A complete set of reagents for detecting the interaction between post-translational modified proteins and their ligands.

Abstract

This invention discloses a complete set of reagents for detecting the interaction between post-translational modified proteins and their ligands. The complete set of reagents of this invention consists of four reagents: A, B, C, and D. A is composed of a biomolecule named R linked to a protein named X; B contains a biomolecule named L; R and L may be the same or different and interact with each other, with a phase transition occurring after the interaction between R and L; C is a polymer formed from a C monomer, wherein the C monomer consists of a monomer named mc, a reporter group named α, and a group named Y. C Molecules obtained by linking biomolecules, with two or more mc molecules, can form polymers; D is composed of molecules named X L The modified protein and its name is Y. D It is composed of biomolecules linked together; Y C With Y D There are interactions between them. This invention achieves high enrichment of interacting proteins and ligands in phase change droplets, amplifies weak interaction signals, and makes them easy to detect.

Description

[0001] This application is a divisional application of Chinese invention patent application 201810455238.1, filed on May 14, 2018, entitled "A set of reagents for detecting the interaction between post-translational modified proteins and their ligands". Technical Field

[0002] This invention relates to a set of reagents for detecting the interaction between post-translational modified proteins and their ligands in the field of biotechnology. Background Technology

[0003] Phase transition, as a property of matter, has long been known in the physics community and in daily life. In recent years, scientists have gradually discovered that the phase transition (or phase separation) mechanism also exists widely in biological cells and plays an important biological role in the temporal and terrestrial control of the cell life cycle.

[0004] Current research has found that when multivalent macromolecules in solution interact with their multivalent ligands, they tend to form larger complexes. The solubility of these complexes generally decreases, causing them to separate from the normal solution phase and form a separate liquid phase enriched with the complex. This transition process is called a "liquid-liquid phase transition." The valence of a multivalent macromolecule refers to the number of binding regions within the macromolecule or its ligands that can interact with each other. For protein interactions, multivalent proteins and their multivalent ligands also undergo a "liquid-liquid phase transition" (or simply "phase transition") in vitro, resulting in a normal solution phase and a viscous liquid phase enriched with the protein. Under a microscope, the protein-rich liquid phase contains numerous small droplets (i.e., phase transition droplets), with droplet diameters reaching the micrometer level or even larger. For example, multivalent SH3 (SRC homology 3domain) and its multivalent ligand PRM (proline-rich motif) can undergo a phase transition at certain concentrations, while PRMH, which has a higher affinity for SH3, can undergo an even stronger phase transition.

[0005] In living organisms, the interaction between proteins and their ligands is the primary way proteins perform their functions. Under physiological conditions, protein-protein interactions exist in a dynamic equilibrium, and the dissociation constant (Kd) is typically used to characterize the strength of these interactions. Based on the Kd value, protein interactions are generally classified into stable interactions (with Kd values ​​ranging from picomoles to micromoles) and transient interactions (with Kd values ​​greater than 1 micromole). Essentially, transient protein-protein interactions can be considered weak interactions. The interactions between modified proteins (including methylation / demethylation, acetylation / deacetylation, phosphorylation / dephosphorylation, ubiquitination / deubiquitination, glycosylation / deglycosylation, etc.) and their ligands are mostly weak interactions. These weak interactions play important roles in cell signal transduction and cell cycle regulation. For example, the weak interaction between phosphorylated proteins and their ligands enables the transfer of phosphate groups in signaling pathways, and the weak interaction between methylated histones and their ligands regulates gene expression. Therefore, studying weak protein-protein interactions helps in understanding important cellular processes. However, weak interactions between proteins remain difficult to detect. Summary of the Invention

[0006] The technical problem this invention aims to solve is how to detect weak interactions between proteins, especially the interactions between post-translational modified proteins and their ligands. Post-translational modification refers to the covalent modification process that occurs on specific amino acid residues of a protein after translation. Currently, more than 300 types of post-translational modifications have been discovered, including common ones such as methylation, acetylation, phosphorylation, ubiquitination, and glycosylation. The opposite of post-translational modification is protein demodification, such as demethylation, deacetylation, dephosphorylation, deubiquitination, and deglycosylation.

[0007] To solve the above-mentioned technical problems, the present invention first provides a method for detecting a protein named X and a protein named X. L A set of reagents for determining whether modified proteins interact with each other is designated as reagent set 1, which consists of four reagents named A, B, C, and D.

[0008] A is formed by connecting a biomolecule named R and X;

[0009] The B contains a biomolecule named L;

[0010] The R and the L may be the same or different and they interact with each other; after the R and the L interact, a phase transition occurs.

[0011] The C is a polymer formed from C monomers, and the C monomers are the following c1) or c2):

[0012] c1) A molecule obtained by linking a monomer named mc, a reporter group named A, and a biomolecule named Y C Two or more of the mc can form a polymer;

[0013] c2) A molecule obtained by linking a tag to c1);

[0014] The D is formed by linking the X L and a biomolecule named Y D ;

[0015] The Y C interacts with the Y D ;

[0016] In the above-mentioned kit 1, the Y C and the Y D can both be proteins;

[0017] In the above-mentioned kit 1, the Y C can be Y11), Y12), or Y13):

[0018] Y11) A protein whose amino acid sequence is shown at positions 362-465 of SEQ ID NO: 7;

[0019] Y12) A protein obtained by substituting and / or deleting and / or adding one or several amino acid residues to the amino acid sequence shown at positions 362-465 of SEQ ID NO: 7 in the sequence list and having the same function;

[0020] Y13) A fusion protein obtained by linking a tag to the N-terminus or / and C-terminus of Y11) or Y12);

[0021] The Y D can be Y21), Y22), or Y23):

[0022] Y21) A protein whose amino acid sequence is shown at positions 22-29 of SEQ ID NO: 11;

[0023] Y22) A protein obtained by substituting and / or deleting and / or adding one or several amino acid residues to the amino acid sequence shown at positions 22-29 of SEQ ID NO: 11 in the sequence list and having the same function;

[0024] Y23) A fusion protein obtained by linking a tag to the N-terminus or / and C-terminus of Y21) or Y22);

[0025] Specifically, KKETPV, represented by positions 22-29 of sequence 11, interacts with PDZ, represented by positions 362-465 of sequence 7.

[0026] In the above-mentioned reagent kit 1, R contains a binding region named binding region 1; L contains a binding region named binding region 2; the interaction between R and L can be carried out through the binding region 1 and the binding region 2, and the number of the binding region 1 in R and the number of the binding region 2 in L can be greater than or equal to 2.

[0027] Wherein, both binding region 1 and binding region 2 are binding regions, and a binding region is the smallest unit of interaction between biomolecules through non-covalent bonds. When there are two or more binding regions between R and L, if the binding regions in R are not completely identical, binding region 1 is a collective term for all binding regions in R; if the binding regions in L are not completely identical, binding region 2 is a collective term for all binding regions in L.

[0028] Both R and L are multivalent molecules. The valence of multivalent molecules refers to the number of binding regions in a molecule that can bind to another molecule during molecular interaction. For R, the valence of R is the number of binding regions 1, and for L, the valence of L is the number of binding regions 2.

