Protein phase separation modulation system and applications thereof

Abstract

The present application relates to a protein phase separation regulation system and application thereof. The protein phase separation regulation system comprises: a target protein having a phase separation characteristic; a regulation tag connected to the target protein, the regulation tag comprising a SUMO tag; and an enzyme cutting site between the regulation tag and the target protein, the enzyme cutting site comprising a TEV enzyme cutting site. In the above protein phase separation regulation system, the design of the SUMO tag can effectively inhibit the non-specific aggregation of the phase separation element, the SUMO tag has the characteristics of a natural protein, has strong biological compatibility, reduces the toxic effect on cells, the introduction of the TEV enzyme cutting site makes the induction of phase separation have timeliness and controllability, can accurately induce phase separation at the required moment, has good flexibility, the construction and operation of the system are relatively simple, easy to implement, and can realize the response to the dynamic change in cells.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to protein phase separation regulation systems and their applications. Background Technology

[0002] Protein phase separation is an important intracellular biological process that regulates cellular function and signal transduction by forming liquid-liquid phase separators (such as intracellular bodies and nucleoli). This process plays a crucial role in cellular stress response, metabolic regulation, and signal transmission. For example, when responding to environmental stress, cells can form stress granules through phase separation to isolate unwanted molecules and concentrate key signal transduction molecules, thereby improving cellular survival. Recent studies have found a close correlation between phase separation and various diseases, such as neurodegenerative diseases and cancer. Therefore, regulating protein phase separation is not only significant for basic biological research but also provides new insights into disease treatment.

[0003] Currently, several technical solutions have been proposed for regulating protein phase separation. The following are some representative implementation schemes: ① Small molecule induction: This method regulates the aggregation state of proteins using small molecule compounds (such as chemical inducers). For example, researchers use certain drugs to induce the aggregation of specific proteins, thereby forming phase separators. The disadvantage of this method is that small molecules may be toxic to cells, and the temporal and spatial precision of regulation is low. ② Temperature regulation: Some studies induce phase separation through temperature changes, such as placing proteins under high or low temperature conditions to regulate their aggregation state. The limitation of this method is that temperature changes may affect other physiological processes of the cell, and it is difficult to achieve a rapid response. ③ RNA-mediated phase separation: Some researchers use RNA molecules as mediators to induce phase separation through the interaction between RNA and proteins. For example, RNA-binding proteins (RBPs) can promote the formation of phase separators by binding to specific RNA sequences. Although this method has a certain degree of flexibility, it is still difficult to achieve real-time regulation. ④ Synthetic biology-based regulatory systems: Some researchers have proposed using synthetic biology methods to regulate phase separation by designing gene circuits. These systems typically include gene expression regulatory elements, signal sensing elements, etc., which can achieve relatively precise regulation within cells, but they are complex to construct and difficult to implement.

[0004] Existing phase separation control methods mainly rely on exogenous small molecule compounds or changes in environmental factors (such as temperature and pH), lacking precise, real-time control of the phase separation process. This often leads to uncertainty and instability in the control results, affecting the practical application of phase separation. These limitations of existing technologies make the search for a novel, biocompatible, and highly controllable phase separation control method particularly important. Summary of the Invention

[0005] Based on this, this application provides a protein phase separation regulation system and its application. The protein phase separation regulation system is used for phase separation regulation processes and has high controllability and biocompatibility.

[0006] A protein phase separation regulation system, the protein phase separation regulation system comprising:

[0007] The target protein has phase separation properties;

[0008] A regulatory tag linked to the target protein, wherein the regulatory tag includes a SUMO tag;

[0009] The restriction enzyme site is located between the regulatory tag and the target protein, and the restriction enzyme site includes the TEV restriction enzyme site.

[0010] In the aforementioned protein phase separation regulation system, the SUMO tag design effectively inhibits the non-specific aggregation of phase separation elements. The SUMO tag possesses the characteristics of natural proteins, exhibiting strong biocompatibility and reducing cytotoxic effects. The introduction of the TEV restriction site makes phase separation induction timely and controllable, allowing for precise induction at the desired time, demonstrating good flexibility. The system is relatively simple to construct and operate, easy to implement, and capable of responding to dynamic changes within the cell. This protein phase separation regulation system, used for phase separation regulation processes, exhibits high controllability and biocompatibility.

