Method for regulating and controlling phase separation capacity of protein and application of method

Abstract

The invention relates to a method for regulating and controlling the phase separation capacity of protein and application thereof. The method comprises the following steps: mixing a first protein element and a second protein element to obtain a mixture; detecting the phase separation capacity of the mixture; wherein the first protein element comprises an IDR structural domain of the scaffold protein, and the second protein element comprises a self-assembly structural domain of the scaffold protein or the IDR structural domain of the scaffold protein. According to the method, the IDR structural domain of the scaffold protein is used as the first protein element, the self-assembly structural domain of the scaffold protein or the IDR structural domain of the scaffold protein is used as the second protein element, and the first protein element and the second protein element interact with each other, so that the phase separation strength of the protein elements can be accurately regulated and controlled; and physical or chemical factor intervention is not needed, so that the compatibility between the regulation process and the normal physiological function of the cell is ensured. The method for regulating and controlling the phase separation capacity of the protein has relatively high controllability and biocompatibility.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to methods for regulating the phase separation ability of proteins and their applications. Background Art

[0002] Biomolecular phase separation is an important biophysical process in cells, which regulates various biological functions by forming membraneless biomolecular condensates. These phase-separated structures play a role similar to membranous organelles in cells, can concentrate specific biomolecules, and regulate the progress of biochemical reactions. By precisely regulating the intensity of phase separation, this method is expected to reduce the occurrence of abnormal cell functions, providing new research ideas and treatment strategies for the fields of human health and disease treatment. This innovative technology is expected to have a profound impact on the biomedical field, expanding the research directions in the fields of cell biology and disease treatment.

[0003] Currently, the methods for regulating the intensity of phase separation mainly include the following categories: 1) Physical regulation: by changing physical conditions such as temperature, pH value, etc.; 2) Chemical regulation: using chemical reagents to change protein interactions; 3) Genetic regulation: by changing the protein sequence to affect the phase separation ability.

[0004] Existing phase separation regulation methods often rely on the intervention of physical or chemical factors. Such a regulation method may interfere with the normal physiological functions of cells; the regulation specificity is insufficient, which may affect other cell processes; moreover, the existing regulation methods often target specific protein systems, lacking wide applicability; it is difficult to achieve standardized regulation effects in different biological systems; and mainly rely on the changes of exogenous small molecule compounds or environmental factors (such as temperature, pH value, etc.), lacking precise and real-time control of the phase separation process. This method often leads to the uncertainty and instability of the regulation results, affecting the practical application of phase separation. The limitations of these existing technologies make it particularly important to find a phase separation regulation method with biocompatibility and high controllability. Summary of the Invention

[0005] Based on this, the present application provides a method for regulating the phase separation ability of proteins, which has biocompatibility and high controllability.

[0006] A method for regulating the phase separation ability of proteins, comprising the following steps:

[0007] Mix a first protein element and a second protein element to obtain a mixture;

[0008] Detect the phase separation ability of the mixture;

[0009] Among them, the first protein element includes the intrinsically disordered region (IDR) domain of a scaffolding protein, and the second protein element includes the self-assembly domain of a scaffolding protein or the IDR domain of a scaffolding protein.

[0010] In the above method for regulating the protein phase separation ability, the IDR domain of a scaffolding protein is used as the first protein element, and the self-assembly domain of a scaffolding protein or the IDR domain of a scaffolding protein is used as the second protein element. The interaction between the first protein element and the second protein element can precisely regulate the strength of protein element phase separation, and without the intervention of physical or chemical factors, ensuring that the regulation process is compatible with the normal physiological functions of cells. The above method for regulating the protein phase separation ability has high controllability and biocompatibility.

[0011] In some embodiments, the second protein element contains the IDR domain of a scaffolding protein, and the second protein element is different from the first protein element.

[0012] In some embodiments, the first protein element is selected from one of SpmX IDR , PodJ IDR and PopZ IDR , and the second protein element is selected from one of SpmX IDR , PodJ IDR and PopZ IDR .

