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WO2018171747A1 - 一种体外DNA-to-Protein(D2P)的合成体系、制剂、试剂盒及制备方法 - Google Patents

一种体外DNA-to-Protein(D2P)的合成体系、制剂、试剂盒及制备方法 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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French (fr)
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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

提供了一种体外DNA-to-Protein（D2P）的合成系统，是将DNA复制、转录、翻译偶联的无细胞蛋白质合成系统。还提供了相应的制剂、试剂盒以及体外无细胞合成蛋白质的方法。

Description

一种体外DNA-to-Protein(D2P)的合成体系、制剂、试剂盒及制备方法 技术领域
本发明涉及生物技术领域，具体地，涉及一种体外DNA-to-Protein(D2P)的合成体系、制剂、试剂盒及制备方法。
背景技术
传统的生物合成系统是指通过模式生物细菌、真菌、植物细胞或动物细胞等表达外源基因的一种分子生物学技术。随着科学技术的发展，无细胞表达体系，也称为体外蛋白合成系统，应运而生。体外蛋白合成系统是指以外源目的mRNA或DNA为模板，通过人工控制补加蛋白质合成所需的底物和转录、翻译相关蛋白因子等物质，能实现目的蛋白质的合成。体外蛋白合成系统表达蛋白质无需进行质粒构建、转化、细胞培养、细胞收集和破碎步骤，是一种相对快速、省时、便捷的蛋白质表达方式。
商业上的体外合成系统有两种：一种以预先体外转录获得的mRNA作为模板翻译合成蛋白质，另一种是将体外转录与体外翻译同时进行，以DNA为模板合成mRNA然后合成蛋白质。这两种系统都比较复杂且实验要求较高，需要使用者预先进行体外转录实验以获得足够多的mRNA模板，或者需要预先提供大量的DNA或者高浓度的质粒作为模板(模板的质量甚至大于合成蛋白质的质量)。这些弊端增加了使用者的操作时间、制造成本、及实验复杂性，同时也大大限制了生物合成的效率和产量。因此，开发一种更简单、方便、高效率、高产量、低成本的体外生物合成系统已成为基础生物研究领域的迫切需要。
发明内容
本发明提供了一种同时在体外进行DNA,RNA,蛋白质生物合成的理论模型和设计。
本发明提供了一种简单、方便、高效率、高产量、低成本的体外合成系统。
本发明提供一种以DNA模板进行体外合成DNA,RNA,蛋白质方法的建立。
本发明提供一种以微量DNA模板进行体外合成蛋白质方法的建立及优化，以克服现有技术所存在的缺陷与不足。
本发明的第一个目的是偶联DNA复制，转录及翻译的体外合成系统的理论构 建；本发明的第二个目的是偶联DNA复制，转录及翻译的体外合成系统的建立及优化；本发明的第三个目的是提供一种简单可行的偶联DNA-to-Protein(D2P)的体外生物合成制剂。本发明的第四个目的是提供一种简单、高产量的体外蛋白质合成试剂盒。
本发明第一方面提供了一种体外的无细胞的合成体系理论的建立，所述无细胞的合成体系包括：
(p1)体外外源DNA合成体系；
(p2)合成的DNA-to-mRNA合成体系；
(p3)合成的mRNA-to-Protein合成体系；
(p4)关联p1-p2-p3的DNA序列设计，酶的设计；
(p5)实现体外无细胞DNA-to-Protein(D2P)合成的系统性理论和定量评估；
(p6)D2P体系实现高效蛋白质合成的理论模型建立。
本发明第二方面提供了一种偶联DNA复制，转录及翻译的体外合成系统，所述无细胞的合成体系包括：
(a)DNA聚合酶；
(b)解旋酶；
(c)DNA结合蛋白；
(d)用于合成DNA的底物；
(e)RNA聚合酶；
(f)用于合成RNA的底物；
(g)用于合成蛋白的底物；
(h)细胞提取物；
在另一优选例中，所述无细胞的蛋白合成体系还包括选自下组的组分：
(j1)镁离子；
(j2)钙离子；
(j2)缓冲剂；
(j3)能量再生系统；
(j4)聚乙二醇；
(j5)任选的外源蔗糖；
(j6)任选的溶剂，所述溶剂为水或水性溶剂。
在另一优选例中，所述的DNA聚合酶包括：phi29 DNA聚合酶、T7 DNA聚合酶、Bst DNA聚合酶、Ecoli DNA聚合酶、DNA聚合酶I的Klenow片段等用于恒温扩增的聚合酶及突变体，并不局限于此。
在另一优选例中，所述的解旋酶包括：T7噬菌体复制系统的解旋酶(4B)、UvrD解旋酶等。
在另一优选例中，所述的DNA结合蛋白包括：T4噬菌体基因32蛋白、RB49噬菌体基因32蛋白、T7噬菌体复制系统的单链结合蛋白，DNA结合蛋白7等。
所述的合成DNA的底物包括：脱氧核苷单磷酸、脱氧核苷三磷酸、或其组合。
所述的合成RNA的底物包括：核苷单磷酸、核苷三磷酸、或其组合。
在另一优选例中，所述的合成蛋白的底物包括：1-20种天然氨基酸、以及非天然氨基酸。
在另一优选例中，所述无细胞的蛋白合成体系还包括外源的用于指导蛋白质合成的DNA分子。
在另一优选例中，所述的DNA分子为线性的。
在另一优选例中，所述的DNA分子为环状的。
在另一优选例中，所述的DNA分子含有编码外源蛋白的序列。
在另一优选例中，所述的编码外源蛋白的序列包括基因组序列、cDNA序列。
在另一优选例中，所述的编码外源蛋白的序列还含有启动子序列、5'非翻译序列、3'非翻译序列。
在另一优选例中，所述无细胞的蛋白合成体系包括选自下组的成分：聚乙二醇、蔗糖、4-羟乙基哌嗪乙磺酸、醋酸钾、醋酸镁、核苷三磷酸、氨基酸、磷酸肌酸，二硫苏糖醇(DTT)、磷酸肌酸激酶、T7RNA聚合酶、或其组合。
本发明第三个方面提供了提供一种简单可行的、偶联DNA-to-Protein(D2P)的体外生物合成制剂，包括：
(i)体外合成系统中所述的细胞提取物，无细胞合成体系，DNA复制体系,DNA转录体系的混合水溶液或者冻干粉；
(ii)在适合的条件，从而合成DNA，RNA及蛋白。
(iii)也可孵育DNA复制体系经过T1，再与细胞提取物及无细胞合成体系结合，从而合成DNA，RNA及蛋白。
在另一优选例中，所述步骤(i)和(ii)中，反应温度为20-30℃，反应时间为2-12h。
在另一优选例中，所述步骤(iii)中，反应温度为25-65℃。
在另一优选例中，所述步骤(iii)中，T1反应时间为2-6h。
本发明第四个方面提供了一种用于体外无细胞合成蛋白的试剂盒，包括：
(k1)第一容器，以及位于第一容器内的酵母细胞提取物；
(k2)第二容器，以及位于第二容器内的DNA聚合酶、解旋酶、DNA结合蛋白；
(kt)标签或说明书。
在另一优选例中，所述的第一容器、第二容器和第三容器是同一容器或不同容器。
在另一优选例中，所述试剂盒还包括任选的选自下组的一个或多个容器：
(k3)第三容器，以及位于第三容器的用于合成DNA的底物；
(k4)第四容器，以及位于第四容器的用于合成RNA的底物；
(k5)第五容器，以及位于第五容器的用于合成蛋白的底物；
(k6)第六容器，以及位于第六容器的镁离子；
(k7)第七容器，以及位于第七容器的钾离子；
(k8)第八容器，以及位于第八容器的缓冲剂。
(K9)第九容器，以及位于第九容器的聚乙二醇和蔗糖等；
本发明第五方面提供了一种无细胞合成体系，所述的合成体系将DNA复制、RNA转录和蛋白翻译偶联或整合在一个合成体系中。
在另一优选例中，所述的合成体系包括：
(i)DNA聚合体系；
(ii)RNA转录体系；和
(iii)蛋白质翻译体系。
在另一优选例中，所述无细胞合成体系还包括：
(iv)糖类；和
(v)磷酸化合物。
在另一优选例中，所述无细胞合成体系还包括：
(j1)镁离子；
(j2)钙离子；
(j3)缓冲剂；
(j4)能量再生系统。
(j5)聚乙二醇；
(j6)任选的外源蔗糖；和
(j7)任选的溶剂，所述溶剂为水或水性溶剂。
在另一优选例中，所述无细胞合成体系还包括任选的模板DNA。
在另一优选例中，所述的(i)DNA聚合体系包括：(a)DNA聚合酶；(b)任选的解旋酶；(c)任选的DNA结合蛋白；和(d)用于合成DNA的底物。
在另一优选例中，所述的(ii)RNA转录体系包括：(e)RNA聚合酶；和(f)用于合成RNA的底物。
在另一优选例中，所述的(iii)蛋白质翻译体系包括：(g)用于合成蛋白的底物；和(h)细胞提取物。
在另一优选例中，所述细胞提取物的细胞来源选自下组的一种或多种类型的细胞：原核细胞和真核细胞。
在另一优选例中，所述细胞提取物的细胞来源选自下组的一种或多种类型的细胞：大肠杆菌、细菌、哺乳动物细胞(如HF9、Hela、CHO、HEK293)、植物细胞、酵母细胞、或其组合。
在另一优选例中，所述的细胞提取物包括酵母细胞提取物。
在另一优选例中，所述酵母细胞选自下组：酿酒酵母、毕氏酵母、克鲁维酵母、或其组合；较佳地，所述的酵母细胞包括：克鲁维酵母，更佳地为乳酸克鲁维酵母。
在另一优选例中，所述的酵母细胞提取物为对酵母细胞的水性提取物。
在另一优选例中，所述酵母细胞提取物不含酵母内源性的长链核酸分子。
在另一优选例中，所述镁离子来源于镁离子源，所述镁离子源选自下组：醋酸镁、谷氨酸镁、或其组合。
在另一优选例中，所述钾离子来源于钾离子源，所述钾离子源选自下组：醋酸钾、谷氨酸钾、或其组合。
在另一优选例中，所述缓冲剂选自下组：4-羟乙基哌嗪乙磺酸、三羟甲基氨基甲烷、或其组合。