[0011] In some embodiments, the target protein includes PodJ_NΔCC1-3.

[0012] In some embodiments, the protein phase separation regulation system further includes a luminescent tag that is linked to the target protein;

[0013] Furthermore, the luminescent tag is a fluorescent tag;

[0014] Furthermore, the luminescent tag includes a YFP fluorescent tag. It should be noted that the fluorescent tag is not limited to a YFP fluorescent tag; it can also be a protein selected from red fluorescent protein, yellow fluorescent protein, mCherry, mTAGBFP2, mCLOVER3, EGFP, Venus, mTagBFP2, mIFP, and TagRFP657 as the fluorescent tag.

[0015] A coding sequence for encoding the protein phase separation regulation system described above.

[0016] A recombinant vector carrying the aforementioned coding sequence.

[0017] In some embodiments, the recombinant vector is the expression vector of the protein phase separation regulation system.

[0018] A method for regulating the phase separation of a target protein includes the following steps:

[0019] Construct the expression vector for the protein phase separation regulation system described above;

[0020] The expression vector was transferred into a host bacterium for expression to obtain the protein phase separation regulation system;

[0021] TEV enzyme is added to the protein phase separation regulation system and reacted, and the formation and changes of the target protein phase separation are monitored using a monitoring instrument.

[0022] In some embodiments, the host bacterium includes Escherichia coli;

[0023] Further, the step of transferring the expression vector into the host bacteria to obtain the protein phase separation regulation system includes: transforming the expression vector into Escherichia coli host bacteria to obtain a transformed strain; culturing and inducing the transformed strain, and then purifying the culture to obtain the protein phase separation regulation system.

[0024] The above-described protein phase separation regulation system, or the above-described coding sequence, or the above-described recombinant vector, or the above-described method, are used to regulate protein phase separation in organisms.

[0025] The application of the protein phase separation regulation system described above, or the coding sequence described above, or the recombinant vector described above, or the method described above in regulating intracellular signal transduction. Attached Figure Description

[0026] Figure 1 This is a schematic diagram illustrating the regulatory principle of the protein phase separation process in this application;

[0027] Figure 2 This is a fluorescence change graph of the protein in vitro phase separation experiment in Example 1;

[0028] Figure 3 This is a graph showing the in vitro phase separation ability of proteins after TEV enzyme digestion in an in vitro phase separation experiment.

[0029] Figure 4 This is a fluorescence change graph of the protein in vivo phase separation experiment in Example 2;

[0030] Figure 5 This is a diagram showing the sub-cellular localization analysis of proteins in an in vivo phase separation experiment after TEV enzyme digestion. Detailed Implementation

[0031] The present application will be further described in detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a more complete understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0032] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in this application and in its specification is for descriptive purposes only and is not intended to be limiting of the application.

[0033] the term

[0034] Unless otherwise stated or in case of conflict, the terms or phrases used in this application shall have the following meanings:

[0035] The terms "and / or," "or / and," and "and / or" as used in this application encompass any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected using at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes solutions connected by "logical AND," and also undoubtedly includes solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0036] In this application, terms such as "preferred," "better," "more suitable," and "ideal" are merely used to describe implementation methods or embodiments that achieve better results, and should be understood not to limit the scope of protection of this application.

[0037] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0038] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0039] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0040] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0041] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0042] All references to this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the purpose and / or technical solution of this application, all cited references are incorporated herein by reference in their entirety and for all purposes. When references are cited in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. Examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0043] Existing methods for regulating protein phase separation have the following drawbacks: ① Insufficient regulatory precision: Current techniques typically rely on chemical induction or changes in environmental conditions (such as temperature and pH), lacking precise control over the phase separation process. This makes it difficult to determine the timing and extent of phase separation, thus affecting the reliability of experimental results. ② Poor biocompatibility: Many phase separation regulation methods use small molecules or compounds that may be toxic to cells, affecting normal cellular physiological functions and interfering with experimental data. This is particularly problematic in drug development and cell biology research. ③ Difficulty in achieving reproducibility: Because phase separation is affected by various external factors, existing regulation methods often struggle to maintain consistency across different experiments, resulting in poor reproducibility of experimental results. ④ High complexity: Many existing schemes (such as gene circuits based on synthetic biology) require complex design and construction, making them difficult to operate. This limits the application of these technologies to specific research teams and hinders their expansion into broader research fields. ⑤ Lack of biofeedback regulation: Existing techniques typically cannot achieve real-time feedback regulation of phase separation, failing to respond promptly to dynamic changes within cells, thus limiting their application in biological research.

