CN109917120B - A complete set of reagents for the detection of interactions between post-translationally modified proteins and their ligands - Google Patents

Abstract

The invention discloses a reagent set for detecting the interaction between a post-translational modified protein and a ligand thereof. The kit of the invention consists of four reagents A, B, C and D; a is formed by connecting a biomolecule with the name of R and a protein with the name of X; b contains a biomolecule designated L; r and L are the same or different and have interaction, and the phase change occurs after the interaction of R and L; c is a multimer formed from C monomers consisting of a monomer named mc, a reporter group named A, and a reporter group named Y _C The obtained molecules are connected, and more than or equal to two mc can form a polymer; d is defined by the name X _L With modifications and the name Y _D Are connected; y is _C And Y _D Have interaction between them. The invention realizes the high enrichment of the interaction protein and the ligand in the phase-change liquid drop, and amplifies the weak interaction signal, so that the detection is easy.

Description

Kit of reagents for detecting interactions between post-translationally modified proteins and their ligands

technical field

The invention relates to a set of reagents for detecting the interaction between a post-translation modified protein and its ligand in the field of biotechnology.

Background technique

As a characteristic of matter, "phase transition" has long been known in the physics world and in daily life. In recent years, scientists have gradually discovered that phase transition (or phase separation) mechanisms also widely exist in biological cells, and in the time and space of the cell life cycle Regulation and other aspects to perform important biological functions.

Current research has found that when multivalent macromolecules in solution interact with their multivalent ligands, larger complexes tend to be produced, and the solubility of the latter generally decreases, thereby separating from the common solution phase to form a complex This transition process is called "liquid-liquid separation phase transition". Wherein, the multivalent valence refers to the number of binding regions contained in a macromolecule or its ligand that can interact with each other. For protein interactions, multivalent proteins and their multivalent ligands also undergo a "liquid-liquid separation phase transition" (referred to as "phase transition") in vitro, that is, a normal solution phase and a normal solution phase can be produced. Protein-rich viscous liquid phase. Under the microscope, it can be seen that the protein-rich liquid phase contains a large number of small droplets (ie, phase-change droplets), and the diameter of the droplets can reach the micron level or even larger. For example, multivalent SH3 (SRC homology 3 domain) and its multivalent ligand PRM (proline-rich motif) can undergo a phase transition at a certain concentration, while PRMH, which has a higher affinity for SH3, can undergo a stronger phase transition with SH3. Change.

In organisms, the interaction between proteins and their ligands is the main way for proteins to perform their functions. Under physiological conditions, the interaction between proteins exists in a form of dynamic equilibrium, and the dissociation constant (Kd) is usually used to characterize the strength of protein interaction. According to different Kd values, protein interactions are usually divided into stable interaction (StableInteraction) and transient interaction (Transient Interaction), the Kd value corresponding to the former is between picomolar to micromolar, and the Kd value corresponding to the latter is greater than 1 micromol. Basically, the transient interaction between proteins can be considered as weak protein interaction. Modified proteins (including methylation/demethylation, acetylation/deacetylation, phosphorylation/dephosphorylation, ubiquitination/deubiquitination, glycosylation/deglycosylation, etc.) Most of the interactions between bodies are weak interactions. This weak interaction plays an important role in cell signal transduction and cell cycle regulation. For example, the weak interaction between phosphorylated proteins and their ligands can realize the transmission of phosphate groups in signaling pathways, and the weak interactions between methylated histones and their ligands can regulate gene expression, etc. It can be seen that the study of weak interactions between proteins helps to understand important cellular processes. However, weak protein-protein interactions are still difficult to detect.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is how to detect the weak interaction between proteins, especially the interaction between a post-translationally modified protein and its ligand. Covalent modification process on the base. At present, more than 300 kinds of post-translational modifications have been found, and the common ones are methylation, acetylation, phosphorylation, ubiquitination, glycosylation, etc. The modification process opposite to protein post-translational modification is protein demodification, such as demethylation sylation, deacetylation, dephosphorylation, deubiquitination, deglycosylation, etc.

In order to solve the above-mentioned technical problems, the present invention firstly provides a set of reagents for detecting whether there is an interaction between the protein named X and the modified protein named _XL , denoted as set reagent 1, the set Reagent 1 is composed of four reagents whose names are A, B, C and D;

The A is formed by linking a biomolecule named R and the X;

said B contains a biomolecule named L;

The R is the same or different from the L and there is an interaction between the two, and a phase transition occurs after the interaction between the R and the L;

The C is a multimer formed by a C monomer, and the C monomer is the following c1) or c2):

c1) A molecule obtained by linking a monomer named mc, a reporter group named _A and a biomolecule named YC, wherein two or more of the mc can form a multimer;

c2) a molecule obtained by attaching a label to c1);

The D is formed by linking the X _L and a biomolecule named Y _D ;

There is an interaction between the Y _C and the Y _D.

In the above kit of reagents 1, both the Y _C and the Y _D can be proteins.

