Method for constructing membrane-free compartment in cell-free system containing exogenously added nickel ions

Abstract

The invention relates to a method for constructing a membrane-free compartment in a cell-free system containing exogenous nickel ions, which comprises the following steps of: constructing a protein expression vector containing a histidine tag (His Tag), introducing the protein expression vector into an expression host, and adding protein obtained by expression and purification into the cell-free system containing the exogenous nickel ions to directly form a protein enrichment phase, namely the membrane-free compartment. The biosynthesis of the quantum dots can be promoted in the zone chamber, the biological enzyme with the histidine tag can be recruited in a high-specificity manner, the enzyme digestion reaction is completed in the zone chamber, and the substances are controllably released.

Description

Technical Field

[0001] The invention relates to a technology in the field of bioengineering, in particular to a method for constructing a membrane-free compartment in a cell-free system containing exogenously added nickel ions. Background Art

[0002] Partitioning plays a vital role in the life process. Cells accurately and efficiently complete various biological functions by partitioning and positioning complex molecules and biochemical reactions within themselves. The membraneless compartments formed by liquid-liquid phase separation play a key role in life activities such as gene transcription due to the lack of membrane structure. Inspired by this phenomenon, researchers have tried to construct artificial membraneless compartments in an in vitro protein synthesis system, i.e., a cell-free system, which already has multiple biological functions, to achieve the partitioning of biochemical reactions, thereby completing more advanced biochemical reactions and improving reaction efficiency.

[0003] There are two main methods for constructing membraneless compartments in vitro. One method is to use electrostatic interaction to form membraneless compartments of macromolecular condensation through the combination of positive and negative charges of biological or chemical macromolecules. The disadvantage of this method is that the electrostatic force is affected by environmental factors, such as solution pH and ionic strength, and the above environments are dynamically changing in cell-free systems, so this method is not applicable. The other is to use short peptide pairs such as RIDD / RIAD for binding. This method requires genetic modification of proteins, which will increase the difficulty and risk of protein purification. The α-helical structure of the peptide pair itself will also affect the function of the modified protein, which will reduce the potential for further functionalization of compartments in the cell-free system. Therefore, this leads to a lack of methods for constructing membraneless compartments in cell-free systems. The only construction cases are achieved by evaporating and concentrating fluorescent proteins or temperature-induced condensation of proteins. The main defects of these two methods are that the degree of compartmentalization is not high and specific temperature treatment is required, and they are poorly compatible with cell-free systems. Especially for the former, the concentrated solution environment often completely terminates the biochemical reactions of the cell-free system itself. Summary of the invention

[0004] In view of the problems encountered in the prior art of constructing membraneless compartments in a cell-free system, such as poor compatibility, poor universality, poor stability, and great difficulty, the present invention proposes a method for constructing membraneless compartments in a cell-free system containing exogenously added nickel ions, which can not only promote the biosynthesis of quantum dots in the compartment, but also can highly specifically recruit biological enzymes with histidine tags, complete enzymatic cleavage reactions in the compartment, and release substances in a controllable manner.

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

[0006] The present invention relates to a method for constructing a membrane-free compartment in a cell-free system containing exogenously added nickel ions. A protein expression vector containing a histidine tag (His Tag) is constructed and introduced into an expression host. The protein obtained by expression and purification is added to the cell-free system containing exogenous nickel ions to directly form a protein-enriched phase, i.e., a membrane-free compartment.

[0007] In the histidine tag (His Tag), the number of histidines connected in series is 4 to 14, preferably 6 to 12.

[0008] The protein includes: an arthropod elastic protein containing a histidine tag (His Tag), an elastic protein-like polypeptide containing a histidine tag (His Tag), or a recombinant spider silk protein containing a histidine tag (His Tag), wherein: an arthropod elastic protein refers to a protein composed of a 10-histidine concatemer and an arthropod elastic protein conserved peptide (GGRPSDSYGAPGGGN) concatemer, and the amino acid sequence is shown in Seq ID No. 1, which is derived from the document Ardell, DH, Andersen, SO Insect biochemistry and molecular biology, 2001, 31, 965-970; an elastic protein-like polypeptide refers to a protein composed of a 10-histidine concatemer and an elastic protein-like polypeptide (GVGVP) concatemer, and the amino acid sequence is shown in Seq ID No. 2, which is derived from the document Song WW., et al. ACS Macro Lett, 2021, 20; 10 (4): 395-400; a recombinant spider silk protein refers to a 12-histidine concatemer and a golden silk spider (Trichonephila clavipes spider dragline silk protein monomer core amino acid sequence MaSpI protein conserved peptide (GRGGLGGQGAGAAAAAGGAGQGGYGGLGSQG) concatemer, the amino acid sequence is shown in Seq ID No. 3, from the literature Hinman, MB, Lewis, RVJ Biol Chem, 1992, 267, 19320-19324.