[0044] Based on this, a first aspect of the embodiments of this application provides a protein phase separation regulation system and its application, which is used for phase separation regulation processes and has high controllability and biocompatibility.

[0045] Specifically, the protein phase separation regulation system includes: a target protein having phase separation properties; a regulatory tag linked to the target protein, the regulatory tag including a SUMO tag; and an enzyme cleavage site located between the regulatory tag and the target protein, the enzyme cleavage site including a TEV enzyme cleavage site.

[0046] In the aforementioned protein phase separation regulation system, the SUMO (small ubiquitin-like modified protein) tag design effectively inhibits the non-specific aggregation of phase separation elements. The SUMO tag possesses the characteristics of natural proteins, exhibiting strong biocompatibility and reducing cytotoxic effects. The introduction of the TEV (enteric thermophilic peptidase) cleavage site makes phase separation induction timely and controllable, allowing for precise induction at the desired time, demonstrating good flexibility. The system is relatively simple to construct and operate, easy to implement, and capable of responding to dynamic changes within the cell. This protein phase separation regulation system, used for phase separation regulation processes, exhibits high controllability and biocompatibility.

[0047] The target protein exhibits phase-separation properties, enabling it to spontaneously form phase-separated molecules after cleavage. In some embodiments, the target protein includes PodJ_NΔCC1-3.

[0048] In some embodiments, the protein phase separation regulation system further includes a luminescent tag linked to the target protein. Linking the luminescent tag facilitates monitoring the formation and dynamic changes of phase separation via luminescence signals. Further, the luminescent tag is a fluorescent tag. Even further, the luminescent tag includes a YFP fluorescent tag. It should be noted that the fluorescent tag is not limited to a YFP fluorescent tag; it can also be a protein selected from red fluorescent protein, yellow fluorescent protein, mCherry, mTAGBFP2, mCLOVER3, EGFP, Venus, mTagBFP2, mIFP, and TagRFP657 as the fluorescent tag.

[0049] In specific examples, the amino acid sequence of the SUMO tag is shown in SEQ ID NO.1; the amino acid sequence of the target phase separation protein PodJ_NΔCC1-3 is shown in SEQ ID NO.2; and the amino acid sequence of the fusion protein SUMO-x-PodJ_NΔCC1-3 is shown in SEQ ID NO.3.

[0050] Specifically, the sequence shown in SEQ ID NO.1 is as follows:

[0051] DSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFA KRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIG.

[0052] The sequence shown in SEQ ID NO.2 is as follows:

[0053] LGAVETANPATGVQEGLDSLAATLTQKMEAARLEMAAKLRESADGRFDRMERKLGEMAAHVQAAEQRSAQAIERMGREIVGVADAFNRRVHAAESRNASAIEQVGGEVARIAASVEHKLNRADSVQAQA LEKLGGEIARITEKLAERIGSAERRNALAIDDVGEQVARVTERLNQRHERSSQELVDRIRQSEERTLRMLEAREKIDSRLSEAQRKLEAAPPSPPPAQAPAPVATAQRPVPPAASPFEDNYFSQAASFS TSEDEADAFDAPPAPARSFEVAEFPAAEPEEPAFAHDDYAIADGFEPESPRYEVEPEVSDFAPAEPSRPMSTRDIIEQARAAARAAAASEGKG.