在另一优选例中，所述的合成RNA的底物包括：核苷单磷酸、核苷三磷酸、或其组合。
在另一优选例中，所述的合成蛋白的底物包括：1-20种天然氨基酸、以及非天然氨基酸。
在另一优选例中，所述的合成体系是DNA复制、RNA转录和蛋白翻译的三合一的合成体系。
在另一优选例中，所述的DNA聚合体系中，模板DNA的用量≤1ng，较佳地≤0.1ng，更佳地≤0.01ng。
在另一优选例中，所述的DNA聚合体系中，模板DNA的用量为0.0001-10ng，较佳地0.001－1ng，更佳地0.001－0.1ng。
在另一优选例中，所述的模板DNA为环状DNA。
在另一优选例中，所述的模板DNA为质粒DNA。
在另一优选例中，所述的质粒DNA包括能够增强蛋白质合成效率的串联DNA元件。
在另一优选例中，所述质粒DNA包括以下元件：串联而成的酵母细胞来源的IRES增强子KlNCE102、Ω序列以及酵母特异性的Kozak序列。
在另一优选例中，所述的合成体系的体积为10-50微升，较佳地，20-40微升。
在另一优选例中，所述的DNA聚合体系中，所述聚合酶选自下组：phi29 DNA聚合酶、T7 DNA聚合酶、Bst DNA聚合酶、E.coli DNA聚合酶、DNA聚合酶I的Klenow、或其组合。
在另一优选例中，所述的DNA聚合体系中，所述聚合酶为phi29 DNA聚合酶。
在另一优选例中，所述的DNA聚合体系中，所述聚合酶的浓度为0.0005－0.5mg/mL，较佳地，0.01－0.2mg/mL，更佳地，0.05－0.1mg/mL。
在另一优选例中，所述的DNA复制、RNA转录和蛋白翻译不包括从合成体系去除不必要蛋白的步骤。
在另一优选例中，所述方法不包括从合成体系去除不必要蛋白(如DNA酶、RNA聚合酶)的步骤。
在另一优选例中，所述聚乙二醇选自下组：PEG3000、PEG8000、PEG6000、PEG3350、或其组合。
在另一优选例中，所述聚乙二醇包括分子量(Da)为200-10000的聚乙二醇，较佳地，分子量为3000-10000的聚乙二醇。
在另一优选例中，所述蛋白合成体系中，聚乙二醇的浓度(w/v，例如g/ml)为0.1-8％，较佳地，0.5-4％，更佳地，1-2％。
在另一优选例中，所述能量再生系统选自下组：磷酸肌酸/磷酸肌酸酶系统、糖酵解途径及其中间产物能量系统、或其组合。
在另一优选例中，所述糖类选自下组：葡萄糖、淀粉、糖原、蔗糖、麦芽糖、环糊精、或其组合。
在另一优选例中，所述糖类的浓度(mmol/L)为10－100mM，较佳地，10-60mM，较佳地，20-50mM，更佳地，20-30mM。
在另一优选例中，所述糖类的的含量(V/V)为1－10％，较佳地，3－8％，更佳地，4－6％，以无细胞合成体系的总体积计。
在另一优选例中，所述磷酸化合物选自下组：磷酸钾、磷酸镁、磷酸铵、磷酸氢二钠、磷酸二氢钠、或其组合。
在另一优选例中，所述磷酸化合物的浓度(v/v)为1－6％，较佳地，2－5％，更佳地，2－3％，以无细胞合成体系的总体积计。
在另一优选例中，所述磷酸化合物的浓度(mmol/L)为10－60mM，较佳地，20－50mM，更佳地，20－30mM。
本发明第六方面提供了一种体外无细胞合成蛋白的方法，包括步骤：
(a)提供一混合体系，包括本发明第五方面所述的无细胞合成体系和外源的用于指导蛋白质合成的模板DNA，所述无细胞合成体系包括DNA复制体系和转录、翻译偶联的无细胞合成体系，在适合的条件，孵育所述的DNA复制体系一段时间T1，将转录、翻译偶联的无细胞合成体系与该DNA复制体系结合，经孵育后合成由所述外源DNA编码的蛋白质。
本发明第七方面提供了一种体外无细胞合成蛋白的方法，包括步骤：
提供一本发明第五方面所述的无细胞合成体系和用于指导蛋白质合成的模板DNA，在适合的条件下，孵育所述的无细胞合成体系和用于指导蛋白质合成的模板DNA一段时间T1，从而合成由所述模板DNA编码的蛋白质。
在另一优选例中，所述的模板DNA为环状DNA。
在另一优选例中，所述的模板DNA为质粒DNA。
在另一优选例中，所述的方法还包括：(b)任选地从所述蛋白合成体系中，分离或检测所述的由所述模板编码的蛋白质。
在另一优选例中，所述步骤(a)中，反应温度为20-37℃，较佳地，20-25℃。
在另一优选例中，所述步骤(b)中，反应时间为6-24h，较佳地，8-16h。
在另一优选例中，所述模板DNA的用量≤1ng，较佳地≤0.1ng，更佳地≤0.01ng。
在另一优选例中，所述模板DNA的用量为0.0001-10ng，较佳地0.001－1ng， 更佳地0.001－0.1ng。
本发明第八方面提供了一种用于体外无细胞合成的制剂，包括：
(i)本发明第五方面所述的无细胞合成体系；和
(ii)用于无细胞合成的辅助试剂。
在另一优选例中，所述制剂为混合溶液或冻干粉。
在另一优选例中，所述辅助试剂选自下组：
(j1)镁离子；
(j2)钙离子；
(j3)缓冲剂；
(j4)能量再生系统。
(j5)聚乙二醇；
(j6)任选的外源蔗糖；和
(j7)任选的溶剂，所述溶剂为水或水性溶剂。
在另一优选例中，所述辅助试剂还包括：
(a)糖类；和
(b)磷酸化合物。
本发明第九方面提供了一种用于体外无细胞合成的试剂盒，包括：
(k1)第一容器，以及位于第一容器内的本发明第五方面所述的无细胞合成体系；和
(kt)标签或说明书。
在另一优选例中，所述试剂盒还包括任选的选自下组的一个或多个容器：
(k2)第二容器，以及位于第二容器的镁离子；
(k3)第三容器，以及位于第三容器的钾离子；
(k4)第四容器，以及位于第四容器的缓冲剂；
(K5)第五容器，以及位于第五容器的聚乙二醇；
(k6)第六容器，以及位于第六容器的任选的外源蔗糖；
(K7)第七容器，以及位于第七容器的任选的溶剂，所述溶剂为水或水性溶剂；
(k8)第八容器，以及位于第八容器的糖类；和
(k9)第九容器，以及位于第九容器的磷酸化合物。
本发明第十方面提供了一种体外偶联合成DNA,mRNA和蛋白质的方法，包括 步骤：
(i)提供本发明第一方面所述的体外DNA复制体系，包括DNA聚合酶、外源的用于指导蛋白质合成的DNA分子、解旋酶、DNA结合蛋白、用于合成DNA的底物；
(ii)在适合的条件，孵育步骤(i)经过T1，再与转录与翻译偶联的无细胞合成体系结合，从而合成由所述外源DNA编码的DNA,mRNA和蛋白质；或
(iii)在适合的条件，将DNA复制体系、转录体系、翻译体系直接混合，加入外源的用于指导蛋白质合成的DNA分子，经孵育后合成由外源DNA直接编码的蛋白质。
本发明第十一方面提供了一种体外偶联合成DNA,mRNA和蛋白质的方法，包括步骤：
(i)提供本发明第一方面所述的体外DNA复制体系，包括DNA聚合酶、外源
的用于指导蛋白质合成的DNA分子、解旋酶、DNA结合蛋白；
(ii)在适合的条件，孵育步骤(i)经过T1，再与转录与翻译偶联的无细胞合成体系结合，从而合成由所述外源DNA编码的DNA,mRNA和蛋白质；或
(iii)在适合的条件，无需孵育步骤就可以完成外源DNA直接编码合成蛋白质。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
下面结合附图和实施例对本发明进一步说明。
图1是体外DNA-to-Protein(D2P)的合成体系的原理图。DNA复制，转录，翻译的中心法则原理。
图2是在phi29 DNA聚合酶复制体系中不同体积的DNA对于体外蛋白合成体系的影响示意图。phi29 DNA聚合酶的反应温度为30℃，反应时间为6-16h，含有萤光素酶基因的质粒为1ng。NC为负对照，没有DNA的体外合成体系。PC为正对照，DNA来自于PCR反应，约500ng。从图上可以看出，0.5-3微升的phi29 DNA聚合酶复制的DNA均可以用作体外转录与翻译偶联的无细胞表达体系。
图3是在phi29 DNA聚合酶复制体系中，不同反应时间的DNA对于体外蛋白合 成体系的影响示意图。phi29 DNA聚合酶的反应温度为30℃，反应时间为1-28天，含有荧光素酶基因的质粒为1ng。NC为负对照，没有DNA的体外合成体系。PC为正对照，DNA来自于PCR反应，约500ng。从图上可以看出，1-28天的phi29 DNA聚合酶复制的DNA仍可以用作体外转录与翻译偶联的无细胞表达体系。
图4是在复制体系中不同浓度phi29 DNA聚合酶扩增的DNA对于体外蛋白合成体系的影响示意图。phi29 DNA聚合酶的反应温度为30℃，反应时间为6-16h，含有eGFP基因的质粒为1ng，phi29聚合酶的反应浓度为0.5mg/mL-0.8ug/mL。NC为负对照，没有DNA的体外合成体系。PC为正对照，DNA来自于PCR反应，约500ng。从图上可以看出，0.8mg/mL-0.4ug/mL的phi29 DNA聚合酶复制的DNA均可以用作体外转录与翻译偶联的无细胞表达体系。
图5是phi29 DNA扩增体系不同时间点合成的DNA模板对于体外蛋白合成体系的影响示意图。phi29 DNA聚合酶的反应温度为20-30℃，反应时间为0-24h，含有eGFP基因的质粒为1ng。NC为负对照，没有DNA的体外合成体系。PC为正对照，DNA来自于PCR反应，约500ng。从图上可以看出，DNA扩增反应进行到6h时，扩增得到的DNA指导合成的eGFP的量达到最高值，进入平台。
图6是使用琼脂糖凝胶对图5中phi29 DNA扩增体系不同时间点合成的DNA量的分析示意图。从图上可以看出，DNA扩增反应进行到6h时，扩增得到的DNA的量达到最大值，随着时间的增加进入平台，不再有显著增加。