[0013] In some embodiments, the second protein element contains the self-assembly domain of a scaffolding protein, and the second protein element is selected from one of PodJ cc1-3 , PodJ cc4-6 , PodJ cc1-3 and PopZ H2-4 .

[0014] In some embodiments, the first protein element is selected from one of SpmX IDR , PodJ IDR and PopZ IDR .

[0015] In some embodiments, the first protein element is linked to a first luminescent tag, and the second protein element is linked to a second luminescent tag, and the second luminescent tag is different from the first luminescent tag.

[0016] In some embodiments, the first luminescent tag and the second luminescent tag are different fluorescent tags.

[0017] In some embodiments, the first luminescent tag is selected from the group consisting of green fluorescent protein, red fluorescent protein, yellow fluorescent protein, mCherry, mTAGBFP2, mCLOVER3, Venus, mTagBFP2, mIFP, and TagRFP657;

[0018] The second luminescent tag is selected from the group consisting of green fluorescent protein, red fluorescent protein, yellow fluorescent protein, mCherry, mTAGBFP2, mCLOVER3, Venus, mTagBFP2, mIFP, and TagRFP657.

[0019] In some embodiments, the mixing ratio of the first protein element to the second protein element is from 1:2 to 1:1;

[0020] And / or, the step of detecting the phase separation ability of the mixture includes: detecting the phase separation ability of the mixture using a fluorescence microscope.

[0021] Use of the method described above in enhancing protein functionality and / or screening of therapeutic drugs for protein-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a diagram showing the detection result of the phase separation of the scaffold protein in Example 1. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0023] The present application will be further described in detail below in conjunction with the embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present application and not to limit the scope of the present application. The purpose of providing these embodiments and examples is to make the understanding of the disclosed content of the present application more thorough and comprehensive. 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 in the present application. Those skilled in the art can make various modifications or alterations without departing from the connotation of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. In addition, in the following description, a large number of specific details are given to provide a more thorough understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0024] Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as commonly understood by those skilled in the technical field to which the present application belongs. The terms used in the description of the present application in this specification are only for the purpose of describing the embodiments and examples and are not intended to limit the present application.

[0025] TERMS

[0026] Unless otherwise stated or there is a contradiction, the terms or phrases used in the present application have the following meanings:

[0027] As used in this application, the selection scope of the terms "and / or", "or / and", and "and / or" includes any one of two or more related listed items, and also includes any and all combinations of the related listed items. The said any and all combinations include combinations of any two related listed items, any more related listed items, or all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, this technical solution undoubtedly includes the technical solution connected by "logical AND", and also undoubtedly includes the technical solution connected by "logical OR". For example, "A and / or B" includes three parallel solutions: A, B, and A + B. Another example, for the technical solution of "A, and / or, B, and / or, C, and / or, D", it includes any one of A, B, C, and D (that is, the technical solution connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, it includes combinations of any two or any three of A, B, C, and D, and also includes the combination of the four items A, B, C, and D (that is, the technical solution connected by "logical AND").

[0028] In this application, "preferred", "better", "more preferred", and "preferably" are only used to describe embodiments or examples with better effects. It should be understood that they do not constitute a limitation on the protection scope of this application.

[0029] In this application, "further", "furthermore", "especially", etc. are used for descriptive purposes, indicating differences in content, but should not be construed as a limitation on the protection scope of this application.

[0030] In this application, "optionally", "optional", and "option" mean "may or may not", that is, any one of the two parallel solutions of "yes" or "no". If "optional" appears in a technical solution in multiple places, without special instructions and without contradictions or mutual restrictions, each "optional" is independent of each other.

[0031] In this application, in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or quantity, nor can it be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive listing and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0032] In this application, for the technical features described in an open-ended manner, it includes the closed technical solutions composed of the listed features, and also includes the open technical solutions containing the listed features.

[0033] In this application, %(w / w) and wt% both represent weight percentage, %(v / v) represents volume percentage, and %(w / v) represents mass-volume percentage.