[0029] The R and L undergo a phase transition through multivalent interactions.

[0030] In the above-mentioned reagent kit 1, R can be a protein, nucleic acid, or polysaccharide. L can be a protein, nucleic acid, or polysaccharide.

[0031] In the above-mentioned reagent kit 1, A may also be connected to a reporter group named B.

[0032] The B group may also be connected to a reporter group named C.

[0033] In the above-mentioned reagent set 1, B and C may be the same or different.

[0034] The term A may be different from that of B and C.

[0035] In the above-mentioned reagent kit 1, A, B, and C can all be fluorescent reporter groups.

[0036] Furthermore, A, B, and C can all be fluorescent proteins.

[0037] In the above-mentioned reagent set 1, the ratio of the number of X and the number of R in A can be an integer greater than or equal to 1.

[0038] The C monomer, including mc, methyl, and Y.C The molar ratio can be 1:1:1.

[0039] In the C monomer, mc, the methyl group and the Y group C They can be linked through a linker region or a chemical bond. Specifically, methyl methyl ester (MCME) can be mCherry.

[0040] In the above-mentioned reagent kit 1, R can be a polymer formed from R monomers, and each R monomer contains a monomer named mr. Two or more mr monomers can form a polymer.

[0041] The L can be a polymer formed from L monomers, each of which contains a monomer named ml, and two or more ml can form a polymer.

[0042] The mc, the mr, and the ml are the same, or at least two of them are the same, or they are all different from each other.

[0043] In the above-mentioned reagent kit 1, at least one monomer in R may contain the binding region 1.

[0044] At least one monomer in L may contain the binding region 2.

[0045] When only one monomer in R contains the binding region 1, the monomer contains at least two binding regions 1. When two or more monomers in R contain the binding region 1, each monomer contains at least one binding region 1.

[0046] When only one monomer in L contains the binding region 2, the monomer contains at least two binding regions 2. When two or more monomers in L contain the binding region 2, each monomer contains at least one binding region 2.

[0047] In the monomer of R containing the binding region 1, the binding region 1 can be attached to the mr.

[0048] In the monomer containing the binding region 2 of L, the binding region 2 can be attached to the ml.

[0049] The mr may be the same as or different from the ml.

[0050] In the above-mentioned reagent kit 1, each of the R monomers may contain the mr and the binding region 1.

[0051] Each L monomer may contain the ml and the binding region 2.

[0052] In the above-mentioned reagent set 1, in the R monomer, the mr is linked to the binding region 1 or a biomolecule containing the binding region 1 through the linking region or a chemical bond.

[0053] In the L monomer, the ml can be linked to the binding region 2 or a biomolecule containing the binding region 1 via the linker region or a chemical bond.

[0054] In the R monomer, the number of binding regions 1 is at least one.

[0055] In the L monomer, the number of binding regions 2 is at least one.

[0056] In the R and L monomers, regardless of whether there is one or more of each part (i.e., the mr or the ml, the binding region 1 or the binding region 2, the B or C), there are no requirements on the connection order between them, as long as two or more R monomers can form a polymer, two or more L monomers can form a polymer, and the two polymers can interact and cause a phase transition.

[0057] In the above text, the linker region has no special requirements. It only needs to be able to connect the two connected portions of each monomer of R and L without affecting their functions. The linker region can be a polypeptide. In the R monomer, mr, ethyl, and binding region 1 or a biomolecule containing binding region 1 can be sequentially linked through the linker region or by chemical bonds.

[0058] In the L monomer, the ml, the propyl group, and the binding region 2 or a biomolecule containing the binding region 2 can be sequentially linked through the linker region or chemical bonds.

[0059] Each of the R monomers is connected to at least one of the X monomers.

[0060] In one embodiment of the present invention, the C-terminals of the R monomers are all connected to the N-terminals of the X via the connection region.

[0061] In the above-mentioned reagent kit 1, the R monomer may also contain the ethyl group.

[0062] Each of the L monomers may also contain the propylene monomer.

[0063] In the above-mentioned reagent set 1, in the R monomer, the mr, the ethyl group, and the binding region 1 or a biomolecule containing the binding region 1 can be linked through the linking region or a chemical bond.

[0064] In the L monomer, the ml, the propyl group, and the binding region 2 or a biomolecule containing the binding region 2 can be linked through the linker region or a chemical bond.

[0065] In the above-mentioned reagent set 1, the R monomers can all be the same, the L monomers can all be the same, and the C monomers can all be the same.

[0066] Both *mr* and *ml* can be yeast protein SmF. Yeast protein SmF is a core component of the ribonucleoprotein complex, and its crystal structure shows that it exists in the form of a homotetrameric form. Therefore, using SmF as a carrier can achieve the polymerization of target proteins.

[0067] The mc can be Bacillus subtilis protein Hfq. Bacillus subtilis protein Hfq exists in the form of a homohexamer, therefore, using Hfq as a carrier can achieve the polymerization of the target protein.

[0068] The binding region 1 may be the region in SH3 shown at positions 364-431 of sequence 1 that binds to PRMH shown at positions 366-380 of sequence 5; the binding region 2 may be the region in PRMH shown at positions 366-380 of sequence 5 that binds to SH3 shown at positions 364-431 of sequence 1.

[0069] The connection region can be (Gly-Gly-Ser). n Or contains (Gly-Gly-Ser) n The polypeptide, where n is a natural number greater than or equal to 2. Specifically, n can be 4 or 2.

[0070] The protein A can be a red fluorescent protein, such as mCherry. Both protein B and protein C can be green fluorescent proteins, such as GFP.

[0071] In the above-mentioned reagent kit 1, both mr and ml can be yeast SmF represented by positions 17-102 of sequence 1.

[0072] The mc can be Hfq, represented by positions 17-94 of sequence 7.

[0073] The biomolecule containing the binding region 1 may be SH3, represented by positions 364-431 of sequence 1.

[0074] The biomolecule containing the binding region 2 may be PRMH as shown at positions 366-380 of sequence 5.

[0075] In the above-mentioned reagent kit 1, the R monomer can be H1), H2), or H3:

[0076] The amino acid sequence of H1 is the protein shown in positions 17-431 of sequence 1;

[0077] H2) A protein that has the same function as the amino acid sequence shown in sequence 1, positions 17-431 of sequence 1 in the sequence listing, but with one or more amino acid residues replaced and / or deleted and / or added.

[0078] The fusion protein is obtained by attaching a tag to the N-terminus and / or C-terminus of H3) or H1) or H2).

[0079] The L monomer can be I1), I2), or I3:

[0080] I1) The amino acid sequence of the protein is shown in positions 17-380 of sequence 5;

[0081] I2) Proteins that have the same function by substituting and / or deleting and / or adding one or more amino acid residues in the amino acid sequence shown in sequence 5 of the sequence listing.

[0082] The fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of I1) or I2) is a fusion protein.

[0083] The C monomer can be J1), J2), or J3:

[0084] J1) The amino acid sequence of the protein is shown in positions 17-465 of sequence 7;

[0085] J2) A protein that has the same function as the amino acid sequence shown in sequence 7 of the sequence listing, with one or more amino acid residues replaced and / or deleted and / or added.

