WO2018171747A1 - In vitro dna-to-protein (d2p) synthesis system, preparation, reagent kit, and preparation method - Google Patents

Abstract

Provided is an in vitro DNA-to-Protein (D2P) synthesis system, which is a cell-free protein synthesis system for DNA replication, transcription, and translation coupling. Also provided are a corresponding preparation, a reagent kit, and a method for in vitro cell-free synthesis of a protein.

Description

In vitro DNA-to-Protein (D2P) synthesis system, preparation, kit and preparation method thereof Technical field

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.

Background technique

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. With the development of science and technology, cell-free expression systems, also known as in vitro protein synthesis systems, have emerged. 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.

There are two commercial in vitro synthesis systems: one that synthesizes a protein using a pre-in vitro transcription of mRNA as a template, and the other that simultaneously performs in vitro transcription and in vitro translation, synthesizes mRNA using DNA as a template, and then synthesizes a protein. Both systems are complex and highly demanding, requiring the user to perform in vitro transcriptional experiments to obtain enough mRNA templates, or to provide a large amount of DNA or a high concentration of plasmids as templates (the quality of the template is even greater than that of the synthesis). The quality of the protein). These drawbacks increase the user's operating time, manufacturing costs, and experimental complexity, while also greatly limiting the efficiency and yield of biosynthesis. Therefore, the development of an in vitro biosynthesis system that is simpler, more convenient, more efficient, high-yield, and low-cost has become an urgent need in the field of basic biological research.

Summary of the invention

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:

(p1) an exogenous DNA synthesis system in vitro;

(p2) a synthetic DNA-to-mRNA synthesis system;

(p3) a synthetic mRNA-to-Protein synthesis system;

(p4) DNA sequence design associated with p1-p2-p3, enzyme design;

(p5) Systematic theoretical and quantitative evaluation of in vitro cell-free DNA-to-Protein (D2P) synthesis;

(p6) The establishment of a theoretical model for efficient protein synthesis in the D2P system.

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:

(a) DNA polymerase;

(b) helicase;

(c) a DNA binding protein;

(d) a substrate for the synthesis of DNA;

(e) RNA polymerase;

(f) a substrate for the synthesis of RNA;

(g) a substrate for the synthesis of a protein;

(h) a cell extract;

In another preferred embodiment, the cell-free protein synthesis system further comprises a component selected from the group consisting of:

(j1) magnesium ion;

(j2) calcium ions;

(j2) buffer;

(j3) energy regeneration system;

(j4) polyethylene glycol;

(j5) optional exogenous sucrose;

(j6) An optional solvent which is water or an aqueous solvent.

In another preferred embodiment, 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.

In another preferred embodiment, the helicase comprises: a helicase (4B), a UvrD helicase, and the like of the T7 bacteriophage replication system.

In another preferred embodiment, 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.

In another preferred embodiment, the substrate of the synthetic protein comprises: 1-20 natural amino acids, and unnatural amino acids.

In another preferred embodiment, the cell-free protein synthesis system further comprises an exogenous DNA molecule for directing protein synthesis.

In another preferred embodiment, the DNA molecule is linear.

In another preferred embodiment, the DNA molecule is cyclic.

In another preferred embodiment, the DNA molecule contains a sequence encoding a foreign protein.

In another preferred embodiment, the sequence encoding the foreign protein comprises a genomic sequence, a cDNA sequence.

In another preferred embodiment, the sequence encoding the foreign protein further comprises a promoter sequence, a 5' untranslated sequence, and a 3' untranslated sequence.

In another preferred embodiment, 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:

(i) a cell extract, a cell-free synthesis system, a DNA replication system, a mixed aqueous solution of a DNA transcription system or a lyophilized powder as described in the in vitro synthesis system;

(ii) Synthesize DNA, RNA and protein under suitable conditions.

(iii) 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.

In another preferred embodiment, in the steps (i) and (ii), the reaction temperature is 20-30 ° C and the reaction time is 2-12 h.

In another preferred embodiment, in the step (iii), the reaction temperature is 25 to 65 °C.

In another preferred embodiment, in the step (iii), 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:

(k1) a first container, and a yeast cell extract located in the first container;

(k2) a second container, and a DNA polymerase, a helicase, a DNA binding protein located in the second container;

(kt) label or instructions.

In another preferred embodiment, the first container, the second container, and the third container are the same container or different containers.

In another preferred embodiment, the kit further comprises an optional one or more containers selected from the group consisting of:

(k3) a third container, and a substrate for synthesizing DNA in the third container;

(k4) a fourth container, and a substrate for synthesizing RNA in the fourth container;

(k5) a fifth container, and a substrate for synthesizing the protein in the fifth container;

(k6) a sixth container, and magnesium ions in the sixth container;

(k7) a seventh container, and potassium ions in the seventh container;

(k8) an eighth container, and a buffer located in the eighth container.

(K9) a ninth container, and polyethylene glycol and sucrose in the ninth container;

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.

In another preferred embodiment, the synthetic system comprises:

(i) a DNA polymerization system;

(ii) an RNA transcription system; and

(iii) Protein translation system.