In the above set of reagents 1, the Y _C can be Y11), Y12) or Y13):

Y11) the amino acid sequence is the protein shown in the 362-465th position of sequence 7;

Y12) The amino acid sequence shown in the 362-465th position of sequence 7 in the sequence listing has undergone the substitution and/or deletion and/or addition of one or several amino acid residues and has the same function protein;

Y13) A fusion protein obtained by linking a tag at the N-terminal or/and C-terminal of Y11) or Y12).

Said Y _D can be Y21), Y22) or Y23):

Y21) the amino acid sequence is the protein shown in the 22-29th position of sequence 11;

Y22) The amino acid sequence shown in the 22nd-29th position of sequence 11 in the sequence listing has undergone the substitution and/or deletion and/or addition of one or several amino acid residues and has the same function protein;

Y23) A fusion protein obtained by linking a tag at the N-terminal or/and C-terminal of Y21) or Y22).

Among them, the KKETPV shown in the 22-29th position of the sequence 11 has an interaction with the PDZ shown in the 362-465th position of the sequence 7.

In the above set of reagents 1, 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 can be through the binding region 1 and the binding region 2, the number of the binding region 1 in the R and the binding region 2 in the L can be greater than or equal to 2.

Wherein, both the binding domain 1 and the binding domain 2 are binding domains, and the binding domain refers to the smallest unit of interactions between biomolecules through non-covalent bonds. When there are more than or equal to 2 binding domains between the R and the L, if the binding domains in the R are not completely the same, the binding domain 1 is the collective name for each binding domain in the R, as described The binding domains in L are not all the same, and the binding domain 2 is a general term for the binding domains in L.

Both said R and said L are polyvalent molecules. Among them, the multivalent valence refers to the number of binding regions contained in one molecule that can bind to another molecule when molecules interact with each other. For the R, the valence of R is The number of the binding domains 1, for the L, the valence of the L is the number of the binding domains 2.

The R and the L undergo a phase transition through multivalent interactions.

In the above kit of reagents 1, the R can be protein, nucleic acid or polysaccharide. The L can be protein, nucleic acid or polysaccharide.

In the above kit of reagents 1, a reporter group named B may also be connected to A.

A reporting group named C can also be connected to the B.

In the above kit of reagents 1, the B and the C can be the same or different.

Said A may be different from said B and said C.

In the above kit of reagents 1, the A, the B and the C can all be fluorescent reporter groups.

Further, the A, the B and the C can all be fluorescent proteins.

In the above kit of reagents 1, the number ratio of X and R in A may be an integer greater than or equal to 1.

The molar ratio of the mc, the A and the Y _C in the C monomer may be 1:1:1.

In the C monomer, the mc, the A and the Y _C can be connected through a linking region or a chemical bond. The formazan can specifically be mCherry.

In the above set of reagents 1, the R can be a multimer formed by R monomers, and the R monomers all contain a monomer named mr, and two or more of the mr can form a multimer.

The L can be a multimer formed from L monomers, and the L monomers all contain a monomer named ml, and two or more of the ml can form a multimer.

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

In the above kit of reagents 1, at least one monomer in R may contain the binding region 1 .

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

When only one monomer in the R contains the binding region 1, the monomer contains at least two of the binding regions 1; when two or more monomers in the R contain the binding region 1 When 1, each monomer contains at least one binding domain 1 .

When only one monomer in the L contains the binding domain 2, the monomer contains at least two binding domains 2; when two or more monomers in the L contain the binding domain 2 2, each monomer contains at least one binding region 2 .

In the monomer of R containing the binding domain 1, the binding domain 1 may be linked to the mr.

In the monomer of L containing the binding region 2, the binding region 2 may be linked to the ml.

Said mr is the same or different from said ml.

In the above kit of reagents 1, the R monomer can contain the mr and the binding region 1 .

Each of the L monomers may contain the ml and the binding region 2 .

In the above kit of reagents 1, in the R monomer, the mr can be connected with the binding region 1 or the biomolecule containing the binding region 1 through the linking region or chemical bond.

In the L monomer, the ml can be connected with the binding region 2 or the biomolecule containing the binding region 1 through the linking region or a chemical bond.

In the R monomer, the number of the binding domain 1 is at least one.

In the L monomer, the number of the binding region 2 is at least one.

In the R and the L monomers, no matter the number of each part (ie the mr or the ml, the binding region 1 or the binding region 2, the B or C) is one or more , there is no requirement for the connection order between each other, as long as it can meet the requirements that two or more of the R monomers can form a multimer, and two or more of the L monomers can form a multimer, and these two multimers can occur interact and cause a phase transition.

In the above, there is no special requirement for the linking region, as long as the linking region can connect the two connected parts in each monomer of the R and the L without affecting the functions of the two. The connecting region may be a polypeptide. In the R monomer, the mr, the B, and the binding region 1 or the biomolecule containing the binding region 1 can be sequentially connected through the linking region or chemical bond.

In the L monomer, the ml, the C and the binding region 2 or the biomolecule containing the binding region 2 can be sequentially connected through the linking region or chemical bond.

Each of the R monomers is linked with at least one X.

In one embodiment of the present invention, the C-terminals of the R monomers are connected to the N-terminals of the X through the connecting region.

