Mutant of fluorescent protein and application thereof

Abstract

The invention discloses a fluorescent protein mutant and application thereof, and belongs to the fields of cytobiology, chemical biology and molecular biology. The invention provides a fluorescent protein mutant, compared with a wild type mEYFP fluorescent protein sequence, the mutant comprises mutation at at least one position corresponding to 47th, 154th, 164th and 176th positions, and by modifying the wild type mEYFP, the fluorescent lifetime of the mutant has fluorescent lifetime sensitivity to LLPS or liquid-solid phase change, even to the two processes.

Description

Technical Field

[0001] The present invention relates to a mutant of a fluorescent protein and its application. Specifically, it relates to a fluorescent protein for detecting phase separation or phase change and its application, belonging to the fields of cell biology, chemical biology, and molecular biology. Background Art

[0002] Liquid-Liquid Phase Separation (LLPS) of biomolecules is a fundamental cellular process that is crucial for maintaining homeostasis and promoting biochemical activities. On the other hand, abnormal phase separation can alter the fluidity of condensates and lead to their transition from liquid-like condensates to solid-like condensates, which may result in the formation of pathological aggregates commonly seen in neurodegenerative diseases such as Alzheimer's disease and amyotrophic lateral sclerosis. Therefore, being able to rapidly and real-time detect such changes in phase separation is of great significance for studying the impact of phase separation on organism functions, especially for exploring the mechanisms of neurodegenerative diseases and clinical detection. The fluidity of traditional condensates is usually evaluated by Fluorescence Recovery After Photobleaching (FRAP). However, this method requires photobleaching with high-intensity lasers, which has a greater impact on organisms. At the same time, due to the need for photobleaching and the collection of post-bleaching signals, it is difficult to perform real-time dynamic detection of multiple targets. Fluorescence lifetime is defined as the average residence time of a fluorophore in the excited state, usually in the nanosecond range. Fluorescence lifetime is sensitive to the local environment of the fluorophore but insensitive to the fluorophore concentration and illumination intensity. Fluorescence Lifetime Imaging Microscopy (FLIM) using organic fluorophores has been used to detect the microenvironment within protein condensates, including polarity and viscosity, ionic effects within condensates, and aggregations caused by protein misfolding. Forster resonance energy transfer fluorescence lifetime microscopy (FRET-FLIM) has also been used to visualize the regulation of protein binding within condensates of microtubule-associated protein tau (MAPT, simply referred to as Tau). Although the fluorescent protein CFP has been used in FRET-FLIM experiments to reveal the molecular packing density of Tau in cells, whether the fluorescence lifetime of genetically encoded fluorescent proteins can be used to detect LLPS and liquid-solid phase transitions remains unknown. Summary of the Invention

[0003] Problems to be Solved by the Invention

[0004] Due to technical limitations, traditional condensate FRAP detection methods require high-intensity laser for photobleaching, which has a greater impact on organisms. The fluorescence lifetime is not sensitive to fluorophore concentration and illumination intensity, and it is still unable to detect the changes of phase separation quickly and in real time. Moreover, it is still unknown whether the fluorescence lifetime of genetically encoded fluorescent proteins can be used to detect LLPS and liquid-solid phase transitions.

[0005] In view of this, the present invention develops a novel fluorescent protein with phase separation sensitivity of fluorescence lifetime by mutating key amino acids of wild-type mEYFP.

[0006] Solutions for Solving the Problems

[0007] [1]. A fluorescent protein mutant, wherein the fluorescent protein mutant is selected from any one of the groups consisting of the following (i)-(v):

[0008] (i) Compared with the sequence shown in SEQ ID NO:1, the mutant contains mutations at at least one position corresponding to positions 47, 154, 164, and 176 of the sequence shown in SEQ ID NO:1, and compared with the fluorescent protein shown in SEQ ID NO:1, the change in the fluorescent signal of the mutant is more sensitive to the liquid-liquid phase separation and / or liquid-solid phase transition of biomolecules;

[0009] (ii) A mutant having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% sequence identity with the amino acid sequence shown in (i), and not including the mutant of the sequence shown in SEQ ID NO:1;

[0010] (iii) A mutant encoded by a polynucleotide that hybridizes with the polynucleotide shown in (a) or (b) under very high stringency conditions:

[0011] (a) A polynucleotide encoding a mutant with the amino acid sequence shown in (i);

[0012] (b) A polynucleotide that is fully complementary to (a);

[0013] (iv) A fragment of the mutant shown in any one of (i), (ii), or (iii), and the change in the fluorescent signal of the fragment still has enhanced sensitivity to the liquid-liquid phase separation and / or liquid-solid phase transition of biomolecules;

[0014] (v) A polypeptide with one or more amino acids added or deleted at at least one end of the N-terminus and C-terminus of the polypeptide with the amino acid sequence shown in (i), (ii), (iii), or (iv);

[0015] Preferably, the fluorescent protein mutant has a mutated amino acid at at least one of the following positions:

[0016] F47L, M154P, V164G, S176G.

[0017] [2]. The fluorescent protein mutant according to [1], wherein the fluorescent protein mutant corresponds to the sequence shown in SEQ ID NO: 1 and has mutations as shown in (i) - (v):

[0018] (i) F47L, M154P, V164G, S176G;

[0019] (ii) F47L;

[0020] (iii) M154P;

[0021] (iv) V164G;

[0022] (v) S176G.

[0023] [3]. The fluorescent protein mutant according to [1] or [2], wherein the fluorescent protein mutant comprises a polypeptide as shown in any one of SEQ ID NO: 2 - 6, or a polypeptide having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, most preferably at least 99% sequence identity with any one of SEQ ID NO: 2 - 6.

[0024] [4]. A fusion protein, wherein the fusion protein comprises the fluorescent protein mutant according to any one of [1] - [3], and a biomolecule to be detected. Preferably, the biomolecule to be detected includes a protein.

[0025] [5]. An isolated polynucleotide, wherein the polynucleotide comprises a sequence encoding the fluorescent protein mutant according to any one of [1] - [3] or a sequence encoding the fusion protein according to [4].

[0026] [6]. An expression vector, wherein the expression vector comprises the isolated polynucleotide according to [5].

[0027] [7]. A recombinant host cell, wherein the recombinant host cell comprises the fluorescent protein mutant according to any one of [1] - [3], the fusion protein according to [4], the isolated polynucleotide according to [5], or the expression vector according to [6].