In another preferred embodiment, the cell-free synthesis system further comprises:

(iv) sugars; and

(v) a phosphate compound.

In another preferred embodiment, the cell-free synthesis system further comprises:

(j1) magnesium ion;

(j2) calcium ions;

(j3) a buffering agent;

(j4) Energy regeneration system.

(j5) polyethylene glycol;

(j6) optional exogenous sucrose; and

(j7) An optional solvent which is water or an aqueous solvent.

In another preferred embodiment, the cell-free synthesis system further comprises an optional template DNA.

In another preferred embodiment, 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.

In another preferred embodiment, the (ii) RNA transcription system comprises: (e) an RNA polymerase; and (f) a substrate for synthesizing RNA.

In another preferred embodiment, the (iii) protein translation system comprises: (g) a substrate for synthesizing a protein; and (h) a cell extract.

In another preferred embodiment, 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.

In another preferred embodiment, 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.

In another preferred embodiment, the cell extract comprises a yeast cell extract.

In another preferred embodiment, 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.

In another preferred embodiment, the yeast cell extract is an aqueous extract of yeast cells.

In another preferred embodiment, the yeast cell extract is free of yeast endogenous long chain nucleic acid molecules.

In another preferred embodiment, 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.

In another preferred embodiment, 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.

In another preferred embodiment, the buffering agent is selected from the group consisting of 4-hydroxyethylpiperazineethanesulfonic acid, trishydroxymethylaminomethane, or a combination thereof.

In another preferred embodiment, the substrate for the synthetic RNA comprises: a nucleoside monophosphate, a nucleoside triphosphate, or a combination thereof.

In another preferred embodiment, the substrate of the synthetic protein comprises: 1-20 natural amino acids, and unnatural amino acids.

In another preferred embodiment, the synthetic system is a three-in-one synthetic system of DNA replication, RNA transcription, and protein translation.

In another preferred embodiment, the amount of the template DNA in the DNA polymerization system is ≤ 1 ng, preferably ≤ 0.1 ng, more preferably ≤ 0.01 ng.

In another preferred embodiment, 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.

In another preferred embodiment, the template DNA is circular DNA.

In another preferred embodiment, the template DNA is plasmid DNA.

In another preferred embodiment, the plasmid DNA comprises tandem DNA elements capable of enhancing protein synthesis efficiency.

In another preferred embodiment, the plasmid DNA comprises the following elements: a yeast cell-derived IRES enhancer K1NCE102, an Ω sequence, and a yeast-specific Kozak sequence.

In another preferred embodiment, the synthetic system has a volume of from 10 to 50 microliters, preferably from 20 to 40 microliters.

In another preferred embodiment, in the DNA polymerization system, 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.

In another preferred embodiment, in the DNA polymerization system, the polymerase is phi29 DNA polymerase.

In another preferred embodiment, 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. .

In another preferred embodiment, the DNA replication, RNA transcription, and protein translation do not include the step of removing unnecessary proteins from the synthetic system.

In another preferred embodiment, the method does not include the step of removing unnecessary proteins (such as DNase, RNA polymerase) from the synthetic system.

In another preferred embodiment, the polyethylene glycol is selected from the group consisting of PEG3000, PEG 8000, PEG 6000, PEG 3350, or a combination thereof.

In another preferred embodiment, 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.

In another preferred embodiment, 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%.

In another preferred embodiment, 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.

In another preferred embodiment, the saccharide is selected from the group consisting of glucose, starch, glycogen, sucrose, maltose, cyclodextrin, or a combination thereof.

In another preferred embodiment, the concentration of the saccharide (mmol/L) is 10-100 mM, preferably 10-60 mM, preferably 20-50 mM, more preferably 20-30 mM.

In another preferred embodiment, 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.

In another preferred embodiment, 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.

In another preferred embodiment, 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.

In another preferred embodiment, the concentration of the phosphate compound (mmol/L) 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) providing 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:

Providing a cell-free synthesis system according to the fifth aspect of the invention and a template DNA for directing protein synthesis, and incubating the cell-free synthesis system and template DNA for guiding protein synthesis for a period of time T1 under suitable conditions Thereby synthesizing the protein encoded by the template DNA.

In another preferred embodiment, the template DNA is circular DNA.

In another preferred embodiment, the template DNA is plasmid DNA.

In another preferred embodiment, the method further comprises: (b) isolating or detecting the protein encoded by the template, optionally from the protein synthesis system.

In another preferred embodiment, in the step (a), the reaction temperature is 20 to 37 ° C, preferably 20 to 25 ° C.

In another preferred embodiment, in the step (b), the reaction time is 6-24 h, preferably 8-16 h.

In another preferred embodiment, the template DNA is used in an amount of ≤ 1 ng, preferably ≤ 0.1 ng, more preferably ≤ 0.01 ng.

In another preferred embodiment, 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:

(i) the cell-free synthesis system of the fifth aspect of the invention; and

(ii) Auxiliary reagents for cell-free synthesis.

In another preferred embodiment, the preparation is a mixed solution or a lyophilized powder.