In the above kit of reagents 1, the R monomer can also contain the B.

The L monomer may also contain the propane.

In the above kit of reagents 1, in the R monomer, the mr, the B, and the binding region 1 or the biomolecule containing the binding region 1 can be connected through the linking region or chemical bond.

In the L monomer, the ml, the C, and the binding region 2 or biomolecules containing the binding region 2 can be connected through the linking region or chemical bond.

In the above kit of reagents 1, the R monomers can be the same, the L monomers can be the same, and the C monomers can be the same.

Both the mr and the ml can be yeast protein SmF. The yeast protein SmF is the core component of the ribonucleoprotein complex, and its crystal structure shows that it exists in the form of homologous tetramers. Therefore, using SmF as a carrier can realize the multimerization of the target protein.

The mc may be Bacillus subtilis protein Hfq. The Bacillus subtilis protein Hfq exists in the form of a homohexamer, so the multimerization of the target protein can be achieved by using Hfq as a carrier.

The binding region 1 can be the region that binds to the PRMH shown in the 366-380 position of the sequence 5 in SH3 shown in the 364-431 position of the sequence 1; the binding region 2 can be the 366- The region in PRMH indicated at position 380 binds to SH3 indicated at positions 364-431 of SEQ ID NO:1.

The connecting region can be (Gly-Gly-Ser) _n or a polypeptide containing (Gly-Gly-Ser) _n , where n is a natural number greater than or equal to 2. n can be specifically 4 or 2.

The formazan can be a red fluorescent protein, such as mCherry. Said B and said C can be green fluorescent protein, such as GFP.

In the above kit of reagents 1, both the mr and the ml can be the yeast SmF shown in the 17-102 position of sequence 1.

The mc can be Hfq shown in the 17th-94th position of sequence 7.

The biomolecule containing the binding region 1 can be SH3 shown in the 364-431 position of the sequence 1.

The biomolecule containing the binding region 2 can be the PRMH shown in the 366-380 position of sequence 5.

In the above set of reagents 1, the R monomer can be H1) or H2) or H3):

H1) the amino acid sequence is the protein shown in the 17th-431th position of sequence 1;

H2) A protein having the same function as the amino acid sequence shown in the 17-431 position of Sequence 1 in the Sequence Listing through substitution and/or deletion and/or addition of one or several amino acid residues;

H3) A fusion protein obtained by linking a tag at the N-terminal or/and C-terminal of H1) or H2).

The L monomer can be I1) or I2) or I3):

I1) the amino acid sequence is the protein shown in the 17th-380th position of sequence 5;

I2) The amino acid sequence shown in the 17th-380th position of Sequence 5 in the Sequence Listing has undergone the substitution and/or deletion and/or addition of one or several amino acid residues and has the same function protein;

I3) A fusion protein obtained by linking a tag at the N-terminal or/and C-terminal of I1) or I2).

The C monomer can be J1) or J2) or J3):

J1) the amino acid sequence is the protein shown in the 17th-465th position of sequence 7;

J2) The amino acid sequence shown in the 17th-465th position of sequence 7 in the sequence listing has undergone one or several amino acid residue substitutions and/or deletions and/or additions and has the same function protein;

J3) A fusion protein obtained by linking a tag at the N-terminal or/and C-terminal of J1) or J2).

In order to facilitate the purification of the protein in H1) or I1) or J1), the tags shown in Table 1 can be attached to the amino terminus or carboxyl terminus of H1) or I1) or J1).

Table 1. Sequence of tags

For the protein in H2) or I2) or J2) above, the substitution and/or deletion and/or addition of one or several amino acid residues is a substitution and/or deletion and/or addition of no more than 10 amino acid residues .

The above-mentioned protein in H2) or I2) or J2) can be synthesized artificially, or its coding gene can be synthesized first, and then obtained by biological expression.

The coding gene of the protein in the above-mentioned H2) or I2) or J2) can be obtained by combining the DNA sequence encoding the R monomer or the DNA sequence encoding the L monomer or the DNA sequence encoding the C monomer in the present invention One or several amino acid residue codons are deleted, and/or missense mutations of one or several base pairs are carried out, and/or the 5' and/or 3' ends are connected to the codons shown in Table 1 The coding sequence of the tag was obtained.

The present invention also provides a set of reagents for detecting whether there is an interaction between X and _XL , which is denoted as set of reagents 2, and the set of reagents 2 consists of the following X1), X2), X3) and X4) composition:

X1) The biological material related to the R monomer is any one of the following X11) to X14):

X11) a nucleic acid molecule encoding the R monomer;

X12) an expression cassette containing the nucleic acid molecule of 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) A recombinant microorganism containing a nucleic acid molecule described in X11), or a recombinant microorganism containing an expression cassette described in X12), or a recombinant microorganism containing a recombinant vector described in X13);

X2) The biological material related to the L monomer is any one of the following X21) to X24):

X21) a nucleic acid molecule encoding the L monomer;

X22) an expression cassette containing the nucleic acid molecule of 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) A recombinant microorganism containing a nucleic acid molecule described in X21), or a recombinant microorganism containing an expression cassette described in X22), or a recombinant microorganism containing a recombinant vector described in X23);