[0028] [8]. A method for detecting liquid-liquid phase separation and / or liquid-solid phase transition of biomolecules, wherein the method uses the fluorescent protein mutant according to any one of [1]-[3], the fusion protein according to [4], the isolated polynucleotide according to [5], the expression vector according to [6], and the recombinant host cell according to [7].

[0029] [9]. The method according to [8], wherein the method detects the liquid-liquid phase separation and / or liquid-solid phase transition of biomolecules by using the change in the fluorescence signal of the fluorescent protein mutant, wherein the change in the fluorescence signal includes at least one of a change in fluorescence intensity, a change in fluorescence lifetime, a change in fluorescence polarization, and a change in fluorescence spectrum.

[0030] Preferably, the change in the fluorescence signal includes a change in fluorescence lifetime.

[0031]

[10] . Any one of the following uses of the fluorescent protein mutant according to any one of [1]-[3], the fusion protein according to [4], the isolated polynucleotide according to [5], the expression vector according to [6], or the recombinant host cell according to [7]:

[0032] (1) Use in preparing a reagent or kit for detecting liquid-liquid phase separation and / or liquid-solid phase transition of biomolecules;

[0033] (2) Use for detecting liquid-liquid phase separation and / or liquid-solid phase transition of biomolecules.

[0034] Effects of the Invention

[0035] The present invention systematically studied the sensitivity of the fluorescence lifetime of fluorescent proteins to LLPS and liquid-solid phase transition, identified several key residues that can be used to regulate the sensitivity of fluorescence lifetime to phase separation, and modified the wild-type mEYFP with insensitive fluorescence lifetime to phase separation through these identified residues, and invented a new type of mEFYP fluorescent protein mutant with sensitive fluorescence lifetime to phase separation. Description of the Drawings

[0036] Figure 1 Change in the fluorescence lifetime of wild-type mYFP for phase separation. Among them:

[0037] Figure 1 In A, the fluorescence lifetime of wild-type mEYFP is not sensitive to phase transition; Figure 1 In B of, starburst (electron microscopy image) formed by G156E, wherein, in Figure 1 In B of, the left blue frame is the central part, and the red frame is the fiber part. In Figure 1In B of [reference], the two figures on the right show the magnified central part and the fiber structure.

[0038] Figure 2 Detect the sensitivity of LLPS and liquid-solid phase transition for different mEYFP mutants. Among them:

[0039] Figure 2 A in [reference] is a schematic diagram of the mutants of mEYFP; Figure 2 B in [reference] is the sensitivity of the fluorescence lifetime of different mEYFP mutants for the detection of LLPS and liquid-solid phase transition. Detailed implementation manners

[0040] The various exemplary embodiments, features and aspects of the present invention will be described in detail below. The special word "exemplary" here means "serving as an example, embodiment or illustration". Any embodiment described as "exemplary" here does not have to be construed as superior or better than other embodiments.

[0041] In addition, in order to better illustrate the present invention, numerous specific details are given in the following detailed implementation manners. Those skilled in the art should understand that the present invention can also be implemented without some specific details. In other instances, methods, means, equipment and steps well-known to those skilled in the art are not described in detail so as to highlight the gist of the present invention.

[0042] Unless otherwise stated, the units used in this specification are all international standard units, and the numerical values and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0043] In this specification, the meaning expressed by using "may" includes both the meaning of performing a certain process and the meaning of not performing a certain process.

[0044] In this specification, the "some specific / preferred implementation manners / embodiments", "other specific / preferred implementation manners / embodiments", "implementation manners", "embodiments" and the like mentioned refer to the specific elements (for example, features, structures, properties and / or characteristics) related to the implementation manner / embodiment described are included in at least one of the implementation manners / embodiments described here, and may exist in other implementation manners / embodiments or may not exist in other implementation manners / embodiments. In addition, it should be understood that the elements can be combined in various implementation manners / embodiments in any suitable manner.

[0045] In this specification, the numerical range expressed by using "numerical value A~numerical value B" refers to the range including the endpoint numerical values A and B.

[0046] In this specification, the terms "polypeptide", "peptide", and "protein" are used interchangeably herein and refer to amino acid polymers of any length. The polymer can be linear or branched, it can contain modified amino acids, and it can be interrupted by non-amino acids. The term also includes amino acid polymers that have been modified (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeled component).

[0047] As used herein, "wild-type" refers to a naturally occurring amino acid or nucleotide sequence that can be directly found in nature and has not been artificially modified. "Mutant" refers to an amino acid or nucleotide sequence in which one or more amino acids or nucleotides have been substituted, deleted, or inserted compared to the natural amino acid or nucleotide sequence. As used in the present invention, "naturally occurring" and "wild-type" are synonyms.

[0048] In this specification, the term "mutant" refers to a polynucleotide or polypeptide that contains alterations (i.e., substitutions, insertions, and / or deletions) at one or more (e.g., several) positions relative to a "wild-type" or "comparative" polynucleotide or polypeptide, where a substitution refers to replacing a nucleotide or amino acid occupying a position with a different nucleotide or amino acid. A deletion refers to removing a nucleotide or amino acid occupying a position. An insertion refers to adding a nucleotide or amino acid adjacent to and immediately following the nucleotide or amino acid occupying the position.

[0049] In this specification, the terms "amino acid mutation" or "nucleotide mutation" include "substituting, repeating, deleting, or adding one or more amino acids or nucleotides". In the present invention, the term "mutation" refers to a change in a nucleotide sequence or an amino acid sequence.

[0050] In some embodiments, the "mutation" of the present invention can be selected from "conservative mutations". In the present invention, the term "conservative mutation" refers to a mutation that can normally maintain the function of a protein. Representative examples of conservative mutations are conservative substitutions.

[0051] In this specification, the term "conservative substitution" involves replacing an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art and include those having basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine).

[0052] As used in this specification, the terms "sequence identity" or "percent identity" in the comparison of two nucleic acids or polypeptides refer to the degree to which they are the same or have the same percentage of specified sequence when compared and aligned for maximum correspondence using a nucleotide or amino acid residue sequence comparison algorithm or by visual inspection. That is, sequence identity of a nucleotide or amino acid sequence can be defined by the ratio of the number of nucleotides or amino acids that are identical when two or more nucleotide or amino acid sequences are aligned in a manner that maximizes the number of matching nucleotides or amino acids and includes gaps as needed, to the total number of nucleotides or amino acids in the aligned portion.