In another preferred embodiment, the auxiliary agent is selected from the group consisting of:

(j1) magnesium ion;

(j2) calcium ions;

(j3) a buffering agent;

(j4) Energy regeneration system.

(j5) polyethylene glycol;

(j6) optional exogenous sucrose; and

(j7) An optional solvent which is water or an aqueous solvent.

In another preferred embodiment, the auxiliary reagent further comprises:

(a) sugars; and

(b) a phosphoric acid compound.

A ninth aspect of the invention provides a kit for in vitro cell-free synthesis comprising:

(k1) a first container, and the cell-free synthesis system of the fifth aspect of the invention located in the first container;

(kt) label or instructions.

In another preferred embodiment, the kit further comprises an optional one or more containers selected from the group consisting of:

(k2) a second container, and magnesium ions in the second container;

(k3) a third container, and potassium ions in the third container;

(k4) a fourth container, and a buffer in the fourth container;

(K5) a fifth container, and a polyethylene glycol in the fifth container;

(k6) a sixth container, and optionally exogenous sucrose located in the sixth container;

(K7) a seventh container, and an optional solvent in the seventh container, the solvent being water or an aqueous solvent;

(k8) an eighth container, and a saccharide located in the eighth container; and

(k9) a ninth container, and a phosphate compound located in the ninth container.

A tenth aspect of the invention provides a method of in vitro coupling of synthetic DNA, mRNA and protein, comprising the steps of:

(i) providing an in vitro DNA replication system according to the first aspect of the invention, comprising a DNA polymerase, an exogenous DNA molecule for directing protein synthesis, a helicase, a DNA binding protein, a substrate for synthesizing DNA;

(ii) under suitable conditions, 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;

(iii) Directly mixing the DNA replication system, the transcription system, and the translation system under suitable conditions, adding an exogenous DNA molecule for directing protein synthesis, and incubated to synthesize a protein directly 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:

(i) providing an in vitro DNA replication system according to the first aspect of the invention, comprising a DNA polymerase, exogenous

a DNA molecule, a helicase, a DNA binding protein for directing protein synthesis;

(ii) under suitable conditions, 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;

(iii) The foreign DNA can be directly encoded to synthesize the protein under suitable conditions without the need for an incubation step.

It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here.

DRAWINGS

The invention will now be further described with reference to the drawings and embodiments.

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.

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. As can be seen from the figure, 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, 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. As can be seen from the figure, 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, and 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. As can be seen from the figure, 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, and 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, and Lane 2 is the IVDTT system (negative control) to which no DNA template was added. In SDS-PAGE analysis, 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.

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 (RFU) of eGFP and eGFP alone in the fusion protein, and the synthesized eGFP alone as a positive control for the IVDTT system. 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 β2AR-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 (RFU) of eGFP and eGFP alone in the fusion protein, and the synthesized eGFP alone as a positive control for the IVDTT system. The relative fluorescence value of the synthesized β2AR-eGFP was significantly higher than that of the negative control, indicating that the fusion protein β2AR-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 (RFU) of eGFP and eGFP alone in the fusion protein, and the synthesized eGFP alone as a positive control for the IVDTT system. 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-α2A-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 (RFU) of 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 IFN-α2A-eGFP was significantly higher than that of the negative control, indicating that the fusion protein IFN-α2A-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). Relative fluorescence values (RFU) from eGFP and eGFP alone in the fusion protein synthesized after 20 h (shaded histogram), and synthetic eGFP alone as a positive control for the IVDTT system. 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.

detailed description

After extensive and in-depth research, through a large number of practices and explorations, a three-in-one system (DNA-to-Protein, D2P) optimized for DNA replication and cell-free transcription and translation in vitro has been invented. ). 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. Compared to all commercially cell-free protein expression systems, 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. Specifically, with the in vitro cell-free expression system of the present invention, 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. A preparation time of about 4-6 hours, a template cost of about 100-500 times.

I. Theoretical construction of DNA-to-Protein (D2P) coupled biosynthesis system

1. Scientific background

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. However, the synthesis and preparation of DNA, RNA, and especially proteins, which constitute biological core materials, remain at the level of 20 or 30 years ago, which greatly restricts the progress of related biological research and pharmaceutical research and development. The development of a high-yield, low-cost in vitro protein biosynthesis system has become an urgent need worldwide.

At present, 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]. With the development of science and technology, 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.

However, due to the cumbersome preparation of in vitro protein synthesis systems and the complexity of the addition of cofactors, all in vitro protein synthesis products on the market are less active and extremely expensive (100-1000 times more than conventional cell synthesis). It is currently only found in a small number of activity assays and experimental attempts in very few laboratories. The development of efficient, convenient and low-cost protein synthesis methods will undoubtedly become a steam locomotive that will promote the future bio-industry revolution, scientific and technological progress, pharmaceutical research and development, and drug production, which will have a revolutionary effect on the progress of the entire bio-industry.

Based on the inventors' basic theory in the field of protein synthesis, protein synthesis mechanisms in eukaryotic cells, their related enzymes, and catalytic functions, we believe that the most efficient biosynthesis system should come from the design of nature. .