[0034] All documents mentioned in this application are incorporated herein by reference as if each document was individually incorporated by reference. Unless it conflicts with the purpose of this application and / or the technical solution, the cited documents involved in this application are cited in their entirety and for all purposes. When this application refers to a cited document, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited document are also incorporated by reference. When this application refers to a cited document, examples and preferred methods of the relevant technical features cited can also be incorporated as references into this application, but only to the extent that this application can be implemented. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or be amended adaptively according to the description in this application.

[0035] Existing methods for regulating phase separation often rely on the intervention of physical or chemical factors. Such a regulation method may interfere with the normal physiological functions of cells; the lack of specificity in regulation may affect other cellular processes. Existing regulation methods often target specific protein systems, lack broad applicability, and it is difficult to achieve standardized regulation effects in different biological systems.

[0036] The purpose of this application is to overcome the deficiencies of the prior art and propose a more precise, effective and safe biological regulation method to regulate the strength of biomolecular phase separation. By deeply studying phase separation protein components and their interaction rules, this application has discovered components that can enhance or weaken the phase separation process and proposed corresponding regulation strategies.

[0037] ① Provide biocompatible solutions: Establish a regulation method without the intervention of physical or chemical factors to ensure that the regulation process is compatible with the normal physiological functions of cells.

[0038] ② Establish a general regulation system: Develop a phase separation regulation method with good generality to achieve standardized application in different biological systems.

[0039] ③ Provide a controllable regulation mechanism: Through in-depth analysis of phase separation protein components and their interactions, achieve precise regulation of the strength of phase separation, and provide new ideas and methods for solving problems such as protein pathological aggregation.

[0040] Therefore, based on this, in the first aspect of the embodiments of this application, a method for regulating the phase separation ability of proteins is provided, including the following steps S110 - S120:

[0041] S110. Mix a first protein component and a second protein component to obtain a mixture;

[0042] S120, detect the phase separation ability of the mixture;

[0043] Wherein, the first protein element includes the IDR domain of the scaffold protein, and the second protein element includes the self-assembly domain of the scaffold protein or the IDR domain of the scaffold protein.

[0044] In the above method for regulating the protein phase separation ability, using the IDR domain of the scaffold protein as the first protein element and the self-assembly domain of the scaffold protein or the IDR domain of the scaffold protein as the second protein element, the interaction between the first protein element and the second protein element can precisely regulate the strength of protein element phase separation, and without the intervention of physical or chemical factors, ensuring that the regulation process is compatible with the normal physiological functions of cells. The above method for regulating the protein phase separation ability has high controllability and biocompatibility.

[0045] Regarding the research on the biological scaffold protein system, these proteins have two important domains:

[0046] ① Self-assembly domain: capable of forming ordered protein aggregates.

[0047] ② Intrinsically disordered region (IDR): capable of forming liquid-liquid phase separation through weak interactions.

[0048] In some embodiments, the second protein element contains the IDR domain of the scaffold protein and is different from the first protein element. The fusion of different IDRs enhances the phase separation ability, possibly promoting condensation through electrostatic and hydrophobic interactions, which provides a new way to enhance protein functionality. Wherein, the first protein element is selected from SpmX IDR , PodJ IDR and PopZ IDR One of them, and the second protein element is selected from SpmX IDR , PodJ IDR and PopZ IDR One of them.

[0049] Specifically, SpmX IDRThe amino acid sequence is as shown in SEQ ID NO.1, i.e., VPAPSPVLRPKVDYDASCAVPKQTPATVNTRTEGDRVIAQREEGLPLNVVVPEEDAPTATEQSAAAVAARLEAILPETPSAPASMAKPAMRQDDLGLPEPPAPTQPAAMEPAPVLFEAEPQPAPVEPPPAAPVEFTPFRLTPQASEQAEAPVEAPAPTRPAPSEPTLFGAPATGSSVFNLDGFSTSEDATVGVMD.