X3) The biological material related to the C monomer is any one of the following X31) to X34):

X31) a nucleic acid molecule encoding the C monomer;

X32) an expression cassette containing the nucleic acid molecule of 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) A recombinant microorganism containing a nucleic acid molecule described in X31), or a recombinant microorganism containing an expression cassette described in X32), or a recombinant microorganism containing a recombinant vector described in X33);

X4) The biological material related to said _YD is any one of the following X41) to X44):

_X41 ) a nucleic acid molecule encoding said YD;

X42) an expression cassette containing the nucleic acid molecule of 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) The recombinant microorganism containing the nucleic acid molecule described in X41), or the recombinant microorganism containing the expression cassette described in X42), or the recombinant microorganism containing the recombinant vector described in X43).

In the above kit of reagents 2, the nucleic acid molecule described in X11) can be as follows x11) or x12) or x13):

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

x12) has 75% or more identity with the nucleotide sequence defined by x11), and encodes the cDNA molecule or genomic DNA molecule of the R monomer;

x13) A cDNA molecule or a genomic DNA molecule that hybridizes to the nucleotide sequence defined by x11) under stringent conditions and encodes the R monomer.

X21) said nucleic acid molecule can be as follows x21) or x22) or x23):

x21) The coding sequence is the cDNA molecule or DNA molecule at the 62-1153rd position of sequence 6 in the sequence listing;

x22) has 75% or more identity with the nucleotide sequence defined by x21), and encodes the cDNA molecule or genomic DNA molecule of the L monomer;

x23) A cDNA molecule or a genomic DNA molecule that hybridizes to the nucleotide sequence defined by x21) under stringent conditions and encodes the L monomer.

X31) The nucleic acid molecule can be as follows x31) or x32) or x33):

x31) coding sequence is the 51-1400th cDNA molecule or DNA molecule of sequence 8 in the sequence listing;

x32) has 75% or more identity with the nucleotide sequence defined by x31), and encodes the cDNA molecule or genomic DNA molecule of the C monomer;

x33) A cDNA molecule or a genomic DNA molecule that hybridizes to the nucleotide sequence defined by x31) under stringent conditions and encodes the C monomer.

Wherein, 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.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "Identity" includes nucleotide sequences that are 75% or higher, or 85% or higher, or 90% or higher, or 95% or higher identical to the nucleotide sequences of the present invention. Identity can be assessed visually or with computer software. Using computer software, identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

The stringent condition is to hybridize at 68°C in a solution of 2×SSC and 0.1% SDS and wash the membrane twice for 5 min each time, and to hybridize at 68°C in a solution of 0.5×SSC and 0.1% SDS And wash the membrane twice, each time for 15 min; or, in a solution of 0.1×SSPE (or 0.1×SSC), 0.1% SDS, hybridize and wash the membrane at 65°C.

The identity of 75% or more may be 80%, 85%, 90% or more.

X12) The expression cassette containing the nucleic acid molecule encoding the R monomer (R monomer gene expression cassette) refers to the DNA capable of expressing the R monomer in the host cell, and the DNA can not only include the promoter The promoter for the transcription of the R monomeric gene may also include a terminator for terminating the transcription of the R monomeric gene. Further, the expression cassette may also include an enhancer sequence.

X22) The expression cassette containing the nucleic acid molecule encoding the L monomer (L monomer gene expression cassette) refers to the DNA capable of expressing the L monomer in the host cell, and the DNA may not only include the promoter The promoter for the transcription of the L monomeric gene may also include a terminator for terminating the transcription of the L monomeric gene. Further, the expression cassette may also include an enhancer sequence.

X32) The expression cassette containing the nucleic acid molecule encoding the C monomer (C monomer gene expression cassette) refers to the DNA that can express the C monomer in the host cell, and the DNA can not only include the promoter The promoter for the transcription of the C monomeric gene may also include a terminator for terminating the transcription of the C monomeric gene. Further, the expression cassette may also include an enhancer sequence.

Existing vectors can be used to construct recombinant vectors containing the R monomeric gene expression cassette or the L monomeric gene expression cassette or the C monomeric gene expression cassette. The vector can be a plasmid, cosmid, phage or viral vector. Specifically, the plasmid can be a pRSFDuet-1 vector.

X13) The recombinant vector can specifically be pRSFDuet-1-SGS, and the pRSFDuet-1-SGS is to replace the DNA fragment between the NcoI and XhoI recognition sequences of the pRSFDuet-1 vector (comprising the NcoI and XhoI recognition sequences) with the sequence The recombinant vector obtained from the DNA molecule shown in the 12th-1360th position of sequence 2 in the list. The pRSFDuet-1-SGS can express the fusion protein of the R monomer shown in sequence 1 and His-tag.

X23) The recombinant vector can specifically be pRSFDuet-1-SGP, and the pRSFDuet-1-SGP is to replace the DNA fragment between the NcoI and XhoI recognition sequences of the pRSFDuet-1 vector (including the NcoI and XhoI recognition sequences) with the sequence The recombinant vector obtained from the DNA molecule shown in the 12th-1162nd position of sequence 6 in the list. The pRSFDuet-1-SGP can express the fusion protein of the L monomer shown in sequence 5 and His-tag.