[0009] The protein is added at a concentration of 0.2 mg / mL-100 mg / mL, preferably 10 mg / mL-50 mg / mL.

[0010] The expression vector refers to: an expression plasmid vector of 4-64 conserved peptide segments of arthropod elastic protein concatemers, an expression plasmid vector of 4-64 elastin-like polypeptide concatemers, or an expression plasmid vector of 4-64 recombinant spider silk protein repeating peptide segments concatemers.

[0011] The expression host is Escherichia coli.

[0012] The exogenous nickel ions in the cell-free system containing exogenous nickel ions are nickel chloride, nickel sulfate or nickel nitrate, preferably nickel chloride; the cell-free system comprises a prokaryotic cell extract, an energy buffer and an expression vector.

[0013] The concentration of the exogenous nickel ions is 0.5-20 mM, preferably 2.5-10 mM, and more preferably 5 mM.

[0014] The cell-free system is implemented using, but not limited to, the technology described in Kwon, Yong-Chan, and Michael C. Jewett. Scientific reports 5.1 (2015): 8663.

[0015] The present invention relates to the application of the membrane-free compartment for confined synthesis, specifically: using the membrane-free compartment as Cd 2+ and S 2- The ions provide a site for quantum dot synthesis to synthesize CdS quantum dots.

[0016] The present invention relates to another application of the membraneless compartment, which is to use it for biological enzyme catalysis, specifically: releasing compartment cargo in the membraneless compartment by relying on tobacco etch virus (TEV) enzyme cleavage reaction.

[0017] In the TEV enzyme cleavage reaction, 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 additional 15 positive charges, and the cleavage site is the TEV enzyme cleavage site.

[0018] The amino acid sequence of the green fluorescent protein is shown in Seq ID No. 4, which is derived from the document Thompson, David B., et al. Chemistry & biology 19.7 (2012). Technical Effects

[0019] The present invention completes the transformation of the cell-free system to adapt to compartmentalization construction by adding additional nickel ions to the cell-free system; in the complex and dynamically changing cell-free system, the metal coordination effect of histidine-nickel ions formed between nickel ions and proteins containing histidine tags is used to induce protein aggregation and quickly form a soluble protein-enriched phase, i.e., a membrane-free compartment. Compared with the prior art, the present invention does not require the use of concentrated evaporation or temperature induction, and directly and quickly forms a stable soluble protein-enriched phase, i.e., a membrane-free compartment, at a lower protein concentration in the cell-free system. At the same time, in the cell-free system, the biosynthesis of quantum dots in the membrane-free compartment is achieved, the high-specificity recruitment of fluorescent proteins or enzymes with histidine tags in the membrane-free compartment in the cell-free system is achieved, and the enzyme cleavage reaction is completed in the compartment and the substance is released in a controllable manner. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a schematic diagram of the process of adding a protein containing a histidine tag (His Tag) to a cell-free system containing nickel ions in Example 1 to form a membrane-free compartment;

[0021] Figure 2 This is a schematic diagram of adding a recombinant spider silk protein containing a histidine tag (His Tag) to the nickel-containing cell-free system in Example 1 to form a membrane-free compartment;

[0022] Figure 3 This is a schematic diagram of adding an elastin-like polypeptide containing a histidine tag (His Tag) to the nickel-containing cell-free system in Example 1 to form a membrane-free compartment;

[0023] Figure 4 This is a schematic diagram of adding arthropod elastic protein containing a histidine tag (His Tag) to the cell-free system containing nickel ions in Example 1 to form a membrane-free compartment;

[0024] Figure 5 This is a schematic diagram of Example 1, which shows that a cell-free system containing nickel ions can form a membrane-less compartment with a His tag protein at a relatively low concentration;

[0025] Figure 6 This is a schematic diagram of the stability of the membraneless compartment in Example 1;

[0026] Figure 7 (a) and Figure 7 (b) is a schematic diagram of the fluidity of the membraneless compartment in Example 1;

[0027] Figure 8 This is a schematic diagram of the process of biosynthesizing quantum dots in a membrane-free compartment in Example 2;