[0054] The sequence shown in SEQ ID NO.3 is as follows:

[0055] MSYYHHHHHHDYDIPTDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGSENLYFQGASMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKHRSATLGAVETANPATGVQEGLDSLAATLTQKMEAARLEMAAKLRESADGRFDRMERKLGEMAAHVQAAEQRSAQAIERMGREIVGVADAFNRRVHAAESRNASAIEQVGGEVARIAASVEHKLNRADSVQAQALEKLGGEIARITEKLAERIGSAERRNALAIDDVGEQVARVTERLNQRHERSSQELVDRIRQSEERTLRMLEEAREKIDSRLSEAQRKLEAAPPSPPPAQAPAPVATAQRPVPPAASPFEDNYFSQAASFSTSEDEADAFDAPPAPARSFEVAEFPAAEPEEPAFAHDDYAIADGFEPESPRYEVEPEVSDFAPAEPSRPMSTRDIIEQARAAARAAAASEGKG。

[0056] In the aforementioned protein phase separation regulation system, the SUMO tag, acting as a solubilizing tag, fuses with the target protein, enhancing its water solubility, reducing its spontaneous aggregation within cells, and inhibiting phase separation. A TEV restriction site is introduced between the SUMO tag and the phase separation element. The presence of the TEV restriction site allows the inhibitory effect of the SUMO tag to be relieved by TEV cleavage when needed, thereby inducing phase separation of the target protein. This application provides a controllable, biocompatible phase separation regulation system by combining the SUMO tag with the TEV restriction site, overcoming the shortcomings of existing technologies in terms of precision, biocompatibility, and operational complexity. This method not only enables efficient regulation of the phase separation process but also allows for safe intracellular application, providing a new tool for related biological research.

[0057] A second aspect of this application provides a coding sequence for encoding the protein phase separation regulation system described above.

[0058] The aforementioned coding sequence facilitates the acquisition of the protein phase separation regulatory system through genetic engineering, enabling its application in regulating protein phase separation in organisms or modulating intracellular signal transduction processes.

[0059] A third aspect of this application provides a recombinant vector carrying the coding sequence described above.

[0060] In some embodiments, the recombinant vector is an expression vector of the protein phase separation regulation system. Further, the recombinant vector is a prokaryotic expression vector. Even further, the recombinant vector is an *E. coli* expression vector. It should be noted that the recombinant vector is not limited to an *E. coli* expression vector, but can also be other prokaryotic expression vectors. It should also be noted that the recombinant vector is not limited to a prokaryotic expression vector, but can also be a eukaryotic expression vector.

[0061] The aforementioned recombinant vectors facilitate the acquisition of the protein phase separation regulation system through genetic engineering, enabling its application in regulating protein phase separation in organisms or modulating intracellular signal transduction processes.

[0062] A fourth aspect of this application provides a method for regulating the phase separation of a target protein, comprising the following steps S110-S130:

[0063] S110. Construct the expression vector for the protein phase separation regulation system described in the first aspect;

[0064] S120. The expression vector is transferred into a host bacterium for expression to obtain the protein phase separation regulation system;

[0065] S130. Add TEV enzyme to the protein phase separation regulation system and react, and use a monitoring instrument to monitor the formation and changes of the target protein phase separation.

[0066] In some embodiments, in S110, the expression vector is a prokaryotic expression vector. More specifically, the expression vector is an *E. coli* expression vector. It should be noted that the expression vector is not limited to an *E. coli* expression vector, but can also be other prokaryotic expression vectors. It should also be noted that the expression vector is not limited to prokaryotic expression vectors, but can also be a eukaryotic expression vector.

[0067] In some embodiments, the host bacterium includes *Escherichia coli*. It should be noted that the host bacterium is not limited to *Escherichia coli*, but may also be other prokaryotes.

[0068] Further, the step of transferring the expression vector into the host bacteria to obtain the protein phase separation regulation system includes: transforming the expression vector into Escherichia coli host bacteria to obtain a transformed strain; culturing and inducing the transformed strain, and then purifying the culture to obtain the protein phase separation regulation system.

[0069] In some embodiments, the monitoring instrument may be, for example, a fluorescence microscope.