X33) The recombinant vector can specifically be pRSFDuet-1-Hfq-mCherry-PDZ, which is a DNA fragment between the NcoI and XhoI recognition sequences of the pRSFDuet-1 vector (comprising NcoI and The recognition sequence of XhoI) is replaced by the recombinant vector obtained by the DNA molecule shown in Sequence 8 in 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.

The microorganisms may be yeast, bacteria, algae or fungi. Wherein, the bacteria can be Escherichia coli.

In the above reagent kit 1 and reagent kit 2, the modification can be protein post-translational modification or demodification of protein post-translational modification. The protein post-translational modification may be methylation, acetylation, phosphorylation, ubiquitination or glycosylation modification. The demodification of the post-translational modification of the protein may be demethylation, deacetylation, dephosphorylation, deubiquitination or deglycosylation.

The present invention also provides a method for detecting whether there is an interaction between biomolecules, the biomolecules are two biomolecules named X and _XL respectively, the _XL is a modified protein, and the X is a protein , the method includes:

Mix solution A, solution B, solution C and solution D to obtain the test solution, the solution A is the solution containing the A; the solution B is the solution containing the B; the solution C is the solution containing the The solution of C; the solution D is a solution containing the D; the R in the A in the liquid to be tested interacts with the L in the B to undergo a phase change to produce a phase change droplet; according to the Whether the signal of the formazan is contained in the phase-change liquid droplet in the liquid to be tested determines whether there is an interaction between the X and the _XL : if the signal of the formazan is contained in the phase-change liquid droplet in the liquid to be tested, There is or a candidate for interaction between the X and the X _L ; if the phase change droplet in the test liquid does not contain the signal of the first, the X and the X _L do not have or the candidate does not have interactions.

Wherein, whether there is a signal of the first formazan in the phase change liquid droplet in the test liquid refers to whether the signal of the first formazan in the test liquid is enriched in the phase change droplet, so that the phase change liquid The signal of the formazan in the droplet is higher than that of the non-phase-change droplet portion in the liquid to be tested. Specifically, the determining whether there is an interaction between the X and the _XL according to the signal of whether there is the formazan in the phase change droplet in the liquid to be tested may include: The signal of the formazan in the liquid droplet is enriched, and the X and the _XL have or have an interaction with the candidate; for example, the signal of the formazan in the phase change droplet in the liquid to be tested is not enriched , there is no or no candidate interaction between X and X _L .

The solution A can be composed of the A and the solvent, the solution B can be composed of the B and the solvent, the solution C can be composed of the C and the solvent, and the solution D can be composed of the D and the solvent. Said solvent composition, said solvent can dissolve said A, said B, said C and said D.

In one embodiment of the present invention, the solvent is KMEI buffer, KMEI buffer is made up of solvent and solute, solvent is water, solute and its concentration are respectively: 150mM KCl, 1mM MgCl ₂ , 1mM EGTA, 10mM imidazole, 1mM DTT , pH=7.

In one embodiment of the present invention, protein multimerization is achieved by fusion expression of the protein with the known polyvalent yeast protein SmF or Bacillus subtilis protein Hfq, that is, the multivalency of reagent A, reagent B and reagent C . The R monomer is SGS (SGS is the abbreviation of fusion protein SmF-GFP-SH3), the L monomer is SGP (SGP is the abbreviation of fusion protein SmF-GFP-PRMH), SH3 and PRMH interact to cause multivalent The interaction of protein SGS and SGP leads to phase transition to produce phase transition droplets.

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

The present invention also provides any of the following applications of the set of reagents 1 or the set of reagents 2:

Z1) Application in detecting or assisting in detecting whether there is interaction between a modified protein and other biomolecules;

Z2) Application in screening regulatory factors for interaction between modified proteins and other biomolecules;

Z3) Application in identifying or assisting in the identification of regulators of interactions between proteins with modifications and other biomolecules;

Z4) Application in detecting the effect of a substance on the interaction between a modified protein and other biomolecules;

Z5) Application in detecting whether a protein has an enzyme activity involved in post-translational modification of the protein;

Z6) Application in the preparation and detection of whether there is an interaction product between a modified protein and other biomolecules;

Z7) Application in the preparation and screening of regulatory factors for the interaction between modified proteins and other biomolecules;

Z8) Application in the preparation and identification of regulatory factors for the interaction between proteins with modifications and other biomolecules;

Z9) Application in the preparation of products for detecting whether a protein has an enzymatic activity involved in post-translational modification of the protein.

In the above applications, the other biomolecules can be proteins.

In the above application, the modification may be protein post-translational modification or de-modification of protein post-translational modification. The protein post-translational modification may be methylation, acetylation, phosphorylation, ubiquitination or glycosylation modification. The demodification of the post-translational modification of the protein may be demethylation, deacetylation, dephosphorylation, deubiquitination or deglycosylation.