[0028] Fig. 9 Schematic diagram of biosynthesis of quantum dots in membrane-free compartments in Example 2;

[0029] Fig.10 This is a schematic diagram of Example 2 where a membraneless compartment promotes the biosynthesis of quantum dots;

[0030] Fig.11 This is a schematic diagram of highly selective enrichment of histidine-tagged proteins in membraneless compartments in Example 3;

[0031] Fig.12 This is a schematic diagram of the process of releasing substances by controlled enzymatic cleavage reaction in a membraneless compartment in Example 3;

[0032] Fig.13 Schematic diagram of the controlled enzymatic cleavage reaction in the membraneless compartment to release substances in Example 3. DETAILED DESCRIPTION Example 1

[0033] This embodiment relates to a method for constructing a membrane-free compartment using nickel ions and a histidine-tagged protein in a nickel-ion-containing cell-free system. The main purpose is to demonstrate the construction of a nickel-ion-containing cell-free system, the construction of a protein expression vector containing a histidine tag and the introduction into an expression host, the expression and purification of a protein containing a histidine tag (His Tag), and then the exogenous addition of nickel ions to quickly form a stable, high-fluidity soluble membrane-free compartment, specifically including:

[0034] 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.

[0035] 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.

[0036] Step 2: When the optical density of the cells at 600 nm (OD600) reaches 0.6 to 0.8, add isopropyl β-D-1-thiogalactoside (IPTG) at a final concentration of 1 mM to induce the expression of chromosome-encoded T7 RNA polymerase.

[0037] Step 3: When the OD600 of the induced cells 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 the cells are resuspended with fresh S30 buffer (1 mL / 1 g fresh cells), broken with a high-pressure homogenizer, and then 2 mM DTT is added, and centrifuged at 18,000 g for 10 min at 4 ° C, the supernatant is collected, and incubated at 37 ° C and 220 rpm for 60 min to complete the runoff reaction.

[0038] 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.

[0039] 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-1 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).

[0040] For the cell-free system containing nickel ions, 5 mM nickel chloride.

[0041] Step 5, plasmid construction, constructing plasmid pET19b-His-R32 consisting of 10 histidine concatemers and 32 arthropod elastic protein conserved peptides (GGRPSDSYGAPGGGN) concatemers, constructing plasmid pET19b-His-E64 consisting of 10 histidine concatemers and 64 elastin-like polypeptides (GVGVP) concatemers, and constructing plasmid pET28a4-His-I16 consisting of 12 histidine concatemers and 16 spider dragline silk protein monomer core amino acid sequence MaSpI protein conserved peptides (GRGGLGGQGAGAAAAAGGAGQGGYGGLGSQG) concatemers.

[0042] Step 6, protein purification: The expression vectors pET19b-His-R32, pET19b-His-E64, and pET28a4-His-I16 were introduced into E. coli BL21 (DE3) cells for the production of His-R32 protein, His-E64 protein, and His-I16 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 target His-R32 protein, His-E64 protein, and His-I16 protein were purified from the cell lysate using immobilized metal affinity chromatography (IMAC).

[0043] Step 7: Figure 1 As shown, 20 mg / mL His-E64 protein, 20 mg / mL His-I16 protein, and 10 mg / mL His-R32 protein purified in step 6 were added to the cell-free system containing exogenous nickel ions as described in step 4. Through histidine-nickel ion binding, the proteins phase separated to form membraneless compartments. The results are shown in FIG. Figure 2-Figure 4 shown.

[0044] It was also found that membraneless compartments could be formed at very low protein concentrations (0.6 mg / mL His-R32 protein), such as Figure 5 Compared with the cell-free system without nickel ion modification ( Figure 6 , left), this histidine-nickel ion binding improved the stability of the membraneless compartment, which remained stable after incubation at 30°C for 8 hours ( Figure 6 , right); at the same time, the fluorescence recovery after photobleaching (FRAP) experiment found that the fluorescence signal in the compartment recovered quickly, proving that the membraneless compartment has very good fluidity, such as Figure 7 shown.

[0045] In summary, this implementation case demonstrates that in a cell-free system containing exogenous nickel ions, a stable and highly fluid membrane-free compartment can be quickly constructed at a low protein concentration by binding nickel ions to histidine-tagged proteins. Example 2

[0046] The present embodiment relates to an application of a membrane-free cell compartment in a cell-free system containing exogenously added nickel ions constructed based on the above method, which can promote the biosynthesis of quantum dots between membrane-free compartments in a cell-free system containing exogenous nickel ions, specifically comprising: adding 5 mM exogenous nickel ions into the cell-free system, and then adding 10 mg / mL of His-R32 protein, which is a concatemer of 10 histidine tandems and 32 arthropod elastic protein conserved peptide segments (GGRPSDSYGAPGGGN), which will form a membrane-free compartment, i.e., the R32 compartment, through histidine-nickel ion binding.