[0070] The aforementioned method for regulating the phase separation of target proteins provides a controllable and biocompatible phase separation regulation system by binding the SUMO tag to the TEV restriction site, overcoming the shortcomings of existing technologies in terms of precision, biocompatibility, and operational complexity. This method not only enables efficient regulation of the phase separation process but also allows for safe intracellular application, providing a new tool for related biological research.

[0071] like Figure 1 ( Figure 1 As shown in the diagram illustrating the protein phase separation process regulation principle of this application, the protein phase separation regulation system of this application mainly achieves precise control of phase separation by designing fusion proteins and TEV restriction sites. The protein phase separation process regulation principle of this protein phase separation regulation system is as follows:

[0072] (1) Design of fusion proteins: SUMO proteins are used as solubilizing tags and fused with target phase-separating elements (e.g., proteins with phase-separating properties). SUMO tags can improve the water solubility of the target protein, reduce its spontaneous aggregation in cells, and inhibit the formation of phase separation.

[0073] (2) Introduction of TEV restriction site: A TEV restriction site is introduced between the SUMO tag and the phase separation element. The presence of the TEV restriction site allows the inhibition effect of the SUMO tag to be relieved by the cleavage action of the TEV enzyme when needed, thereby inducing phase separation of the target protein.

[0074] (3) Construction of fusion protein: Design and construct a fusion gene containing SUMO tag, TEV restriction site, fluorescent tag YFP and target phase separation protein, and express and purify it.

[0075] (4) Transformation: The constructed plasmid containing the fusion protein is transformed into bacteria to ensure its correct expression in the cell.

[0076] (5) TEV enzyme treatment: Add TEV enzyme at the appropriate time point to induce SUMO tag cleavage, thereby initiating phase separation of the target protein.

[0077] (6) Monitoring and analysis of phase separation: The formation and dynamic changes of phase separation are monitored by fluorescence microscopy.

[0078] The technical solution of this application has the following advantages:

[0079] By designing a SUMO-tagged fusion protein and a TEV restriction site, this paper presents a phase separation system capable of real-time, precise regulation. This system allows researchers to induce or inhibit phase separation at the desired time, achieving high-precision phase separation control. The SUMO tag, as a natural protein, exhibits good biocompatibility, can be safely used within cells with reduced cytotoxicity, and demonstrates high biocompatibility. The system is relatively simple to construct and operate, making it easy to implement and allowing researchers in different laboratories to conveniently apply this technology, simplifying the operational process. Through the design of the fusion protein, it is possible to respond to dynamic changes within the cell, enhancing the flexibility and adaptability of phase separation regulation and achieving dynamic feedback control. This application provides an innovative solution for phase separation research, advancing basic biological research and biomedical applications.

[0080] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0081] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0082] Example 1

[0083] The procedure for the in vitro protein phase separation experiment in this embodiment is as follows:

[0084] 1. Design of fusion proteins

[0085] The fusion protein in this embodiment consists of the following parts:

[0086] SUMO tag: Through its solubilizing properties, it improves the solubility of the target protein and prevents its spontaneous aggregation in E. coli. Its amino acid sequence is: DSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKR QGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIG.

[0087] TEV restriction site: A TEV restriction site is introduced between the SUMO tag and the target protein to allow for cleavage when needed, thus relieving the inhibitory effect of the SUMO tag. The amino acid sequence of the restriction site is: ENLYFQG.

[0088] Target phase separation protein: PodJ_NΔCC1-3, whose amino acid sequence is: LGAVETANPATGVQEGLDSLAATLTQKMEAARLEMAAKLRESADGRFDRMERKLGEMAAHVQAAEQRSAQAIERMGREIVGVADAFNRRVHAAESRNASAIEQVGGEVARIAASVEHKLNRADSVQAQALEKLGGEIARITEKLAERIGSAERR NALAIDDVGEQVARVTERLNQRHERSSQELVDRIRQSEERTLRMLEEAREKIDSRLSEAQRKLEAAPPSPPPAQAPAPVATAQRPVPPAASPFEDNYFSQAASFSTSEDEADAFDAPPAPARSFEVAEFPAAEPEEPAFAHDDYAIADGFEPESPRYEVEPEVSDFAPAEPSRPMSTRDIIEQARAAARAAAASEGKG.

