Application of controllable delivery of multi-membraneless-zone interventricular substances in cell-free system

Abstract

According to the application of the controllable delivery of the multi-membraneless compartment interlamellar substances in the cell-free system, two independent and coexisting protein enrichment phases are rapidly formed in the optimized cell-free system by utilizing the protein expression of a protein expression vector and adding histidine tag (His Tag) protein, namely, after the multi-membraneless compartment is formed, the two independent and coexisting protein enrichment phases are separated from the cell-free system, and the cell-free system can be used for controlling the delivery of the multi-membraneless compartment interlamellar substances in the cell-free system. And controllable delivery of micromolecular substances and macromolecular substances in multiple membrane-free zone chambers is carried out. The multiple membrane-free compartments constructed by the invention can controllably enrich specific active components and biochemical reactions in a cell-free system into the compartments and separate the specific active components and biochemical reactions from other complex metabolic reactions, and due to the high dynamic nature of the compartments, controllable communication between biomolecules and the external environment and among multiple compartments can be realized; further, the accuracy and controllability of metabolic reaction are greatly improved, and a solid foundation is laid for application of a cell-free system in the aspects of complex metabolic pathway exploration, multi-enzyme catalysis, drug delivery and the like.

Description

Technical Field

[0001] The present invention relates to a technology in the field of bioengineering, specifically an application of controllable delivery of substances between multiple membrane-free compartments in a cell-free system. Background Art

[0002] As a popular in vitro system for the synthesis of non-living proteins, the cell-free system plays an irreplaceable role in the fields of toxic protein synthesis, metabolic pathway mining, artificial cells, etc., with its advantages such as no cell wall, strong tolerance and high throughput. However, the lack of some key functions of the cell-free system has seriously weakened its application potential. One of the most important is that cells can achieve multi-partitioning and positioning of complex molecules and biochemical reactions within themselves through multi-compartmentalization and material delivery and communication between compartments, so that they can accurately and efficiently complete their respective biological functions. However, the cell-free system has to go through processes such as cell disruption and high-speed centrifugation during preparation, which causes the cell-free system to lose its multi-compartmental structure, making it impossible to achieve controllable delivery of substances between compartments, thereby limiting its application in biological functions.

[0003] Currently, there is no method to achieve controlled delivery of substances between multiple membraneless compartments in a cell-free system. Because of the complex protein synthesis and metabolic reactions in the cell-free system, the substance delivery between compartments that is mostly completed in a simple chemical buffer system is difficult to replicate in the cell-free system, which means that the current cell-free system is still carried out in a one-pot reaction environment. The optimization of the cell-free system is more focused on the optimization of a certain component, simple protein, or metabolic pathway, and cannot fundamentally solve the problem of the intermingling of many functions such as protein synthesis and metabolism in the cell-free system. This confusion will greatly limit the application of the cell-free system in the exploration of complex metabolic pathways, multi-enzyme catalysis, and drug delivery. Summary of the invention

[0004] In response to the gap in the prior art that is unable to construct multiple membraneless compartments in a complex and dynamically changing cell-free system and to endow the cell-free system with advanced biological functions through the multiple membraneless compartments, the present invention proposes an application of controllable delivery of substances between multiple membraneless compartments in a cell-free system. The constructed multiple 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, controllable communication between biological molecules and the external environment and multiple compartments can also be achieved, thereby greatly improving the accuracy and controllability of metabolic reactions and improving the one-pot reaction mode inherent in traditional cell-free systems.

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

[0006] The present invention relates to an application of controllable delivery of substances between multiple membraneless compartments in a cell-free system, wherein the following are sequentially added into an optimized cell-free system in any order: a. a protein expression vector is added for in vitro protein synthesis, or b. a histidine tag (His Tag) protein is added to obtain two independent coexisting protein-enriched phases, i.e., multiple membraneless compartments, and then controllable delivery of small molecules and macromolecules between the multiple membraneless compartments is performed.

[0007] The protein expression vector comprises: a plasmid pJL1-PopZ of Caulobacter crescentus cell polar organizing protein Z protein or a fusion protein expression vector composed of the plasmid pJL1-PopZ and a target functional protein.