[0053] According to the present invention, "moderate to very high stringency conditions" include "moderate stringency conditions", "medium-high stringency conditions", "high stringency conditions" or "very high stringency conditions", which describe the conditions for nucleic acid hybridization and washing. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which is incorporated herein by reference. Both aqueous and non-aqueous methods are described in this reference and either can be used. For example, specific hybridization conditions are as follows: (1) Low stringency hybridization conditions are in 6× sodium chloride / sodium citrate (SSC) at about 45°C, followed by washing 2 times in 0.2× SSC, 0.1% SDS at at least 50°C (for low stringency conditions, the wash temperature can be increased to 55°C); (2) Moderate stringency hybridization conditions are in 6× SSC at about 45°C, followed by washing 1 or more times in 0.2× SSC, 0.1% SDS at 60°C; (3) High stringency hybridization conditions are in 6× SSC at about 45°C, followed by washing 1 or more times and preferably in 0.2× SSC, 0.1% SDS at 65°C; (4) Very high stringency hybridization conditions are 0.5 M sodium phosphate, 7% SDS at 65°C, followed by washing 1 or more times in 0.2× SSC, 1% SDS at 65°C.

[0054] As used in this specification, the term "expression" includes any step involved in polypeptide production, including but not limited to: transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0055] In this specification, the term "expression vector" refers to a DNA construct that contains a DNA sequence operably linked to appropriate control sequences so as to express a gene of interest in a suitable host. A "recombinant expression vector" refers to a DNA construct used for expressing, for example, a polynucleotide encoding a desired foreign polypeptide. The recombinant expression vector may include, for example, a transcriptional subunit that contains i) a collection of genetic elements that regulate gene expression, such as promoters and enhancers; ii) a structural or coding sequence that is transcribed into mRNA and translated into a protein; and iii) appropriate transcriptional and translational start and stop sequences. The recombinant expression vector is constructed in any suitable manner. The nature of the vector is not important, and any vector can be used, including plasmids, viruses, bacteriophages, and transposons. Possible vectors for use in the present invention include, but are not limited to, chromosomal, non-chromosomal, and synthetic DNA sequences, such as bacterial plasmids, bacteriophage DNA, yeast plasmids, and vectors derived from combinations of plasmid and bacteriophage DNA, DNA from viruses such as vaccinia, adenovirus, fowlpox, baculovirus, SV40, and pseudorabies.

[0056] In this specification, the terms "isolated" and "purified" are used to refer to a molecule (e.g., an isolated nucleic acid, polypeptide, etc.) or other component that has been removed from at least one other component with which it is naturally associated. The term "purified" does not require absolute purity, but is intended as a relative definition.

[0057] In this specification, the term "liquid-liquid phase separation" or "LLPS" refers to the formation of phase-separated droplets with different components and properties by the interaction of biomacromolecules (such as proteins, RNA) inside cells or in vitro.

[0058] In this specification, the term "liquid-solid phase transition" refers to the formation of a solid substance formed by the condensation of phase-separated droplets with different components and properties by the interaction of biomacromolecules (such as proteins, RNA) inside cells or in vitro.

[0059] Unless otherwise defined or clearly indicated by the context, all technical and scientific terms in the present invention have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains.

[0060] The technical solutions of the present invention will be described in detail as follows:

[0061] <First aspect>

[0062] In a first aspect of the present invention, there is provided a fluorescent protein mutant that is sensitive to at least one of the phase separation (e.g., liquid-liquid phase separation) and phase change (e.g., liquid-solid phase transition) of biomolecules.

[0063] In some embodiments, compared with the wild-type fluorescent protein mEYFP shown in SEQ ID NO:1, the change in the fluorescence signal of the mutant is more sensitive to the liquid-liquid phase separation and / or liquid-solid phase transition of biomolecules.

[0064] In some embodiments, the fluorescent protein mutant is selected from any one of the groups consisting of the following (i)-(v):

[0065] (i) Compared with the sequence shown in SEQ ID NO:1, the mutant contains a mutation at at least one position corresponding to positions 47, 154, 164, and 176 of the sequence shown in SEQ ID NO:1, and compared with the fluorescent protein shown in SEQ ID NO:1, the change in the fluorescence signal of the mutant is more sensitive to the liquid-liquid phase separation and / or liquid-solid phase transition of biomolecules.

[0066] (ii) A mutant having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the amino acid sequence shown in (i), and not including the sequence shown in SEQ ID NO:1;

[0067] (iii) A mutant encoded by a polynucleotide that hybridizes with the polynucleotide shown in (a) or (b) under very high stringency conditions:

[0068] (a) A polynucleotide encoding a mutant having the amino acid sequence shown in (i);

[0069] (b) A polynucleotide that is fully complementary to (a);

[0070] (iv) A fragment of the mutant shown in any one of (i), (ii), or (iii), and the change in the fluorescence signal of the fragment still has an increased sensitivity to the liquid-liquid phase separation and / or liquid-solid phase transition of biomolecules;

[0071] (v) A polypeptide having one or more amino acids added or deleted at at least one end of the N-terminus and C-terminus of the polypeptide having the amino acid sequence shown in (i), (ii), (iii), or (iv);

[0072] In some preferred embodiments, the fluorescent protein mutant corresponds to the sequence shown in SEQ ID NO:1 and has mutations in the amino acids at the following at least one position: F47L, M154P, V164G, S176G.

[0073] In some specific embodiments, the fluorescent protein mutant corresponds to the sequence shown in SEQ ID NO:1 and has the mutations shown in (i)-(v):

[0074] (i) F47L, M154P, V164G, S176G (in the embodiments of the present invention, it is FLmEYFP, and its amino acid sequence is shown in SEQ ID No. 2);

[0075] (ii) F47L (labeled as mEYFP in the embodiments of the present invention F47L or FLSmEYFP, and its amino acid sequence is shown in SEQ ID No. 3);

[0076] (iii) M154P (labeled as mEYFP in the embodiments of the present invention M154P , and its amino acid sequence is shown in SEQ ID No. 4);

[0077] (iv) V164G (labeled as mEYFP in the embodiments of the present invention V164G , and its amino acid sequence is shown in SEQ ID No. 5);

[0078] (v) S176G (labeled as mEYFP in the embodiments of the present invention S176G , and its amino acid sequence is shown in SEQ ID No. 6).