The set of reagents for detecting the interaction between the post-translationally modified protein and its ligand and the multivalent recruitment system based on the set of reagents of the present invention realize the interaction between the interacting protein and the ligand in the phase change droplet by using the phase transition mechanism Highly enriched, the originally weak interaction signal is strongly amplified, making it easy to detect. The set of reagents and multivalent recruitment system of the present invention can be used to detect the interaction between proteins and their ligands, especially weak interactions, and can also identify whether a protein has a certain enzyme activity involved in protein post-translational modification, such as phosphoric acid Kinase activity corresponding to methylation modification, methyltransferase activity corresponding to methylation modification, etc., and regulators that can regulate the above enzyme activities can also be identified.

Description of drawings

Figure 1 Analysis of the experimental results of the detection system 1-8 of the interaction between H3K9me3 and CD. A is the observation of the phase change droplet morphology of system 3, and the right picture is the enlarged image of the framed area in the left picture. B is the laser confocal high-content microscopy imaging analysis of systems 1-8. Scale bar = 100 μm.

Figure 2 is the analysis of the experimental results of the detection system 9-20 for the interaction between H3K9me3 and CD. A is the laser confocal high-content microscopy imaging analysis of system 9-20. Scale bar = 100 μm. B is the quantitative analysis of the fluorescence intensity of mCherry in the phase change droplet in 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 proportion of H3K9me3-KKETPV in the mixture of H3K9me3-KKETPV and H3K9-KKETPV, respectively.

Detailed ways

The present invention will be further described in detail below in conjunction with specific embodiments, and the given examples are only for clarifying the present invention, not for limiting the scope of the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. Materials, reagents, instruments, etc. used in the following examples can be obtained from commercial sources unless otherwise specified. Quantitative experiments in the following examples were all set up to repeat the experiments three times, and the results were averaged.

The invention provides a set of reagents for detecting the interaction between biomolecules X and _XL , the set of reagents is composed of four reagents whose names are A, B, C and D;

A is connected by a biomolecule named R and a protein named X. R is a polymer formed by R monomers. The R monomers are all the same. Each monomer contains mr and a fluorescent reporter named B. Group, binding region 1 or biomolecules containing binding region 1, protein X, each part of each monomer is connected by a linking region or a chemical bond, wherein more than two mr can form a multimer;

B contains a biomolecule named L, L is a multimer formed by L monomers, and the L monomers are all the same, each monomer contains ml, a fluorescent reporter group named C, a binding region 2 or a binding region For the biomolecules in zone 2, the parts of each monomer are linked by a linking zone or a chemical bond, wherein more than or equal to 2 ml can form a multimer;

R and L are the same or different and have interaction between them, the interaction between R and L is carried out through the binding region 1 of R and the binding region 2 of L, and the numbers of binding region 1 in R and binding region 2 in L are equal Greater than or equal to 2, phase transition occurs after R interacts with L;

C is a multimer formed by C monomers. C monomers are connected by a monomer named mc, a reporter group named A and a biomolecule named Y _C. More than or equal to two mcs can form Polymer;

_D is composed of a protein with a post-translational modification named _XL and a biomolecule named YD;

There is an interaction between Y _C and Y _D.

In the following, both R and L are proteins to illustrate the interaction of the kit of reagents of the present invention in detecting the interaction between proteins containing post-translational methylation modifications and their ligands. Specifically, both mr and ml are SmF, a fluorescent reporter group Both B and fluorescent reporter group C are GFP, the biomolecule containing binding region 1 is SH3, the biomolecule containing binding region 2 is PRMH, and the fusion protein of SmF, GFP and SH3 is recorded as SGS (that is, R monomer), The fusion protein of SmF, GFP and PRMH is recorded as SGP (that is, L monomer); mc is Hfq, fluorescent reporter group A is mCherry, Y _C is PDZ shown in the 362-465th position of sequence 7, Hfq, mCherry Fuse with PDZ to obtain monomer C; Y _D is KKETPV (position 22-29 of Sequence 11).

Example 1, Verification of the interaction between H3K9me3 and CD

In this embodiment, X _L is H3K9me3, X is CD, and H3K9me3 interacts with CD.

1. Preparation of recombinant vector

1. A recombinant vector expressing the fusion protein of SGS and CD

The DNA fragment (comprising the recognition sequence of NcoI and XhoI) between the NcoI and XhoI recognition sequences of the pRSFDuet-1 vector (Novagen product of Merck) is replaced with the DNA molecule shown in the 12th-1360th position of sequence 2 in the sequence listing, The recombinant vector pRSFDuet-1-SGS was obtained, and pRSFDuet-1-SGS could express the protein shown in Sequence 1 (SGS was fused with His-tag, ie R monomer, denoted as His-SGS).

Among them, the DNA molecule encoding His-SGS shown in the 14th-1354th position of the sequence 2 shown in the sequence 1, the 1344-1349th and 1355-1360th positions of the sequence 2 are the recognition sequences of NcoI and XhoI respectively, and the sequence 1 The 3-8th position of Sequence 1 is the amino acid sequence of His-tag, the 17th-102nd position of Sequence 1 is the amino acid sequence of SmF, the 109-349th position of Sequence 1 is the amino acid sequence of GFP, and the 364-431st position of Sequence 1 It is the amino acid sequence of SH3, and the 103-108, 350-363 and 432-444 of the sequence 1 are the amino acid sequences of the connecting region. His-SGS can form tetradecamers through the action of SmF.