[0008] The amino acid sequence of the cell pole organization Z protein of Caulobacter crescentus is shown in Seq ID No. 1, which is derived from the document Bowman, Grant R., et al. Cell 134.6 (2008): 945-955.

[0009] The amount of the protein expression vector added in the optimized cell-free system is 0.2nM-30nM, preferably 5nM-20nM, more preferably 10nM-18nM.

[0010] The optimized cell-free system refers to: exogenously adding nickel ions to the cell-free system including prokaryotic cell extracts, energy buffer and expression vector.

[0011] The cell-free system, which performs in vitro protein synthesis based on the added target protein expression vector, is derived from the document Kwon, Yong-Chan, and Michael C. Jewett. Scientific reports 5.1 (2015): 8663.

[0012] The concentration of the exogenous nickel ions is 0.5-20 mM, preferably 2.5-10 mM, and more preferably 5 mM.

[0013] The exogenous nickel ions are nickel chloride, nickel sulfate or nickel nitrate, preferably nickel chloride.

[0014] The optimized cell-free system further preferably adds glutamate at a total concentration of 150-250 mM, and more preferably 200 mM.

[0015] The glutamate is potassium glutamate, magnesium glutamate, ammonium glutamate, sodium glutamate or a combination thereof, preferably a combination of potassium glutamate, magnesium glutamate and ammonium glutamate, wherein the ratio of the three is 10:1:1 to 25:1:1, preferably 18:1:1.

[0016] The temperature of the in vitro protein synthesis is 16°C-37°C, preferably 30°C.

[0017] The small molecule substances are fluorescent small molecules, drug small molecules and signal small molecules.

[0018] The macromolecular substances are fluorescent proteins, drug proteins, signal proteins and biological enzymes.

[0019] The controllable mentioned here refers to spatiotemporal control, that is, regulating the positioning of substances in time and space.

[0020] The protein containing a histidine tag (His Tag) includes: an arthropod elastic (His-R32)-like protein containing a histidine tag (His Tag).

[0021] The His-R32 protein containing a histidine tag (His Tag) refers to a protein composed of a 10-histidine concatemer and a conserved peptide segment (GGRPSDSYGAPGGGN) of an arthropod elastic protein. The amino acid sequence is shown in Seq ID No. 2, which is derived from the literature Ardell, DH, Andersen, SO Insect biochemistry and molecular biology, 2001, 31, 965-970.

[0022] The final concentration of the exogenously added protein containing a histidine tag (His Tag) is 0.2 mg / mL-100 mg / mL, preferably 10 mg / mL-50 mg / mL.

[0023] The controllable delivery of the macromolecular substance is achieved by adding tobacco etch virus (TEV) protease for enzymatic cleavage reaction, and the cleaved substance is specifically composed of a 10-histidine concatemer, a connecting peptide (amino acid composition is GGSGGSGGSGGS), a TEV enzyme cleavage site (ENLYFQG) and a green fluorescent protein with additional 15 positive charges, and the cleavage site is the TEV enzyme cleavage site.

[0024] The amino acid sequence of the green fluorescent protein is shown in Seq ID No. 3, which is derived from the document Thompson, David B., et al. Chemistry & biology 19.7 (2012). Technical Effects

[0025] The present invention optimizes the adaptability of the cell-free system to multi-compartmental substance transfer by simultaneously adding nickel ions and glutamate to the cell-free system; and selects the cell pole organization Z protein of Caulobacter crescentus and the arthropod elastic (His-R32) protein containing a histidine tag (His Tag) for the controllable transfer of substances between multiple membrane-free compartments. Compared with the prior art, the present invention realizes the controllable transfer of small molecules between multiple membrane-free compartments in the cell-free system, realizes the controllable transfer of macromolecules between multiple membrane-free compartments in the cell-free system, and realizes the self-regulation of the transfer of macromolecules between multiple membrane-free compartments in the cell-free system through the compartment self-regulation network. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of forming a membrane-free compartment by expressing a protein expression vector in the cell-free system optimized in Example 1;