[0086] J3) is a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of J1) or J2).

[0087] To facilitate the purification of proteins in H1, I1, or J1, tags as shown in Table 1 can be attached to the amino or carboxyl terminus of H1, I1, or J1.

[0088] Table 1. Sequence of Labels

[0089] Label residues sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

[0090] The protein in H2, I2, or J2 mentioned above, wherein the substitution and / or deletion and / or addition of one or more amino acid residues is a substitution and / or deletion and / or addition of no more than 10 amino acid residues.

[0091] The proteins in H2, I2, or J2 mentioned above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0092] The coding genes for the proteins in H2), I2), or J2) mentioned above can be obtained by deleting one or more amino acid residues from the DNA sequence encoding the R monomer, the L monomer, or the C monomer, and / or by performing a missense mutation of one or more base pairs, and / or by attaching the coding sequence of the tag shown in Table 1 to its 5′ end and / or 3′ end.

[0093] The present invention also provides a method for detecting X and X. L A set of reagents that do not interact with each other is denoted as reagent set 2, and reagent set 2 consists of the following X1), X2), X3) and X4):

[0094] X1) The biomaterial associated with the R monomer is any one of X11) to X14) below:

[0095] X11) encodes the nucleic acid molecule of the R monomer;

[0096] X12) contains an expression cassette containing the nucleic acid molecule described in X11;

[0097] X13) A recombinant vector containing the nucleic acid molecule described in X11), or a recombinant vector containing the expression cassette described in X12;

[0098] X14) Recombinant microorganisms containing the nucleic acid molecules described in X11), or recombinant microorganisms containing the expression cassette described in X12), or recombinant microorganisms containing the recombinant vector described in X13);

[0099] X2) The biomaterial associated with the L monomer is any one of X21) to X24) below:

[0100] X21) encodes the nucleic acid molecule of the L monomer;

[0101] X22) contains an expression cassette containing the nucleic acid molecule described in X21);

[0102] X23) A recombinant vector containing the nucleic acid molecule described in X21), or a recombinant vector containing the expression cassette described in X22;

[0103] X24) Recombinant microorganisms containing the nucleic acid molecules described in X21), or recombinant microorganisms containing the expression cassette described in X22), or recombinant microorganisms containing the recombinant vector described in X23);

[0104] X3) The biomaterial associated with the C monomer is any one of X31) to X34) below:

[0105] X31) encodes the nucleic acid molecule of the C monomer;

[0106] X32) contains an expression cassette containing the nucleic acid molecule described in X31;

[0107] X33) A recombinant vector containing the nucleic acid molecule described in X31), or a recombinant vector containing the expression cassette described in X32;

[0108] X34) Recombinant microorganisms containing the nucleic acid molecules described in X31), or recombinant microorganisms containing the expression cassette described in X32), or recombinant microorganisms containing the recombinant vector described in X33);

[0109] X4) and the Y D The relevant biological material is any one of the following X41) to X44):

[0110] X41) encodes the Y D Nucleic acid molecules;

[0111] X42) contains an expression cassette containing the nucleic acid molecule described in X41);

[0112] X43) A recombinant vector containing the nucleic acid molecule described in X41), or a recombinant vector containing the expression cassette described in X42;

[0113] X44) Recombinant microorganisms containing the nucleic acid molecules described in X41), or recombinant microorganisms containing the expression cassette described in X42), or recombinant microorganisms containing the recombinant vector described in X43).

[0114] In the above-mentioned reagent kit 2, the nucleic acid molecule described in X11) can be as follows: x11), x12), or x13):

[0115] x11) The coding sequence is the cDNA molecule or DNA molecule at positions 62-1306 of sequence 2 in the sequence listing;

[0116] The nucleotide sequences defined by x12) and x11) have 75% or more identity and encode the cDNA molecule or genomic DNA molecule of the R monomer;

[0117] x13) hybridizes with the nucleotide sequence defined by x11) under stringent conditions and encodes a cDNA molecule or genomic DNA molecule of the R monomer.

[0118] The nucleic acid molecule described in X21) may be as follows: x21) or x22) or x23):

[0119] (x21) The coding sequence is the cDNA or DNA molecule located at positions 62-1153 of sequence 6 in the sequence listing;

[0120] The nucleotide sequences defined by x22) and x21) have 75% or more identity and encode the cDNA molecule or genomic DNA molecule of the L monomer;

[0121] x23) hybridizes with the nucleotide sequence defined by x21) under stringent conditions and encodes a cDNA molecule or genomic DNA molecule of the L monomer.

[0122] The nucleic acid molecule described in X31) may be as follows: x31), x32), or x33):

[0123] (x31) The coding sequence is the cDNA molecule or DNA molecule located at positions 51-1400 of sequence 8 in the sequence listing;

[0124] The nucleotide sequences defined by x32) and x31) have 75% or more identity and encode the cDNA molecule or genomic DNA molecule of the C monomer;

[0125] x33) hybridizes with the nucleotide sequence defined by x31) under stringent conditions and encodes a cDNA molecule or genomic DNA molecule of the C monomer.

[0126] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0127] The term "identity" as used herein refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 75% or higher, 85% or higher, 90% or higher, or 95% or higher identity with the nucleotide sequences of this invention. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0128] The stringent conditions are: hybridization and washing twice at 68°C in a solution of 2×SSC and 0.1% SDS, each time for 5 min; followed by hybridization and washing twice at 68°C in a solution of 0.5×SSC and 0.1% SDS, each time for 15 min; or hybridization and washing at 65°C in a solution of 0.1×SSPE (or 0.1×SSC) and 0.1% SDS.

[0129] The aforementioned 75% or higher degree of identity can be 80%, 85%, 90%, or 95% or higher degree of identity.

[0130] The expression cassette (R-monomer gene expression cassette) containing a nucleic acid molecule encoding the R-monomer described in X12) refers to DNA capable of expressing the R-monomer in a host cell. This DNA may include not only a promoter to initiate transcription of the R-monomer gene but also a terminator to terminate transcription of the R-monomer gene. Furthermore, the expression cassette may also include an enhancer sequence.

[0131] The expression cassette (L-monomer gene expression cassette) containing a nucleic acid molecule encoding the L-monomer, as described in X22, refers to DNA capable of expressing the L-monomer in a host cell. This DNA may include not only a promoter to initiate transcription of the L-monomer gene but also a terminator to terminate transcription of the L-monomer gene. Furthermore, the expression cassette may also include an enhancer sequence.

[0132] The expression cassette (C monomer gene expression cassette) containing a nucleic acid molecule encoding the C monomer, as described in X32, refers to DNA capable of expressing the C monomer in a host cell. This DNA may include not only a promoter to initiate transcription of the C monomer gene but also a terminator to terminate transcription of the C monomer gene. Furthermore, the expression cassette may also include an enhancer sequence.

[0133] Recombinant vectors containing the R monomer gene expression cassette, the L monomer gene expression cassette, or the C monomer gene expression cassette can be constructed using existing vectors. The vector can be a plasmid, granule, bacteriophage, or viral vector. Specifically, the plasmid can be the pRSFDuet-1 vector.