2. Basic principles

In nature, cells will continue to divide, replicate, grow, and produce DNA, RNA, and protein in large quantities without interruption. Taking simple Escherichia coli (E. coli) as an example, according to their replication speed of 20-30 minutes, the cell volume is 1 cubic micron, the weight is 1 pg, the dry weight is 0.4 pg, the DNA is 0.017 pg, and the RNA is 0.10 pg. The rate of biosynthesis of protein 0.20 pg) was: 34 μg/mL/hour DNA, 200 μg/mL/hour RNA, 400 μg/mL/hour protein [6-8]. The copy amplification ratio was DNA: RNA: Protein = 1:5.9:12. 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 μg/mL/hour DNA, 36 μg/mL/hour RNA, 182 μg/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. For example, a virus is the most efficient replication in the biological world. However, when there is no host, it cannot completely complete its own DNA replication (RNA virus cannot complete RNA to DNA, then DNA replication), and the growth of the virus is completely stagnant. . This can also be seen from different species or from the amplification ratio of DNA to RNA to Protein: the replication amplification ratio of E. coli cells is DNA: RNA: Protein = 1:5.9:12, and the copying ratio of yeast cells is DNA. : RNA: Protein = 1:64:330, the ratio of replication of human cells is DNA: RNA: Protein = 1: 2: 20 [8]. If it is considered that about 75% and 80% of the genomes in S. cerevisiae and human cells can be transcribed, respectively, and in S. cerevisiae and human cells, non-coding RNA is transcribed. The proportion of RNA in excess of 95% and 98%, respectively, less than 5% (yeast) and 2% (human) RNA-encoded protein, the yeast cell and human replication amplification ratio is DNA: mRNA: Protein = 1: 4.27 : 440 and DNA: mRNA: Protein = 1:0.05:25 [13-16]. Therefore, it is only possible to maximize the efficiency of biosynthesis and the yield of final protein preparation by fully utilizing the entire "central rule" and establishing a complete DNA-to-Protein pathway.

Based on this, we propose a new DNA-to-Protein (D2P) coupled in vitro cell-free biosynthesis theory, which will copy three of the "central rules": DNA replication, DNA to RNA transcription, RNA to protein The translations are all in vitro, and the synergistic synthesis of DNA, RNA and Protein is carried out in a coupled manner to achieve the purpose of improving biosynthesis efficiency.

3. Technical architecture

Implementations of D2P technology include: nucleic acid amplification technology, RNA polymerization technology, and in vitro protein translation technology. 3a. 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. However, 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. Compared with PCR technology, 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. At the same time, 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.

The phi29 DNA polymerase used in the D2P in vitro synthesis system, 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. Molecular engineering, fusion with single-stranded DNA binding proteins, or a combination of several isothermal polymerases, is efficiently applied to the D2P synthesis system to create a highly efficient D2P coupled biosynthesis system.

3b. 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.

3c. In summary, the construction of D2P-coupled in vitro cell-free biosynthesis system to achieve the synthesis of trace DNA (nock-microgram) to a large number of proteins (microgram-mg) is theoretically feasible.

2. DNA-to-Protein (D2P) coupled in vitro cell-free biosynthesis system

In an embodiment, the in vitro synthesis system provided by the present invention 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.

In the present invention, 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.

In the present invention, 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.

In the present invention, 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.

In the present invention, 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.

Third, DNA-to-Protein (D2P) coupled in vitro cell-free synthesis system preparation

The present invention provides a cell-free synthetic system preparation for in vitro coupled DNA replication, transcription and translation, the formulation comprising:

(i) an aqueous solution or lyophilized powder of the cell extract;

(ii) an aqueous solution or lyophilized powder of the DNA replication reaction system;

(iii) an aqueous solution or lyophilized powder of the DNA transcription reaction system;

(iv) an aqueous solution or lyophilized powder of a cell-free synthesis system;

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.

Cell extract, cell-free synthesis system, DNA replication system, mixed lyophilized powder of DNA transcription reaction or combination of one or more lyophilized powder systems and aqueous solution system, under suitable conditions, thereby synthesizing DNA, RNA and protein . 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.

In the step, the temperature of the DNA replication reaction system is 25-65 ° C, and the reaction time of T1 is 2-6 h.

DNA-to-Protein (D2P)-conjugated in vitro cell-free protein synthesis kit

The invention provides a DNA-to-Protein (D2P) coupled kit for in vitro cell-free synthesis, comprising:

(k1) a first container, and a cell extract located in the first container;

(k2) a second container, and a DNA replication reaction system located in the second container;

(k3) an optional third container, and a DNA transcription reaction system located in the third container;

(kt) label or instructions.

In a preferred embodiment, 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.

In vitro expression 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.

In the present invention, 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).

Cell-free synthesis system in vitro

In a preferred embodiment, 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.

In the present invention, 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).

In the present invention, Kluyveromyces cerevisiae (e.g., Kluyveromyces lactis) is not particularly limited, and includes any Kluvi (e.g., Kluyveromyces lactis) strain capable of improving the efficiency of synthetic proteins.

In the present invention, the cell-free in vitro synthesis system comprises:

(i) a DNA polymerization system;

(ii) an RNA transcription system; and

(iii) Protein translation system.