[0079] In some embodiments, the change in the fluorescence signal of the fluorescent protein mutant includes at least one of the change in fluorescence intensity, the change in fluorescence lifetime, the change in fluorescence polarization, and the change in fluorescence spectrum.

[0080] In some specific embodiments, the change in the fluorescence signal is a change in fluorescence lifetime.

[0081] In some preferred embodiments, the fluorescent protein mutant corresponds to the sequence shown in SEQ ID NO: 1 and has the mutations shown in (i) and / or (ii). Specifically, compared with the wild-type mEYFP, the fluorescence lifetime of FLSmEYFP is more sensitive to the liquid-solid phase transition of biomolecules (i.e., liquid-solid phase transition); compared with the wild-type mEYFP, the fluorescence lifetime of FLmEYFP is more sensitive to the LLPS and / or liquid-solid phase transition of biomolecules.

[0082] The above mutation sites enable the fluorescent protein of the present invention to have a good sensitivity fluorescence lifetime and can quickly detect phase separation and phase transition.

[0083] <Second aspect>

[0084] In the second aspect of the present invention, a fusion protein is provided, and the fusion protein comprises the fluorescent protein mutant described in the first aspect of the present invention and a biomolecule to be detected.

[0085] In some preferred embodiments, the biomolecule to be detected includes a protein.

[0086] In some exemplary embodiments, the biomolecule to be detected can be a biomolecule that may undergo LLPS and / or liquid-solid phase transition.

[0087] In some exemplary embodiments, the biomolecule to be detected can be a biomolecule (marker) that may undergo LLPS and / or liquid-solid phase transition in neurodegenerative diseases.

[0088] In some embodiments, the neurodegenerative disease includes an acute neurodegenerative disease or a chronic neurodegenerative disease.

[0089] In some specific embodiments, the neurodegenerative disease includes amyotrophic lateral sclerosis (ALS) or frontotemporal lobar degeneration (FTLD) disease.

[0090] In some specific embodiments, the biomolecule marker is the fused in sarcoma (FUS) or its mutant related to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD).

[0091] In some embodiments, the fusion protein further contains a tag.

[0092] In this specification, the term "tag" refers to such a short peptide that is fused or linked to a target protein (such as the fluorescent protein mutant of the present invention or a fusion protein containing the fluorescent protein mutant), and thereby promotes the soluble expression, detection, and / or purification of the recombinant protein. The tag can be fused or linked to the N-terminus and / or C-terminus of the target protein (optionally through a linker or a protease cleavage site), and in some embodiments, the tag can also be fused or linked to the interior of the target protein.

[0093] In some exemplary embodiments, the tag includes a maltose binding protein (MBP) tag, and in some specific embodiments, the tag sequence is as shown in SEQ ID No. 9.

[0094] In some exemplary embodiments, the tag includes a 6×His tag.

[0095] According to the present invention, the term "protease cleavage site" refers to a site that can be specifically recognized and cleaved by a protease. Various specific proteases and their recognition sites are well known to those skilled in the art and are described in many prior art documents. Those skilled in the art can, according to the actual situation, use a suitable protease cleavage site in the fusion protein and cleave it with the corresponding protease. The use of protease cleavage sites can be advantageous. For example, it can be used to excise signal peptides and / or tags from the fusion protein to obtain a mature protein with the desired activity.

[0096] In some specific embodiments, the protease cleavage site sequence is as shown in SEQ ID No. 10.

[0097] In some embodiments, the various parts in the fusion protein (such as fluorescent protein mutants, biomolecules to be detected, tags, protease cleavage sites, etc.) can be connected by optional linkers.

[0098] According to the present invention, the term "linker" or "Linker" refers to a connection between two molecules or parts, such as two domains of a fusion protein. Typically, the linker is located between or flanks two groups, molecules, or other parts and is covalently bonded to each to connect the two. In some embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker can be a polynucleotide. In some embodiments, the linker can be a DNA molecule. In some embodiments, the linker can be an RNA molecule. In some embodiments, the linker can include an aptamer capable of binding a ligand. In some embodiments, the linker is an amino acid or multiple amino acids (such as a peptide or protein).

[0099] <The third aspect>

[0100] In the third aspect of the present invention, there is provided an isolated polynucleotide encoding a fluorescent protein mutant as described in the first aspect of the present invention or a fusion protein as described in the second aspect of the present invention.

[0101] The polynucleotide of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or synthetic DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0102] The polynucleotide encoding the mutant or fusion protein of the present invention includes: a coding sequence encoding only the mutant or fusion protein; the coding sequence of the mutant or fusion protein and various additional coding sequences; the coding sequence of the mutant or fusion protein (and optionally additional coding sequences) and non-coding sequences.

[0103] <The fourth aspect>

[0104] In a fourth aspect of the present invention, there is provided an expression vector, which comprises the polynucleotide described in the third aspect of the present invention.

[0105] In some embodiments, the polynucleotide described in the third aspect is operably linked to one or more heterologous regulatory sequences that control gene expression to produce a polynucleotide capable of expressing a polypeptide.

[0106] In some exemplary embodiments, the expression vector can be constructed by genetically engineering means to link the expression vector with a biomolecule to be detected and / or a fluorescent protein mutant.

[0107] In some specific embodiments, the recombinant expression vector includes a coding sequence of a fluorescent protein mutant, a coding sequence of a biomolecule to be detected (such as a protein to be detected), and a coding sequence of a tag.

[0108] <Fifth aspect>

[0109] In a fifth aspect of the present invention, there is provided a recombinant host cell, wherein the recombinant host cell comprises the fluorescent protein mutant described in the first aspect of the present invention, the fusion protein described in the second aspect of the present invention, the isolated polynucleotide described in the third aspect of the present invention, or the expression vector described in the fourth aspect of the present invention.

[0110] In some alternative embodiments, an expression vector containing a polynucleotide encoding a fluorescent protein mutant or a fusion protein is introduced into a suitable host cell to express the corresponding fluorescent protein mutant or fusion protein.

[0111] <Sixth aspect>

[0112] In a sixth aspect of the present invention, there is provided a method for detecting liquid-liquid phase separation and / or liquid-solid phase transition of a biomolecule, wherein the method uses the fluorescent protein mutant described in the first aspect of the present invention, the fusion protein described in the second aspect of the present invention, the polynucleotide described in the third aspect of the present invention, the expression vector described in the fourth aspect of the present invention, or the recombinant host cell described in the fifth aspect of the present invention.