图7是使用Western Blot的方法检测IVDTT合成的eGFP。泳道1是加入含有eGFP编码序列的质粒的IVDTT体系，泳道2是不加入DNA模板的IVDTT体系(阴性对照)。Western Blot使用了eGFP蛋白的一抗，泳道1条带的分子量大小与理论分子量(26.7KDa)非常接近，这表明合成的目标蛋白是正确的eGFP蛋白。
图8是使用荧光SDS-PAGE成像方法检测IVDTT合成的eGFP蛋白。泳道1是加入含有eGFP编码序列的质粒的IVDTT体系，泳道2是不加入DNA模板的IVDTT体系(阴性对照)。在SDS-PAGE分析中，eGFP蛋白中的荧光基团在不完全变性的情况下还能够受激发并发出荧光。泳道1中检测到的荧光条带的分子量大小与eGFP的理论分子量(26.7KDa)非常接近，这表明合成的eGFP蛋白能够受激发并发出荧光。
图9是使用IVDTT体系合成的融合蛋白Ubiquitin-eGFP和单独eGFP受激后发出的相对荧光值(RFU)，测量了体外合成体系孵育3h(空白的柱状图)和20h(带有阴影的柱状图)后合成的融合蛋白中eGFP和单独的eGFP发出的相对荧光值 (RFU)，合成的单独eGFP作为IVDTT体系的正对照。合成的Ubiquitin-eGFP发出的相对荧光值比阴性对照(NC)的相对荧光值显著高，表明IVDTT体系合成了融合蛋白Ubiquitin-eGFP。
图10是使用IVDTT体系合成的融合蛋白p53-eGFP和单独eGFP受激后发出的相对荧光值，测量了体外合成体系孵育3h(空白的柱状图)和20h(带有阴影的柱状图)后合成的融合蛋白中eGFP和单独的eGFP发出的相对荧光值(RFU)，合成的单独eGFP作为IVDTT体系的正对照。合成的p53-eGFP发出的相对荧光值比阴性对照的相对荧光值显著高，表明IVDTT体系合成了融合蛋白p53-eGFP。
图11是使用IVDTT体系合成的融合蛋白β2AR-eGFP和单独eGFP受激后发出的相对荧光值，测量了体外合成体系孵育3h(空白的柱状图)和20h(带有阴影的柱状图)后合成的融合蛋白中eGFP和单独的eGFP发出的相对荧光值(RFU)，合成的单独eGFP作为IVDTT体系的正对照。合成的β2AR-eGFP发出的相对荧光值比阴性对照的相对荧光值显著高，表明IVDTT体系合成了融合蛋白β2AR-eGFP。
图12是使用IVDTT体系合成的融合蛋白AQP1-eGFP和单独eGFP受激后发出的相对荧光值，测量了体外合成体系孵育3h(空白的柱状图)和20h(带有阴影的柱状图)后合成的融合蛋白中eGFP和单独的eGFP发出的相对荧光值(RFU)，合成的单独eGFP作为IVDTT体系的正对照。合成的AQP1-eGFP发出的相对荧光值比阴性对照的相对荧光值显著高，表明IVDTT体系合成了融合蛋白AQP1-eGFP。
图13是使用IVDTT体系合成的融合蛋白IFN-α2A-eGFP和单独eGFP受激后发出的相对荧光值，测量了体外合成体系孵育3h(空白的柱状图)和20h(带有阴影的柱状图)后合成的融合蛋白中eGFP和单独的eGFP发出的相对荧光值(RFU)，合成的单独eGFP作为IVDTT体系的正对照。合成的IFN-α2A-eGFP发出的相对荧光值比阴性对照的相对荧光值显著高，表明IVDTT体系合成了融合蛋白IFN-α2A-eGFP。
图14和图15是使用IVDTT体系分别合成的融合蛋白ate-H-eGFP、ate-L-eGFP和单独eGFP受激后发出的相对荧光值，测量了体外合成体系孵育3h(空白的柱状图)和20h(带有阴影的柱状图)后合成的融合蛋白中eGFP和单独的eGFP发出的相对荧光值(RFU)，合成的单独eGFP作为IVDTT体系的正对照。合成的ate-H-eGFP和ate-L-eGFP发出的相对荧光值均比阴性对照的相对荧光值显著高，表明IVDTT体系合成了融合蛋白ate-H-eGFP和ate-L-eGFP。
图16是体外DNA-to-Protein(D2P)的合成体系的优势图。
具体实施方式
经过广泛而深入的研究，通过大量实践和摸索，首次发明了一种经优化的可将DNA复制与体外无细胞转录、翻译相偶联或整合的三合一体系(DNA-to-Protein,D2P)。D2P体系可用极微量的模板量(用量可降低2-4个数量级或者更多)，既可以有效地降低成本，可同时高效快速合成DNA,RNA和蛋白质，大大降低了无细胞合成系统的使用复杂性与成本。与商业上的所有无细胞蛋白表达体系相比，本发明提供的D2P体外无细胞表达系统可以利用极少量的(纳克-微克)DNA，连续、简便、高效地合成特定蛋白质。具体地，用本发明的体外无细胞表达系统，所合成的荧光素酶活性的相对光单位值可高达目前商业化体系(如兔子网织红细胞体外表达体系)的约60倍，节省了使用者约4-6h的准备时间，100-500倍左右的模板成本。
一、DNA-to-Protein(D2P)偶联生物合成体系的理论构建
1.科学背景
“中心法则”是地球上生物发生发展的基本原理。它是遗传信息从DNA传递给DNA，即完成DNA的复制过程，以及从DNA传递给RNA，再从RNA传递给蛋白质，即完成遗传信息的转录和翻译的过程。这是所有有细胞结构的生物所遵循的核心法则。现代生物学的进步在很大程度上是建立在对于这一法则的理解和应用上的。比如核酸扩增技术(PCR)，分子克隆，基因组改造，细胞信号调控，神经网络，疾病原理和治疗，以及重组蛋白质的表达，等等。近20年来，随着基因测序，组学，计算机和互联网技术的发展，生物学研究也取得了大量革命性的进步。然而，组成生物核心物质的DNA，RNA,特别是蛋白质的合成和制备却依然停留在二三十年以前的水平，大大制约了相关生物学研究和和医药研发的进步。开发一种高产量、低成本的体外蛋白质生物合成系统，已成为世界范围的迫切需要。
目前蛋白质的合成方法可以分为两种：传统的细胞蛋白质合成和体外无细胞蛋白合成。传统的蛋白质合成方法起源于1970年代，是指通过模式生物细菌、真菌、植物细胞或动物细胞等表达外源基因的一种分子生物学技术[1-2]。随着科学技术的发展，无细胞表达体系也称为体外蛋白合成系统在1990年代应运而生[3-5]，其是以外源目的mRNA或DNA为蛋白质合成模板，通过人工控制补加蛋白质合成所需的底物和转录、翻译相关蛋白因子等物质，能实现目的蛋白质的合成。体 外翻译系统中表达蛋白质无需进行质粒构建、转化、细胞培养、细胞收集和破碎步骤，是一种相对快速、省时、便捷的蛋白质表达方式。
然而，由于体外蛋白质合成体系的制备繁琐，添加辅助因子复杂，使得目前市场上所有的体外蛋白质合成产品都活性较低，且极其昂贵(100-1000倍于传统细胞合成)。目前仅见于极个别实验室中少量的活性测定和实验尝试中。发展高效、便捷、低成本的蛋白质合成方法，无疑将成为推动未来生物工业革命，科技进步，医药研发，药物生产的蒸汽机车，对于整个生物产业的进步具有革命性的作用。
基于发明人在蛋白质合成领域基础理论、真核细胞中蛋白质合成机制、及其相关酶、和催化功能，等的超过20年的研究，我们认为最高效的生物合成系统应该来自于大自然的设计。
2.基本原理
大自然中细胞会保持持续的分裂，复制，生长，不间断地，大量地制造DNA,RNA和蛋白质。以简单的大肠杆菌(Escherichia coli,E.coli)为例，依照它们20-30分钟的复制速度，它(细胞体积1立方微米，重量1pg,干重0.4pg,DNA 0.017pg,RNA 0.10pg,蛋白质0.20pg)生物合成的速度是：34μg/mL/hour DNA,200μg/mL/hour RNA,400μg/mL/hour蛋白质[6-8]。复制放大比例为DNA:RNA:Protein＝1:5.9:12。真核的酵母细胞(Saccharomyces cerevisiae，Sc)，依照它们90分钟的复制速度，它(细胞体积73立方微米，重量79pg,干重40pg,DNA 0.06pg,RNA 4pg,蛋白质20pg)生物合成的速度是：0.55μg/mL/hour DNA,36μg/mL/hour RNA,182μg/mL/hour蛋白质[9-12]。无论合成蛋白质的速度，还是从DNA到RNA到Protein的放大比例均远大于现有的体外蛋白质合成体系。
而这些制造过程的核心基础就是DNA-RNA-Protein的“中心法则”。其中每一步都是必需的，不能缺少。而实现高效率生物合成最基础的便是第一步的DNA的自我复制。例如，病毒是生物界中的复制效率最高的物种，然而当没有宿主时，其由于无法完成其自身DNA的复制(RNA病毒无法完成RNA到DNA,然后DNA复制)，病毒的生长是完全停滞的。这一点也可以从不同物种中还是从DNA到RNA到Protein的放大比例看出：E.coli细胞的复制放大比例为DNA:RNA:Protein＝1:5.9:12，酵母细胞的复制放大比例为DNA:RNA:Protein＝1:64:330，人细胞的复制放大比例为DNA:RNA:Protein＝1:2:20[8]。如果考虑到酿酒酵母(S.cerevisiae)和人细胞中分别有约75％和80％的基因组能够被转录，而且在酿酒酵母 和人细胞中，非编码RNA(non-coding RNA)在转录出的RNA中的比例分别超过95％和98％，只有少于5％(酵母)和2％(人)RNA编码蛋白质，则酵母细胞和人的复制放大比例分别为DNA:mRNA:Protein＝1:4.27:440和DNA:mRNA:Protein＝1:0.05:25[13-16]。因此只有充分利用整个“中心法则”，完整的建立DNA到RNA到Protein全部路径，才有可能最大限度的提示生物合成的效率和最终蛋白质制备的产量。
以此为依据，我们这里提出了全新的DNA-to-Protein(D2P)偶联体外无细胞生物合成理论，将“中心法则”中的三部：DNA复制，DNA到RNA的转录，RNA到蛋白质的翻译，全部放在体外，以偶联的方式，进行DNA,RNA,Protein的协同合成，以达到提高生物合成效率的目的。
