Method for constructing multiple membraneless compartments in cell-free system

Abstract

A method for constructing multiple membraneless compartments in a cell-free system is characterized in that at least two membraneless compartments are constructed by adding phase separation protein into the cell-free system or by means of biosynthesis of the cell-free system. The membrane-free zone chamber constructed by the invention can controllably enrich specific active components and biochemical reactions in a cell-free system into the zone chamber and separate the specific active components and biochemical reactions from other complex metabolic reactions. Due to the high dynamic nature of the compartments, cooperation of the compartments can be achieved through the multiple membrane-free compartments, and regulation and control of the collection amount of compartment products are jointly completed.

Description

Technical Field

[0001] The present invention relates to a technology in the field of bioengineering, specifically a method for constructing multiple membrane-free compartments in a cell-free system. Background Art

[0002] The cell-free expression system (CFE) for in vitro synthesis of non-living proteins plays a pivotal role in the expansion of nucleic acid tools, complex protein production and artificial cell construction due to its openness, high throughput, rapidity and convenience. However, the existing CFE system will inevitably destroy the original compartmental state of the cell during the preparation process, so that the molecules and proteins originally located in each compartment are released into the solution environment, which leads to the mixing of its protein synthesis and metabolism and other functions. This "chaos" greatly limits its potential in the development of fine functions such as compartmental collaboration and spatiotemporal regulation.

[0003] At present, the optimization of cell-free systems is still mainly carried out in a “one-pot” environment. Many improvements, such as optimizing magnesium ions, still cannot solve the problem of mutual influence between different biochemical reactions, which makes it difficult for cell-free systems to achieve more precise spatiotemporal regulation and construct complex biochemical reaction networks. As the most promising solution to this problem, the construction methods of membrane-free compartments in cell-free systems are still extremely scarce. For example, the construction of a single membrane-free compartment is mainly achieved by evaporating and concentrating fluorescent proteins or temperature-induced condensation of proteins. The main drawbacks of these two methods are that the degree of compartmentalization is not high and specific temperature treatment is required, which leads to a decrease in the biological activity of the cell-free system, and the functional compatibility of the membrane-free compartment with the cell-free system is poor. As for the multi-membrane-free compartments, which are twice as difficult, no one has successfully constructed them in a cell-free system. Summary of the invention

[0004] The present invention aims to solve the problem that the existing technology cannot construct multiple membraneless compartments in a complex and dynamically changing cell-free system, and proposes a method for constructing multiple membraneless compartments in a cell-free system. The constructed membraneless compartments can controllably enrich specific active components and biochemical reactions in the cell-free system into the compartments and separate them from other complex metabolic reactions. Due to the high dynamics of the compartments, the multiple membraneless compartments can also achieve inter-compartmental collaboration and jointly complete the regulation of the collection amount of compartment products.

[0005] The present invention is achieved through the following technical solutions:

[0006] The present invention relates to a method for constructing multiple membrane-free compartments in a cell-free system, wherein at least two membrane-free compartments are constructed by adding phase separation proteins to the cell-free system or by biosynthesis of the cell-free system.

[0007] The cell-free system includes: prokaryotic cell extract, energy buffer and expression vector, which are derived from the document Kwon, Yong-Chan, and Michael C. Jewett. Scientific reports 5.1 (2015): 8663.

[0008] The added phase separation protein includes: adding arthropod-like elastic protein, Caulobacter crescentus cell pole organization Z protein (PopZ) or elastic protein-like polypeptide.

[0009] The phase separation protein is a protein with phase separation ability, including intrinsic disordered proteins and self-assembling proteins.

[0010] The exogenous addition concentration of the phase separation protein is 0.2 mg / mL-100 mg / mL, preferably 10 mg / mL-50 mg / mL.

[0011] The biosynthesis is carried out by adding the expression vectors of the cell pole organization Z (PopZ) protein of Caulobacter crescentus, recombinant spider silk protein, arthropod elastic protein and / or elastic protein polypeptide into the cell-free system for in vitro protein synthesis.

[0012] The temperature of the in vitro protein synthesis is 16°C-37°C, preferably 30°C.

[0013] The amount of the expression vector added in the cell-free system is 2.5ng-480ng, preferably 60ng-360ng, and more preferably 120ng-240ng.