[0113] In some embodiments, the detection method includes labeling a biomolecule to be detected with a fluorescent protein mutant to form a fusion protein (as described in the second aspect above).

[0114] In some embodiments, the detection method detects liquid-liquid phase separation and / or liquid-solid phase transition of a biomolecule by using changes in the fluorescence signal of the fluorescent protein mutant, wherein the changes in the fluorescence signal include at least one of changes in fluorescence intensity, changes in fluorescence lifetime, changes in fluorescence polarization, and changes in fluorescence spectrum.

[0115] In some preferred embodiments, the change in the fluorescence signal includes a change in fluorescence lifetime.

[0116] In some specific embodiments, the fluorescent protein mutant labels the biomolecule to be detected. When the molecule to be labeled undergoes phase separation or phase transition, the fluorescence lifetime of the fluorescent protein mutant changes, enabling rapid real-time detection of changes in the state of the biomolecule to be detected.

[0117] In some exemplary embodiments, the FUS mutant G156E protein labeled with the fluorescent protein mutant. When the FUS protein undergoes liquid-liquid phase separation and / or liquid-solid phase transition, the fluorescence lifetime of the fluorescent protein mutant changes. By observing the fluorescence lifetime and using mathematical fitting, further phase separation and / or phase transition information can be obtained.

[0118] In some specific embodiments, the fluorescence lifetime can be observed by confocal microscopy and measured based on a fluorescence lifetime imaging system and / or a bioluminescence resonance energy transfer system.

[0119] In some embodiments, the fluorescence lifetime imaging includes using a fluorescence lifetime imaging microscope, such as a single-photon counting confocal microscope, a single-molecule time-resolved confocal fluorescence microscopy system, a FLIM microscopic fluorescence lifetime imaging system, etc.

[0120] In some embodiments, the bioluminescence resonance energy transfer system includes a BRET (Bioluminescence Resonance Energy Transfer) system and a FRET (Fluorescence Resonance Energy Transfer) system.

[0121] <Seventh aspect>

[0122] In the seventh aspect of the present invention, there is provided any one of the following uses of the fluorescent protein mutant according to the first aspect of the present invention, the fusion protein according to the second aspect, the polynucleotide according to the third aspect, the expression vector according to the fourth aspect, the recombinant host cell according to the fifth aspect, or the method according to the sixth aspect of the present invention:

[0123] (1) Use for detecting liquid-liquid phase separation and / or liquid-solid phase transition of biomolecules;

[0124] (2) Use in the preparation of reagents or kits for detecting liquid-liquid phase separation and / or liquid-solid phase transition of biomolecules.

[0125] In some specific embodiments, FLSmEYFP can be used in the preparation of reagents and / or kits for detecting liquid-liquid phase separation of biomolecules; and / or in the preparation of reagents and / or kits for detecting liquid-solid phase transitions of biomolecules. FLmEYFP can be used in the preparation of reagents and / or kits for detecting liquid-solid phase transitions of biomolecules.

[0126] Example

[0127] The embodiments of the present invention will be described in detail below in conjunction with examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the examples, the operations are carried out under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments not indicated by the manufacturer are all conventional products that can be obtained commercially.

[0128] Example 1

[0129] The present invention studied the sensitivity of the fluorescence lifetime of fluorescent proteins to LLPS and liquid-solid phase transitions and found that the fluorescence lifetime of wild-type mEYFP is not sensitive to changes in phase separation (as shown in A below). In the present invention, the fusion protein (Fused in sarcoma, FUS) related to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) was used as a model. The patient-derived FUS mutant G156E (abbreviation: FUS(G156E)) was reported to have a stronger aggregation tendency and accelerate the liquid-to-solid phase transition. Therefore, the present invention uses FUS(G156E) fused with fluorescent protein to test whether the lifetime of the fluorescent protein can reflect the change of at least one state of phase separation and phase transition when liquid-like condensates and solid-like condensates are completely formed. The following are the specific experimental steps. Figure 1 I. Experimental materials

[0130] Tris-HCl (Sigma-Aldrich), KCl (Sigma-Aldrich), NaCl (Sigma-Aldrich), MgCl

[0131] (Sigma-Aldrich), PMSF (Amresco), imidazole (Sigma-Aldrich), EGTA (Biodee), TCEP (Goldbio), glycerol (GPC biotech), BL21(DE3) competent cells (TransGen Biotech), TEV protease (laboratory purification). 2 (Sigma-Aldrich), PMSF (Amresco), imidazole (Sigma-Aldrich), EGTA (Biodee), TCEP (Goldbio), glycerol (GPC biotech), BL21(DE3) competent cells (TransGen Biotech), TEV protease (laboratory purification).

[0132] II. Experimental steps

[0133] 1. Protein preparation steps

[0134] The specific acquisition process of the FUS(G156E) protein labeled with mEYFP and its mutant proteins is as follows: Taking the FUS(G156E) protein labeled with mEYFP as an example, using the initial expression plasmid MBP-GFP-FUS(G156E) (provided by the laboratory of Teacher Lipeng Li from Tsinghua University), through polymerase chain reaction (PCR) and homologous recombination, construct the expression plasmid of the FUS(G156E) protein labeled with mEYFP: MBP-mEYFP-FUS(G156E). The amino acid sequence of the protein expressed by it is shown in SEQ ID No. 8. After confirming that the constructed plasmid sequence is correct by sequencing (Table 1), subsequent protein purification is carried out. The plasmid for expressing the FUS(G156E) protein labeled with mEYFP introduces a maltose-binding protein (MBP) tag at the N-terminus of the fluorescent protein, and an amino acid sequence recognized and cleaved by TEV protease is introduced between the fluorescent protein and the MBP tag, so as to remove the MBP tag in subsequent experiments; the C-terminus of the fluorescent protein is connected to the FUS(G156E) protein, and there is a 6×His tag between the two for affinity purification with Ni-NTA agarose purification resin.