3.技术架构
D2P技术的实现包括：核酸扩增技术，RNA聚合技术，和体外蛋白翻译技术。3a.核酸扩增技术包括非等温扩增技术和等温扩增技术。聚合酶链反应是核酸扩增技术的典型代表。但是PCR技术对于温度循环有依赖性，往往需要较高的温度对DNA模板进行变性和新合成DNA分子的扩增延伸，而高温能够引起体外合成体系中蛋白质因子的变性失活，所以不适合用于体外合成体系。与PCR技术相比，核酸等温扩增的特点是在特定的、比较温和的温度条件下实现核酸的扩增，因而可以将DNA复制、mRNA转录与蛋白合成进行体外偶联。同时，用于核酸等温扩增的DNA聚合酶，包括phi29 DNA聚合酶，T7 DNA聚合酶等在温度和扩增效率上都有较大的优势，这样就无需预先准备大量DNA分子，只需少量的DNA模板就可以实现蛋白质的体外合成。
该D2P体外合成体系中使用的phi29 DNA聚合酶，T7 DNA聚合酶并非是纯粹的野生型或者突变型，将根据体外合成体系的需要，进行DNA聚合酶的保真性，合成效率，延伸能力等方面的分子改造，与单链DNA结合蛋白的融合，或者几种等温聚合酶的组合，高效地应用于D2P的合成体系中，创造出高效的D2P偶联生物合成体系。
3b.该D2P体外合成体系中使用的T7RNA聚合酶具有特异性高和转录效率高的特性，能够快速高效地从DNA模板转录出大量mRNA分子。T7RNA聚合酶配合高效的体外蛋白翻译系统，又进一步的降低了对DNA分子的量的要求。
3c.综上所述，构建D2P偶联体外无细胞生物合成体系，实现由微量DNA(纳克-微克级)到大量蛋白质(微克-毫克级)的合成，理论上是可行的。
二、DNA-to-Protein(D2P)偶联的体外无细胞生物合成体系
在实施方式中，本发明提供的体外合成体系包括：细胞提取物，4-羟乙基哌嗪乙磺酸，醋酸钾，醋酸镁，腺嘌呤核苷三磷酸(ATP)，鸟嘌呤核苷三磷酸(GTP)，胞嘧啶核苷三磷酸(CTP)，胸腺嘧啶核苷三磷酸(TTP)，氨基酸混合物，磷酸肌酸，二硫苏糖醇(DTT)，磷酸肌酸激酶，RNA酶抑制剂，RNA聚合酶，亚精胺，血红素，DNA聚合酶，解旋酶，DNA结合蛋白等。
在本发明中，RNA聚合酶没有特别限制，可以选自一种或多种RNA聚合酶，典型的RNA聚合酶为T7RNA聚合酶。
在本发明中，DNA聚合酶没有特别限制，能够用于恒温扩增的聚合酶，可以选自一种或多种DNA聚合酶，典型的DNA聚合酶为phi29 DNA聚合酶、T7 DNA聚合酶、Bst DNA聚合酶等，并不局限于此。
在本发明中，解旋酶可以选自一种或多种，典型的解旋酶为T7噬菌体复制系统的解旋酶(4B)、UvrD解旋酶等，并不局限于此。
在本发明中，DNA结合蛋白可以选自一种或多种：T4噬菌体基因32蛋白、RB49噬菌体基因32蛋白、T7噬菌体复制系统的单链结合蛋白，DNA结合蛋白7、等，并不局限于此。
三、DNA-to-Protein(D2P)偶联的体外无细胞合成体系制剂
本发明提供了一种体外的偶联DNA复制、转录及翻译的无细胞的合成体系制剂，所述制剂包括：
(i)细胞提取物的水溶液或冻干粉；
(ii)DNA复制反应体系的水溶液或冻干粉；
(iii)DNA转录反应体系的水溶液或冻干粉；
(iv)无细胞合成体系的水溶液或冻干粉；
无细胞合成体系包括：4-羟乙基哌嗪乙磺酸，醋酸钾，醋酸镁，氨基酸混合物，磷酸肌酸，二硫苏糖醇(DTT)，磷酸肌酸激酶，RNA酶抑制剂，聚乙二醇等反应物。
细胞提取物，无细胞合成体系，DNA复制体系，DNA转录反应的混合冻干粉或者其中一种或多种冻干粉体系与水溶液体系的组合，在适合的条件，从而合成DNA，RNA及蛋白。也可DNA复制体系经过孵育时间T1，再与细胞提取物，DNA转录体系及无细胞合成体系结合，从而合成所述DNA，RNA及蛋白。
所述步骤中，DNA复制反应体系的温度为25-65℃，T1的反应时间为2-6h.
四、DNA-to-Protein(D2P)偶联的体外无细胞蛋白质合成试剂盒
本发明提供了一种DNA-to-Protein(D2P)偶联的体外无细胞合成的试剂盒，包括：
(k1)第一容器，以及位于第一容器内的细胞提取物；
(k2)第二容器，以及位于第二容器内的DNA复制反应体系；
(k3)任选的第三容器，以及位于第三容器的DNA转录反应体系；
(kt)标签或说明书。
在一优选实施方式中，所述的第一容器、第二容器和第三容器是同一容器或不同容器。
一种特别优选的体外蛋白质合成的试剂盒包含一个体外合成体系，包括：细胞提取物，4-羟乙基哌嗪乙磺酸，醋酸钾，醋酸镁，腺嘌呤核苷三磷酸(ATP)，鸟嘌呤核苷三磷酸(GTP)，胞嘧啶核苷三磷酸(CTP)，胸腺嘧啶核苷三磷酸(TTP)，氨基酸混合物，磷酸肌酸，二硫苏糖醇(DTT)，磷酸肌酸激酶，RNA酶抑制剂，T7RNA聚合酶，亚精胺，血红素，DNA聚合酶，RNA聚合酶，解旋酶，DNA结合蛋白。
体外表达系统
酵母(yeast)兼具培养简单、高效蛋白质折叠、和翻译后修饰的优势。其中酿酒酵母(Saccharomyces cerevisiae)和毕氏酵母(Pichia pastoris)是表达复杂真核蛋白质和膜蛋白的模式生物，酵母也可作为制备体外翻译系统的原料。
克鲁维酵母(Kluyveromyces)是一种子囊孢子酵母，其中的马克斯克鲁维酵母(Kluyveromyces marxianus)和乳酸克鲁维酵母(Kluyveromyces lactis)是工业上广泛使用的酵母。与其他酵母相比,乳酸克鲁维酵母具有许多优点,如超强的分泌能力,更好的大规模发酵特性、食品安全的级别、以及同时具有蛋白翻译后修饰的能力等。
在本发明中，酵母体外表达系统不受特别限制，一种优选的酵母体外表达系统为克鲁维酵母表达系统(更佳地，乳酸克鲁维酵母表达系统)。
体外的无细胞的合成体系
在一优选实施方式中，本发明的体外的无细胞的合成体系包括酵母体外合成体系。
酵母(yeast)兼具培养简单、高效蛋白质折叠、和翻译后修饰的优势。其中酿酒酵母(Saccharomyces cerevisiae)和毕氏酵母(Pichia pastoris)是表达复杂真核蛋白质和膜蛋白的模式生物，酵母也可作为制备体外翻译系统的原料。
克鲁维酵母(Kluyveromyces)是一种子囊孢子酵母，其中的马克斯克鲁维酵母(Kluyveromyces marxianus)和乳酸克鲁维酵母(Kluyveromyces lactis)是工业上广泛使用的酵母。与其他酵母相比，乳酸克鲁维酵母具有许多优点,如超强的分泌能力,更好的大规模发酵特性、食品安全的级别、以及同时具有蛋白翻译后修饰的能力等。
在本发明中，酵母体外合成体系不受特别限制，一种优选的酵母体外合成体系为克鲁维酵母表达系统(更佳地，乳酸克鲁维酵母表达系统)。
在本发明中，克鲁维酵母(如乳酸克鲁维酵母)不受特别限制，包括任何一种能够提高合成蛋白效率的克鲁维(如乳酸克鲁维酵母)菌株。
在本发明中，所述无细胞体外合成体系包括：
(i)DNA聚合体系；
(ii)RNA转录体系；和
(iii)蛋白质翻译体系。
在另一优选例中，所述无细胞合成体系还包括：
(iv)糖类；和
(v)磷酸化合物。
在另一优选例中，所述糖类选自下组：葡萄糖、淀粉、糖原、蔗糖、麦芽糖、环糊精、或其组合。
在另一优选例中，所述糖类的浓度(mmol/L)为10－100mM，较佳地，10-60mM，较佳地，20-50mM，更佳地，20-30mM。
在另一优选例中，所述糖类的的含量(V/V)为1－10％，较佳地，3－8％，更佳地，4－6％，以无细胞合成体系的总体积计。
在另一优选例中，所述磷酸化合物选自下组：磷酸钾、磷酸镁、磷酸铵、磷酸氢二钠、磷酸二氢钠、或其组合。
在另一优选例中，所述磷酸化合物的浓度(v/v)为1－6％，较佳地，2－5％，更佳地，2－3％，以无细胞合成体系的总体积计。
在本发明中，所述酵母细胞提取物在体外合成体系中的比例不受特别限制，通常所述酵母细胞提取物的含量(wt％)为10％－95％，较佳地，20％－80％， 更佳地，40％－60％，以所述合成体系的总重量计。
在本发明中，所述的酵母细胞提取物不含完整的细胞，典型的酵母细胞提取物包括用于蛋白翻译的核糖体、转运RNA、氨酰tRNA合成酶、蛋白质合成需要的起始因子和延伸因子以及终止释放因子。此外，酵母提取物中还含有一些源自酵母细胞的细胞质中的其他蛋白，尤其是可溶性蛋白。
在本发明中，所述的酵母细胞提取物所含蛋白含量为20-100mg/mL,较佳为50-100mg/mL。所述的测定蛋白含量方法为考马斯亮蓝测定方法。
在本发明中，所述的酵母细胞提取物的制备方法不受限制，一种优选的制备方法包括以下步骤：
(i)提供酵母细胞；
(ii)对酵母细胞进行洗涤处理，获得经洗涤的酵母细胞；
(iii)对经洗涤的酵母细胞进行破细胞处理，从而获得酵母粗提物；
(iv)对所述酵母粗提物进行固液分离，获得液体部分，即为酵母细胞提取物。
在本发明中，所述的固液分离方式不受特别限制，一种优选的方式为离心。
在一优选实施方式中，所述离心在液态下进行。
在本发明中，所述离心条件不受特别限制，一种优选的离心条件为5000-100000g，较佳地，8000-30000g。
在本发明中，所述离心时间不受特别限制，一种优选的离心时间为0.5min－2h，较佳地，20min－50min。
在本发明中，所述离心的温度不受特别限制，优选的，所述离心在1-10℃下进行，较佳地，在2-6℃下进行。
在本发明中，所述的洗涤处理方式不受特别限制，一种优选的洗涤处理方式为采用洗涤液在pH为7-8(较佳地，7.4)下进行处理，所述洗涤液没有特别限制，典型的所述洗涤液选自下组：4-羟乙基哌嗪乙磺酸钾、醋酸钾、醋酸镁、或其组合。