[0014] The recombinant spider silk protein refers to a protein composed of several concatemers of the core amino acid sequence of the MaSpI protein conservative peptide segment (GRGGLGGQGAGAAAAAGGAGQGGYGGLGSQG) of the spider dragline silk protein monomer of the golden silk spider (Trichonephila clavipes).

[0015] The arthropod elastic protein-like protein refers to a protein composed of several concatemers of the arthropod elastic protein-like conserved peptide segment (GGRPSDSYGAPGGGN).

[0016] The elastin-like polypeptide refers to a protein composed of several concatemers of elastin-like polypeptide (GVGVP).

[0017] The expression vector of the elastin-like polypeptide refers to: an expression plasmid vector of 4-64 elastin-like polypeptide concatemers or a fusion protein expression vector composed of the elastin-like polypeptide concatemers and fluorescent proteins, RNA binding domains (RBDs) or enzymes. Technical Effects

[0018] The present invention realizes the construction of multiple membraneless compartments in a cell-free system by appropriately selecting and utilizing multiple phase separation proteins (cell pole organization Z protein of Caulobacter crescentus, recombinant spider silk protein, arthropod elastic protein, and elastic protein-like polypeptide), exogenously adding or biosynthesizing two or more of the above phase separation proteins in a cell-free system. The present invention controllably enriches specific active components and biochemical reactions in the environment into multiple different compartments in a cell-free system, and separates them from other complex metabolic reactions; realizes inter-compartmental collaboration of multiple membraneless compartments in a cell-free system, specifically, completing the regulation of the amount of product collected by one compartment on another compartment. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a schematic diagram of the process of forming multiple membraneless compartments by exogenously adding two phase separation proteins in Example 1;

[0020] Figure 2 This is a schematic diagram of multiple membraneless compartments formed by exogenously added phase separation protein R32 and PopZ in Example 1;

[0021] Figure 3 This is a schematic diagram of multiple membraneless compartments formed by exogenously adding phase separation proteins R32 and E64 in Example 1;

[0022] Figure 4 This is a schematic diagram of the process of forming multiple membraneless compartments with exogenously added phase separation proteins and biosynthesized phase separation proteins in Example 2;

[0023] Figure 5 This is a schematic diagram of multiple membraneless compartments formed by exogenously added phase separation protein R32 and biosynthetic phase separation protein I16 in Example 2;

[0024] Figure 6 This is a schematic diagram of the process of biosynthesizing two phase-separated proteins to form multiple membraneless compartments in Example 3;

[0025] Figure 7 Schematic diagram of multiple membraneless compartments formed by biosynthetic phase separation proteins R32 and E64 in Example 3;

[0026] Figure 8 Schematic diagram of multiple membraneless compartments formed by the biosynthetic phase separation protein R32 and PopZ in Example 3;

[0027] Fig. 9 Schematic diagram of multiple membraneless compartments formed by biosynthetic phase separation protein I16 and PopZ in Example 3;

[0028] Fig.10 Schematic diagram of the three membrane-less compartments of Example 4;

[0029] Fig.11 Schematic diagram of the enrichment and isolation of complex reactions between biomolecules and the outside world by multiple membraneless compartments in Example 5;

[0030] Fig.12 This is a schematic diagram of the design of the multi-membraneless compartment self-regulatory network regulating the amount of compartment protein signal collection in Example 5;

[0031] Fig.13 This is a schematic diagram of the cooperation between multiple membraneless compartments in Example 5;

[0032] Fig.14 This is a schematic diagram of the quantitative data of the cooperative effect between multiple membraneless compartments in Example 5. DETAILED DESCRIPTION Example 1

[0033] This example relates to a method for constructing multiple membraneless compartments in a cell-free system, and its main purpose is to demonstrate the first specific implementation method, that is, by exogenously adding two phase separation proteins to the cell-free system, such as arthropod elastic protein and cell pole organization Z protein of Caulobacter crescentus or arthropod elastic protein and elastic protein-like polypeptide, specifically including:

[0034] Step 1, construct plasmid pET19b-R32 of 32 arthropod elastic conserved peptide segments (GGRPSDSYGAPGGGN) concatemers, construct plasmid pET28a-popZ of the cell pole organization Z protein of Caulobacter crescentus, and construct plasmid pET19b-E64 of 64 elastic protein-like polypeptide (GVGVP) concatemers. The three expression vectors were introduced into Escherichia coli BL21 (DE3) cells for the production of R32 protein, PopZ protein and E64 protein, and the harvested cells were suspended in 20mM Tris-HCl buffer (pH=8.0) containing 150mM NaCl and 5mM imidazole, and then lysed using a high-pressure homogenizer. Subsequently, immobilized metal affinity chromatography (IMAC) was used to purify the target R32 protein, PopZ protein and E64 protein from the cell lysate.