[0135] Table 1 Protein Amino Acid Sequence

[0136]

[0137]

[0138]

[0139] (Note: The mutation sites are indicated by italics + underlines in the table)

[0140] (1) Prepare solutions: The buffers used in the protein purification process are as follows (Table 2). Prepare them in advance, filter them with a 0.22 μm filter membrane, and store them at 4°C. TCEP needs to be prepared and added to the buffer on the day of each protein purification to prevent the loss of the ability to reduce the disulfide bonds in proteins due to oxidation after being placed in the air for a long time.

[0141] Table 2 Buffer Components in Protein Purification

[0142]

[0143] (2) Transformation and induction: Transform the plasmid expressing the protein into BL21(DE3) competent cells, spread it on an LB solid medium plate containing 100 μg / mL ampicillin with a spreading rod, and place it in an incubator at 37 °C for overnight culture. The next day, pick 5 - 10 monoclonal colonies on the plate and put them into 5 mL of LB liquid medium containing 100 μg / mL ampicillin, and culture them in a shaker at 37 °C at a rotation speed of 220 rpm for 5 h until the bacterial liquid becomes turbid. Then transfer all the bacterial liquid to 1 L of LB liquid medium containing 100 μg / mL ampicillin, and culture it in a shaker at 37 °C at a rotation speed of 220 rpm until the optical density value (OD600) of the bacterial liquid at a wavelength of 600 nm is in the range of 0.6 - 0.8. Then cool down both the shaker and the bacterial liquid to 16 °C, add IPTG (Goldbio) to the bacterial liquid to a final concentration of 0.5 mM, induce protein expression for 16 - 18 h, and then collect the bacterial cells with a high-speed centrifuge, quickly freeze them in liquid nitrogen, and store them in a -80 °C refrigerator for subsequent purification.

[0144] (3) Protein purification: Place the quickly frozen bacterial cells on ice to thaw, resuspend them with 40 mL of lysis buffer until no visible bacterial clumps remain, and then transfer them to a 50 mL beaker. Under an ice-water bath environment, use an ultrasonic crusher to break the bacterial liquid under the ultrasonic condition of 350 W power, working for 2 s and then pausing for 5 s, with a total ultrasonic duration of 30 min. Transfer the broken bacterial liquid to a high-speed centrifuge tube, and centrifuge it at 12,000 rpm for 1 h with a high-speed centrifuge at 4 °C. After centrifugation, pipette the supernatant into a 50 mL centrifuge tube, add 1 - 2 mL of Ni-NTA agarose purification resin (Cytiva) washed with lysis buffer, incubate it in a cold room at 4 °C for 1.5 - 2 h, centrifuge it at 5000 rpm for 10 min at 4 °C, separate the Ni-NTA agarose purification resin bound to the target protein and transfer it to an empty gravity column, and elute the target protein successively with washing buffer and elution buffer, and collect the eluted protein. Subsequently, use a chromatographic column Superdex TM 200Increase 10 / 300 column (GE healthcare) for protein purification, elute the chromatographic column with a molecular sieve elution buffer for 1 column volume (25 mL). During the elution process, collect the eluted protein in separate tubes according to the change in absorbance at 280 nm, and perform SDS-PAGE identification on each tube of protein to determine whether the purity and size of the protein are correct. Refer to the volume position where the protein elutes after loading on the chromatographic column to confirm the protein in the corresponding tube finally selected for storage, combine them, concentrate them to a final concentration of 50 μM with a 30K ultrafiltration centrifuge tube (Amicon-Ultra), aliquot the protein at 5 μL per tube, quickly freeze it in liquid nitrogen, and store it in a -80 °C refrigerator to obtain the mEYFP-labeled FUS(G156E) protein.

[0145] 2. Liquid-liquid phase separation and liquid-solid phase transition steps

[0146] To initiate the phase separation of the mEYFP-labeled FUS(G156E) protein obtained in Step 1, the aliquoted mEYFP-labeled FUS(G156E) protein was taken, and a phase separation reaction system (Table 3) was prepared, mixed evenly, and added to a glass-bottom 384-well plate (Cellvis). At this time, the protein state is called "before phase separation" or "before liquid-liquid phase separation"; after the mEYFP-labeled FUS(G156E) protein before phase separation was allowed to stand at room temperature for 2 h, the mEYFP-labeled FUS(G156E) protein formed droplets (larger than 200 nm) that could be observed under an optical microscope for experimental observation. At this time, the protein state is called "2-hour condensate". Subsequently, the glass-bottom 384-well plate containing the droplets (2-hour condensate) of the mEYFP-labeled FUS(G156E) protein that had undergone liquid-liquid phase separation was fixed on a multi-purpose decolorizing shaker, and the rotation speed was adjusted to approximately 200 rpm / min, and the sample was shaken horizontally for 24 h to obtain a solid condensate of the mEYFP-labeled FUS(G156E) protein after liquid-solid phase transition, including 24-hour fiber-free condensate or 24-hour fibrous condensate (both are products obtained by liquid-solid phase transition, only different in morphology), for subsequent experiments.

[0147] Table 3 FUS(G156E) liquid-liquid phase separation reaction system

[0148]

[0149]

[0150] 3. Data acquisition and analysis steps

[0151] The FLIM data acquisition and analysis system (Picoquant, Symphotime) used in this experiment was coupled with an FV1200 confocal microscope (Olympus), with both using independent optical paths and software. In the experiment, a 100x oil immersion objective was used on the FV1200 to perform imaging first, then switched to the FLIM data acquisition system to collect relevant data, and finally data analysis was carried out on the software. The software used for FLIM data acquisition and analysis was SymPhoTime 64, and the specific process was as follows: Locate the area and appropriate focal plane for FLIM observation on the FV1200, magnify the target field of view for the FLIM experiment to an appropriate size, take a picture of the target field of view with a size of 512×512 pixels, and save the image. Subsequently, adjust the optical path on the FV1200 software to the optical path of the FLIM system so that the emitted light enters the fluorescence lifetime detector directly without passing through the confocal detector. Then, set the FLIM data acquisition parameters to make the imaging conditions on the FLIM software consistent with the image resolution on the FV1200, and then optimize the FLIM acquisition conditions. By previewing the real-time TSCPC curve under the test conditions, change the laser attenuation to adjust the laser intensity. After confirming that the laser intensity is appropriate and all parameter settings are correct, turn on the laser key on the laser driver, click the control button on the FLIM software, select XY repetitive scanning of the target field of view on the FV1200 software, start collecting fluorescence lifetime images. After recording enough photons, stop data acquisition and perform subsequent data analysis on the obtained FLIM images.