[0089] 2. Specific experimental procedure:

[0090] Design and Cloning: Using molecular cloning technology, an expression vector for the SUMO-TEV target protein was designed. The target gene was amplified by PCR and ligated with the SUMO and TEV sequences to construct the in vitro expression vector pTEV5-SUMO-x-YFP-PodJ_NΔCC1-3.

[0091] Transformation of Escherichia coli: The recombinant expression vector was transformed into Escherichia coli BL21(DE3), cultured in a medium containing antibiotics, and positive strains were screened.

[0092] Expression and induction: Transformed strains were cultured in LB medium and protein expression was induced by 0.5 mM IMPTG (isopropyl-β-D-thiogalactoside) for 12 hours.

[0093] Purification of fusion protein: Impurities were removed by Ni-NTA affinity chromatography and dialysis to obtain high-purity fusion protein.

[0094] TEV enzyme treatment: 5 μL of 13.5 μM SUMO-x-YFP-PodJ_NΔCC1-3 was mixed with 0.5 μL of 0.5 μM TEV enzyme. After adding TEV enzyme, the reaction was carried out at room temperature for 10 min. After the reaction, the formation and dynamic changes of phase-separated particles of the target protein were observed using a fluorescence microscope. The detection results are as follows: Figure 2 and Figure 3 As shown. Figure 2 This is a fluorescence change diagram of the protein in vitro phase separation experiment in Example 1. Figure 3 This is a graph showing the in vitro phase separation capability analysis of proteins after TEV enzyme digestion in an in vitro phase separation experiment.

[0095] from Figure 2 and Figure 3 As can be seen, in this embodiment, the SUMO tag binds to the TEV restriction site. After TEV restriction, the phase separation element of the fusion protein can rapidly form phase separation droplets in vitro. This technology is stable in living cells and provides a reliable experimental tool for phase separation research.

[0096] Example 2

[0097] The procedure for the in vivo protein phase separation experiment in this embodiment is as follows:

[0098] 1. Design of fusion proteins

[0099] Construct an in vivo expression vector for Escherichia coli: TEV enzyme and pCDF-SUMO-x-PodJ_NΔCC1-3.

[0100] The fusion protein in this embodiment consists of the following parts:

[0101] SUMO tag: Through its solubilizing properties, it improves the solubility of the target protein and prevents its spontaneous aggregation in E. coli.

[0102] TEV restriction site: A TEV restriction site is introduced between the SUMO tag and the target protein to allow for cleavage when needed, thus relieving the inhibitory effect of the SUMO tag.

[0103] Target phase-separating protein: PodJ_NΔCC1-3.

[0104] The amino acid sequence of fusion protein SUMO-x-PodJ_NΔCC1-3 is: MSYYHHHHHHDYDIPTDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAH REQIGSENLYFQGASMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNR IELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKHRSATLGAVETANPATGVQEGL DSLAATLTQKMEAARLEMAAKLRESADGRFDRMERKLGEMAAHVQAAEQRSAQAIERMGREIVGVADAFNRRVHAAESRNASAIEQVGGEVARIAASVEHKLNRADSVQAQALEKLGGEIARITEKLAERIGSAERRN ALAIDDVGEQVARVTERLNQRHERSSQELVDRIRQSEERTLRMLEEAREKIDSRLSEAQRKLEAAPPSPPPAQAPAPVATAQRPVPPAASPFEDNYFSQAASFTSSEDEADAFDAPPAPARSFEVAEFPAAEPEEPAFAHDDYAIADGFEPESPRYEVEPEVSDFAPAEPSRPMSTRDIIEQARAAARAAAASEGKG.

[0105] 2. Specific experimental procedure:

[0106] Design and Cloning: Using molecular cloning technology, an expression vector for the SUMO-TEV target protein was designed. The target gene was amplified by PCR and ligated with the SUMO and TEV sequences to construct the in vivo expression vector pCDF-SUMO-x-YFP-PodJ_NΔCC1-3.

[0107] Transformation of Escherichia coli: The recombinant expression vector was transformed into Escherichia coli BL21(DE3), cultured in antibiotic-containing medium, and positive strains were screened.