[0035] Step 2: Figure 1 As shown, multiple membraneless compartments are constructed in a cell-free system by adding exogenous phase separation proteins, which can be achieved by any of the following methods:

[0036] A. Add R32 protein and PopZ protein at a final concentration of 20 mg / mL into the cell-free system. R32 protein and PopZ protein can form independent multi-membraneless compartments, such as Figure 2 As shown, the red color represents the membraneless compartment formed by the R32 protein, and the green color represents the membraneless compartment formed by the PopZ protein. It can be observed that the respective membraneless compartments exist independently of each other.

[0037] B. Add 20 mg / mL of R32 protein and 20 mg / mL of E64 protein into the cell-free system. R32 protein and E64 protein can form independent multi-membraneless compartments, such as Figure 3 As shown, the red color represents the membraneless compartment formed by the R32 protein, and the green color represents the membraneless compartment formed by the E64 protein. It can be observed that the respective membraneless compartments exist independently of each other. Example 2

[0038] This example relates to a method for constructing multiple membraneless compartments in a cell-free system, and its main purpose is to demonstrate the second specific implementation method, namely, biosynthesis of a phase separation protein in a cell-free system + exogenous addition of a phase separation protein or biosynthesis of a recombinant spider silk protein + exogenous addition of arthropod elastic protein, specifically including:

[0039] Step 1, construct plasmid pET19b-R32 of 32 conserved peptide segments (GGRPSDSYGAPGGGN) concatemers of arthropod elastic proteins. The expression vectors were introduced into Escherichia coli BL21 (DE3) cells for producing R32 protein, and the harvested cells were suspended in 20mM Tris-HCl buffer (pH=8.0) containing 150mM NaCl and 5mM imidazole, and then lysed using a high-pressure homogenizer. Immobilized metal affinity chromatography (IMAC) was then used to purify the target R32 protein from the cell lysate.

[0040] Step 2, respectively construct a protein expression vector pJL1-popZ composed of the cell pole organization Z protein of Caulobacter crescentus, whose amino acid sequence is shown in Seq ID No. 1, which is derived from the document Bowman, Grant R., et al. Cell 134.6 (2008): 945-955, and whose expression vector is a PopZ protein expression vector or a fusion protein expression vector composed of it and the target functional protein; and construct a protein expression vector pJL1-I16 composed of 16 concatemers of the core amino acid sequence of the MaSpI protein conserved peptide segment (GRGGLGGQGAGAAAAAGGAGQGGYGGLGSQG) of the spider dragline silk protein monomer of the golden silk spider (Trichonephila clavipes), whose amino acid sequence is shown in Seq ID No. 2, which is derived from the document Hinman, MB, Lewis, RVJ Biol Chem, 1992, 267, 19320-19324.

[0041] Step 3: Figure 4As shown in FIG. 1 , multiple membraneless compartments were constructed in a cell-free system. Specifically, 20 mg / mL of R32 protein and 120 ng of protein expression vector pJL1-I16 were added to the cell-free system and incubated at 30°C for 8 h to allow protein synthesis in the cell-free system, thereby constructing multiple membraneless compartments. Multiple membraneless compartments were observed by the above method, where red represents the R32 compartment and green represents the I16 compartment, as shown in FIG. Figure 5 shown. Example 3

[0042] This example relates to a method for constructing multiple membraneless compartments in a cell-free system, and its main purpose is to demonstrate a third specific implementation method, namely, biosynthesis of two phase-separated proteins, such as arthropod-like elastic protein and elastic protein-like polypeptide; arthropod-like elastic protein and cell pole organization Z protein of Caulobacter crescentus; recombinant spider silk protein and cell pole organization Z protein of Caulobacter crescentus, by a cell-free system, specifically including:

[0043] Step 1, construct the protein expression vector pJL1-R32 of 32 arthropod elastic protein conserved peptides (GGRPSDSYGAPGGGN) concatemers, construct the protein expression vector pJL1-E64 of 64 elastic protein-like polypeptides (GVGVP) concatemers, construct the protein expression vector pJL1-I16 of 16 spider dragline silk protein monomer core amino acid sequence MaSpI protein conserved peptides (GRGGLGGQGAGAAAAAGGAGQGGYGGLGSQG) concatemers, and construct the protein expression vector pJL1-popZ of the cell pole organization Z protein of Bacillus crescentus.