[0027] Figure 2 This is a schematic diagram of the fluidity of membraneless compartments formed by PopZ protein in Example 1;

[0028] Figure 3 Flow chart for constructing multiple membraneless compartments for Example 1;

[0029] Figure 4 Schematic diagram of multiple membrane-less compartments constructed in the cell-free system optimized in Example 1;

[0030] Figure 5 Schematic diagram of the selective uptake of small molecules by two single membraneless compartments in Example 1;

[0031] Figure 6 This is a schematic diagram of the selective uptake of small molecules by multiple membraneless compartments in Example 1;

[0032] Figure 7 This is a schematic diagram of the spatiotemporal controllable material transfer of small molecules between multiple membraneless compartments in Example 1;

[0033] Figure 8 This is a flow chart of the controllable delivery of macromolecular substances between multiple membraneless compartments in Example 2;

[0034] Fig. 9 This is a schematic diagram of the spatiotemporal controllable transfer of macromolecular substances between multiple membraneless compartments in Example 2;

[0035] Fig.10 Schematic diagram of macromolecular substance transfer kinetics between multiple membraneless compartments in Example 2;

[0036] Fig.11 This is a flow chart of Example 3 of the multi-membraneless compartment self-regulatory network regulating the transfer of macromolecular substances between compartments;

[0037] Fig.12 This is a confocal microscope photograph of the multi-membraneless compartment self-regulatory network regulating the transfer of macromolecular substances between compartments in Example 3;

[0038] Fig.13 This is a schematic diagram of the quantitative data of the inter-compartment macromolecular substance transfer of the multi-membraneless inter-compartment self-regulatory network in Example 3. DETAILED DESCRIPTION Example 1

[0039] This embodiment relates to an application of spatiotemporally controllable small molecule substance delivery between multiple membrane-less compartments, including optimizing a cell-free system, constructing multiple membrane-less compartments in the system, and demonstrating spatiotemporally controllable small molecule substance delivery between multiple membrane-less compartments, including:

[0040] Step 1: Construct an optimized cell-free system. First, prepare a prokaryotic cell extract. Specifically, culture Escherichia coli BL21 (DE3) cells in a 2L baffled Erlenmeyer flask containing 1L 2×YTPG medium in a shaking incubator at 37°C and 220rpm.

[0041] The 2×YTPG culture medium contains 10 g / L yeast extract, 16 g / L tryptone, 5 g / L NaCl, 7 g / L K2HPO4, 3 g / L KH2PO4 and 18 g / L glucose.

[0042] Step 2: When the optical density of the cells at 600 nm (OD 600 ) reached 0.6-0.8, isopropyl β-D-1-thiogalactoside (IPTG) was added at a final concentration of 1 mM to induce the expression of chromosome-encoded T7 RNA polymerase.

[0043] Step 3: When the OD of the induced cells 600 When the pH reaches 3.0-4.0, the cells are harvested by centrifugation. The cells are washed three times with pre-cooled S30 buffer (10 mM Tris, 14 mM magnesium glutamate, 60 mM potassium glutamate, pH adjusted to 8.2 with acetic acid) containing 2 mM dithiothreitol (DTT), and then resuspended with fresh S30 buffer (1 mL / 1 g fresh cells), broken with a high-pressure homogenizer, and then 2 mM DTT is added. Centrifuge at 18,000 g for 10 min at 4 ° C, collect the supernatant, and incubate at 37 ° C, 220 rpm for 60 min to complete the runoff reaction.

[0044] Step 4. After centrifugation at 12,000 g for 10 min at 4°C, the supernatant was collected as the cell extract, which was snap-frozen in liquid nitrogen and stored at -80°C until use.

[0045] The components of the S30 buffer are: 57mM HEPES-KOH (pH=7.5), 1.2mM ATP, and 0.85mM GTP, UTP, and CTP; 34.0μg / mL calcium folinate; 170.0μg / mL Escherichia coli tRNA mixture; 2mM each of 20 typical amino acids; 0.33mM nicotinamide adenine dinucleotide (NAD); 0.27mM coenzyme A (CoA); 1.5mM spermidine; 1mM putrescine; 2.7mM sodium oxalate; and 33mM phosphoenolpyruvate (PEP).