[0134] X13) The recombinant vector may specifically be pRSFDuet-1-SGS, wherein pRSFDuet-1-SGS is a recombinant vector obtained by replacing the DNA fragment (containing the recognition sequences of NcoI and XhoI) between the NcoI and XhoI recognition sequences of the pRSFDuet-1 vector with the DNA molecule shown in positions 12-1360 of sequence 2 in the sequence listing. The pRSFDuet-1-SGS can express the fusion protein of the R monomer and His-tag shown in sequence 1.

[0135] X23) The recombinant vector may specifically be pRSFDuet-1-SGP, which is a recombinant vector obtained by replacing the DNA fragment (containing the recognition sequences of NcoI and XhoI) between the NcoI and XhoI recognition sequences of the pRSFDuet-1 vector with the DNA molecule shown in positions 12-1162 of sequence 6 in the sequence listing. The pRSFDuet-1-SGP can express the fusion protein of the L monomer and His-tag shown in sequence 5.

[0136] X33) The recombinant vector may specifically be pRSFDuet-1-Hfq-mCherry-PDZ, wherein pRSFDuet-1-Hfq-mCherry-PDZ is a recombinant vector obtained by replacing the DNA fragment (containing the recognition sequences of NcoI and XhoI) between the NcoI and XhoI recognition sequences of the pRSFDuet-1 vector with the DNA molecule shown in Sequence 8 of the sequence listing. The pRSFDuet-1-Hfq-mCherry-PDZ can express the fusion protein formed by Hfq, mCherry, PDZ and His-tag shown in Sequence 7.

[0137] The microorganism may be yeast, bacteria, algae, or fungi. Among them, the bacteria may be Escherichia coli.

[0138] In the aforementioned reagent kits 1 and 2, the modification can be a post-translational modification of the protein or a demodification of a post-translational modification of the protein. The post-translational modification of the protein can be methylation, acetylation, phosphorylation, ubiquitination, or glycosylation. The demodification of the post-translational modification of the protein can be demethylation, deacetylation, dephosphorylation, deubiquitination, or deglycosylation.

[0139] This invention also provides a method for detecting whether there is an interaction between biomolecules, wherein the biomolecules are named X and X. L Two biomolecules, namely X L X is a modified protein, and the method includes:

[0140] Solution A, solution B, solution C, and solution D are mixed to obtain the test solution, wherein solution A is a solution containing A; solution B is a solution containing B; solution C is a solution containing C; and solution D is a solution containing D. In the test solution, the R in A and the L in B interact to undergo a phase transition, producing phase change droplets. The X and X' are determined based on whether the phase change droplets in the test solution contain the signal of A. L Whether there is an interaction between them: If the phase change droplet in the test liquid contains the signal of A, X and X L The X and Xcan have or are candidate for interaction; if the phase change droplets in the test liquid do not contain the signal of A, the X and the Xcan have interaction; L There is no interaction between the candidates or between the candidates.

[0141] Wherein, whether the phase change droplets in the test liquid contain the signal of A refers to whether the signal of A in the test liquid is enriched in the phase change droplets, so that the signal of A in the phase change droplets is higher than that in the non-phase change droplets of the test liquid. Specifically, determining X and X based on whether the phase change droplets in the test liquid contain the signal of A. L Whether there is an interaction between them may include: as the signal of A in the phase change droplet in the test liquid is enriched, X and X L The X and Xcan have or are candidate for interaction; if the signal of A in the phase change droplets in the test liquid is not enriched, the X and the Xcan have interaction; L There is no interaction between the candidates or between the candidates.

[0142] Solution A may be composed of A and a solvent, solution B may be composed of B and the solvent, solution C may be composed of C and the solvent, and solution D may be composed of D and the solvent, wherein the solvent is capable of dissolving A, B, C and D.

[0143] In one embodiment of the present invention, the solvent is a KMEI buffer, which is composed of a solvent and a solute. The solvent is water, and the solute and its concentration are 150mM KCl, 1mM MgCl2, 1mM EGTA, 10mM imidazole, 1mM DTT, and pH=7.

[0144] In one embodiment of the present invention, protein multiplication is achieved by fusing the protein with a known multivalent yeast protein SmF or Bacillus subtilis protein Hfq, i.e., multivalent expression of reagents A, B, and C. The R monomer is SGS (SGS is an abbreviation for the fusion protein SmF-GFP-SH3), and the L monomer is SGP (SGP is an abbreviation for the fusion protein SmF-GFP-PRMH). The interaction between SH3 and PRMH induces the interaction between the multivalent proteins SGS and SGP, leading to a phase transition and the generation of phase-transition droplets.

[0145] In the above method, the modification can be a post-translational modification of the protein or a demodification of a post-translational modification of the protein. The post-translational modification of the protein can be methylation, acetylation, phosphorylation, ubiquitination, or glycosylation. The demodification of the post-translational modification of the protein can be demethylation, deacetylation, dephosphorylation, deubiquitination, or deglycosylation.

[0146] The present invention also provides any of the following applications of the reagent kit 1 or the reagent kit 2:

[0147] Z1) Applications in detecting or assisting in the detection of whether modified proteins interact with other biomolecules;

[0148] Application of Z2 in screening for regulatory factors of interactions between modified proteins and other biomolecules;

[0149] Application of Z3 in identifying or assisting in the identification of regulatory factors of interactions between modified proteins and other biomolecules;

[0150] Application of Z4 in detecting the effects of substances on the interactions between modified proteins and other biomolecules;

[0151] Z5) Application in detecting whether a protein has enzyme activity involved in post-translational modifications;

[0152] Z6) Application in the preparation of products for detecting whether modified proteins interact with other biomolecules;

[0153] Application of Z7 in the preparation of products that screen modified proteins for regulatory factors of interactions between other biomolecules;

[0154] Application of Z8 in the preparation and identification of products that regulate the interaction between modified proteins and other biomolecules;

[0155] Z9) is used in the preparation of enzyme products for detecting whether proteins have enzyme activity involved in post-translational modifications.

[0156] In the above applications, the other biomolecules may be proteins.

[0157] In the above applications, the modification can be a post-translational modification of the protein or a demodification of a post-translational modification of the protein. The post-translational modification of the protein can be methylation, acetylation, phosphorylation, ubiquitination, or glycosylation. The demodification of the post-translational modification of the protein can be demethylation, deacetylation, dephosphorylation, deubiquitination, or deglycosylation.

[0158] The present invention provides a complete set of reagents for detecting the interaction between post-translational modified proteins and their ligands, and a multivalent recruitment system based on these reagents. Utilizing a phase transition mechanism, the interacting proteins and ligands are highly enriched in phase-change droplets, strongly amplifying the originally weak interaction signal and facilitating detection. The complete set of reagents and the multivalent recruitment system of the present invention can be used to detect interactions between proteins and their ligands, especially weak interactions. They can also identify whether a protein possesses the activity of an enzyme involved in post-translational modification, such as kinase activity corresponding to phosphorylation modification or methyltransferase activity corresponding to methylation modification, and can also identify regulators that can modulate the above enzyme activities. Attached Figure Description

[0159] Figure 1Analysis of experimental results for the detection of the interaction between H3K9me3 and CD in systems 1-8. A shows the morphological observation of the phase change droplets in system 3; the right image is a magnified view of the area selected in the left image. B shows the laser confocal high-content microscopy imaging analysis of systems 1-8. Scale bar = 100 μm.