[0044] The amino acid sequence of the protein of 32 arthropod elastic protein-like conserved peptide segments (GGRPSDSYGAPGGGN) is shown in Seq ID No. 3, which is derived from the literature Ardell, DH, Andersen, SO Insect biochemistry and molecular biology, 2001, 31, 965-970.

[0045] The amino acid sequence of the protein of the 64 elastin-like polypeptide (GVGVP) concatemer is shown in SeqID No. 4, which is derived from the document Song WW., et al. ACS Macro Lett, 2021, 20; 10(4): 395-400.

[0046] Step 2: Figure 6 As shown, the phase separation protein plasmid expression vector is added into the cell-free system to construct multiple membrane-free compartments, which can be achieved by any of the following methods:

[0047] A. 120 ng of protein expression vector pJL1-R32 and 120 ng of protein expression vector pJL1-E64 were added to the cell-free system and incubated at 30°C for 8 h to allow protein synthesis in the cell-free system, thereby constructing multiple membraneless compartments.

[0048] Multiple membraneless compartments were observed by the above method, where red represents the E64 compartment and green represents the R32 compartment. Figure 7 shown.

[0049] B. Add 120 ng of protein expression vector pJL1-R32 and 120 ng of protein expression vector pJL1-popZ to the cell-free system and incubate at 30°C for 8 h to allow protein synthesis in the cell-free system, thereby constructing multiple membraneless compartments.

[0050] Multiple membraneless compartments were observed by the above method, where red represents the R32 compartment and blue represents the PopZ compartment, as shown in Figure 8 shown.

[0051] C. Add 60 ng of protein expression vector pJL1-I16 and 60 ng of protein expression vector pJL1-popZ to the cell-free system and incubate at 30°C for 8 h to allow protein synthesis in the cell-free system, thereby constructing multiple membraneless compartments.

[0052] Multiple membraneless compartments were observed by the above method, where green represents the I16 compartment and purple represents the PopZ compartment, as shown in Fig. 9 shown. Example 4

[0053] This example relates to a method for constructing multiple membraneless compartments in a cell-free system. The main purpose is to demonstrate the construction of three independent membraneless compartments in a cell-free system. The specific implementation method is to biosynthesize two phase-separated proteins, such as recombinant spider silk protein and cell pole organization Z protein of Caulobacter crescentus, through the cell-free system, and then exogenously add a phase-separated protein, such as arthropod elastic protein, to the cell-free system, specifically including:

[0054] Step 1, construct a protein expression vector pJL1-I16 consisting of 16 concatemers of the core amino acid sequence of the MaSpI protein conserved peptide segment (GRGGLGGQGAGAAAAAGGAGQGGYGGLGSQG) of the spider dragline silk protein monomer of the golden silk spider (Trichonephila clavipes), and construct a protein expression vector pJL1-popZ consisting of the cell pole organization Z protein of Caulobacter crescentus.

[0055] Step 2, construct plasmid pET19b-R32 of 32 conserved peptide segments (GGRPSDSYGAPGGGN) concatemers of arthropod elastic protein. The expression vectors were introduced into Escherichia coli BL21 (DE3) cells for the production of R32 protein, and the harvested cells were suspended in 20mM Tris-HCl buffer (pH=8.0) containing 150mM NaCl and 5mM imidazole, and then lysed using a high-pressure homogenizer. The target R32 protein was then purified from the cell lysate using immobilized metal affinity chromatography (IMAC).