[0108] Culture and induction: Two transformant strains were cultured together in LB medium, and protein expression was induced by 0.1 mM IPTG (isopropyl-β-D-thiogalactoside) for 2 hours.

[0109] Test results as follows Figure 4 and Figure 5 As shown. Figure 4 This is a fluorescence change diagram of the protein in vivo phase separation experiment in Example 2. Figure 5 This is a diagram showing the sub-cellular localization analysis of proteins in an in vivo phase separation experiment after TEV enzyme digestion.

[0110] like Figure 4 and Figure 5 It is known that the SUMO tag binds to the TEV restriction site, and after TEV cleavage, the phase separation element of the fusion protein can rapidly form phase separation droplets in vivo, providing a reliable experimental tool for phase separation research.

[0111] In summary, this application provides a phase separation system capable of real-time and precise regulation by designing a fusion protein based on the SUMO tag and a TEV restriction site. This allows researchers to induce or inhibit phase separation at the desired time, achieving high-precision phase separation control. The SUMO tag, as a natural protein, exhibits good biocompatibility, can be safely used within cells, reduces toxic effects on cells, and demonstrates high biocompatibility.

[0112] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0113] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A protein phase separation regulation system, characterized in that, The protein phase separation regulation system includes a fusion protein, which is mainly composed of the following components: The target protein, which has phase-separation properties, is PodJ_NΔCC1-3 with the amino acid sequence shown in SEQ ID NO.2; A regulatory tag linked to the target protein, wherein the regulatory tag is a SUMO tag with an amino acid sequence as shown in SEQ ID NO.1; The restriction enzyme site located between the regulatory tag and the target protein, the restriction enzyme site including the TEV restriction enzyme site; The amino acid sequence of the fusion protein is shown in SEQ ID NO.

3.

2. A protein phase separation regulation system, characterized in that, The protein phase separation regulation system includes a fusion protein having the following components: The target protein, which has phase-separation properties, is PodJ_NΔCC1-3 with the amino acid sequence shown in SEQ ID NO.2; A regulatory tag linked to the target protein, wherein the regulatory tag is a SUMO tag with an amino acid sequence as shown in SEQ ID NO.1; The restriction enzyme site located between the regulatory tag and the target protein, the restriction enzyme site including the TEV restriction enzyme site; A luminescent tag, wherein the luminescent tag is attached to the target protein, and the luminescent tag is a fluorescent tag.

3. The protein phase separation regulation system according to claim 2, characterized in that, The luminescent tag is selected from one of the following proteins: YFP, red fluorescent protein, yellow fluorescent protein, mCherry, mTAGBFP2, mCLOVER3, EGFP, Venus, mTagBFP2, mIFP, and TagRFP657.

4. A recombinant vector, characterized in that, The recombinant vector carries the coding sequence of the protein phase separation regulation system according to any one of claims 1-3.

5. The recombinant vector according to claim 4, characterized in that, The recombinant vector is the expression vector of the protein phase separation regulation system.

6. A method for regulating the phase separation of a target protein, characterized in that, Includes the following steps: Construct an expression vector for the protein phase separation regulation system according to any one of claims 1-3; The expression vector was transferred into a host bacterium for expression to obtain the protein phase separation regulation system; TEV enzyme is added to the protein phase separation regulation system and reacted, and the formation and changes of the target protein phase separation are monitored using a monitoring instrument.

7. The method according to claim 6, characterized in that, The host bacterium includes Escherichia coli. The step of transferring the expression vector into the host bacterium for expression to obtain the protein phase separation regulation system includes: transforming the expression vector into the Escherichia coli host bacterium to obtain a transformed strain; culturing and inducing the transformed strain, and then purifying the culture to obtain the protein phase separation regulation system.

8. The application of the protein phase separation regulation system according to any one of claims 1-3, or the recombinant vector according to any one of claims 4-5, or the method according to any one of claims 6-7 in regulating protein phase separation in organisms.

9. The application of the protein phase separation regulation system according to any one of claims 1-3, or the recombinant vector according to any one of claims 4-5, or the method according to any one of claims 6-7 in regulating intracellular signal transduction.

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