[0160] Figure 2 Analysis of experimental results for the detection of the interaction between H3K9me3 and CD in system 9-20. A shows the laser confocal high-content microscopy imaging analysis of system 9-20. Scale bar = 100 μm. B shows the quantitative analysis of mCherry fluorescence intensity in the phase change droplets in Figure A. H3K9me3 and H3K9 represent H3K9me3-KKETPV and H3K9-KKETPV, respectively; 0, 0.2, 0.4, 0.6, 0.8, and 1.0 represent the proportions of H3K9me3-KKETPV in the mixture of H3K9me3-KKETPV and H3K9-KKETPV, respectively. Detailed Implementation

[0161] The present invention will be further described in detail below with reference to specific embodiments. The embodiments given are only for illustrating the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the materials, reagents, instruments, etc. used in the following embodiments are all commercially available. In the quantitative experiments in the following embodiments, three replicate experiments were set up, and the results were averaged.

[0162] This invention provides a method for detecting biomolecules X and X. L A set of reagents for inter-interaction, consisting of four reagents named A, B, C, and D;

[0163] A is composed of a biomolecule named R and a protein named X. R is a polymer formed by R monomers. All R monomers are the same. Each monomer contains mr, a fluorescent reporter group named B, a binding region 1 or a biomolecule containing a binding region 1, and protein X. The parts of each monomer are connected by a linker or chemical bond. Among them, more than or equal to 2 mr can form a polymer.

[0164] B contains a biomolecule named L, which is a polymer formed from L monomers. All L monomers are identical. Each monomer contains ml, a fluorescent reporter group named C, and a binding region 2 or a biomolecule containing a binding region 2. The parts of each monomer are connected by a linker or chemical bond. Among them, more than or equal to 2 ml can form a polymer.

[0165] R and L are the same or different and interact with each other. The interaction between R and L is carried out through the binding region 1 of R and the binding region 2 of L. The number of binding regions 1 in R and the number of binding regions 2 in L are both greater than or equal to 2. After the interaction between R and L, a phase transition occurs.

[0166] C is a polymer formed from C monomers. C monomers consist of a monomer named mc, a reporter group named methyl, and a group named Y. C It is composed of biomolecules linked together, and two or more mc molecules can form a polymer;

[0167] D is named X L The protein with post-translational modifications and its name is Y. D It is composed of biomolecules linked together;

[0168] Y C With Y D There is an interaction between them.

[0169] The following example, where both R and L are proteins, illustrates how the reagent kit of this invention detects the interaction between proteins containing post-translational methylation modifications and their ligands. Specifically, mr and ml are both SmF, fluorescent reporter group B and fluorescent reporter group C are both GFP, the biomolecule containing binding region 1 is SH3, and the biomolecule containing binding region 2 is PRMH. The fusion protein of SmF, GFP, and SH3 is denoted as SGS (i.e., R monomer), and the fusion protein of SmF, GFP, and PRMH is denoted as SGP (i.e., L monomer); mc is Hfq, fluorescent reporter group A is mCherry, and Y... C The PDZ shown in positions 362-465 of sequence 7, Hfq, mCherry, and PDZ are fused to obtain the C monomer; Y D It is KKETPV (positions 22-29 of sequence 11).

[0170] Example 1: Verification of the interaction between H3K9me3 and CD

[0171] In this embodiment, X L H3K9me3 is H3K9me3, X is CD, and H3K9me3 and CD interact.

[0172] I. Preparation of Recombinant Vectors

[0173] 1. Recombinant vector expressing the SGS and CD fusion protein

[0174] The DNA fragment between the NcoI and XhoI recognition sequences of the pRSFDuet-1 vector (a Novagen product from Merck) was replaced with the DNA molecule shown in positions 12-1360 of sequence 2 in the sequence listing to obtain the recombinant vector pRSFDuet-1-SGS. pRSFDuet-1-SGS can express the protein shown in sequence 1 (SGS fused to His-tag, i.e., R monomer, denoted as His-SGS).

[0175] In this sequence, positions 14-1354 of sequence 2 represent the DNA molecule encoding His-SGS as shown in sequence 1. Positions 1344-1349 and 1355-1360 of sequence 2 are the recognition sequences for NcoI and XhoI, respectively. Positions 3-8 of sequence 1 are the amino acid sequence of the His-tag. Positions 17-102 of sequence 1 are the amino acid sequence of SmF. Positions 109-349 of sequence 1 are the amino acid sequence of GFP. Positions 364-431 of sequence 1 are the amino acid sequence of SH3. Positions 103-108, 350-363, and 432-444 of sequence 1 are the amino acid sequences of the linker region. His-SGS can form a tetrademer through the action of SmF.

[0176] The DNA fragment between the NcoI and XhoI recognition sequences of pRSFDuet-1-SGS (containing the NcoI and XhoI recognition sequences) was replaced with the DNA molecule shown in positions 1-212 of sequence 4 in the sequence listing to obtain the recombinant vector pRSFDuet-1-SGS-CD. pRSFDuet-1-SGS-CD expresses the fusion protein of His-SGS shown in sequence 1 and CD shown in sequence 3 in the sequence listing (denoted as SGS-CD).

[0177] In particular, positions 9-203 of sequence 4 encode the CD shown in sequence 3. SGS-CD can form a tetrademer through the action of SmF.

[0178] 2. Recombinant vectors expressing SGP

[0179] The DNA fragment between the NcoI and XhoI recognition sequences of the pRSFDuet-1 vector (containing the recognition sequences of NcoI and XhoI) was replaced with the DNA molecule shown in positions 12-1162 of sequence 6 in the sequence listing to obtain the recombinant vector pRSFDuet-1-SGP. pRSFDuet-1-SGP can express the protein shown in sequence 5 (SGP fused with His-tag, denoted as His-SGP, also known as L monomer).

[0180] In particular, the DNA molecule encoding sequence 5, shown at positions 14-1156 of sequence 6, contains the His-SGP amino acid sequence, positions 3-8 of sequence 5 contain the His-tag amino acid sequence, positions 17-102 of sequence 5 contain the SmF amino acid sequence, positions 109-349 of sequence 5 contain the GFP amino acid sequence, positions 366-380 of sequence 5 contain the PRMH amino acid sequence, and positions 103-108 and 350-365 of sequence 5 contain the linker amino acid sequence.

[0181] His-SGP can form a tetrademer through the action of SmF.

[0182] 3. Recombinant vectors expressing monomer C (Hfq-mCherry-PDZ)

[0183] The DNA fragment between the NcoI and XhoI recognition sequences of the pRSFDuet-1 vector (containing the recognition sequences of NcoI and XhoI) was replaced with the DNA molecule shown in Sequence 8 of the sequence listing to obtain the recombinant vector pRSFDuet-1-Hfq-mCherry-PDZ. pRSFDuet-1-Hfq-mCherry-PDZ can express the protein shown in Sequence 7 (the fusion protein formed by Hfq, mCherry, PDZ and His-tag, denoted as Hfq-mCherry-PDZ).