In another preferred embodiment, the cell-free synthesis system further comprises:

(iv) sugars; and

(v) a phosphate compound.

In another preferred embodiment, the saccharide is selected from the group consisting of glucose, starch, glycogen, sucrose, maltose, cyclodextrin, or a combination thereof.

In another preferred embodiment, the concentration of the saccharide (mmol/L) is 10-100 mM, preferably 10-60 mM, preferably 20-50 mM, more preferably 20-30 mM.

In another preferred embodiment, 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.

In another preferred embodiment, 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.

In another preferred embodiment, 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.

In the present invention, 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.

In the present invention, 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. In addition, the yeast extract contains some other proteins in the cytoplasm derived from yeast cells, especially soluble proteins.

In the present invention, 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.

In the present invention, the preparation method of the yeast cell extract is not limited, and a preferred preparation method comprises the following steps:

(i) providing yeast cells;

(ii) washing the yeast cells to obtain washed yeast cells;

(iii) subjecting the washed yeast cells to cell disruption to obtain a crude yeast extract;

(iv) solid-liquid separation of the crude yeast extract to obtain a liquid fraction, that is, a yeast cell extract.

In the present invention, the solid-liquid separation method is not particularly limited, and a preferred mode is centrifugation.

In a preferred embodiment, the centrifugation is carried out in a liquid state.

In the present invention, 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.

In the present invention, 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.

In the present invention, the temperature of the centrifugation is not particularly limited. Preferably, the centrifugation is carried out at 1-10 ° C, preferably at 2-6 ° C.

In the present invention, 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.

In the present invention, 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. In the present invention, 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.

In a preferred embodiment, 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.).

In a preferred embodiment, the in vitro synthesis system further comprises sucrose. The 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. .

In a preferred embodiment, the in vitro synthesis system further contains heme. The 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.

In a preferred embodiment, the in vitro synthesis system further comprises spermidine. The 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.

In a preferred embodiment, 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. . In the present invention, 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). In the present invention, the type and content of the buffer are not particularly limited. Typically, 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 sub Spermine, 1%-10% polyethylene glycol, 10-100 mM glucose, 10-60 mM potassium phosphate.

Coding sequence of foreign protein (template DNA)

As used herein, 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. In the present invention, the DNA molecule is circular or plasmid DNA. The DNA molecule contains a sequence encoding a foreign protein. In the present invention, 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.

In the present invention, the selection of the exogenous DNA is not particularly limited. Usually, 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.

In a preferred embodiment, 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 (β-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.

In the present invention, 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 main advantages of the invention include:

1) The first three-step amplification coupling reaction from DNA to RNA to protein was performed in a system in vitro.

2) The system of the present invention can be used to simultaneously synthesize DNA, RNA, and protein in vitro.

3) The system of the present invention can be used to rapidly generate a target protein directly from a very small amount of DNA template.

4) 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.

5) 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.

6) 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.

7) 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.

8) 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. Compared with the traditional in vitro protein expression system, 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.

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. Experimental methods in which the specific conditions are not indicated in the following examples are generally carried out according to the conditions described in conventional conditions, for example, Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturing conditions. The conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight and parts by weight.

Unless otherwise stated, the materials and reagents used in the examples of the present invention are all commercially available products.

Example 1: Amplification of a plasmid template using phi29 DNA polymerase

1.1 Preparation of DNA amplification system: random primer with a final concentration of 20-30 μM, plasmid template of 0.05-0.15 μg/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).

1.2 Amplification reaction of plasmid template DNA in vitro: The above reaction system is placed in an environment of 20-30 ° C, and allowed to stand for 6-12 h, usually overnight reaction.

Example 2: In vitro protein synthesis using amplified template DNA

2.1 In vitro protein synthesis system: final concentration of 22 mM 4-hydroxyethyl piperazine ethanesulfonic acid, pH 7.4, 30-150 mM potassium acetate, 1.0-5.0 mM magnesium acetate, 1.5-4 mM mixture of nucleoside triphosphates (adenine nucleus) Glycoside triphosphate, guanosine triphosphate, cytidine triphosphate and uridine triphosphate), 0.08-0.24 mM amino acid mixture (glycine, alanine, valine, leucine, isoluminescence) Acid, phenylalanine, valine, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, Lysine, arginine and histidine), 25 mM creatine phosphate, 1.7 mM dithiothreitol, 0.27 mg/mL phosphocreatine kinase, 0.027-0.054 mg/mL T7 RNA polymerase, 1%-4 % polyethylene glycol, 0.5%-2% sucrose, and finally 50% by volume of yeast cell extract;

2.2 in vitro protein synthesis reaction: adding 0.5-3 μL of the amplified template DNA to the above 30 μL reaction system, placed in an environment of 20-30 ° C, and allowed to stand for 2-20 h;

2.3 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.

2.4 Determination of green fluorescent protein (eGFP) activity: After the reaction was completed, 10 μL of the reaction system was added to a 384-well blackboard and placed in a Tecan Infinite F200 fluorescence spectrophotometer to detect the relative fluorescence unit value emitted by the green fluorescent protein after excitation ( Relative Fluorescence Unit (RFU) is used as the unit of activity, as shown in Figures 4 and 5.