[0050] PodJ IDR The amino acid sequence is as shown in SEQ ID NO.2, i.e., QELVDRIRQSEERTLRMLEEAREKIDSRLSEAQRKLEAAPPSPPPAQAPAPVATAQRPVPPAASPFEDNYFSQAASFSTSEDEADAFDAPPAPARSFEVAEFPAAEPEEPAFAHDDYAIADGFEPESPRYEVEPEVSDFAPAEPSRPMSTRDIIEQARAAARAAAASEGKG.

[0051] PopZ IDR The amino acid sequence is as shown in SEQ ID NO.3, i.e., DAPAEPAAEAAPPPPPEPEPEPVSFDDEVLELTDPIAPEPELPPLETVGDIDVYS PPEPESEPAYTPPPAAPVFDRDE.

[0052] In some embodiments, the second protein element contains the self-assembly domain of the scaffold protein. The interaction between IDR and the self-assembled protein significantly improves the phase separation ability. This mode can not only enhance functional aggregation but also may inhibit pathological aggregation, providing new ideas for the treatment of protein-related diseases. Among them, the second protein element is selected from one of PodJ cc1-3 、PodJ cc4-6 、PodJ cc1-3 and PopZ H2-4 One of them. The first protein element is selected from one of SpmX IDR 、PodJ IDR and PopZ IDR One of them.

[0053] Specifically, PodJ cc1-3The amino acid sequence of [object] is shown in SEQ ID NO.4, i.e., MTAASPWSVKGIDPKAREVAKDLARRSGMTLGEWLNRMIIEGDGQTADPRLAGDDVPNRAYLEIVKDDAPPRIEIAEHPADEVGRVALALDRLTQRIEAAEGRNAAAITGIDHSVRDALTRLGASEREQIAVAARFEGAVDELKTEQARATERLRRIESEAAGPRSAEALRALEGALGKVAGHLYEGEARTREAIATLEAKLNQQSSGDPSALVEAVVARLGERLEAAETRTSDALRELGASFQALDQR。

[0054] PodJ cc4-6 The amino acid sequence of [object] is shown in SEQ ID NO.5, i.e., LGAVETANPATGVQEGLDSLAATLTQKMEAARLEMAAKLRESADGRFDRMERKLGEMAAHVQAAEQRSAQAIERMGREIVGVADAFNRRVHAAESRNASAIEQVGGEVARIAASVEHKLNRADSVQAQALEKLGGEIARITEKLAERIGSAERRNALAIDDVGEQVARVTERLNQRHERSS。

[0055] PopZ H2-4 The amino acid sequence of [object] is shown in SEQ ID NO.6, i.e., VAEQLVGVSAASAAASAFGSLSSALLMPKDGRTLEDVVRELLRPLLKEWLDQ NLPRIVETKVEEEVQRISRGRGA。

[0056] In some embodiments, the first protein element is connected with a first luminescent tag, the second protein element is connected with a second luminescent tag, and the second luminescent tag is different from the first luminescent tag.

[0057] Further, the first luminescent tag and the second luminescent tag are different fluorescent tags.

[0058] Still further, the first luminescent tag is selected from the group consisting of green fluorescent protein, red fluorescent protein, yellow fluorescent protein, mCherry, mTAGBFP2, mCLOVER3, Venus, mTagBFP2, mIFP, and TagRFP657;

[0059] The second fluorescent tag is selected from one of green fluorescent protein, red fluorescent protein, yellow fluorescent protein, mCherry, mTAGBFP2, mCLOVER3, Venus, mTagBFP2, mIFP, and TagRFP657.

[0060] In some embodiments, the mixing ratio of the first protein element and the second protein element is 1:2 to 1:1.

[0061] In some embodiments, before the step of S110, the following steps are further included: constructing a first protein element and a second protein element. Specifically, the first protein element and the second protein element are constructed by using genetic engineering techniques.

[0062] Compared with the prior art, the present application has the following advantages:

[0063] ① Universal regulation system: The regulation method of the present application enables it to be standardized and applied in different biological systems. This universality greatly improves the scope of application of the method, and can solve different types of phase separation problems more widely compared with the specific applications of the prior art.