[0184] In particular, the DNA molecule shown in positions 3-1397 of sequence 8 encodes Hfq-mCherry-PDZ as shown in sequence 7. Positions 3-8 of sequence 7 are the amino acid sequence of His-tag, positions 17-94 of sequence 7 are the amino acid sequence of Hfq, positions 101-340 of sequence 7 are the amino acid sequence of mCherry, positions 362-465 of sequence 7 are the amino acid sequence of PDZ, and positions 95-100 and 341-361 of sequence 7 are the amino acid sequences of the linker region.

[0185] Hfq-mCherry-PDZ can form a hexamer through the action of Hfq.

[0186] Preparation of a recombinant vector expressing Hfq fusion protein: The DNA fragment between the NcoI and XhoI recognition sequences of the pRSFDuet-1 vector (containing the recognition sequences of NcoI and XhoI) was replaced with the DNA molecule shown in positions 11-1143 of sequence 10 in the sequence listing, resulting in the recombinant vector pRSFDuet-1-mCherry-PDZ. pRSFDuet-1-mCherry-PDZ can express the protein shown in sequence 9 (the fusion protein formed by mCherry, PDZ and His-tag, denoted as mCherry-PDZ, which is used as a control below).

[0187] In particular, the DNA molecule encoding sequence 9, shown at positions 13-1134 of sequence 10, contains the amino acid sequence of the His-tag at positions 3-8, the amino acid sequence of mCherry at positions 17-256, the amino acid sequence of PDZ at positions 271-374, and the amino acid sequence of the linker region at positions 257-270.

[0188] 4. Preparation of Reagent D

[0189] Chemical Synthesis X L Connect Y D Reagent D is a methylated protein obtained by trimethylating the 4th lysine residue of H3K9-KKETPV shown in sequence 11 of the sequence listing. It is denoted as H3K9me3-KKETPV. The sequence of H3K9me3-KKETPV is as follows: ARTK(Me)3QTARGGSGGSGGSWGGSKKETPVAV.

[0190] II. Fusion Protein Expression and Purification

[0191] The pRSFDuet-1-SGS, pRSFDuet-1-SGS-CD, pRSFDuet-1-SGP, pRSFDuet-1-Hfq-mCherry-PDZ, and pRSFDuet-1-mCherry-PDZ from step one were introduced into Escherichia coli competent cells BL21(DE3) (Tiangen Biotech (Beijing) Co., Ltd.) to obtain recombinant strains BL21-pRSFDuet-1-SGS, BL21-pRSFDuet-1-SGS-CD, BL21-pRSFDuet-1-SGP, BL21-pRSFDuet-1-Hfq-mCherry-PDZ, and BL21-pRSFDuet-1-mCherry-PDZ.

[0192] The His-tagged fusion proteins expressed by recombinant strains BL21-pRSFDuet-1-SGS, BL21-pRSFDuet-1-SGS-CD, BL21-pRSFDuet-1-SGP, BL21-pRSFDuet-1-Hfq-mCherry-PDZ, and BL21-pRSFDuet-1-mCherry-PDZ were purified according to the following method:

[0193] (1) Bacterial culture and protein induction expression: The above recombinant strain was inoculated into 1L LB medium. It was cultured at 37℃ and 200rpm until the OD600 was about 0.8-1 (about 8-9 hours). The bacterial culture was transferred to 18℃ and cooled for 1 hour. IPTG was added to a final concentration of 0.5mM to induce protein expression overnight (about 16 hours) to obtain the culture medium.

[0194] (2) Bacterial resuspension and disruption: Centrifuge the culture medium obtained in step (1), discard the supernatant, resuspend the bacterial pellet in 40 mL binding buffer (40 mM Tris-Cl, 500 mM NaCl, pH 8.0 or 7.4) and sonicate to disrupt the pellet. Centrifuge the disrupted product at 20,000 rpm for 1 hour and collect the supernatant (containing the target fusion protein).

[0195] (3) Ni column purification: Prepare a Ni column in advance and equilibrate it with binding buffer. Pour the supernatant obtained in step (2) into the Ni column. When the liquid is almost dry, add wash buffer to wash for 2-3 column volumes, then add elution buffer to elute the target fusion protein and collect the eluent.

[0196] Wash buffer: 40mM Tris-HCl, 500mM NaCl, 40mM imidazole, pH same as binding buffer.

[0197] elution buffer: 40mM Tris-HCl, 500mM NaCl, 500mM imidazole, pH same as binding buffer.

[0198] (4) Ion exchange purification: Select a suitable ion exchange column based on the isoelectric point of the protein. Dilute the effluent from step (3) with 40 mM Tris-Cl buffer to reduce the ion concentration and obtain a protein dilution. Install the ion exchange column into the ATKA protein purification system (GE) and load the protein dilution. Elute the protein bound to the column by gradually increasing the salt ion concentration and collect the target fusion protein. The elution buffer used consists of solution A and solution B, and the ratio between the two is adjusted according to the specific situation: Solution A: 40 mM Tris-Cl, pH same as binding buffer; Solution B: 40 mM Tris-Cl, 2 M NaCl, pH same as binding buffer.

[0199] (5) Gel filtration purification: After ultrafiltration concentration of the target fusion protein obtained in step (4), it is separated and purified by a preset molecular sieve program to obtain a further purified target fusion protein.

[0200] The KMEI buffer used for column equilibration and elution consists of a solvent and a solute. The solvent is water, and the solutes and their concentrations are as follows: 150 mM KCl, 1 mM MgCl2, 1 mM EGTA, 10 mM imidazole, 1 mM DTT, pH=7.

[0201] (6) Detection and preservation of purified proteins: SDS-PAGE was used to detect His-SGS, SGS-CD, His-SGP, Hfq-mCherry-PDZ and mCherry-PDZ of BL21-pRSFDuet-1-SGS-CD, His-SGP, Hfq-mCherry-PDZ and mCherry-PDZ of BL21-pRSFDuet-1-SGP-PAGE obtained in the above steps, and after confirming that the size of the above fusion proteins all met the expectations, the proteins were concentrated and frozen at -80℃ for later use.

[0202] III. Detection of the interaction between H3K9me3 and CD

[0203] The solutions (all in KMEI buffer) of His-SGS, SGS-CD, His-SGP, Hfq-mCherry-PDZ, mCherry-PDZ (as a control), H3K9me3-KKETPV, and H3K9-KKETPV (as a control) obtained in step two were aliquoted into 384 microplates according to the systems shown in Table 2, with one system per well. The concentrations of SGS-CD, His-SGP, Hfq-mCherry-PDZ, mCherry-PDZ, and the mixture of H3K9me3-KKETPV and H3K9-KKETPV in each system were 1 μM.