[0047] like Figure 8 As shown, Cd 2+ and S 2- ions were added to the compartments, incubated at 37°C for 30 min, and observed by confocal microscopy. Fig. 9 As shown in the figure, the synthesis of CdS quantum dots (CdS-QDs) in the membraneless compartment can be observed. The round droplets in the figure are membraneless compartments, and the blue ones are CdS quantum dots. Then, the microplate reader was used to detect that the existence of membraneless compartments can promote the synthesis of CdS quantum dots.

[0048] like Fig.10 As shown in the figure, the characteristic peak of CdS quantum dots is at ~550nM, and the intensity of the fluorescence value on the ordinate represents the yield. The black line in the figure represents the control with only no membrane compartment, and the red line represents the control with only Cd added. 2+ and S 2- Ion control, blue represents the addition of Cd in the membraneless compartment 2+ and S 2- ion experimental group, it can be clearly observed that the yield of CdS quantum dots has been significantly improved under the condition of no membrane compartment. Example 3

[0049] The present embodiment relates to an application of a membrane-free cell compartment in a cell-free system containing exogenously added nickel ions constructed based on the above method, which can recruit fluorescent proteins or enzymes with histidine tags with high specificity through the membrane-free compartment, complete the enzyme cleavage reaction in the compartment and release the substance in a controllable manner, specifically including: adding 5mM exogenous nickel ions into the cell-free system, and then adding 10mg / mL of His-R32 protein, which is a concatemer of 10 histidine concatemers and 32 arthropod elastic protein conserved peptide segments (GGRPSDSYGAPGGGN), which will form a membrane-free compartment, i.e., the R32 compartment, through the combination of histidine and nickel ions. The protein expression vector pJL1-his-gfp containing 15 additional positively charged green fluorescent proteins (sequence as shown in Seq ID No. 4, from the literature Thompson, David B., et al. Chemistry & Biology 19.7 (2012): 831-843.) with and without 10 histidine concatemers were constructed respectively. 15+ with pJL1-gfp 15+ 240 ng of protein expression vector pJL1-his-gfp 15+ with pJL1-gfp 15+Add to the cell-free system and incubate at 30°C for 4 h to synthesize the corresponding fluorescent protein. After confocal microscopy, it was found that the fluorescent protein with histidine tag was highly specifically enriched in the membrane-free compartment, while the one without histidine tag was excluded from the compartment. Fig.11 As shown, the circular droplets in the figure are membraneless compartments, green is fluorescent protein, and the right figure is quantitative data of the enrichment level of fluorescent protein in the membraneless compartments. It can be observed that whether or not there is a histidine tag will lead to a difference of dozens of times in the enrichment level, highlighting the high specific enrichment of histidine-tagged proteins in membraneless compartments.

[0050] like Fig.12 As shown, it is the enzyme digestion reaction in the membraneless compartment, which specifically includes: 15+ The protein was modified to construct a green fluorescent protein HisTEVs-GFP with 10 histidine tandems, a connecting peptide (amino acid composition of GGSGGSGGSGGS), a TEV enzyme cleavage site (ENLYFQG) and an additional 15 positively charged 15+ Protein expression vector pJL1-his-TEVs-gfp 15+ . 120 ng of the expression vector was added to the cell-free system containing the above-mentioned membrane-free compartments, and incubated at 30°C for 2 hours to synthesize the corresponding fluorescent protein. After that, confocal microscopy revealed that the membrane-free compartments highly specifically enriched the fluorescent protein. After that, the TEV enzyme with the same histidine tag was added, and the compartments also enriched the enzyme. The TEV enzyme would recognize the TEV enzyme cleavage site in the membrane-free compartments and perform cleavage, thereby making the green fluorescent protein HisTEVs-GFP. 15+ GFP 15+ , released by membraneless compartments, e.g. Fig.13 As shown, the red circular droplets represent membraneless compartments, the green fluorescent signal represents fluorescent protein, and the right side is the quantitative data of this process. It can be seen that the TEV enzyme cleavage reaction is carried out in the membraneless compartment, releasing the guest protein therein, thereby achieving controllable material spatial regulation by conducting confined enzymatic cleavage reaction in the membraneless compartment.