[0046] In the optimized cell-free system, the optimized glutamate is 180 mM potassium glutamate; 10 mM ammonium glutamate; 10 mM magnesium glutamate, and contains 5 mM nickel chloride.

[0047] Step 5: construct a protein expression vector pJL1-PopZ composed of the polar organizing protein Z of Caulobacter crescentus cells, add 10.55 nM of the plasmid into the cell-free system optimized in step 4 for in vitro protein synthesis, and observe in situ under a confocal microscope that the modified cell-free system can biosynthesize membrane-free compartments very quickly within 15 minutes, such as Figure 1 ; Fusion between membraneless compartments was observed, demonstrating the fluidity of the compartments, e.g. Figure 2 shown.

[0048] Step 6: Figure 3 As shown, a plasmid pET19b-His-R32 containing 10 histidine concatemers and 32 arthropod elastic protein conserved peptide segments (GGRPSDSYGAPGGGN) concatemers was constructed. The expression vector was introduced into Escherichia coli BL21 (DE3) cells for the production of His-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. Subsequently, the target His-R32 protein was purified from the cell lysate using immobilized metal affinity chromatography (IMAC), and then 10mg / mL of the protein was added to the optimized cell-free system with a single membraneless compartment in step 5 to form another membraneless compartment through histidine-nickel ion binding.

[0049] like Figure 4 As shown, the green droplets are the PopZ membraneless compartments composed of PopZ protein, and the red droplets are the R32 membraneless compartments composed of R32 protein. A multi-compartment structure in which the two compartments exist independently can be observed.

[0050] Step 7: Add positively charged Rh 6G small molecules, negatively charged SRh B and Pyranine small molecules at concentrations of 5 μM, 50 μM and 200 μM into the cell-free system with different membrane-free compartments to test the localization differences of different membrane-free compartments for small molecules with different charges, such as Figure 5 shown.

[0051] When multiple membraneless compartments coexisted, it was observed that the multiple membraneless compartments had different selective uptake abilities for small molecules, and there were large differences between the different compartments, thereby achieving the multi-membraneless compartment localization of different small molecules in the cell-free system, such as Figure 6 Then, by making the two membrane-free compartments appear alternately at different time sequences, the precise delivery of small molecules in multiple membrane-free compartments in the cell-free system was achieved, as shown in Figure 2. Figure 7 As shown, the left figure shows the material transfer of negatively charged small molecules and positively charged small molecules between compartments under two spatiotemporal controlled modes, and the right figure shows the quantitative data of the changes in fluorescence signals before and after the small molecule material transfer under different modes. Example 2

[0052] This embodiment relates to an application of spatiotemporally controllable macromolecular substance delivery between multiple membrane-less compartments, including optimizing a cell-free system, constructing multiple membrane-less compartments in the system, and demonstrating spatiotemporally controllable macromolecular substance delivery between multiple membrane-less compartments, including:

[0053] Step 1: Construct an optimized cell-free system. First, prepare a prokaryotic cell extract. Specifically, culture Escherichia coli BL21 (DE3) cells in a 2L baffled Erlenmeyer flask containing 1L 2×YTPG medium in a shaking incubator at 37°C and 220rpm.

[0054] The 2×YTPG culture medium contains 10 g / L yeast extract, 16 g / L tryptone, 5 g / L NaCl, 7 g / L K2HPO4, 3 g / L KH2PO4 and 18 g / L glucose.

[0055] Step 2: When the optical density of the cells at 600 nm (OD 600 ) reached 0.6-0.8, isopropyl β-D-1-thiogalactoside (IPTG) was added at a final concentration of 1 mM to induce the expression of chromosome-encoded T7 RNA polymerase.