[0064] ② Biocompatibility: The regulation method of the present application does not require the introduction of physical or chemical factors, which ensures the compatibility of the regulation process with the normal physiological functions of cells. Compared with the regulation means that often rely on external factors in the prior art, the present invention can better maintain the physiological state of cells and reduce the interference with cell functions.

[0065] ③ Controllability and precise regulation: Through in-depth analysis of the phase separation protein elements and their interactions, the regulation method of the present application realizes precise regulation of the strength of phase separation. This precise regulation ability enables researchers to more effectively intervene in specific biological problems (such as protein pathological aggregation), improves the efficiency of research and application, and has a more significant effect compared with the fuzzy regulation of the prior art.

[0066] ④ Cost reduction and efficiency improvement: Since the regulation method of the present application does not rely on external physical or chemical factors, it reduces the material cost and experimental complexity, and at the same time improves the repeatability and reliability of the experiment. This enables researchers to save time and resources when conducting relevant experiments and improves the overall research efficiency.

[0067] The implementation solutions of the present application will be described in detail below in conjunction with embodiments. It should be understood that these embodiments are only used to illustrate the present application and not to limit the scope of the present application. For the experimental methods without specific conditions noted in the following embodiments, priority is given to the guidance given in the present application, and it can also be carried out according to the experimental manuals or conventional conditions in the art, or according to the conditions recommended by the manufacturer, or by referring to the experimental methods known in the art.

[0068] In the following specific embodiments, regarding the measurement parameters of raw material components, without special instructions, there may be slight deviations within the weighing accuracy range. Regarding temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0069] Example 1

[0070] The experimental operation process of this example is as follows:

[0071] Through preliminary experimental research of the present invention, the results show that the proposed interaction mode can effectively affect the phase separation ability of the scaffolding protein. The specific situation is as follows:

[0072] Experimental design: Three different interaction modes were used for research: self-assembly + self-assembly, IDR + IDR, self-assembly + IDR. Through systematic analysis of these modes, their effects on phase separation behavior were evaluated.

[0073] 1. The experimental process of the self-assembly + self-assembly mode is as follows:

[0074] (1) Experimental grouping:

[0075] Self-assembly experimental group, self-assembly control group 1, and self-assembly control group 2. The experimental objects of each group are as follows:

[0076] Self-assembly experimental group: The experimental objects are two self-assembly proteins, namely PodJ cc1-3 and PopZ H2-4 , which are mixed at a concentration mixing ratio of 1:1 by area (the concentration of each protein before mixing is 10 μM, and the final concentration of each protein after mixing is 5 μM).

[0077] Self-assembly control group 1: The experimental object is only one self-assembly protein, PodJ cc1-3 .

[0078] Self-assembly control group 2: The experimental object is only one self-assembly protein, PopZ H2-4 .

[0079] Among them, the fluorescent protein linked to PodJ cc1-3 is YEP, and the fluorescent protein linked to PopZ H2-4 is mChy.

[0080] (2) Experimental steps:

[0081] Self-assembly experimental group: Two self-assembly protein components were mixed at a concentration mixing ratio of 1:1 (the concentration of each protein was 10 μM before mixing, and the final concentration of each protein was 5 μM after mixing). After mixing, it was ice-bathed for 10 min, and the states of the two protein components were directly observed using a fluorescence microscope at room temperature, and the proportion of the luminescent area of each protein was counted.

[0082] Self-assembly control group 1 and self-assembly control group 2: The corresponding self-assembly protein components with a final concentration of 5 μM were ice-bathed for 10 min, and the states of the protein components were directly observed using a fluorescence microscope at room temperature, and the proportion of the luminescent area of each protein was counted.

[0083] The experimental results are shown in Figure 1 (i.e., Figure 1 a). As can be seen from Figure 1 a, the interaction between (identical or different) self-assembly components not only fails to promote phase separation but may also lead to protein denaturation.