在本发明中，所述破细胞处理的方式不受特别限制，一种优选的所述的破细胞处理包括高压破碎、冻融(如液氮低温)破碎。
所述体外蛋白质合成体系中的核苷三磷酸混合物为腺嘌呤核苷三磷酸、鸟嘌呤核苷三磷酸、胞嘧啶核苷三磷酸和尿嘧啶核苷三磷酸。在本发明中，各种单核苷酸的浓度没有特别限制，通常每种单核苷酸的浓度为0.5-5mM，较佳地为1.0-2.0 mM。
所述体外合成体系中的氨基酸混合物可包括天然或非天然氨基酸，可包括D型或L型氨基酸。代表性的氨基酸包括(但并不限于)20种天然氨基酸：甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、蛋氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸和组氨酸。每种氨基酸的浓度通常为0.01-0.5mM，较佳地0.02-0.2mM，如0.05、0.06、0.07、0.08mM。
在一优选实施方式中，所述体外合成体系还含有聚乙二醇类似物。
在本发明中，代表性的PEG例子包括(但并不限于)：PEG3000，PEG8000，PEG6000和PEG3350。应理解，本发明的体系还可包括其他各种分子量的聚乙二醇(如PEG200、400、1500、2000、4000、6000、8000、10000等)。
在优选例中，所述体外合成体系还含有蔗糖。蔗糖的浓度没有特别限制，通常，蔗糖的浓度(w/v)为0.2-4％，较佳地，0.5-4％，更佳地，0.5-1％，以所述合成体系的总体积计。
在优选例中，所述体外合成体系还含有血红素。血红素的浓度没有特别限制，通常，血红素的浓度为0.01-0.1mM，较佳地，0.02-0.08mM，更佳地，0.03-0.05mM，最佳地，0.04mM。
在优选例中，所述体外合成体系还含有亚精胺。亚精胺的浓度没有特别限制，通常，亚精胺的浓度为0.05-1mM，较佳地，0.1-0.8mM，更佳地，更佳地，0.2-0.5mM，更佳地，0.3-0.4mM，最佳地，0.4mM。
在优选例中，所述体外合成体系还含有缓冲剂，所述缓冲剂的成分不受特别限制，一种优选的缓冲剂含有4-羟乙基哌嗪乙磺酸、和/或Tris缓冲液。在本发明中，所述缓冲剂还可含有其他缓冲成分，如醋酸钾、醋酸镁，从而形成pH为6.5-8.5(优选7.0-8.0)的反应液或反应缓冲液。在本发明中，缓冲剂的类型和含量不受特别限制。通常，缓冲剂的浓度为1-200mM或1-100mM，较佳地，5-50mM。
一种特别优选的体外合成体系，除了酵母提取物，还含有选自下组的一种或多种或全部成分：0.05-0.1mg/mL phi29DNA聚合酶，0.01-0.05mg/mL RNA聚合酶，22mM，pH为7.4的4-羟乙基哌嗪乙磺酸，30-150mM醋酸钾，1.0-5.0mM醋酸镁，1.5-4mM核苷三磷酸混合物，0.08-0.24mM的氨基酸混合物，25mM磷酸肌酸，1.7mM二硫苏糖醇，0.27mg/mL磷酸肌酸激酶，0.5％-2％ 蔗糖，0.027-0.054mg/mL T7RNA聚合酶，0.03-0.04mM的血红素，0.3-0.4mM的亚精胺，1％-10％聚乙二醇，10-100mM葡萄糖，10－60mM磷酸钾。
外源蛋白的编码序列(模板DNA)
如本文所用，术语“外源蛋白的编码序列”与“外源模板DNA”、“模板DNA”可互换使用，均指外源的用于指导蛋白质合成的DNA分子。在本发明中，所述的DNA分子为环状的或质粒DNA。所述的DNA分子含有编码外源蛋白的序列。在本发明中，所述的编码外源蛋白的序列的例子包括(但并不限于)：基因组序列、cDNA序列。所述的编码外源蛋白的序列还含有启动子序列、5’非翻译序列、3’非翻译序列。
在本发明中，所述外源DNA的选择没有特别限制，通常，外源DNA选自下组：编码荧光素蛋白、或荧光素酶(如萤火虫荧光素酶)、绿色荧光蛋白、黄色荧光蛋白、氨酰tRNA合成酶、甘油醛-3-磷酸脱氢酶、过氧化氢酶、肌动蛋白、抗体的可变区域的外源DNA、萤光素酶突变体的DNA、或其组合。
外源DNA还可以选自下组：编码α-淀粉酶、肠道菌素A、丙型肝炎病毒E2糖蛋白、胰岛素前体、干扰素αA、白细胞介素-1β、溶菌酶素、血清白蛋白、单链抗体段(scFV)、甲状腺素运载蛋白、酪氨酸酶、木聚糖酶的外源DNA、或其组合。
在一优选实施方式中，所述外源DNA编码选自下组的蛋白：绿色荧光蛋白(enhanced GFP,eGFP)、黄色荧光蛋白(YFP)、大肠杆菌β-半乳糖苷酶(β-galactosidase,LacZ)、人赖氨酸-tRNA合成酶(Lysine-tRNA synthetase)、人亮氨酸-tRNA合成酶(Leucine-tRNA synthetase)、拟南芥甘油醛3-磷酸脱氢酶(Glyceraldehyde-3-phosphate dehydrogenase)、鼠过氧化氢酶(Catalase)、或其组合。
在本发明中，可以将有价值的模板DNA包括在本发明的无细胞合成体系中，也可以根据感兴趣的外源蛋白在本发明的无细胞合成体系中加入相应的外源模板DNA。
本发明的主要优点包括：
1)首次在体外的一个体系中完成从DNA到RNA到蛋白质的三步扩增偶联反应。
2)本发明的体系可以用于同时体外合成DNA,RNA,蛋白质。
3)本发明的体系可以用于快速的由极微量的DNA模板直接生成目标蛋白质。
4)本发明的制剂可以由微量DNA为模板直接完成体外蛋白质合成，比用RNA或者DNA为模板的体外蛋白表达更简单快捷，且高效。
5)本发明的制剂可以用于大量目标蛋白的合成，同时易保存，易使用，不需要额外的添加剂。
6)本发明的试剂盒可以用于微量DNA或者质粒为模板的体外蛋白质合成，比用RNA或者DNA为模板的体外蛋白表达更简单快捷，且高效。
7)本发明的D2P体外表达系统可以用于表达多种复杂蛋白，且能够获得较高的蛋白含量。
8)本发明的D2P体外表达系统操作简单，快捷，且高效地表达多种蛋白，便于高通量蛋白质的快速高效合成，远优于现有的体外合成试剂盒和传统的细胞内蛋白合成体系。
与传统的细胞蛋白表达系统相比，本发明的D2P体外无细胞生物合成系统省略了耗时耗力的大量分子克隆，转化，细胞培养过程。与传统的体外蛋白表达系统相比，本发明的D2P体外无细胞生物合成系统省略了大量DNA样本的制备和浓缩过程，省略了mRNA制备过程,极大地提高了工作效率，提升了合成效率，合成的蛋白质更易于提纯，为使用者节省大量时间和成本，并使得大规模，高通量的蛋白质制造成为可能。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
如无特别说明，则本发明实施例中所用的材料和试剂均为市售产品。
实施例1：使用phi29 DNA聚合酶对质粒模板进行扩增
1.1DNA扩增体系的配制：终浓度为20-30μM的随机引物，0.05-0.15μg/mL的质粒模板，0.5-1mM的dNTP，2×BSA，0.05-0.1mg/mL的phi29 DNA聚合酶，1×phi29反应缓冲液(成分为50mM Tris-HCl,10mM MgCl 2,10mM(NH 4) 2SO 4,4mM DTT,pH7.5)。
1.2体外质粒模板DNA的扩增反应：将上述的反应体系放置于20-30℃的环境中，静置6-12h，通常为过夜反应。
实施例2：使用扩增的模板DNA进行体外蛋白质的合成
2.1体外蛋白质合成体系：终浓度为22mM pH为7.4的4-羟乙基哌嗪乙磺酸，30-150mM醋酸钾，1.0-5.0mM醋酸镁，1.5-4mM核苷三磷酸混合物(腺嘌呤核苷三磷酸、鸟嘌呤核苷三磷酸、胞嘧啶核苷三磷酸和尿嘧啶核苷三磷酸)，0.08-0.24mM的氨基酸混合物(甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、蛋氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸和组氨酸)，25mM磷酸肌酸，1.7mM二硫苏糖醇，0.27mg/mL磷酸肌酸激酶，0.027-0.054mg/mL T7 RNA聚合酶，1％-4％的聚乙二醇，0.5％-2％的蔗糖，最后加入50％体积的酵母细胞提取物；
2.2体外蛋白质合成反应：向上述30μL反应体系中加入0.5-3μL扩增后的模板DNA，放置在20-30℃的环境中，静置反应2-20h；
2.3萤火虫萤光素酶(Firefly luciferase,Fluc)活性测定：反应结束后，在96孔白板或384孔白板中加入等体积的底物荧光素(luciferine)，立即放置于Envision 2120多功能酶标仪(Perkin Elmer),读数，检测萤火虫荧光素酶活性，相对光单位值(Relative Light Unit，RLU)作为活性单位，如图2和图3所示。
2.4绿色荧光蛋白(eGFP)活性测定：反应结束后，在384孔黑板中加入10μL反应体系，置于Tecan Infinite F200荧光分光光度计中读数，检测绿色荧光蛋白受激发后放出的相对荧光单位值(Relative Fluorescence Unit，RFU)作为活性单位，如图4和图5所示。
实施例3：不同体积的phi29扩增体系对于体外蛋白合成效率的影响
3.1使用phi29 DNA聚合酶扩增含有Fluc编码DNA序列的模板质粒，置于20-30℃的环境中，过夜静置反应；
3.2分别取0.5、1、2和3μL的上述DNA扩增体系，加入到30μL体外蛋白合成体系中，同时加入使用普通PCR的方法制备的模板DNA作为阳性对照(positive control,PC)，不加入任何模板的体外蛋白合成体系作为阴性对照(negative control,NC)，每个实验组设置3个平行实验，置于20-30℃的环境中，反应2-20h；