[0056] Step 3: When the OD of the induced cells 600When the pH reaches 3.0-4.0, the cells are harvested by centrifugation. The cells are washed three times with pre-cooled S30 buffer (10 mM Tris, 14 mM magnesium glutamate, 60 mM potassium glutamate, pH adjusted to 8.2 with acetic acid) containing 2 mM dithiothreitol (DTT), and then resuspended with fresh S30 buffer (1 mL / 1 g fresh cells), broken with a high-pressure homogenizer, and then 2 mM DTT is added. Centrifuge at 18,000 g for 10 min at 4 ° C, collect the supernatant, and incubate at 37 ° C, 220 rpm for 60 min to complete the runoff reaction.

[0057] Step 4. After centrifugation at 12,000 g for 10 min at 4°C, the supernatant was collected as the cell extract, which was snap-frozen in liquid nitrogen and stored at -80°C until use.

[0058] The components of the S30 buffer are: 57mM HEPES-KOH (pH=7.5), 1.2mM ATP, and 0.85mM GTP, UTP, and CTP; 34.0μg / mL calcium folinate; 170.0μg / mL Escherichia coli tRNA mixture; 2mM each of 20 typical amino acids; 0.33mM nicotinamide adenine dinucleotide (NAD); 0.27mM coenzyme A (CoA); 1.5mM spermidine; 1mM putrescine; 2.7mM sodium oxalate; and 33mM phosphoenolpyruvate (PEP).

[0059] In the optimized cell-free system, the optimized glutamate is 180 mM potassium glutamate; 10 mM ammonium glutamate; 10 mM magnesium glutamate, and contains 5 mM nickel chloride.

[0060] Step 5, construct a plasmid pET19b-His-R32 containing 10 histidine concatemers and 32 arthropod elastic protein conserved peptide segments (GGRPSDSYGAPGGGN) concatemers. The expression vector was introduced into Escherichia coli BL21 (DE3) cells for the production of His-R32 protein. The harvested cells were suspended in 20 mM Tris-HCl buffer (pH = 8.0) containing 150 mM NaCl and 5 mM imidazole, and then lysed using a high-pressure homogenizer. Subsequently, the His-R32 protein was purified from the cell lysate using immobilized metal affinity chromatography (IMAC).

[0061] Step 6, constructing a protein expression vector pJL1-PopZ composed of the polar organizing protein Z of Caulobacter crescentus cells;

[0062] Step 7: Construct an expression vector pJL1-His-linker-TEVs-gfp containing 10 histidine concatemers, a linker peptide (amino acid composition: GGSGGSGGSGGS), a TEV enzyme cleavage site (ENLYFQG), and a green fluorescent protein with an additional 15 positive charges. 15+ .

[0063] The amino acid sequence of the green fluorescent protein is shown in Seq ID No. 3, which is derived from the document Thompson, David B., et al. Chemistry & biology 19.7 (2012).

[0064] Step 8: Figure 8 As shown, first, the His-R32 protein in step 5 is added to the optimized cell-free system in step 4 to form the first membrane-free compartment, i.e., the R32 membrane-free compartment. Then, the protein expression vector pJL1-PopZ in step 6 is added. After protein expression in the cell-free system, the second membrane-free compartment, i.e., the PopZ membrane-free compartment, is formed. Then, TEV enzyme is added for enzyme cleavage reaction. By controlling the enzyme cleavage reaction, the controllable transfer of macromolecules between multiple membrane-free compartments is achieved. Specifically, 10 mg / ml of the His-R32 protein in step 5 and 4.77 nM of the expression vector pJL1-His-linker-TEVs-gfp in step 7 are added to the optimized cell-free system. 15+ , incubated at 30°C for 2h to enrich the protein macromolecules in the R32 compartment, then added 10.55nM of the expression vector pJL1-PopZ in step 7 and incubated at 30°C for 4h; then added 8.82μM TEV enzyme and incubated at 30°C for 2h to allow the TEV enzyme to cleave the protein macromolecules His-linker-TEVs-GFP in the R32 compartment. 15+ The enzyme cleavage reaction is carried out, and the protein macromolecules are released and transferred to another PopZ compartment, completing the controllable transfer of macromolecules between multiple membraneless compartments.