[0152] 4. Observation steps for the internal structure of condensates

[0153] In order to confirm the internal structure (including the central part (referred to as the center) and the surrounding fibers) of the fibrous aggregates (i.e., 24-hour fibrous aggregates) of the mEYFP-labeled FUS (G156E) protein after the liquid-solid phase transition, a transmission electron microscope (Hitachi HT7800) was used for observation. The specific steps are as follows: the liquid-liquid phase separation reaction system of the mEYFP-labeled FUS (G156E) protein was prepared (the specific method is the same as step 2 above), and the liquid-solid phase transition experiment was performed in the center of a single-grid confocal culture dish (the specific method is the same as step 2 above), and the laser confocal microscope FV1200 was used for imaging. After confirming the formation of fibrous aggregates, the corresponding area was marked on the bottom of the dish with a marker. Then, an equal volume of 2.5 (v / v)% glutaraldehyde fixative (Yuanye Bio) was added, and after fixing at room temperature for 20 minutes, 1 mL of 2.5 (v / v)% glutaraldehyde was added to fill the central area of ​​the culture dish, and fixed at 4°C overnight for subsequent electron microscopy sample preparation. The sample is embedded with resin, polymerized and sliced ​​in the target area, and the sample is picked up with a grid for staining, then washed with distilled water, and after absorbing excess water, it is placed at room temperature for 5 hours until the sample is dry, and then it can be used for electron microscope observation. When observing the sample with a transmission electron microscope, first select the target structure to be photographed, and then adjust the focal length of the objective lens and the size and brightness of the electron beam spot to perform imaging at a voltage of 80kV.

[0154] 5. Image processing steps

[0155] Select the image file to be processed on the FLIM software and open it with the image fitting program that comes with the software. In order to improve the fitting quality, the image is merged twice, and the image becomes 256×256 pixels. Circle the target area for fluorescence lifetime analysis on the image and perform fitting. Set the parameters in the fitting area of ​​the software. Select "n-Exponential Reconvolution" as the fitting model, select the circled target area as the fitting object, and fit the n value in the model to 1. The TCSPC curve will be displayed in the software. If the fitted curve does not coincide well with the TCSPC curve, or if the reference parameter χ used as the goodness of fit is used, 2 If the value is obviously inappropriate (the closer it is to 1, the better the fitting quality), the n value is increased and the same fitting process is performed again until a good quality fitting result is obtained. In the present invention, a sufficiently satisfactory fitting result can be obtained when the n value is 2, and the fluorescence lifetime τ based on the average fluorescence intensity of the target area is obtained. AvInt (Intensity weighted average lifetime, hereinafter referred to as fluorescence lifetime or intensity weighted average lifetime) (Formula 1-1).

[0156]

[0157] where τ AvInt represents the fluorescence lifetime averaged based on fluorescence intensity, I represents the fluorescence intensity of each photon, τ represents the fluorescence lifetime, and I sum represents the total fluorescence intensity, k represents a specific photon, and n represents the total number of photons.

[0158] Furthermore, the sensitivities of the fluorescence lifetime of mEYFP or its mutants to liquid-liquid phase separation and to liquid-solid phase transition are obtained by calculating the fluorescence lifetime ratios of different mobility products. The sensitivity of the fluorescence lifetime of mEYFP or its mutants to liquid-liquid phase separation is: the ratio of the fluorescence lifetime of the 2-hour condensate of the FUS(G156E) protein labeled with mEYFP or its mutants to the fluorescence lifetime of the FUS(G156E) protein labeled with mEYFP or its mutants before phase separation; the sensitivity of the fluorescence lifetime of mEYFP or its mutants to liquid-solid phase transition is: the ratio of the fluorescence lifetime of the central part of the 24-hour fibrous condensate of the FUS(G156E) protein labeled with mEYFP or its mutants to the fluorescence lifetime of the 2-hour condensate of the FUS(G156E) protein labeled with mEYFP or its mutants. The closer the above sensitivity value is to 1, the lower the sensitivity.

[0159] III. Experimental Results

[0160] As Figure 1 shown, the intensity-weighted average lifetimes (mean ± standard error, n > 9, from three independent experiments) of the unmodified mEYFP-labeled FUS(G156E) protein undergoing free diffusion before phase separation, 2-hour condensate, 24-hour non-fibrous condensate, and 24-hour fibrous condensate (i.e., the starburst with fibers formed by FUS(G156E), see Figure 1 B therein, which includes a central part, abbreviated as "24-hour center", and surrounding fibers, abbreviated as "24-hour fibers"). The statistical significance between the 24-hour center and the 24-hour fibers was evaluated by a two-tailed paired t-test, and the others were evaluated by a two-tailed unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. By the fluorescence lifetime ratio of the 2-hour condensate to the 24-hour center, it was found that the change in fluorescence lifetime of the 2-hour condensate was smaller compared to the 24-hour center, and it can be seen from Figure 1 that the fluorescence lifetime of the unmodified mEYFP was less sensitive to the fluorescence lifetime change from liquid to solid.

[0161] Example 2

[0162] In the present invention, key amino acids of wild-type mEYFP were mutated, and the modified mEYFP mutant was used to label the FUS(G156E) protein, and the fluorescence lifetime during phase separation and phase transition was detected, and a novel fluorescent protein with at least one sensitivity of fluorescence lifetime to phase separation and phase transition was discovered.

[0163] I. Experimental procedures and reagents

[0164] The specific experimental procedures and reagents are similar to those in Step 1 and Step 2 of Example 1, except that in the plasmid MBP-mEYFP-FUS(G156E) expressing the FUS(G156E) protein labeled with mEYFP, mEYFP was replaced with the corresponding mEYFP mutant.