3.3合成的Fluc活性的测定。
实施例4：测定反应不同天数的phi29 DNA扩增体系对体外蛋白合成的影响
4.1将DNA扩增体系置于20-30℃的环境中1、7、14、21和28天，在65℃环境中加热10min来终止反应，并将终止的反应体系置于-20℃保存；
4.2向30μL体外蛋白合成体系中加入0.5-3μL的反应不同天数的DNA扩增样品，并设置对照和平行实验，置于20-30℃的环境中，反应2-20h；
4.3合成的Fluc活性的测定。
实施例5：不同浓度的phi29 DNA聚合酶扩增的DNA模板对体外蛋白合成体系的影响
5.1将DNA扩增体系中的phi29的浓度最高设置为0.8mg/mL，按照2/3的比例向低浓度稀释，共设置个20个不同的phi29浓度，利用这20个DNA扩增体系对含有eGFP编码DNA序列的模板质粒进行扩增，置于20-30℃的环境中，过夜静置反应；
5.2向30μL体外蛋白合成体系中加入0.5-3μL上述DNA扩增体系，放置在20-30℃的环境中，静置反应2-20h；
5.3合成的eGFP活性的测定。
实施例6：测定不同反应时间点的phi29 DNA扩增体系对体外蛋白合成体系的影响
6.1将以含有eGFP编码DNA序列的质粒为模板的phi29 DNA扩增体系在不同的反应时间点终止反应，并保存于-20℃待用，不同的反应时间点分别为0min、5min、10min、30min、1h、2h、4h、6h、8h、12h、16h和24h；
6.2向30μL体外蛋白合成体系中加入0.5-3μL上述不同反应时间点终止的DNA扩增体系，并设置对照和平行实验，置于在20-30℃的环境中，静置反应2-20h；
6.3合成的eGFP蛋白活性的测定。
实施例7：使用Western Blot的方法检测IVDTT合成的目标蛋白
7.1将用于合成eGFP蛋白的IVDTT体系通过加入Loading Buffer和加热的方 法完全变性；
7.2将上述变性的样品进行SDS-PAGE电泳，并转移到PVDF膜上；
7.3PVDF膜经过封闭，孵育一抗和二抗等步骤后，进行压片曝光处理，其中使用的一抗是特异性识别eGFP蛋白的抗体；
7.4对Western Blot的结果进行分析。
实施例8：使用荧光SDS-PAGE的方法检测IVDTT合成的目标蛋白
8.1将用于合成eGFP蛋白的IVDTT体系通过加入Loading Buffer的方法非完全变性；
8.2将上述变性的样品进行SDS-PAGE电泳；
8.3将上述SDS-PAGE胶置于凝胶成像系统中，并对其进行激发，拍摄荧光图像；
8.4对上述荧光SDS-PAGE图像进行分析。
实施例9：使用IVDTT体系合成蛋白Ubiquitin
9.1Ubiquitin蛋白是含有76个氨基酸残基，通过共价方式连接到其他蛋白上，构成一种重要的翻译后修饰，可以介导包括促进蛋白降解等多种不同的细胞进程。人体细胞中的Ubiquitin是由四种基因编码的，其中UBA52和RPS27A中含有单拷贝的Ubiquitin的编码序列，而另外两个基因UBB和UBC则含有多拷贝的Ubiquitin编码序列。
此处我们选择来源于基因RPS27A的Ubiquitin编码序列，cDNA序列是从Hela细胞转录组逆转录后产物中扩增得到的，并将其构建到IVDTT表达质粒中。
9.2在IVDTT表达质粒中，我们构建了一个eGFP的编码序列，位于目标蛋白编码序列的3’端，使表达出的蛋白为目标蛋白和eGFP的融合蛋白，中间使用含有9个氨基酸残基的肽段相连，可以通过检测合成的eGFP的量来快速确定目标蛋白的表达量。在本实施例中，该融合蛋白则为Ubiquitin和eGFP的融合蛋白，我们命名为Ubiquitin-eGFP。
9.3向含有phi29 DNA聚合酶的扩增体系中加入含有Ubiquitin-eGFP和eGFP编码序列的质粒，并将反应体系置于20-30℃的环境中，过夜反应。
9.4取1μL的上述DNA扩增体系，加入到30μL体外蛋白合成体系中，将不加入任何模板的体外蛋白合成体系作为阴性对照(negative control,NC)，每 个实验组设置3个平行实验，置于20-30℃的环境中，反应3-20h；
9.5合成的eGFP活性的测定。
实施例10：使用IVDTT体系合成蛋白p53的核心结构域
10.1蛋白p53是肿瘤抑制因子，能够与非常多种蛋白相互作用，在细胞中发挥非常重要的作用，本身也具有比较复杂的结构。P53核心结构域含有221个氨基酸残基，在很多种类的癌症细胞中，几乎所有的能够使p53失活的突变都位于核心结构域中，所以对这一结构域的研究对于了解癌症的发生具有重要作用。
10.2将p53核心结构域的编码序列构建入IVDTT的表达质粒中，编码含有eGFP的融合蛋白p53-eGFP。
10.3向含有phi29 DNA聚合酶的扩增体系中加入含有p53-eGFP和eGFP编码序列的质粒，并将反应体系置于20-30℃的环境中，过夜反应。
10.4取1μL的上述DNA扩增体系，加入到30μL体外蛋白合成体系中，将不加入任何模板的体外蛋白合成体系作为阴性对照(negative control,NC)，每个实验组设置3个平行实验，置于20-30℃的环境中，反应3-20h；
10.5合成的eGFP活性的测定。
实施例11：使用IVDTT体系合成膜蛋白β2AR
11.1G蛋白偶联受体(G protein coupled receptor,GPCR)是一类含有7个跨膜区的膜蛋白，能够接受外界信号，并将其传导到细胞内，通过下游的G蛋白复合物引起一系列不同的细胞进程，是一类非常重要的膜蛋白分子，是很多种药物的靶点蛋白，针对这类蛋白的研究显得至关重要。Beta2肾上腺素受体(Beta2-adrenergic receptor,β2AR)是被研究的比较多的一种GPCR蛋白，含有413个氨基酸残基，Robert J.Lefkowitz和Brian K.Kobilka因为对其研究被授予2012年诺贝尔化学奖。
11.2将β2AR的编码序列构建入IVDTT的表达质粒中，编码含有eGFP的融合蛋白β2AR-eGFP。
11.3向含有phi29 DNA聚合酶的扩增体系中加入含有β2AR-eGFP和eGFP编码序列的质粒，并将反应体系置于20-30℃的环境中，过夜反应。
11.4取1μL的上述DNA扩增体系，加入到30μL体外蛋白合成体系中，将不加入任何模板的体外蛋白合成体系作为阴性对照(negative control,NC)，每 个实验组设置3个平行实验，置于20-30℃的环境中，反应3-20h；
11.5合成的eGFP活性的测定。
实施例12：使用IVDTT体系合成膜蛋白水通道蛋白AQP1
12.1水通道蛋白1(Aquaporin1,AQP1)是一种含有6个跨膜螺旋的膜蛋白，含有269个氨基酸残基，一般会形成四聚体，但是每个单独的AQP1都能形成独立的水通道。水通道能够允许水分子沿着渗透压梯度快速转移，对于维持细胞渗透压具有重要作用。Peter Agre由于对AQP1的研究被授予2003年诺贝尔化学奖。
12.2将AQP1的编码序列构建入IVDTT的表达质粒中，编码含有eGFP的融合蛋白AQP1-eGFP。
12.3向含有phi29 DNA聚合酶的扩增体系中加入含有AQP1-eGFP和eGFP编码序列的质粒，并将反应体系置于20-30℃的环境中，过夜反应。
12.4取1μL的上述DNA扩增体系，加入到30μL体外蛋白合成体系中，将不加入任何模板的体外蛋白合成体系作为阴性对照(negative control,NC)，每个实验组设置3个平行实验，置于20-30℃的环境中，反应3-20h；
12.5合成的eGFP活性的测定。
实施例13：使用IVDTT合成干扰素IFN-α2A
13.1干扰素(interferon,IFN)是人体内一种重要的细胞因子，在一些病原体存在时，体内某些细胞能够合成和分泌干扰素，能够引起干扰素分泌的病原体包括病毒、细菌、寄生虫和癌细胞等。除了抵御病原体入侵外，干扰素还有其他功能，如活化一些免疫细胞和提高病原体的呈递效率。在临床上干扰素可以用于抗病毒和晚期癌症的治疗，所以对干扰素的研究和生产对于科研和临床应用均有非常重要的作用。在本实施例中，我们利用IVDTT体系合成干扰素IFN-α2A。
13.2将IFN-α2A的编码序列构建入IVDTT的表达质粒中，编码含有eGFP的融合蛋白IFN-α2A-eGFP。
13.3向含有phi29 DNA聚合酶的扩增体系中加入含有IFN-α2A-eGFP和eGFP编码序列的质粒，并将反应体系置于20-30℃的环境中，过夜反应。
13.4取1μL的上述DNA扩增体系，加入到30μL体外蛋白合成体系中，将不加入任何模板的体外蛋白合成体系作为阴性对照(negative control,NC)，每个实验组设置3个平行实验，置于20-30℃的环境中，反应3-20h；
13.5合成的eGFP活性的测定。
实施例14：使用IVDTT体系合成anti-PD-L1抗体Atezolizumab
14.1PD1(programmed death 1)和其两个底物PD-L1和PD-L2是T细胞介导的免疫应答的共抑制分子。在2016年和2017年，FDA批准了几个针对PD-L1的单克隆抗体应用到临床几种癌症的治疗上，如atezolizumab、durvalumab和avelumab等，而且越来越多的针对PD1和PD-L1的单克隆抗体进入临床实验。抗体等蛋白质药物在临床上的应用越来越广泛，针对蛋白质药物的生产的研究也越来越受到重视，如何高效和低成本的生产蛋白药物成为研究的热门方向。在本实施例中，我们利用IVDTT体系分别表达Atezolizumab的重链(Heavy chain,ate-H)和轻链(light chain,ate-L)。