[0065] The experimental results are as follows. It was observed that by regulating the formation of compartments and the addition time of TEV enzyme, the transfer process of macromolecular substances can be controlled in time and space, so as to achieve the spatiotemporal and directional transfer of protein macromolecular substances between multiple membrane-free compartments in the cell-free system. Fig. 9 As shown in the figure, it is the transfer result under the time-space control mode, such as Fig.10c shows the quantitative data of the difference in protein signals before and after transfer. The kinetic data clearly reflects the relationship between the amount of macromolecules transferred between compartments and the transfer time. The quantitative data in the upper right corner of the figure show that the transfer rate of macromolecules between compartments is TEV enzyme-dependent, which once again proves that by regulating the enzyme cleavage time and addition amount of TEV enzyme, we can achieve a high degree of spatiotemporal control over the transfer of macromolecules between membraneless compartments. Example 3

[0066] This embodiment relates to an application of a self-regulatory network between multiple membraneless compartments to regulate the transfer of macromolecules, including optimizing a cell-free system, constructing multiple membraneless compartments in the system, and demonstrating the construction of a complex regulatory network between multiple membraneless compartments, specifically referring to the regulation of the self-regulatory network between multiple membraneless compartments on the transfer of macromolecules between compartments, including:

[0067] Step 1: Construct an optimized cell-free system. First, prepare a prokaryotic cell extract. Specifically, culture Escherichia coli BL21 (DE3) cells in a 2L baffled Erlenmeyer flask containing 1L 2×YTPG medium in a shaking incubator at 37°C and 220rpm.

[0068] The 2×YTPG culture medium contains 10 g / L yeast extract, 16 g / L tryptone, 5 g / L NaCl, 7 g / L K2HPO4, 3 g / L KH2PO4 and 18 g / L glucose.

[0069] Step 2: When the optical density of the cells at 600 nm (OD 600 ) reached 0.6-0.8, isopropyl β-D-1-thiogalactoside (IPTG) was added at a final concentration of 1 mM to induce the expression of chromosome-encoded T7 RNA polymerase.

[0070] Step 3: When the OD of the induced cells 600 When the pH reaches 3.0-4.0, the cells are harvested by centrifugation. The cells are washed three times with pre-cooled S30 buffer (10 mM Tris, 14 mM magnesium glutamate, 60 mM potassium glutamate, pH adjusted to 8.2 with acetic acid) containing 2 mM dithiothreitol (DTT), and then resuspended with fresh S30 buffer (1 mL / 1 g fresh cells), broken with a high-pressure homogenizer, and then 2 mM DTT is added. Centrifuge at 18,000 g for 10 min at 4 ° C, collect the supernatant, and incubate at 37 ° C, 220 rpm for 60 min to complete the runoff reaction.

[0071] Step 4. After centrifugation at 12,000 g for 10 min at 4°C, the supernatant was collected as the cell extract, which was snap-frozen in liquid nitrogen and stored at -80°C until use.

[0072] The components of the S30 buffer are: 57mM HEPES-KOH (pH=7.5), 1.2mM ATP, and 0.85mM GTP, UTP, and CTP; 34.0μg / mL calcium folinate; 170.0μg / mL Escherichia coli tRNA mixture; 2mM each of 20 typical amino acids; 0.33mM nicotinamide adenine dinucleotide (NAD); 0.27mM coenzyme A (CoA); 1.5mM spermidine; 1mM putrescine; 2.7mM sodium oxalate; and 33mM phosphoenolpyruvate (PEP).

[0073] In the optimized cell-free system, the optimized glutamate is 180 mM potassium glutamate; 10 mM ammonium glutamate; 10 mM magnesium glutamate, and contains 5 mM nickel chloride.