[0084] 2. The experimental procedure for the IDR+IDR mode is as follows:

[0085] (1) Experimental grouping:

[0086] IDR experimental group 1, IDR experimental group 2, IDR control group 1, IDR control group 2, and IDR control group 3. The experimental subjects for each group are as follows:

[0087] IDR experimental group 1: The experimental subjects are two IDR proteins, namely SpmX IDR , PodJ IDR , and the two protein components were mixed at a concentration mixing ratio of 1:1 (the concentration of each protein was 10 μM before mixing, and the final concentration of each protein was 5 μM after mixing).

[0088] IDR experimental group 2: The experimental subjects are two IDR proteins, namely SpmX IDR , PopZ IDR , and the two protein components were mixed at a concentration mixing ratio of 1:1 (the concentration of each protein was 10 μM before mixing, and the final concentration of each protein was 5 μM after mixing).

[0089] IDR control group 1: The experimental subject is only one IDR protein, SpmX IDR .

[0090] IDR control group 2: The experimental subject is only one IDR protein, PodJ IDR .

[0091] IDR control group 3: The experimental subject is only one IDR protein, PopZ IDR .

[0092] Among them, SpmX IDR is linked to the fluorescent protein CFP, PodJ IDR is linked to the fluorescent protein YFP, PopZ IDR is linked to the fluorescent protein mChy.

[0093] (2) Experimental steps:

[0094] IDR experimental group 1, IDR experimental group 2: Mix the two IDR protein components in a concentration mixing ratio of 1:1 by area (the concentration of each protein before mixing is 10 μM, and the final concentration of each protein after mixing is 5 μM). After mixing, incubate on ice for 10 min, and directly observe the states of the two protein components using a fluorescence microscope at room temperature, and count the proportion of the luminescent area of each protein.

[0095] IDR control group 1, IDR control group 2, and IDR control group 3: Incubate the corresponding IDR protein components with a final concentration of 5 μM on ice for 10 min, directly observe the state of the protein components using a fluorescence microscope at room temperature, and count the proportion of the luminescent area of each protein.

[0096] The experimental results are shown in Figure 1 (i.e., Figure 1 b). It can be seen from Figure 1 b that the fusion between different IDR protein components enhances the phase separation ability, and may promote condensation through electrostatic and hydrophobic interactions, which provides a new way to enhance protein functionality.

[0097] 3. The experimental process of the IDR + self-assembly mode is as follows:

[0098] (1) Experimental grouping:

[0099] IDR + self-assembly experimental group 1, IDR + self-assembly experimental group 2, control group 1, control group 2, and control group 3. The experimental objects of each group are as follows:

[0100] IDR + self-assembly experimental group 1: The experimental objects are SpmX IDR and PodJ cc1-3 , and the two protein components are mixed in a concentration mixing ratio of 1:1 by area (the concentration of each protein before mixing is 10 μM, and the final concentration of each protein after mixing is 5 μM).

[0101] IDR + self-assembly experimental group 2: The experimental objects are SpmX IDR and PodJ cc4-6 , and the two protein components are mixed in a concentration mixing ratio of 1:1 by area (the concentration of each protein before mixing is 10 μM, and the final concentration of each protein after mixing is 5 μM).

[0102] Control group 1: The experimental subject is only one IDR protein, SpmX IDR .

[0103] Control group 2: The experimental subject is only one self-assembling protein, PodJ cc1-3 .

[0104] Control group 3: The experimental subject is only one self-assembling protein, PodJ cc4-6 .

[0105] Among them, the fluorescent protein linked to SpmX IDR is CFP, and the fluorescent protein linked to PodJ cc1-3 is YFP, and the fluorescent protein linked to PodJ cc4-6 is YFP.

[0106] (2) Experimental steps:

[0107] IDR + self-assembly experimental group 1, IDR + self-assembly experimental group 2: Mix the IDR protein component and the self-assembly protein component according to a concentration mixing ratio of 1:1 (the concentration of each protein before mixing is 10 μM, and the final concentration of each protein after mixing is 5 μM). After mixing, ice-bath for 10 min, and directly observe the states of the two protein components with a fluorescence microscope at room temperature, and count the proportion of the luminescent area of each protein.