14.2将ate-H和ate-L的编码序列构建入IVDTT的表达质粒中，编码含有eGFP的融合蛋白ate-H-eGFP和ate-L-eGFP。
14.3向含有phi29 DNA聚合酶的扩增体系中加入含有ate-H-eGFP、ate-L-eGFP和eGFP编码序列的质粒，并将反应体系置于20-30℃的环境中，过夜反应。
14.4取1μL的上述DNA扩增体系，加入到30μL体外蛋白合成体系中，将不加入任何模板的体外蛋白合成体系作为阴性对照(negative control,NC)，每个实验组设置3个平行实验，置于20-30℃的环境中，反应3-20h；
14.5合成的eGFP活性的测定。
实验结果
1.体外DNA-to-Protein(D2P)的合成体系的原理。
如图1所示，DNA复制，mRNA转录和蛋白翻译是自然界中心法则主要遗传信息传递途径，也是此专利的核心理论。
2.不同体积的phi29扩增体系对于体外蛋白合成效率的影响
从图2可以看出，0.5-3μL的Phi29 DNA聚合酶扩增的DNA均可以用作体外转录与翻译偶联的体外蛋白合成体系。其中，无论是0.5μL还是3μL的DNA扩增体系用于体外蛋白合成的结果与正对照无明显差异，相对光单位值(Relative Light Unit，RLU)达到1×10e9。
3.测定反应不同天数的phi29 DNA扩增体系对体外蛋白合成的影响
从图3可以看出，phi29 DNA聚合酶反应时间为1-28天，含有荧光素酶基因的质粒为1ng，所合成的DNA并没有因时间长而产量增加或是降低。NC为负对照，没有DNA的体外合成体系。PC为正对照，DNA来自于PCR反应，约500ng。1-28天的phi29 DNA聚合酶复制的DNA仍可以用作转录与翻译偶联的体外蛋白合成体系。
4.不同浓度的phi29 DNA聚合酶扩增的DNA模板对体外蛋白合成体系的影响
从图4可以看出，当phi29 DNA聚合酶反应浓度为0.5mg/mL-0.8ug/mL时，扩增得到的DNA模板均可以用作转录与翻译偶联的体外蛋白合成体系，尤其0.8mg/mL-0.4ug/mL的phi29 DNA聚合酶复制的DNA与正对照无显著差异。
5.测定不同反应时间点的phi29 DNA扩增体系对体外蛋白合成体系的影响
从图5可以看出，随着反应时间的延长，phi29扩增出的DNA量经历一个逐渐增加然后到达平台的过程，显示为合成的eGFP的量逐渐增加，然后达到一个平台。从合成的eGFP的量来判断，在反应进行6h后扩增的DNA量就已经达到平台。从图6可以看出，phi29扩增产生的DNA量同样在6h达到了最大值，与合成的eGFP产量的趋势类似。所以通常可以把phi29 DNA扩增体系的反应时间最少定为6h，通常为过夜反应。
6.使用Western Blot和荧光SDS-PAGE的方法检测IVDTT合成的目标蛋白
从图7可以看出，在加入含有eGFP编码序列质粒的IVDTT体系的泳道1中，Western Blot检测到的条带的分子量大小与eGFP的理论分子量(26.7KDa)非常接近，而且使用的是抗eGFP的抗体，综上IVDTT合成的目标蛋白是eGFP。从图8可以看出，在加入含有eGFP编码序列质粒的IVDTT体系的泳道1中，检测到的荧光条带的分子量大小与eGFP的理论分子量(26.7KDa)非常接近，而且使用的激发光和受激后的发射光的波长特征与eGFP类似，这表明IVDTT体系中检测到的荧光信号是由合成的eGFP蛋白发出的。综上所述，IVDTT体系能够合成正确折叠并能受激发出荧光的有活性的eGFP蛋白，而eGFP蛋白可以用于IVDTT体系合成目标蛋白的指示标签。
7.IVDTT体系能够合成蛋白质Ubiquitin
从图9可以看出，合成的融合蛋白Ubiquitin-eGFP受激发后发出的荧光值在3小时和20小时分别达到72RFU和88RFU，而阴性对照(NC)的荧光值分别为22RFU和35RFU，表明IVDTT体系中合成了Ubiquitin-eGFP。单独的eGFP蛋白作为阳性对照，其荧光值在3小时和20小时分别达到了207RFU和232RFU。
8.IVDTT体系能够合成p53核心结构域
从图10可以看出，合成的融合蛋白p53-eGFP受激发后发出的荧光值在3小时和20小时分别达到256RFU和354RFU，而阴性对照(NC)的荧光值分别为16RFU和35RFU，表明IVDTT体系中合成了p53-eGFP。单独的eGFP蛋白作为阳性对照，其荧光值在3小时和20小时分别达到了231RFU和363RFU。
9.IVDTT体系能够合成膜蛋白β2AR
从图11可以看出，合成的融合蛋白β2AR-eGFP受激发后发出的荧光值在3小时和20小时分别达到271RFU和362RFU，而阴性对照(NC)的荧光值分别为16RFU和35RFU，表明IVDTT体系中合成了β2AR-eGFP。单独的eGFP蛋白作为阳性对照，其荧光值在3小时和20小时分别达到了231RFU和363RFU。
10.IVDTT体系能够合成膜蛋白AQP1
从图12可以看出，合成的融合蛋白AQP1-eGFP受激发后发出的荧光值在3小时和20小时分别达到331RFU和491RFU，而阴性对照(NC)的荧光值分别为16RFU和35RFU，表明IVDTT体系中合成了AQP1-eGFP。单独的eGFP蛋白作为阳性对照，其荧光值在3小时和20小时分别达到了231RFU和363RFU。
11.IVDTT体系能够合成干扰素IFN-α2A
从图13可以看出，合成的融合蛋白IFN-α2A-eGFP受激发后发出的荧光值在3小时和20小时分别达到399 RFU和562 RFU，而阴性对照(NC)的荧光值分别为16RFU和35RFU，表明IVDTT体系中合成了IFN-α2A-eGFP。单独的eGFP蛋白作为阳性对照，其荧光值在3小时和20小时分别达到了231RFU和363RFU。
12.IVDTT体系能够分别合成anti-PD-L1的单克隆抗体Atezolizumab的重链 和轻链
从图14和图15可以看出，合成的融合蛋白ate-H-eGFP受激发后发出的荧光值在3小时和20小时分别达到96RFU和179RFU，合成的融合蛋白ate-L-eGFP受激发后发出的荧光值在3小时和20小时分别达到378RFU和544RFU，而阴性对照(NC)的荧光值分别为16RFU和35RFU，表明IVDTT体系中分别合成了ate-H-eGFP和ate-L-eGFP。单独的eGFP蛋白作为阳性对照，其荧光值在3小时和20小时分别达到了231RFU和363RFU。
13.体外DNA-to-Protein(D2P)的合成体系的优势
从图16和表1可以看出，体外DNA-to-Protein(D2P)的合成体系的优势在于用微量的DNA当模板，常温下快速地实现了体外蛋白的表达，并能长期保存，大大降低DNA的成本，缩短制备DNA模板所需的时间、步骤，提高体外蛋白质合成的效率。
表1.D2P技术的IVDTT与传统的以PCR制备DNA模板的IVTT的比较
Figure PCTCN2018080322-appb-000001
参考文献：
1.Wingfield,P.T.Overview of the purification of recombinant proteins.Current protocols in protein.science,2015；80:611-35.
2.Structural Genomics,C.,et al.Protein production and purification.Nature methods,2008；5:135-146.
3.Jermutus,L.,et al.Recent advances in producing and selecting  functional proteins by using cell-free translation.Current opinion in biotechnology,1998；9:534-548.
4.Katzen,F.,et al.The past,present and future of cell-free protein synthesis.Trends in biotechnology,2005；23:150-156.
5.Endo Y.,et al.Cell-Free Protein Production:Methods and Protocols,Methods in Molecular Biology,2010；vol.607.
6.Kubitschek,H.E.,and Friske,J.A.Determination of bacterial cell volume with the Coulter Counter.Journal of bacteriology,1986；168:1466-1467.
7.Philips R.,Physical biology of cell,2009；26.
8.https://www.thermofisher.com/cn/zh/home/references/ambion-tech-support/rna-tools-and-calculators/macromolecular-components-of-e.html
9.Sherman,F.Getting started with yeast.Methods in enzymology,2002；350:3-41.
10.Haddad,S.A.,and Lindegren,C.C.A method for determining the weight of an individual yeast cell.Applied microbiology,1953；1:153-156.
