmid was 1 ng.
  • the synthesized DNA did not increase or decrease in yield due to long time.
  • NC is a negative control with no in vitro synthesis of DNA.
  • PC is a positive control and DNA is derived from a PCR reaction of approximately 500 ng.
  • the 1-28 day phi29 DNA polymerase-replicated DNA can still be used as an in vitro protein synthesis system coupled to transcription and translation.
  • the concentration of phi29 DNA polymerase is 0.5mg/mL-0.8ug/mL
  • the amplified DNA template can be used as an in vitro protein synthesis system coupled with transcription and translation, especially 0.8mg.
  • the DNA replicated by phi29 DNA polymerase at /mL-0.4ug/mL was not significantly different from the positive control.
  • the reaction time of the phi29 DNA amplification system can usually be set at least 6 hours, usually overnight.
  • the molecular weight of the band detected by Western Blot is very close to the theoretical molecular weight of eGFP (26.7 KDa), and anti-eGFP is used.
  • the antibody, the target protein synthesized by IVDTT is eGFP.
  • the molecular weight of the detected fluorescent band was very close to the theoretical molecular weight of eGFP (26.7 KDa), and the excitation light and the received light were used.
  • the wavelength characteristics of the excited emitted light are similar to those of eGFP, indicating that the fluorescent signal detected in the IVDTT system is emitted by the synthetic eGFP protein.
  • the IVDTT system is capable of synthesizing an active eGFP protein that is correctly folded and capable of being excited by fluorescence, and the eGFP protein can be used as an indicator for the synthesis of a target protein in the IVDTT system.
  • IVDTT system can synthesize protein Ubiquitin
  • the fluorescence value of the synthesized fusion protein Ubiquitin-eGFP was 72RFU and 88RFU at 3 hours and 20 hours, respectively, while the negative control (NC) fluorescence values were 22RFU and 35RFU, respectively, indicating IVDTT.
  • Ubiquitin-eGFP was synthesized in the system. Separate eGFP protein was used as a positive control, and the fluorescence values reached 207 RFU and 232 RFU at 3 hours and 20 hours, respectively.
  • IVDTT system can synthesize p53 core domain
  • the fluorescence value of the synthesized fusion protein p53-eGFP was 256 RFU and 354 RFU at 3 hours and 20 hours, respectively, while the negative control (NC) fluorescence values were 16 RFU and 35 RFU, respectively, indicating IVDTT.
  • p53-eGFP was synthesized in the system. Separate eGFP protein was used as a positive control, and the fluorescence values reached 231 RFU and 363 RFU at 3 hours and 20 hours, respectively.
  • IVDTT system can synthesize membrane protein ⁇ 2AR
  • the fluorescence value of the synthesized fusion protein ⁇ 2AR-eGFP was 271RFU and 362RFU at 3 hours and 20 hours, respectively, while the fluorescence values of the negative control (NC) were 16RFU and 35RFU, respectively, indicating IVDTT.
  • ⁇ 2AR-eGFP was synthesized in the system. Separate eGFP protein was used as a positive control, and the fluorescence values reached 231 RFU and 363 RFU at 3 hours and 20 hours, respectively.
  • 10.IVDTT system can synthesize membrane protein AQP1
  • the fluorescence value of the synthesized fusion protein AQP1-eGFP was 331 RFU and 491 RFU at 3 hours and 20 hours, respectively, while the negative control (NC) fluorescence values were 16 RFU and 35 RFU, respectively, indicating IVDTT.
  • AQP1-eGFP was synthesized in the system. Separate eGFP protein was used as a positive control, and the fluorescence values reached 231 RFU and 363 RFU at 3 hours and 20 hours, respectively.
  • IVDTT system can synthesize interferon IFN- ⁇ 2A
  • the fluorescence value of the synthesized fusion protein IFN- ⁇ 2A-eGFP was 399 RFU and 562 RFU at 3 hours and 20 hours, respectively, while the negative control (NC) fluorescence value was 16 RFU and 35RFU, indicating that IFN- ⁇ 2A-eGFP was synthesized in the IVDTT system.
  • Separate eGFP protein was used as a positive control, and the fluorescence values reached 231 RFU and 363 RFU at 3 hours and 20 hours, respectively.
  • the IVDTT system is capable of synthesizing the heavy and light chains of the anti-PD-L1 monoclonal antibody Atezolizumab, respectively.
  • the fluorescence value of the synthesized fusion protein ate-H-eGFP reached 96RFU and 179RFU at 3 hours and 20 hours, respectively, and the synthesized fusion protein ate-L-eGFP was stimulated.
  • the fluorescence values emitted reached 378 RFU and 544 RFU at 3 hours and 20 hours, respectively, while the negative control (NC) fluorescence values were 16 RFU and 35 RFU, respectively, indicating that ate-H-eGFP and ate-L-eGFP were synthesized in the IVDTT system, respectively.
  • Separate eGFP protein was used as a positive control, and the fluorescence values reached 231 RFU and 363 RFU at 3 hours and 20 hours, respectively.
  • the in vitro DNA-to-Protein (D2P) synthesis system has the advantage of using a small amount of DNA as a template to rapidly express the protein in vitro at room temperature, and can be preserved for a long time, greatly reducing The cost of DNA shortens the time and steps required to prepare a DNA template and improves the efficiency of protein synthesis in vitro.

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Abstract

L'invention concerne un système de synthèse in vitro d'ADN en protéine (D2P), qui est un système de synthèse de protéine acellulaire pour la réplication d'ADN, la transcription et le couplage de traduction. L'invention concerne également une préparation correspondante, un kit de réactif et un procédé de synthèse acellulaire in vitro d'une protéine.
PCT/CN2018/080322 2017-03-23 2018-03-23 Système de synthèse in vitro d'adn en protéine (d2p), préparation, kit de réactif et procédé de préparation WO2018171747A1 (fr)

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