Example 3: Effect of different volumes of phi29 amplification system on in vitro protein synthesis efficiency

3.1 Amplification of a template plasmid containing the Lucc-encoding DNA sequence using phi29 DNA polymerase, placed in an environment of 20-30 ° C, and allowed to stand overnight;

3.2 Take 0.5, 1, 2 and 3 μL of the above DNA amplification system, add to 30 μL of in vitro protein synthesis system, and add template DNA prepared by ordinary PCR method as positive control (PC), without adding any The in vitro protein synthesis system of the template was used as a negative control (NC), and each experiment group was set up with 3 parallel experiments, placed in an environment of 20-30 ° C, and reacted for 2-20 h;

3.3 Determination of the synthetic Fluc activity.

Example 4: Effect of phi29 DNA amplification system for different days of reaction on protein synthesis in vitro

4.1 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;

4.2 Adding 0.5-3 μL of DNA amplification samples with different days of reaction to 30 μL of in vitro protein synthesis system, and setting up control and parallel experiments, placed in an environment of 20-30 ° C, and reacting for 2-20 h;

4.3 Determination of the synthesized Fluc activity.

Example 5: Effect of different concentrations of phi29 DNA polymerase amplified DNA template on in vitro protein synthesis system

5.1 Set the concentration of phi29 in the DNA amplification system to 0.8mg/mL, dilute to a low concentration in a ratio of 2/3, set a total of 20 different phi29 concentrations, and use the 20 DNA amplification systems to contain The template plasmid of the eGFP-encoding DNA sequence is amplified, placed in an environment of 20-30 ° C, and allowed to stand overnight;

5.2 Add 0.5-3 μL of the above DNA amplification system to a 30 μL in vitro protein synthesis system, place in an environment of 20-30 ° C, and let the reaction stand for 2-20 h;

5.3 Determination of synthetic eGFP activity.

Example 6: Effect of phi29 DNA amplification system at different reaction time points on in vitro protein synthesis system

6.1 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;

6.2 Adding 0.5-3 μL of DNA amplification system terminated at different reaction time points to 30 μL of in vitro protein synthesis system, and setting up a control and parallel experiment, placed in an environment of 20-30 ° C, and allowed to react for 2-20 h;

6.3 Determination of the activity of the synthesized eGFP protein.

Example 7: Detection of target protein synthesized by IVDTT using Western Blot

7.1 The IVDTT system for the synthesis of eGFP protein is completely denatured by the addition of Loading Buffer and heating;

7.2 The above denatured sample is subjected to SDS-PAGE electrophoresis and transferred to a PVDF membrane;

7.3 After the PVDF membrane is blocked, 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;

7.4 Analysis of the results of Western Blot.

Example 8: Detection of target protein synthesized by IVDTT using fluorescent SDS-PAGE

8.1 The IVDTT system used to synthesize eGFP protein is not completely denatured by the method of adding Loading Buffer;

8.2 performing the above denatured sample by SDS-PAGE electrophoresis;

8.3 placing the above SDS-PAGE gel in a gel imaging system and exciting it to take a fluorescent image;

8.4 Analysis of the above fluorescent SDS-PAGE image.

Example 9: Synthesis of protein Ubiquitin using the IVDTT system

The 9.1 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.

Here we selected the Ubiquitin coding sequence derived from the gene RPS27A, which was amplified from the reverse transcription product of the Hela cell transcriptome and constructed into the IVDTT expression plasmid.

9.2 In 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. In this example, the fusion protein is a fusion protein of Ubiquitin and eGFP, which we named Ubiquitin-eGFP.

9.3 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.

9.4 Take 1 μL of the above DNA amplification system, add to 30 μL of in vitro protein synthesis system, use in vitro protein synthesis system without any template as negative control (NC), set up 3 parallel experiments in each experimental group. In the environment of 20-30 ° C, the reaction is 3-20h;

9.5 Determination of synthetic eGFP activity.

Example 10: Synthesis of the core domain of protein p53 using the IVDTT system

10.1 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.

10.2 The coding sequence of the p53 core domain was constructed into the expression plasmid of IVDTT, encoding the fusion protein p53-eGFP containing eGFP.

10.3 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.

10.4 Take 1 μL of the above DNA amplification system, add to 30 μL of in vitro protein synthesis system, and use in vitro protein synthesis system without any template as negative control (NC). Set up 3 parallel experiments in each experimental group. In the environment of 20-30 ° C, the reaction is 3-20h;

10.5 Determination of synthetic eGFP activity.

Example 11: Synthesis of membrane protein β2AR using IVDTT system

11.1G G protein coupled receptor (GPCR) 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 (β2AR) 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.

11.2 The coding sequence of β2AR was constructed into the expression plasmid of IVDTT, encoding the fusion protein β2AR-eGFP containing eGFP.