11.https://www.idtdna.com/pages/docs/educational-resources/molecular-facts-and-figures.pdf？sfvrsn＝4
12.http://book.bionumbers.org/what-is-the-macromolecular-composition-of-the-cell/
13.Morris,K.V.,and Mattick,J.S.The rise of regulatory RNA.Nature reviews.Genetics,2014；15:423-437
14.Wahlestedt,C.Targeting long non-coding RNA to therapeutically upregulate gene expression.Nature reviews.Drug discovery,2013；12:433-446
15.Samanta,M.P.,et al.Global identification of noncoding RNAs in Saccharomyces cerevisiae by modulating an essential RNA processing pathway.Proceedings of the National Academy of Sciences of the United States of America,2006；103:4192-4197
16.Niederer,R.O.,et al.sLong Noncoding RNAs in the Yeast S.cerevisiae.Advances in experimental medicine and biology,2017；1008: 119-132
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (14)

  1. 一种将DNA复制、转录、翻译偶联的无细胞合成体系，其特征在于，所述无细胞的合成体系包括：
    (a)DNA聚合酶；
    (b)解旋酶；
    (c)DNA结合蛋白；
    (d)用于合成DNA的底物；
    (e)RNA聚合酶；
    (f)用于合成RNA的底物；
    (g)用于合成蛋白的底物；
    (h)酵母细胞提取物。
  2. 如权利要求1所述的生物合成体系，其特征在于，所述无细胞的合成体系还包括选自下组的一种或多种组分：
    (j1)镁离子；
    (j2)钙离子；
    (j3)缓冲剂；
    (j4)能量再生系统。
    (j5)聚乙二醇；
    (j6)任选的外源蔗糖；和
    (j7)任选的溶剂，所述溶剂为水或水性溶剂。
  3. 如权利要求1所述的合成体系，其特征在于，所述的DNA聚合酶选自下组：phi29 DNA聚合酶、T7 DNA聚合酶、Bst DNA聚合酶、E.coli DNA聚合酶、DNA聚合酶I的Klenow片段、或其组合。
  4. 如权利要求1所述的合成体系，其特征在于，所述的解旋酶选自下组：T7噬菌体复制系统的解旋酶(4B)、UvrD解旋酶、或其组合。
  5. 如权利要求1所述的合成体系，其特征在于，所述的DNA结合蛋白选自下组：T4噬菌体基因32蛋白、RB49噬菌体基因32蛋白、T7噬菌体复制系统的单链结合蛋白，DNA结合蛋白7、或其组合。
  6. 如权利要求1所述的合成体系，其特征在于，所述的DNA聚合酶，解旋酶 及DNA结合蛋白包含DNA聚合酶的保真性，延伸速度，延伸能力等特性的分子改造，多种蛋白或者结构域的融合体，及根据应用于体外合成体系的需要所做出的改造或者多种组合。
  7. 一种体外偶联合成DNA,mRNA和蛋白质的方法，其特征在于，包括步骤：
    (i)提供一体外DNA复制体系，包括DNA聚合酶、外源的用于指导蛋白质合成的DNA分子、解旋酶、DNA结合蛋白、用于合成DNA的底物；
    (ii)在适合的条件，孵育步骤(i)经过T1，再与一转录与翻译偶联的无细胞合成体系结合，从而合成由所述外源DNA编码的DNA,mRNA和蛋白质；或
    (iii)在适合的条件，将所述DNA复制体系、转录体系、翻译体系直接混合，加入外源的用于指导蛋白质合成的DNA分子，经孵育后合成由外源DNA直接编码的蛋白质。
  8. 一种用于体外无细胞生物合成的试剂盒，其特征在于，包括：
    (k1)第一容器，以及位于第一容器内的细胞提取物；
    (k2)第二容器，以及位于第二容器内的DNA聚合酶、解旋酶、DNA结合蛋白；
    (kt)标签或说明书。
  9. 如权利要求8所述的试剂盒，其特征在于，还包括任选的选自下组的一个或多个容器：
    (k3)第三容器，以及位于第三容器的用于合成DNA的底物；
    (k4)第四容器，以及位于第四容器的用于合成RNA的底物；
    (k5)第五容器，以及位于第五容器的用于合成蛋白的底物；
    (k6)第六容器，以及位于第六容器的镁离子；
    (k7)第七容器，以及位于第七容器的钾离子；
    (k8)第八容器，以及位于第八容器的缓冲剂；
    (K9)第九容器，以及位于第九容器的聚乙二醇和蔗糖等。
  10. 一种无细胞合成体系，其特征在于，所述的合成体系将DNA复制、RNA转录和蛋白翻译偶联或整合在一个合成体系中。
  11. 一种体外无细胞合成蛋白的方法，其特征在于，包括步骤：
    (a)提供一混合体系，包括权利要求10所述的无细胞合成体系和外源的用于指导蛋白质合成的模板DNA，所述无细胞合成体系包括DNA复制体系和转录、翻译偶联的无细胞合成体系，在适合的条件，孵育所述的DNA复制体系一段时间T1， 将转录、翻译偶联的无细胞合成体系与该DNA复制体系结合，经孵育后合成由所述外源DNA编码的蛋白质。
  12. 一种体外无细胞合成蛋白的方法，其特征在于，包括步骤：
    (a)提供一权利要求10所述的无细胞合成体系和用于指导蛋白质合成的模板DNA，在适合的条件下，孵育所述的无细胞合成体系和用于指导蛋白质合成的模板DNA一段时间T1，从而合成由所述模板DNA编码的蛋白质。
  13. 一种用于体外无细胞合成的制剂，其特征在于，包括：
    (i)权利要求10所述的无细胞合成体系；和
    (ii)用于无细胞合成的辅助试剂。
  14. 一种用于体外无细胞合成的试剂盒，其特征在于，包括：
    (k1)第一容器，以及位于第一容器内的权利要求10所述的无细胞合成体系；和
    (kt)标签或说明书。
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113564214A (zh) * 2020-04-28 2021-10-29 康码（上海）生物科技有限公司 蛋白质体外合成纯化一体化制备方法、试剂盒及其应用
WO2024051855A1 (zh) * 2022-09-09 2024-03-14 康码（上海）生物科技有限公司 一种核酸构建物以及在ivtt体系中的应用

Families Citing this family (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111378706B (zh) * 2018-12-27 2022-07-19 康码（上海）生物科技有限公司 通过Edc3基因敲除改变体外蛋白合成能力的方法及其应用
CN111378708B (zh) * 2018-12-28 2022-07-19 康码（上海）生物科技有限公司 一种体外无细胞蛋白合成体系及其应用
CN111378707B (zh) * 2018-12-28 2022-06-21 康码（上海）生物科技有限公司 一种体外无细胞蛋白合成体系及其应用
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CN111484998B (zh) 2019-05-30 2023-04-21 康码（上海）生物科技有限公司 体外定量共表达多种蛋白的方法及其应用
EP4066931A4 (en) 2019-11-30 2024-02-14 Kangma-Healthcode (Shanghai) Biotech Co., Ltd BIOMAGNETIC MICROSPHERES AND PRODUCTION PROCESSES THEREOF AND USE THEREOF
CN110904135B (zh) * 2019-12-30 2021-08-13 苏州珀罗汀生物技术有限公司 蛋白质基础表达体系、合成系统及制备方法
CN113403360A (zh) * 2020-03-16 2021-09-17 康码（上海）生物科技有限公司 一种基于疏水界面的体外无细胞蛋白合成方法、d2p试剂盒及相关应用
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CN113493801A (zh) * 2020-04-08 2021-10-12 康码（上海）生物科技有限公司 一种含外源镁离子的体外无细胞蛋白合成体系和试剂盒及其应用
WO2021237410A1 (zh) * 2020-05-25 2021-12-02 深圳先进技术研究院 多蛋白体系的共生产方法、多蛋白体系的共生产系统及应用
WO2021237411A1 (zh) * 2020-05-25 2021-12-02 中国科学院深圳先进技术研究院 多蛋白体系的共生产方法、多蛋白体系的循环共生产系统及应用
CN116769048A (zh) 2021-12-31 2023-09-19 康码（上海）生物科技有限公司 聚合分子、包括其的单一结构和多聚结构
CN115197966A (zh) * 2022-08-15 2022-10-18 深圳源兴基因技术有限公司 一种利用生物反应器大规模生产重组痘苗病毒载体的方法
IL321137A (en) 2022-11-30 2025-07-01 Kangma Healthcode Shanghai Biotech Co Ltd Recombinant hemoglobin

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102732548A (zh) * 2012-05-16 2012-10-17 中国农业大学 一种表达蛇毒激肽原酶的高效麦胚无细胞蛋白合成系统的建立及应用
CN105734046A (zh) * 2014-12-12 2016-07-06 中国检验检疫科学研究院 无细胞表达系统制备Armored RNA的方法
CN106011163A (zh) * 2016-05-19 2016-10-12 武汉华美生物工程有限公司 一种无细胞表达信号蛋白的方法及表达系统

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100519758C (zh) * 2002-09-20 2009-07-29 新英格兰生物实验室公司 核酸的解旋酶依赖性扩增

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102732548A (zh) * 2012-05-16 2012-10-17 中国农业大学 一种表达蛇毒激肽原酶的高效麦胚无细胞蛋白合成系统的建立及应用
CN105734046A (zh) * 2014-12-12 2016-07-06 中国检验检疫科学研究院 无细胞表达系统制备Armored RNA的方法
CN106011163A (zh) * 2016-05-19 2016-10-12 武汉华美生物工程有限公司 一种无细胞表达信号蛋白的方法及表达系统

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
GAN, RUI ET AL.: "A Combined Cell -Free Transcription-Translation System from Saccharomyces Cerevisiae for Rapid and Robust Protein Synthesis", BIOTECHNOLOGY JOURNAL, vol. 9, no. 5, 31 May 2014 (2014-05-31), pages 641 - 651, XP055377623, ISSN: 1860-6768 *
YANG, YINGCHAO ET AL.: "Advances in Cell -free Protein Biosynthesis", CHINESE PHARMACEUTICAL AFFAIRS, vol. 25, no. 8, 31 August 2011 (2011-08-31), pages 832 - 835, ISSN: 1002-7777 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113564214A (zh) * 2020-04-28 2021-10-29 康码（上海）生物科技有限公司 蛋白质体外合成纯化一体化制备方法、试剂盒及其应用
WO2024051855A1 (zh) * 2022-09-09 2024-03-14 康码（上海）生物科技有限公司 一种核酸构建物以及在ivtt体系中的应用

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