[0056] Step 3: Construct three independent coexisting multi-membraneless compartments in the cell-free system. Add 120 ng of protein expression vector pJL1-I16, 120 ng of protein expression vector pJL1-popZ and 20 mg / mL of R32 protein into the cell-free system, and incubate at 30°C for 8 h to allow protein synthesis in the cell-free system, thereby constructing three multi-membraneless compartments. Three multi-membraneless compartments were observed by the above method, where red represents the R32 compartment, green represents the I16 compartment, and purple represents the PopZ compartment, as shown in Figure 2. Fig.10 shown. Example 5

[0057] This embodiment involves the application of multiple membraneless compartments in a cell-free system. The main purpose is to demonstrate that multiple membraneless compartments can controllably enrich specific active components and biochemical reactions in the environment into multiple different compartments in a cell-free system and separate them from other complex reactions. Secondly, by using this property, the inter-compartmental collaboration of multiple membraneless compartments is achieved in a cell-free system, specifically to complete the regulation of the amount of product collected by one compartment on another compartment, specifically including:

[0058] Step 1, construct plasmid pET19b-R32 of 32 arthropod elastic protein conserved peptide segments (GGRPSDSYGAPGGGN) concatemers, and construct plasmid pET28a-popZ of the cell pole organization Z protein of Caulobacter crescentus. The two expression vectors were introduced into Escherichia coli BL21 (DE3) cells for the production of R32 protein, PopZ protein and E64 protein, respectively. The harvested cells were suspended in 20mM Tris-HCl buffer (pH=8.0) containing 150mM NaCl and 5mM imidazole, and then lysed using a high-pressure homogenizer. Subsequently, immobilized metal affinity chromatography (IMAC) was used to purify the target R32 protein and PopZ protein from the cell lysate.

[0059] Step 2: Construct multiple membraneless compartments in the cell-free system by adding exogenous phase separation proteins. That is, add R32 protein and PopZ protein at a final concentration of 10 mg / mL to the cell-free system to construct multiple membraneless compartments independent of R32 protein and PopZ protein. Add 50 μM positively charged fluorescent small molecule Rh 6G, 2 μM biologically active lysozyme (Lys), and 240 ng of fluorescent protein protein expression vector pJL1-mTagBFP2. It was found that the PopZ compartment was enriched with the fluorescent small molecule Rh 6G, and the R32 compartment was enriched with lysozyme (Lys), and both compartments separated the fluorescent protein synthesized by the cell-free system outside the compartment, so that the biological molecules in the compartment were not affected by external complex reactions, such as Fig.11 As shown, red is the fluorescent small molecule Rh 6G, green is lysozyme (Lys), blue is the fluorescent protein synthesized by the cell-free system reaction, and the right figure is the quantitative data of the distribution of biomolecules.

[0060] The amino acid sequence of the fluorescent protein is shown in Seq ID No. 5, which is derived from the document Stark, JC et al. Adv. 7154, eaat5107 (2018).

[0061] Step 3. Construct an expression vector pJL1-popZRBD with the Caulobacter crescentus cell pole organizing protein Z protein (Polar Organizing Protein Z), a connecting peptide (amino acid composition of GGSGGSGGSGGS) and RNA-binding domains (RBDs), and add it to the modified cell-free system for in vitro protein synthesis to form the first membraneless compartment; add the R32 protein, a concatemer of 32 arthropod elastic protein conserved peptides (GGRPSDSYGAPGGGN), to the modified cell-free system to form the second membraneless compartment. Construct an expression vector pJL1-His-gfp for the fusion protein of 10 histidines and GFPmut3 protein. The design principle is that when the cell-free system expresses the pJL1-popZRBD plasmid to form a membraneless compartment, the compartment will restrict the localization of mRNA in the environment to its compartment, thereby not only self-regulating its own protein synthesis rate, but also affecting the collection amount of protein signals in another compartment. The experimental process is as follows. Fig.12 The above expression vector and protein were introduced into the modified cell-free system, reacted at 30°C for 4 hours, and the enrichment of the fluorescent protein in the membrane-free compartment was detected, as shown in FIG. Fig.13 As shown, the red color represents the membrane-free compartment formed by PopZ protein fused with RBD, and the green fluorescence represents the protein signal product of the cell-free system collected in another R32 compartment. The quantitative results are shown in Fig.14 As shown, the results clearly show that the self-regulatory network of the first membraneless compartment can regulate the collection amount of protein signal products in another membraneless compartment, thereby realizing inter-compartmental cooperation of multiple membraneless compartments in a cell-free system.