11.3 A plasmid containing the coding sequence of β2AR-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.

11.4 Take 1 μL of the above DNA amplification system, add to 30 μL of in vitro protein synthesis system, and use in vitro protein synthesis system without any template as negative control (NC). Set up 3 parallel experiments in each experimental group. In the environment of 20-30 ° C, the reaction is 3-20h;

11.5 Determination of synthetic eGFP activity.

Example 12: Synthesis of membrane protein aquaporin AQP1 using the IVDTT system

12.1 Aquaporin 1 (AQP1) 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.

12.2 The coding sequence of AQP1 was constructed into the expression plasmid of IVDTT, encoding the fusion protein AQP1-eGFP containing eGFP.

12.3 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.

12.4 Take 1 μL of the above DNA amplification system, add to 30 μL of in vitro protein synthesis system, and use in vitro protein synthesis system without any template as negative control (NC). Set up 3 parallel experiments in each experimental group. In the environment of 20-30 ° C, the reaction is 3-20h;

12.5 Determination of synthetic eGFP activity.

Example 13: Synthesis of interferon IFN-α2A using IVDTT

13.1 Interferon (IFN) is an important cytokine in the human body. In the presence of some pathogens, certain cells in the body can synthesize and secrete interferon, and pathogens that cause interferon secretion include viruses, bacteria, parasites and Cancer cells, etc. 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. In this example, we synthesized the interferon IFN-α2A using the IVDTT system.

13.2 The coding sequence of IFN-α2A was constructed into the expression plasmid of IVDTT, encoding the fusion protein IFN-α2A-eGFP containing eGFP.

13.3 A plasmid containing the IFN-α2A-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.

13.4 Take 1 μL of the above DNA amplification system, add to 30 μL of in vitro protein synthesis system, and use in vitro protein synthesis system without any template as negative control (NC). Set up 3 parallel experiments in each experimental group. In the environment of 20-30 ° C, the reaction is 3-20h;

13.5 Determination of synthetic eGFP activity.

Example 14: Synthesis of anti-PD-L1 antibody Atezolizumab using IVDTT system

14.1 PD1 (programmed death 1) and its two substrates, PD-L1 and PD-L2, are co-inhibitory molecules of T cell-mediated immune responses. In 2016 and 2017, 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. In this example, we used the IVDTT system to express the heavy chain (Healy chain, ate-H) and light chain (ate-L) of Atezolizumab, respectively.

14.2 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.

14.3 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.

14.4 Take 1 μL of the above DNA amplification system, add to 30 μL of in vitro protein synthesis system, and use in vitro protein synthesis system without any template as negative control (NC). Set up 3 parallel experiments in each experimental group. In the environment of 20-30 ° C, the reaction is 3-20h;

14.5 Determination of synthetic eGFP activity.

Experimental result

1. Principle of in vitro DNA-to-Protein (D2P) synthesis system.

As shown in Figure 1, 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.

2. Effect of different volume of phi29 amplification system on protein synthesis efficiency in vitro

As can be seen from Figure 2, 0.5-3 μL of Phi29 DNA polymerase amplified DNA can be used as an in vitro protein synthesis system coupled with in vitro transcription and translation. Among them, the results of the protein amplification system of 0.5 μL or 3 μL for in vitro protein synthesis were not significantly different from those of the positive control, and the relative light unit (RLU) reached 1×10e9.

3. Determination of the effect of phi29 DNA amplification system on protein synthesis in vitro on different days of reaction

As can be seen from Fig. 3, 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.

4. Effects of different concentrations of phi29 DNA polymerase amplified DNA template on in vitro protein synthesis system

As can be seen from Figure 4, when 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.

5. Determination of the effect of phi29 DNA amplification system on protein synthesis system in vitro at different reaction time points

It can be seen from Fig. 5 that as the reaction time prolongs, the amount of DNA amplified by phi29 undergoes a process of gradually increasing and then reaching the platform, showing that the amount of synthetic eGFP is gradually increased, and then reaches a platform. Judging from the amount of synthesized eGFP, the amount of DNA amplified after 6 hours of reaction has reached the platform. As can be seen from Figure 6, the amount of DNA produced by phi29 amplification also reached a maximum at 6 h, similar to the trend in synthetic eGFP production. Therefore, the reaction time of the phi29 DNA amplification system can usually be set at least 6 hours, usually overnight.

6. Detection of target proteins synthesized by IVDTT using Western Blot and fluorescent SDS-PAGE

As can be seen from Figure 7, in lane 1 of the IVDTT system containing the plasmid containing the eGFP coding sequence, 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. As can be seen from Fig. 8, in the lane 1 of the IVDTT system to which the plasmid containing the eGFP coding sequence was added, 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. In summary, 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.

7. IVDTT system can synthesize protein Ubiquitin

As can be seen from Figure 9, 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.

8. IVDTT system can synthesize p53 core domain

As can be seen from Fig. 10, 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.

9. IVDTT system can synthesize membrane protein β2AR

As can be seen from Fig. 11, the fluorescence value of the synthesized fusion protein β2AR-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. β2AR-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

As can be seen from Fig. 12, 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.

11. IVDTT system can synthesize interferon IFN-α2A

As can be seen from Fig. 13, the fluorescence value of the synthesized fusion protein IFN-α2A-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-α2A-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.