[0074] Step 5, construct a plasmid pET19b-His-R32 containing 10 histidine concatemers and 32 arthropod elastic protein conserved peptide segments (GGRPSDSYGAPGGGN) concatemers. The expression vector was introduced into Escherichia coli BL21 (DE3) cells for the production of His-R32 protein. The harvested cells were suspended in 20 mM Tris-HCl buffer (pH = 8.0) containing 150 mM NaCl and 5 mM imidazole, and then lysed using a high-pressure homogenizer. Subsequently, the His-R32 protein was purified from the cell lysate using immobilized metal affinity chromatography (IMAC).

[0075] Step 6, construct a protein expression vector pJL1-PopZRBD with the cell pole organization Z protein of Caulobacter crescentus, a connecting peptide (amino acid composition of GGSGGSGGSGGS) and RNA binding domains (RBDs), that is, a phase separation protein-regulatory fusion protein expression vector.

[0076] The amino acid sequence of the RNA binding domain is as shown in Seq ID No. 4, which is derived from the document Simon, Joseph R., et al. Molecular cell 75.1 (2019): 66-75.

[0077] The core sequence of the protein expression vector pJL1-PopZRBD is the PopZ sequence, and the connected RBD sequence is the target functional protein sequence.

[0078] Step 7: Construct a green fluorescent protein expression vector pJL1-His-linker-TEVs-gfp with 10 histidine concatemers, a linker peptide (amino acid composition: GGSGGSGGSGGS), a TEV enzyme cleavage site (ENLYFQG), and an additional 15 positively charged green fluorescent protein 15+ .

[0079] Step 8: construct a protein expression vector pJL1-His-TEV protease containing 10 histidine tandems and tobacco etch virus (TEV) protease (the sequence is shown in Seq ID No. 5, from the literature Lau K, Bouchri B, Pojer F. protocols.io).

[0080] Step 9: Fig.11 As shown, first, the His-R32 protein in step 5 is added to the optimized cell-free system in step 4 to form the first membrane-free compartment, i.e., the R32 membrane-free compartment, and then the phase separation protein-regulatory fusion protein expression vector pJL1-PopZRBD in step 6 is added. After the protein is expressed in the cell-free system, the second membrane-free compartment, i.e., the PopZ membrane-free compartment, is formed; at the same time, the PopZ compartment will regulate the expression of the protein expression vector pJL1-His-TEVprotease in step 8 through the self-regulatory network, thereby regulating the enzyme cleavage reaction efficiency of the synthesized TEV enzyme, and then the self-regulatory network between multiple membrane-free compartments regulates the transfer of macromolecular substances between compartments, specifically: adding 10 mg / ml His-R32 protein in step 5 and 9.54 nM expression vector pJL1-His-linker-TEVs-gfp in step 7 15+ Incubate at 30°C for 2 h in the optimized cell-free system in step 4 to synthesize the protein macromolecule His-linker-TEVs-GFP 15+， And locate it in the R32 membraneless compartment, then add the protein expression vector pJL1-PopZRBD in step 6 at a concentration of 8.79nM and the protein expression vector pJL1-His-TEV protease in step 8 at 2.03nM. When the cell-free system expresses the pJL1-PopZRBD plasmid to form the PopZ membraneless compartment, the compartment will restrict the positioning of mRNA in the environment to its compartment, so that it can not only self-regulate its own protein synthesis rate, but also affect the synthesis amount of His-TEV enzyme. The transfer of macromolecules between compartments depends on the concentration of TEV enzyme, that is, the transfer of macromolecules between compartments can be self-regulated.

[0081] like Fig.12 As shown in the figure, green is the fluorescent signal of the transferred macromolecules, and red is the R32 membraneless compartment. The image shows that at different times, the signal intensity in the R32 membraneless compartment changes from strong to weak, while the PopZ compartment receives the macromolecules, and the green fluorescent signal represented by it changes from weak to strong. This regulation process is completed by the PopZ compartment. This shows that the membraneless compartment self-regulates the transfer of macromolecules between compartments. The quantitative data shows the whole process more clearly, such as Fig.13 shown.