[0001] Compared with the prior art, the present invention induces protein aggregation in a cell-free system containing exogenous nickel ions by utilizing the metal coordination effect of histidine-nickel ions formed between nickel ions and proteins containing histidine tags, and quickly forms a soluble protein-enriched phase, i.e., a membrane-free compartment. The above-mentioned technical means do not need to use concentrated evaporation or temperature induction, and directly and quickly form a stable soluble protein-enriched phase, i.e., a membrane-free compartment, at a lower protein concentration in a cell-free system. At the same time, the compartment can not only promote the biosynthesis of quantum dots within the compartment, but also recruit fluorescent proteins or enzymes with histidine tags with high specificity, complete the enzymatic reaction within the compartment, and release substances in a controllable manner, thereby achieving higher biological reaction efficiency and more advanced biochemical reaction regulation functions, and laying a solid foundation for multi-enzyme catalysis, 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 a membrane-free compartment in a cell-free system containing exogenously added nickel ions, characterized in that: By constructing a protein expression vector containing a histidine tag and introducing it into an expression host, the protein obtained by expression and purification is added to a cell-free system containing exogenous nickel ions to directly form a protein-enriched phase, i.e., a membrane-free compartment; The protein includes: arthropod elastic protein containing a histidine tag (His Tag), elastic protein polypeptide containing a histidine tag (HisTag) or recombinant spider silk protein containing a histidine tag (His Tag); The exogenous nickel ions in the cell-free system containing exogenous nickel ions are nickel chloride, nickel sulfate or nickel nitrate, preferably nickel chloride; the cell-free system comprises a prokaryotic cell extract, an energy buffer and an expression vector.

2. The method for constructing a membrane-free compartment in a cell-free system containing exogenously added nickel ions according to claim 1, characterized in that: In the histidine tag (His Tag), the number of histidines connected in series is 4 to 14.

3. The method for constructing a membrane-free compartment in a cell-free system containing exogenously added nickel ions according to claim 1, characterized in that: The arthropod elastic protein refers to a protein composed of a 10-histidine concatemer and an arthropod elastic protein conserved peptide segment (GGRPSDSYGAPGGGN) concatemer, and the amino acid sequence is shown in Seq ID No.

1.

4. The method for constructing a membrane-free compartment in a cell-free system containing exogenously added nickel ions according to claim 1, characterized in that: The elastin-like polypeptide is a protein composed of a 10-histidine concatemer and an elastin-like polypeptide (GVGVP) concatemer, and the amino acid sequence is shown in Seq ID No.

2.

5. The method for constructing a membrane-free compartment in a cell-free system containing exogenously added nickel ions according to claim 1, characterized in that: The recombinant spider silk protein refers to a protein composed of a 12-histidine concatemer and a concatemer of a core amino acid sequence of the MaSpI protein conservative peptide segment (GRGGLGGQGAGAAAAAGGAGQGGYGGLGSQG) of the spider dragline silk protein monomer of the golden web spider (Trichonephilaclavipes), and the amino acid sequence is shown in Seq ID No.

3.

6. The method for constructing a membrane-free compartment in a cell-free system containing exogenously added nickel ions according to any one of claims 1 to 5, characterized in that: The protein is added at a concentration of 0.2 mg / mL-100 mg / mL.

7. The method for constructing a membrane-free compartment in a cell-free system containing exogenously added nickel ions according to claim 1, characterized in that: The expression vector refers to: an expression plasmid vector of 4-64 conserved peptide segments of arthropod elastic protein concatemers, an expression plasmid vector of 4-64 elastin-like polypeptide concatemers, or an expression plasmid vector of 4-64 recombinant spider silk protein repeating peptide segments concatemers.

8. The method for constructing a membrane-free compartment in a cell-free system containing exogenously added nickel ions according to claim 1, characterized in that: The concentration of the exogenous nickel ions is 0.5-20 mM.

9. An application of a membrane-free compartment constructed according to any one of claims 1 to 8, characterized in that: It is used for confined synthesis, specifically: the membrane-free compartment is used as Cd 2+ and S 2- The ions provide a site for quantum dot synthesis to synthesize CdS quantum dots.

10. Use of a membrane-free compartment constructed according to any one of claims 1 to 8, characterized in that: It was used for bioenzyme catalysis, specifically: compartment cargo release in membraneless compartments through tobacco etch virus (TEV) enzyme cleavage reaction; In the TEV enzyme cleavage reaction, 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.4.

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