[0165] II. Experimental results

[0166] In the present invention, four residues affecting the fluorescence lifetime sensitivity were determined by analysis, corresponding to positions 47, 154, 164, and 176 in mEYFP (Table 1). To further verify whether these sites actually affect the lifetime sensitivity, four residues in mEYFP were mutated in the present invention ( Figure 2 A in). Different from the FUS(G156E) protein labeled with mEYFP before modification (mEYFP-WT), the FUS(G156E) protein labeled with the mEYFP F47L / M154P / V164G / S176G mutant (hereinafter referred to as FLmEYFP) was much more sensitive to LLPS and liquid-solid phase transition ( Figure 2 B in), and its fluorescence lifetime decreased from 2.75±0.01 ns in solution (before phase separation, that is, before liquid-liquid phase separation) to 2.535±0.003 ns in liquid condensates (2-hour condensates), and further decreased to 2.281±0.005 ns in the center of 24-hour fibrous condensates. When these residues were mutated one by one, the FUS(G156E) protein labeled with the mEYFP F47L mutant (hereinafter referred to as FLSmEYFP) showed the highest sensitivity to liquid-solid phase transition, while the lifetimes of the FUS(G156E) proteins labeled with mEYFP M154P , mEYFP V164G and mEYFP S176G mutants were mainly affected by LLPS and were almost or very little sensitive to liquid-solid phase transition ( Figure 2 B in, Table 4).

[0167] Table 4 Total fluorescence lifetimes of mEYFP mutants during liquid-solid phase transition of the FUS(G156E) protein labeled with mEYFP mutants

[0168]

[0169]

[0170] In summary, the transition from liquid condensates to solid condensates plays a key role in neurodegenerative diseases. Imaging after fluorescence recovery after photobleaching (FRAP) is a widely used tool for observing changes in fluidity within condensates. However, due to technical limitations, FRAP is mainly used to examine one or a few condensates at the micron scale simultaneously. It is not feasible in FRAP and other commonly used methods to non-destructively and in real-time parallelly detect changes in the fluidity of multiple condensates without relying on fluorescence intensity. Therefore, the system of the present invention systematically studies the fluorescence lifetime of fluorescent proteins for real-time detection of liquid-liquid phase separation (LLPS) and liquid-solid phase transitions, and enhances their fluorescence lifetime sensitivity to LLPS or liquid-solid phase transitions, or even to both processes, by modifying wild-type mEYFP.

[0171] It should be noted that although the technical solutions of the present invention are introduced with specific examples, those skilled in the art can understand that the present invention should not be limited thereto.

[0172] The various embodiments of the present invention have been described above. The above description is exemplary and not exhaustive, and is also not limited to the disclosed embodiments. Many modifications and variations are obvious to those of ordinary skill in the art in the technical field without departing from the scope and spirit of the described embodiments. The choice of terms used herein is intended to best explain the principles of the embodiments, practical applications, or improvements to technologies in the market, or to enable other ordinary skilled persons in the technical field to understand the disclosed embodiments.

Claims

1. A fluorescent protein mutant, characterized in that: The fluorescent protein mutant is selected from any one of the following groups (i)-(v): (i) the mutant comprises a mutation at at least one of positions 47, 154, 164, and 176 corresponding to the sequence of SEQ ID NO: 1, compared to the sequence of SEQ ID NO: 1, and the change in the fluorescence signal of the mutant is more sensitive to liquid-liquid phase separation and / or liquid-solid phase transition of biological molecules compared to the fluorescent protein of SEQ ID NO: 1; (ii) having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with the amino acid sequence shown in (i), and excluding mutants of the sequence shown in SEQ ID NO: 1; (iii) a mutant encoded by a polynucleotide that hybridizes to the polynucleotide shown in (a) or (b) under very high stringency conditions: (a) a polynucleotide encoding a mutant of the amino acid sequence shown in (i); (b) a polynucleotide complementary to the full length of (a); (iv) a fragment of the mutant shown by any one of (i), (ii) or (iii), wherein the change in the fluorescence signal of the fragment still has an improved sensitivity to liquid-liquid phase separation and / or liquid-solid phase transition of biomolecules; (v) a polypeptide having an amino acid sequence as shown in (i), (ii), (iii) or (iv) with one or more amino acids added or deleted at at least one of the N-terminus and the C-terminus; Preferably, the fluorescent protein mutant has a mutated amino acid at at least one of the following positions: F47L, M154P, V164G, S176G.

2. The fluorescent protein mutant according to claim 1, characterized in that The fluorescent protein mutant corresponds to the sequence shown in SEQ ID NO: 1, and has mutations as shown in (i) to (v): (i)F47L, M154P, V164G, S176G; (ii) F47L; (iii) M154P; (iv) V164G; (v)S176G.

3. The fluorescent protein mutant according to claim 1 or 2, characterized in that The fluorescent protein mutant comprises a polypeptide as shown in any one of SEQ ID NOs: 2-6, or a polypeptide having at least 90%, optionally at least 95%, preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity with any one of SEQ ID NOs: 2-6.

4. A fusion protein, characterized in that The fusion protein comprises the fluorescent protein mutant according to any one of claims 1 to 3, and a biological molecule to be detected. Preferably, the biological molecule to be detected comprises a protein.

5. An isolated polynucleotide, characterized in that The polynucleotide comprises a sequence encoding the fluorescent protein mutant according to any one of claims 1 to 3 or a sequence encoding the fusion protein according to claim 4.

6. An expression vector, characterized in that: The expression vector comprises the isolated polynucleotide according to claim 5.

7. A recombinant host cell, characterized in that The recombinant host cell comprises the fluorescent protein mutant according to any one of claims 1 to 3, the fusion protein according to claim 4, the isolated polynucleotide according to claim 5, or the expression vector according to claim 6.

8. A method for detecting liquid-liquid phase separation and / or liquid-solid phase transition of biomolecules, characterized in that: The method uses the fluorescent protein mutant according to any one of claims 1 to 3, the fusion protein according to claim 4, the isolated polynucleotide according to claim 5, the expression vector according to claim 6, and the recombinant host cell according to claim 7.

9. The method according to claim 8, characterized in that The method uses the change of the fluorescence signal of the fluorescent protein mutant to detect the liquid-liquid phase separation and / or liquid-solid phase transition of the biological molecule, wherein the change of the fluorescence signal includes at least one of the change of fluorescence intensity, the change of fluorescence lifetime, the change of fluorescence polarization, and the change of fluorescence spectrum. Preferably, the change in the fluorescence signal includes a change in fluorescence lifetime.

10. Any of the following uses of the fluorescent protein mutant according to any one of claims 1 to 3, the fusion protein according to claim 4, the isolated polynucleotide according to claim 5, the expression vector according to claim 6, or the recombinant host cell according to claim 7: (1) Use in the preparation of reagents or kits for detecting liquid-liquid phase separation and / or liquid-solid phase transition of biomolecules; (2) Use in detecting liquid-liquid phase separation and / or liquid-solid phase transition of biomolecules.

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