[0204] Table 2. Detection system for the interaction between H3K9me3 and CD

[0205]

[0206]

[0207] In Table 2, "+" indicates the presence of the substance; "-" indicates the absence of the substance; and "*" indicates a mixture of H3K9me3-KKETPV and H3K9-KKETPV. In systems 9-14, the molar percentages of H3K9me3-KKETPV in the mixture of H3K9me3-KKETPV and H3K9-KKETPV are 0, 0.2, 0.4, 0.6, 0.8, and 1.0, respectively. In systems 15-20, the molar percentages of H3K9me3-KKETPV in the mixture of H3K9me3-KKETPV and H3K9-KKETPV are 0, 0.2, 0.4, 0.6, 0.8, and 1.0, respectively.

[0208] The above systems were incubated at 4°C until the phase change droplets in the system that underwent phase change completely settled to the bottom of the well plate. Images were acquired using a laser confocal high-content imaging microscope. The results ( Figure 1 (B) shows that the solutions in systems 1 and 2 remained unchanged, and no fluorescent signal aggregation regions were found; phase change droplets were generated in systems 3 and 4, and the green fluorescent signal (fluorescent signal emitted by GFP) aggregated in the phase change droplets, and its signal intensity was much higher than that in the solution. Figure 1 In systems A), no changes were observed in the solutions of systems 5 and 6, and no fluorescent signal aggregation regions were found. In systems 7 and 8, phase change droplets were generated, with green fluorescent signals aggregation within the droplets, and their signal intensity was significantly higher than that in the solution. No red fluorescent signal aggregation was observed in the droplets. In systems 9-14, phase change droplets were generated, with green fluorescent signals aggregation within the droplets, and their signal intensity was significantly higher than that in the solution. No significant red fluorescent signal aggregation was observed in the droplets. Figure 2 In system 15-20, phase change droplets were generated, and green fluorescence signals accumulated in the droplets, with a signal intensity much higher than that in the solution. It was also found that when the mixture consisted entirely of H3K9-KKETPV (i.e., the molar percentage of H3K9me3-KKETPV in the mixture was 0), no obvious red fluorescence (mCherry fluorescence signal) was observed in the phase change droplets. However, as the proportion of H3K9me3 in the mixture of H3K9me3 and H3K9 increased, the intensity of the red fluorescence signal in the phase change droplets also increased. The intensity of the red fluorescence signal in the phase change droplets reached its maximum when the mixture consisted entirely of H3K9me3-KKETPV (i.e., the molar percentage of H3K9me3-KKETPV in the mixture was 1.0). Figure 2 ).

[0209] This indicates that SGS can bind with SGP to generate phase transition droplets, which can be identified by the fluorescent signal emitted by GFP. In systems where mCherry-PDZ is present but Hfq-mCherry-PDZ is absent, regardless of whether the system contains H3K9me3-KKETPV or H3K9-KKETPV, the phase transition droplets in these systems cannot accumulate red fluorescent signals. In systems where mCherry-PDZ is absent but Hfq-mCherry-PDZ is present, the content of H3K9me3 is positively correlated with the enrichment of red fluorescent signals in the phase transition droplets. This indicates that CD in SGS-CD can recruit H3K9me3-KKETPV into the phase change droplet through the interaction between CD and H3K9me3. KKETPV in H3K9me3-KKETPV can further recruit Hfq-mCherry-PDZ emitting red fluorescence signals into the phase change droplet, thereby causing the phase change droplet to emit an enriched red fluorescence signal. If Hfq-mCherry-PDZ is replaced with mCherry-PDZ, the red fluorescence signal in the phase change droplet is not obvious, indicating that Hfq-mCherry-PDZ, after forming a hexavalent state (i.e., hexamer) through Hfq, is more suitable than monovalent mCherry-PDZ for detecting the interaction between CD and H3K9me3.

[0210] In summary, the phase transition-based reagent kit and multivalent recruitment system of this invention amplifies the interaction signal and improves the detection sensitivity of H3K9me3 and CD interactions, providing a simple and easy new method for detecting weak interactions between post-translational modified proteins and their ligands.

Claims

1. A set of reagents for detecting whether there is an interaction between protein X and a modified protein XL, consisting of four reagents named A, B, C and D respectively; The A is composed of a biomolecule named R and a protein named X linked together. The B contains a biomolecule named L; The R and the L may be the same or different and they interact with each other; after the R and the L interact, a phase transition occurs. The C is a polymer formed from the C monomer, and the C monomer is either c1) or c2) below: c1 ) a molecule obtained by linking a biological molecule of the name Y C with a monomer of the name mc, a reporter group of the name Me and a molecule of the name Y c2) a molecule obtained by linking a biological molecule of the name Y C with a monomer of the name mc, a reporter group of the name Me and a molecule of the name Y c2) The molecule obtained by attaching a tag to c1); The D is connected to a protein with the name X L with a modified protein and a biomolecule with the name Y D ​ The Y C has an interaction with the Y D has an interaction with the Y The Y C With the Y D are proteins, wherein, The Y C For Y12) or Y13): Y12) is a protein having the amino acid sequence shown in positions 362-465 of sequence 7; The fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of Y13) is a protein obtained by linking a tag to the N-terminus and / or C-terminus of Y12. And / or, the Y D For Y22) or Y23): Y22) is a protein having the amino acid sequence shown at positions 22-29 of sequence 11; The fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of Y23) is a protein obtained by linking a tag to the N-terminus and / or C-terminus of Y22. The R is a nucleic acid or a polysaccharide; and / or the L is a nucleic acid or a polysaccharide.

2. The complete set of reagents according to claim 1, characterized in that: The R contains a binding region named binding region 1; the L contains a binding region named binding region 2; the interaction between the R and the L is carried out through the binding region 1 and the binding region 2, and the number of the binding region 1 in the R and the number of the binding region 2 in the L are both greater than or equal to 2.

3. The complete set of reagents according to claim 1 or 2, characterized in that: A is also connected to a reporter group named B; And / or, B is further connected to a reporter group named C.

4. The complete set of reagents according to claim 3, characterized in that: B and C may be the same or different; And / or, the A is different from the B and the C.

5. The complete set of reagents according to claim 4, characterized in that: The groups A, B, and C are all fluorescent reporter groups.

6. The complete set of reagents according to claim 5, characterized in that: The fluorescent reporter group is a fluorescent protein.

7. The complete set of reagents according to claim 1, characterized in that: The ratio of the number of X and the number of R in A is an integer greater than or equal to 1.

8. The complete set of reagents according to claim 1, characterized in that: R is a polymer formed from R monomers, and each R monomer contains a monomer named mr. Two or more mr monomers can form a polymer. And / or, the L is a polymer formed from L monomers, each of the L monomers contains a monomer named ml, and two or more ml can form a polymer; The mc, the mr, and the ml are the same, or at least two of them are the same, or they are all different from each other.

9. The complete set of reagents according to claim 8, characterized in that: At least one monomer in R contains a binding region 1; And / or, at least one monomer in the L contains a binding region 2.

10. The complete set of reagents according to claim 9, characterized in that: Each of the R monomers contains the mr and the binding region 1; And / or, each of the L monomers contains the ml and binding region 2.

11. The complete set of reagents according to claim 10, characterized in that: In the R monomer, the mr is connected to the binding region 1 or a biomolecule containing the binding region 1 through a linker or chemical bond; And / or, in the L monomer, the ml is connected to the binding region 2 or a biomolecule containing the binding region 2 through the linker region or a chemical bond.