[0108] Control group 1, Control group 2 and Control group 3: Ice-bath the corresponding protein components with a final concentration of 5 μM for 10 min, and directly observe the states of the protein components with a fluorescence microscope at room temperature, and count the proportion of the luminescent area of each protein.

[0109] The experimental results are shown in Figure 1 (i.e., Figure 1 c). It can be seen from Figure 1 c that the interaction between IDR and the self-assembling protein significantly improves the phase separation ability. This mode can not only enhance functional aggregation but also may inhibit pathological aggregation, providing new ideas for the treatment of protein-related diseases.

[0110] In summary, the experimental results of Example 1 show that effective phase separation has not formed between (the same or different) self-assembling proteins, but instead has led to protein denaturation and precipitation, presumably forming irregular inclusion bodies. This is consistent with the hypothesis of the interaction of self-assembling proteins in this application. After obtaining the image results, the results are statistically analyzed by observing the state of the phase separation droplets (whether regular round droplets are formed) and counting the proportion of the area of the phase separation droplets( Figure 1 b - c). On the one hand, the IDR condensates of different scaffold proteins can completely fuse, indicating that their phase separation ability has been enhanced. This finding supports our theory that IDR interaction enhances the phase separation ability.

[0111] In the method for regulating the protein phase separation ability of the present application, the interaction between the first protein element and the second protein element can achieve precise regulation of the strength of protein element phase separation, and without the intervention of physical or chemical factors, it ensures that the regulation process is compatible with the normal physiological functions of cells, has high controllability and biocompatibility, provides a valuable data basis for subsequent in-depth research, and lays a solid foundation for further exploring the phase separation regulation mechanism and its biological applications.

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

[0113] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the invention patent should be subject to the appended claims.

Claims

1. A method for regulating the protein phase separation ability, characterized in that Comprising the following steps: Mix the first protein element and the second protein element to obtain a mixture; Detect the phase separation ability of the mixture; Wherein, the first protein element comprises the IDR domain of a scaffolding protein, and the second protein element comprises the self-assembly domain of a scaffolding protein or the IDR domain of a scaffolding protein.

2. The method according to claim 1, characterized in that The second protein element contains the IDR domain of a scaffolding protein, and the second protein element is different from the first protein element.

3. The method according to claim 2, characterized in that, The first protein element is selected from SpmX IDR , PodJ IDR and PopZ IDR One of them, and the second protein element is selected from SpmX IDR , PodJ IDR and PopZ IDR One of them.

4. The method according to claim 1, wherein The second protein element contains the self-assembly domain of the scaffolding protein, and the second protein element is selected from PodJ cc1-3 , PodJ cc4-6 , PodJ cc1-3 and PopZ H2-4 and is one of them.

5. The method according to claim 4, wherein The first protein element is selected from SpmX IDR , PodJ IDR and PopZ IDR and is one of them.

6. The method according to claim 1, characterized in that, The first protein element is linked with a first luminescent tag, and the second protein element is linked with a second luminescent tag, and the second luminescent tag is different from the first luminescent tag.

7. The method according to claim 6, wherein The first luminescent tag and the first luminescent tag are different fluorescent tags.

8. The method according to claim 7, wherein The first luminescent tag is selected from one of green fluorescent protein, red fluorescent protein, yellow fluorescent protein, mCherry, mTAGBFP2, mCLOVER3, Venus, mTagBFP2, mIFP and TagRFP657; The second luminescent tag is selected from one of green fluorescent protein, red fluorescent protein, yellow fluorescent protein, mCherry, mTAGBFP2, mCLOVER3, Venus, mTagBFP2, mIFP and TagRFP657.

9. The method according to any one of claims 1-8, characterized in that, The mixing ratio of the first protein element and the second protein element is 1:2 to 1:1; And / or, the step of detecting the phase separation ability of the mixture includes: detecting the phase separation ability of the mixture by using a fluorescence microscope.

10. Use of the method according to any one of claims 1-9 in enhancing protein functionality and / or screening of therapeutic drugs for protein-related diseases.

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