Replace the DNA fragment between the NcoI and XhoI recognition sequences of pRSFDuet-1-SGS (comprising the recognition sequences of NcoI and XhoI) with the DNA molecule shown in the 1-212th position 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).

Among them, the 9th-203rd position of sequence 4 encodes the CD shown in sequence 3. SGS-CD can form tetradecamers through the action of SmF.

2. Recombinant vector expressing SGP

The DNA fragment between the NcoI and XhoI recognition sequences of the pRSFDuet-1 vector (comprising the recognition sequences of NcoI and XhoI) is replaced with the DNA molecule shown in the 12th-1162th position 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 is fused with His-tag, denoted as His-SGP, that is, L monomer).

Wherein, the DNA molecule encoding His-SGP shown in the 14th-1156th position of the sequence 6 is the His-SGP shown in the sequence 5, the 3rd-8th position of the sequence 5 is the amino acid sequence of the His-tag, and the 17th-102nd position of the sequence 5 is The amino acid sequence of SmF, the 109-349th position of sequence 5 is the amino acid sequence of GFP, the 366-380th position of sequence 5 is the amino acid sequence of PRMH, the 103-108th and 350-365th position of sequence 5 are the connecting region amino acid sequence.

His-SGP can form tetradecamers through the action of SmF.

3. Recombinant vector expressing C monomer (Hfq-mCherry-PDZ)

The DNA fragment between the NcoI and XhoI recognition sequences of the pRSFDuet-1 vector (comprising the recognition sequences of NcoI and XhoI) was replaced with the DNA molecule shown in sequence 8 in 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).

Among them, the DNA molecule encoding Hfq-mCherry-PDZ shown in the 3-1397th position of the sequence 8 is the Hfq-mCherry-PDZ shown in the sequence 7, the 3-8th position of the sequence 7 is the amino acid sequence of the His-tag, and the 17th-94th position of the sequence 7 The amino acid sequence of Hfq, the 101-340th of sequence 7 is the amino acid sequence of mCherry, the 362-465th of sequence 7 is the amino acid sequence of PDZ, the 95th-100th and 341-361th of sequence 7 are Amino acid sequence of the linker region.

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

Prepare and express the recombinant vector that does not contain Hfq fusion protein: replace the DNA fragment (comprising the recognition sequence of NcoI and XhoI) between the NcoI and XhoI recognition sequences of the pRSFDuet-1 vector with the 11th-1143th position of sequence 10 in the sequence listing The DNA molecules of the recombinant vector pRSFDuet-1-mCherry-PDZ are obtained, and 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, the following as comparison).

Among them, the DNA molecule encoding mCherry-PDZ shown in the 13-1134th position of the sequence 10 shown in the sequence 9, the 3-8th position of the sequence 9 is the amino acid sequence of the His-tag, and the 17th-256th position of the sequence 9 is For the amino acid sequence of mCherry, the 271-374th position of sequence 9 is the amino acid sequence of PDZ, and the 257th-270th position of sequence 9 is the amino acid sequence of the connecting region.

4. Preparation of Reagent D

chemically synthesized reagent D for linking X _L to Y _D , reagent D is a methylated modified protein obtained by trimethylating the 4th lysine of H3K9-KKETPV shown in Sequence 11 in the sequence listing, It is denoted as H3K9me3-KKETPV, and the sequence of H3K9me3-KKETPV is as follows: ARTK(Me)3QTARGGSGGSGGSWGGSKKETPVAV.

2. Fusion protein expression and purification

Introduce pRSFDuet-1-SGS, pRSFDuet-1-SGS-CD, pRSFDuet-1-SGP, pRSFDuet-1-Hfq-mCherry-PDZ and pRSFDuet-1-mCherry-PDZ into Escherichia coli competent cells BL21 ( DE3) (Tiangen Biochemical Technology (Beijing) Co., Ltd.), obtained 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.

According to the following method, the recombinant strains BL21-pRSFDuet-1-SGS, BL21-pRSFDuet-1-SGS-CD, BL21-pRSFDuet-1-SGP, BL21-pRSFDuet-1-Hfq-mCherry-PDZ and BL21-pRSFDuet- The fusion protein containing His tag expressed by 1-mCherry-PDZ was purified:

(1) Bacterial culture and protein induction expression: the above-mentioned recombinant strains were inoculated into 1L LB medium. Cultivate at 37°C, 200rpm to an OD600 of about 0.8-1 (about 8-9hr). Transfer the bacterial solution to 18°C to cool down for 1 hr, add IPTG to a final concentration of 0.5 mM to induce protein expression overnight (about 16 hr), and obtain a culture solution.