[0082] Compared with the prior art, by simultaneously adding nickel ions and glutamate to the cell-free system, after completing the optimization of the cell-free system's ability to adapt to multi-compartmental substance transfer, the cell pole tissue Z protein of Caulobacter crescentus and the arthropod elastic (His-R32) protein containing a histidine tag (His Tag) can be selected for the controllable transfer of substances between multiple membrane-free compartments. This model realizes the controllable transfer of small molecules and macromolecules between multiple membrane-free compartments in the cell-free system; it realizes the self-regulation of the transfer of macromolecules between multiple membrane-free compartments through the compartment self-regulation network in the cell-free system. The present invention improves the inherent one-pot reaction mode in the traditional cell-free system, and the reliable multi-compartmental substance highly controllable transfer mode established will lay a solid foundation for the application of cell-free systems in complex metabolic pathway exploration, multi-enzyme catalysis, drug delivery, etc.

[0083] 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. An application of controllable delivery of substances between multiple membrane-less compartments in a cell-free system, wherein: a. adding a protein expression vector to perform in vitro protein synthesis, or b. adding a histidine tag (His Tag) protein to an optimized cell-free system in any order, thereby constructing two independent coexisting protein-enriched phases, i.e., multiple membrane-less compartments, and then performing controllable delivery of small molecules and macromolecules between the multiple membrane-less compartments; The optimized cell-free system refers to: exogenously adding nickel ions to the cell-free system including prokaryotic cell extract, energy buffer and expression vector; The small molecule substances are fluorescent small molecules, drug small molecules and signal small molecules; The macromolecular substances are fluorescent proteins, drug proteins, signal proteins and biological enzymes.

2. The use of the controllable delivery of substances between multiple membrane-free compartments in a cell-free system according to claim 1, characterized in that: The protein expression vector includes: the plasmid pJL1-PopZ of the polar organizing protein Z protein of Caulobacter crescentus or a fusion protein expression vector composed of the plasmid pJL1-PopZ and the target functional protein; The amino acid sequence of the cell pole organization Z protein of Caulobacter crescentus is shown in Seq ID No.

1.

3. The use of the controllable delivery of substances between multiple membrane-free compartments in a cell-free system according to claim 1 or 2, characterized in that: The added amount of the protein expression vector in the optimized cell-free system is 0.2nM-30nM.

4. The use of the controllable delivery of substances between multiple membrane-free compartments in a cell-free system according to claim 1, characterized in that: The concentration of the exogenous nickel ions is 0.5-20 mM; The exogenous nickel ions are nickel chloride, nickel sulfate or nickel nitrate.

5. The use of the controllable delivery of substances between multiple membrane-free compartments in a cell-free system according to claim 1, characterized in that: The total concentration of glutamate is 150-250 mM; The glutamate is potassium glutamate, magnesium glutamate, ammonium glutamate, sodium glutamate or a combination thereof.

6. The use of the controllable delivery of substances between multiple membrane-free compartments in a cell-free system according to claim 1, characterized in that: The protein containing a histidine tag (His Tag) includes: an arthropod elastic (His-R32)-like protein containing a histidine tag (His Tag); The His-R32 protein containing a histidine tag (His Tag) refers to a protein composed of a 10-histidine concatemer and a conserved peptide segment (GGRPSDSYGAPGGGN) of an arthropod elastic protein, and the amino acid sequence is shown in SeqIDNo.

2.

7. The use of the controllable delivery of substances between multiple membrane-free compartments in a cell-free system according to claim 1 or 6, characterized in that: The final concentration of the exogenously added protein containing a histidine tag (His Tag) is 0.2 mg / mL-100 mg / mL.

8. The use of the controllable delivery of substances between multiple membrane-free compartments in a cell-free system according to claim 1, characterized in that: The controllable delivery of the macromolecular substance is achieved by adding tobacco etch virus (TEV) protease to perform an enzymatic cleavage reaction, wherein the cleaved substance specifically consists of a 10-histidine concatemer, a connecting peptide (amino acid composition is GGSGGSGGSGGS), a TEV enzyme cleavage site (ENLYFQG) and a green fluorescent protein with an additional 15 positive charges, and the cleavage site is the TEV enzyme cleavage site; The amino acid sequence of the green fluorescent protein is shown in Seq ID No.3.

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