[0062] The amino acid sequence of the RBDs is shown in Seq ID No. 6, which is derived from the document Simon, Joseph R., et al. Molecular cell 75.1 (2019): 66-75.

[0001] Compared with the prior art, the present invention realizes the construction of multiple membraneless compartments in the cell-free system by selecting appropriate multiple phase separation proteins (Caulobacter crescentus cell pole organization Z protein, recombinant spider silk protein, arthropod elastic protein, and elastin-like polypeptide), exogenously adding or biosynthesizing two or more of the above phase separation proteins at appropriate concentrations and time in the cell-free system, which can make up for the deficiency of the cell-free system in moving towards advanced structural functions that rely on multi-compartmental collaboration, and also lays a solid foundation for the cell-free system to undertake fine spatiotemporal regulation and complex biochemical reaction networks.

[0002] The above-mentioned specific implementation can be partially adjusted in different ways by those skilled in the art without departing from the principle and purpose of the present invention. The protection scope of the present invention shall be based on the claims and shall not be limited by the above-mentioned specific implementation. Each implementation scheme within its scope shall be subject to the constraints of the present invention.

Claims

1. A method for constructing multiple membrane-free compartments in a cell-free system, characterized in that: At least two membrane-free compartments are constructed by adding phase separation proteins to the cell-free system and / or by biosynthesis in the cell-free system; The cell-free system comprises: prokaryotic cell extract, energy buffer and expression vector; The added phase separation protein includes: adding arthropod elastic protein, Caulobacter crescentus cell pole organizing Z protein (PopZ) and / or elastin-like polypeptide; The biosynthesis is carried out by adding the expression vectors of the cell pole organization Z protein of Caulobacter crescentus, recombinant spider silk protein, arthropod elastic protein and / or elastic protein polypeptide into the cell-free system to perform in vitro protein synthesis.

2. The method for constructing multiple membrane-free compartments in a cell-free system according to claim 1, characterized in that: The amino acid sequence of the cell pole organization Z protein of Caulobacter crescentus is shown in Seq ID No.

1.

3. The method for constructing multiple membrane-free compartments in a cell-free system according to claim 1, characterized in that: The recombinant spider silk protein refers to a protein composed of several concatemers of the core amino acid sequence of the MaSpI protein conservative peptide segment (GRGGLGGQGAGAAAAAGGAGQGGYGGLGSQG) of the spider dragline silk protein monomer of the golden silk spider (Trichonephila clavipes).

4. The method for constructing multiple membrane-free compartments in a cell-free system according to claim 1, characterized in that: The arthropod elastic protein-like protein refers to a protein composed of several concatemers of the arthropod elastic protein-like conserved peptide segment (GGRPSDSYGAPGGGN).

5. The method for constructing multiple membrane-free compartments in a cell-free system according to claim 1, characterized in that: The elastin-like polypeptide refers to a protein composed of several concatemers of elastin-like polypeptide (GVGVP).

6. The method for constructing multiple membrane-free compartments in a cell-free system according to claim 3 or 4, characterized in that: The number of concatemers in the recombinant spider silk protein and arthropod elastic protein-like protein is 4-64.

7. The method for constructing multiple membrane-free compartments in a cell-free system according to claim 5, characterized in that: The expression vector of the elastin-like polypeptide refers to: an expression plasmid vector of 4-64 elastin-like polypeptide concatemers or a fusion protein expression vector composed of the elastin-like polypeptide concatemers and fluorescent proteins, RNA binding domains (RBDs) or enzymes.

8. The method for constructing multiple membrane-free compartments in a cell-free system according to claim 1, characterized in that: The amino acid sequence of the recombinant spider silk protein is shown in Seq ID No. 2; The amino acid sequence of the arthropod elastic protein is shown in Seq ID No. 3; The amino acid sequence of the elastin-like polypeptide is shown in Seq ID No.

4.

9. The method for constructing multiple membrane-free compartments in a cell-free system according to claim 7, characterized in that: The amino acid sequence of the fluorescent protein is shown in Seq ID No.5; the amino acid sequence of the RBDs is shown in Seq ID No.

6.

10. The method for constructing multiple membrane-free compartments in a cell-free system according to claim 1, characterized in that: The exogenous addition concentration of the phase separation protein is 0.2 mg / mL-100 mg / mL; The amount of the expression vector added in the cell-free system is 2.5ng-480ng.

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