12. The IVDTT system is capable of synthesizing the heavy and light chains of the anti-PD-L1 monoclonal antibody Atezolizumab, respectively.

As can be seen from Fig. 14 and Fig. 15, 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.

13. Advantages of in vitro DNA-to-Protein (D2P) synthesis system

As can be seen from Figure 16 and Table 1, 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.

Table 1. Comparison of IVDTT of D2P technology with traditional IVTT for PCR preparation of DNA template

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5. Endo Y., et al. Cell-Free Protein Production: Methods and Protocols, Methods in Molecular Biology, 2010; vol.

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12.http://book.bionumbers.org/what-is-the-macromolecular-composition-of-the-cell/

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All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims (14)

1. A cell-free synthesis system for DNA replication, transcription, and translation coupling, wherein the cell-free synthesis system comprises:

   (a) DNA polymerase;

   (b) helicase;

   (c) a DNA binding protein;

   (d) a substrate for the synthesis of DNA;

   (e) RNA polymerase;

   (f) a substrate for the synthesis of RNA;

   (g) a substrate for the synthesis of a protein;

   (h) Yeast cell extract.
2. The biosynthetic system according to claim 1 wherein said cell-free synthesis system further comprises one or more components selected from the group consisting of:

   (j1) magnesium ion;

   (j2) calcium ions;

   (j3) a buffering agent;

   (j4) Energy regeneration system.

   (j5) polyethylene glycol;

   (j6) optional exogenous sucrose; and

   (j7) An optional solvent which is water or an aqueous solvent.
3. The synthesis system according to claim 1, wherein said DNA polymerase is selected from the group consisting of phi29 DNA polymerase, T7 DNA polymerase, Bst DNA polymerase, E. coli DNA polymerase, and DNA polymerase. a Klenow fragment of I, or a combination thereof.
4. The synthetic system according to claim 1 wherein said helicase is selected from the group consisting of a helicase (4B), a UvrD helicase, or a combination thereof of the T7 bacteriophage replication system.
5. The synthetic system according to claim 1, wherein said DNA binding protein is selected from the group consisting of T4 phage gene 32 protein, RB49 phage gene 32 protein, T7 phage replication system single-stranded binding protein, DNA binding protein 7, or a combination thereof.
6. The synthetic system according to claim 1, wherein said DNA polymerase, helicase and DNA-binding protein comprise molecular fibrils of properties such as fidelity, elongation rate, and elongation ability of DNA polymerase, and various proteins. Or a fusion of domains, and modifications or combinations made according to the needs of the in vitro synthesis system.
7. A method for in vitro coupling of synthetic DNA, mRNA and protein, comprising the steps of:

   (i) providing 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;

   (ii) under suitable conditions, 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 exogenous DNA;

   (iii) directly mixing the DNA replication system, transcription system, and translation system under appropriate conditions, adding an exogenous DNA molecule for directing protein synthesis, and culturing a protein directly encoded by the foreign DNA after incubation.
8. A kit for in vitro cell-free biosynthesis, comprising:

   (k1) a first container, and a cell extract located in the first container;

   (k2) a second container, and a DNA polymerase, a helicase, a DNA binding protein located in the second container;

   (kt) label or instructions.
9. The kit of claim 8 further comprising an optional one or more containers selected from the group consisting of:

   (k3) a third container, and a substrate for synthesizing DNA in the third container;

   (k4) a fourth container, and a substrate for synthesizing RNA in the fourth container;

   (k5) a fifth container, and a substrate for synthesizing the protein in the fifth container;

   (k6) a sixth container, and magnesium ions in the sixth container;

   (k7) a seventh container, and potassium ions in the seventh container;

   (k8) an eighth container, and a buffer in the eighth container;

   (K9) a ninth container, and polyethylene glycol and sucrose in the ninth container.
10. A cell-free synthesis system characterized in that the synthetic system couples or integrates DNA replication, RNA transcription and protein translation into a synthetic system.
11. A method for cell-free synthesis of proteins in vitro, comprising the steps of:

    (a) providing a mixed system comprising the cell-free synthesis system of claim 10 and exogenous template DNA for directing protein synthesis, the cell-free synthesis system comprising a DNA replication system and no transcription or translation coupling a cell synthesis system, incubated the DNA replication system for a period of time T1 under suitable conditions, and combining a transcriptionally and translationally coupled cell-free synthesis system with the DNA replication system, and incubated to encode the foreign DNA encoding protein.
12. A method for cell-free synthesis of proteins in vitro, comprising the steps of:

    (a) providing a cell-free synthesis system according to claim 10 and a template DNA for directing protein synthesis, and incubating said cell-free synthesis system and template DNA for guiding protein synthesis for a period of time under suitable conditions T1, thereby synthesizing the protein encoded by the template DNA.
13. A preparation for cell-free synthesis in vitro, comprising:

    (i) the cell-free synthesis system of claim 10;

    (ii) Auxiliary reagents for cell-free synthesis.
14. A kit for in vitro cell-free synthesis, comprising:

    (k1) a first container, and the cell-free synthesis system of claim 10 located in the first container;

    (kt) label or instructions.

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