12. The complete set of reagents according to claim 10 or 11, characterized in that: Each of the R monomers also contains a reporter group named B; And / or, each of the L monomers further contains a reporter group named propyl.

13. The complete set of reagents according to claim 12, characterized in that: In the R monomer, the mr, the ethyl group, and the binding region 1 or a biomolecule containing the binding region 1 are linked by a linker or chemical bond. And / or, in the L monomer, the ml, the propyl group, and the binding region 2 or a biomolecule containing the binding region 2 are linked by a linker region or a chemical bond.

14. The complete set of reagents according to claim 13, characterized in that: All R monomers are the same; all L monomers are the same; all C monomers are the same. And / or, the mr and the ml are both yeast protein SmF; and / or, the mc is Bacillus subtilis protein Hfq; And / or, the binding region 1 is the region in SH3 shown at positions 364-431 of sequence 1 that binds to PRMH shown at positions 366-380 of sequence 5; the binding region 2 is the region in PRMH shown at positions 366-380 of sequence 5 that binds to SH3 shown at positions 364-431 of sequence 1; And / or, the connection region is (Gly-Gly-Ser). n Or contains (Gly-Gly-Ser) n The polypeptide, where n is a natural number greater than or equal to 2; And / or, wherein A is a red fluorescent protein; And / or, both B and C are green fluorescent proteins.

15. The complete set of reagents according to claim 11, characterized in that: Both mr and ml are yeast SmF represented by positions 17-102 of sequence 1; And / or, the mc is the Hfq shown in positions 17-94 of sequence 7; And / or, the biomolecule containing the binding region 1 is SH3 as shown at positions 364-431 of sequence 1; And / or, the biomolecule containing the binding region 2 is PRMH represented by positions 366-380 of sequence 5.

16. The complete set of reagents according to claim 8, characterized in that: The R monomer is either H2 or H3. H2) is a protein having the amino acid sequence shown in positions 17-431 of sequence 1; The fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of H2) in H3); And / or, the L monomer is I2) or I3): I2) A protein having the amino acid sequence shown in positions 17-380 of sequence 5; I3) The fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of I2); And / or, the C monomer is J2) or J3): J2) A protein having the amino acid sequence shown in positions 17-465 of sequence 7; The fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of J2) is obtained by J3).

17. A kit of reagents for detecting whether there is an interaction between a protein named X and a modified protein named XL, comprising the following X1), X2), X3), and X4): X1) is any one of the following X11) to X14): X11) encodes a nucleic acid molecule that forms a fusion protein A by linking the R monomer and the X protein; X12) contains an expression cassette containing the nucleic acid molecule described in X11; X13) A recombinant vector containing the nucleic acid molecule described in X11), or a recombinant vector containing the expression cassette described in X12; X14) Recombinant microorganisms containing the nucleic acid molecules described in X11), or recombinant microorganisms containing the expression cassette described in X12), or recombinant microorganisms containing the recombinant vector described in X13); X2) is any one of X21) to X24) below: X21) encodes a nucleic acid molecule containing the L monomer; X22) contains an expression cassette containing the nucleic acid molecule described in X21); X23) A recombinant vector containing the nucleic acid molecule described in X21), or a recombinant vector containing the expression cassette described in X22; X24) Recombinant microorganisms containing the nucleic acid molecules described in X21), or recombinant microorganisms containing the expression cassette described in X22), or recombinant microorganisms containing the recombinant vector described in X23); X3) is any one of the following X31) to X34): X31) encodes a nucleic acid molecule containing the C monomer; X32) contains an expression cassette containing the nucleic acid molecule described in X31; X33) A recombinant vector containing the nucleic acid molecule described in X31), or a recombinant vector containing the expression cassette described in X32; X34) Recombinant microorganisms containing the nucleic acid molecules described in X31), or recombinant microorganisms containing the expression cassette described in X32), or recombinant microorganisms containing the recombinant vector described in X33); X4) is any one of the following X41) to X44): X41) encoding Y D and the modified X L A nucleic acid molecule consisting of a protein fusion protein D, formed by the linkage of proteins; X42) contains an expression cassette containing the nucleic acid molecule described in X41); X43) A recombinant vector containing the nucleic acid molecule described in X41), or a recombinant vector containing the expression cassette described in X42; X44) Recombinant microorganisms containing the nucleic acid molecule described in X41), or recombinant microorganisms containing the expression cassette described in X42), or recombinant microorganisms containing the recombinant vector described in X43). in, The R monomer contains a monomer named mr and a reporter group named ethyl. Two or more mr groups can form a polymer. The R monomer contains a binding region 1, and the mr, the ethyl group, and the binding region 1 are linked by a linker region or chemical bonds. The L monomer contains a monomer named ml and a reporter group named propyl. Two or more ml can form a polymer. The L monomer contains a binding region 2 or a biomolecule containing a binding region 2. The ml, the propyl, and the binding region 2 or the biomolecule containing the binding region 2 are linked by a linker region or a chemical bond. The mr may be the same as or different from the ml, and the binding region 1 and the binding region 2 may interact to cause a phase transition in the expression products of X1) and X2); The C monomer is either c1) or c2) as follows: c1) A molecule obtained by linking a monomer named mc, a reporter group named A, and a biomolecule named Y, and at least two of the mc can form a polymer; C ​ c2) The molecule obtained by attaching a tag to c1); The Y D To be with the Y C Biomolecules that interact with each other.

18. The complete set of reagents according to claim 17, characterized in that: X11) The nucleic acid molecule described has the nucleotide sequence from position 62 to 1306 of sequence 2; And / or, X21) the nucleic acid molecule has the nucleotide sequence from position 62 to 1153 of sequence 6; And / or, X31) the nucleic acid molecule has the nucleotide sequence from position 51 to 1400 of sequence 8.

19. The complete set of reagents according to claim 1 or 17, characterized in that: The modification is a post-translational modification of a protein or a demodification of a post-translational modification of a protein.

20. The reagent kit according to claim 19, wherein, The protein is post-translational modified by methylation, acetylation, phosphorylation, ubiquitination, or glycosylation. The demodification of the protein post-translational modifications includes demethylation, deacetylation, dephosphorylation, deubiquitination, or deglycosylation.

21. Any of the following applications of the kit of reagents described in any of claims 1-20: Z1) Applications in detecting or assisting in the detection of whether modified proteins interact with other biomolecules; Application of Z2 in screening for regulatory factors of interactions between modified proteins and other biomolecules; Application of Z3 in identifying or assisting in the identification of regulatory factors of interactions between modified proteins and other biomolecules; Application of Z4 in detecting the effects of substances on the interactions between modified proteins and other biomolecules; Z5) Application in detecting whether a protein has enzyme activity involved in post-translational modifications; Z6) Application in the preparation of products for detecting whether modified proteins interact with other biomolecules; Application of Z7 in the preparation of products that screen modified proteins for regulatory factors of interactions between other biomolecules; Application of Z8 in the preparation and identification of products that regulate the interaction between modified proteins and other biomolecules; Z9) is used in the preparation of enzyme products for detecting whether proteins have enzyme activity involved in post-translational modifications.

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