(2) Bacterial resuspension and crushing: Centrifuge the culture medium obtained in step (1), discard the supernatant, resuspend the bacterial pellet with 40mM bindingbuffer (40mM Tris-Cl, 500mM NaCl, pH 8.0 or 7.4) and perform ultrasonication To crush, the crushed product was ultracentrifuged at 20,000 rpm for 1 hr, and the supernatant (containing the fusion protein of interest) was collected.

(3) Ni column purification: Prepare the Ni column in advance and equilibrate it with binding buffer. The supernatant obtained in step (2) was poured into a Ni column. When the liquid dries up quickly, add wash buffer to wash 2-3 column volumes, then add elution buffer to elute the target fusion protein, and collect the effluent.

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

Elution buffer: 40mM Tris-HCl, 500mM NaCl, 500mM imidazole, pH same as binding buffer.

(4) Ion exchange purification: according to the isoelectric point of the protein, select a suitable ion exchange column. Dilute the effluent from step (3) with 40 mM Tris-Cl buffer to reduce the ion concentration to obtain protein dilution. Install the ion exchange column to the ATKA protein purification system (GE Company) and complete the loading of the protein dilution. The protein bound to the column was eluted by gradually increasing the concentration of salt ions and the fusion protein of interest was collected. The eluent used for elution is composed of A solution and B solution, and the ratio between the two is adjusted according to the specific situation: A solution: 40mM Tris-Cl, pH is the same as the binding buffer; B solution: 40mM Tris-Cl, 2M NaCl, pH Same as binding buffer.

(5) Purification by gel filtration: After the target fusion protein obtained in step (4) is concentrated by ultrafiltration, it is separated and purified by a preset molecular sieve program to obtain a further purified target fusion protein.

The KMEI buffer used for column equilibration and elution is composed of solvent and solute, the solvent is water, the solute and its concentration are: 150mM KCl, 1mM MgCl ₂ , 1mM EGTA, 10mM imidazole, 1mM DTT, pH=7.

(6) Detect and save the purified protein: use SDS-PAGE to analyze the His-SGS expressed by BL21-pRSFDuet-1-SGS, the SGS-CD expressed by BL21-pRSFDuet-1-SGS-CD, and the BL21- His-SGP expressed by pRSFDuet-1-SGP, Hfq-mCherry-PDZ expressed by BL21-pRSFDuet-1-Hfq-mCherry-PDZ, and mCherry-PDZ expressed by BL21-pRSFDuet-1-mCherry-PDZ were detected. After the size of the fusion protein was in line with the expectation, the protein was concentrated and frozen at -80°C for later use.

3. Detection of the interaction between H3K9me3 and CD

The solution 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 2 (solvents are KMEI buffer ) were distributed in 384 microwell plates according to the system shown in Table 2, one system per well, in the system SGS-CD, His-SGP, Hfq-mCherry-PDZ, mCherry-PDZ and H3K9me3-KKETV and H3K9- The concentration of the KKETPV mixture in the corresponding system is 1 μM:

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

In Table 2, "+" indicates that the substance is contained; "-" indicates that the substance is not contained; "*" indicates the mixture of H3K9me3-KKETPV and H3K9-KKETPV, and H3K9me3-KKETPV accounts for the proportion of H3K9me3-KKETPV and H3K9me3-KKETPV in systems 9-14. The molar percentages of the mixture of H3K9-KKETPV are 0, 0.2, 0.4, 0.6, 0.8 and 1.0, respectively, and the molar percentages of H3K9me3-KKETPV in the mixture of H3K9me3-KKETPV and H3K9-KKETPV in systems 15-20 are 0, 0.2, 0.4, respectively , 0.6, 0.8, and 1.0.

The above systems were incubated statically at 4°C until the phase change droplets in the phase transition system completely settled to the bottom of the well plate, and images were collected with a laser confocal high-content imaging microscope. The results (B in Figure 1) showed that, The solutions in System 1 and System 2 did not change, and no fluorescent signal aggregation area was found; in System 3 and System 4, phase-change droplets were generated, and the green fluorescent signal (fluorescent signal from GFP) was gathered in the phase-change droplets. And its signal intensity is much higher than the signal intensity in the solution (A in Figure 1); the solution in system 5 and system 6 has not changed, and no fluorescent signal aggregation area has been found; both system 7 and system 8 produce phase change fluid Droplets, the green fluorescent signal is concentrated in the phase-change droplet, and its signal intensity is much higher than that in the solution, and no red fluorescent signal is found in the droplet; the system 9-14 produces a phase-change droplet, The green fluorescent signal is concentrated in the phase change droplet, and its signal intensity is much higher than that in the solution, and no obvious red fluorescent signal is found in the droplet (Figure 2); system 15-20 produces a phase transition Droplets, the green fluorescent signal gathers in the phase-change droplets, and its signal intensity is much higher than that in the solution; it is also found that when the mixture is completely composed of H3K9-KKETPV (that is, the molar percentage of H3K9me3-KKETPV in the mixture is 0), there is no obvious red fluorescence (fluorescence signal from mCherry) aggregation in the phase-change droplets; and with the increase of the proportion of H3K9me3 in the mixture of H3K9me3 and H3K9, the red fluorescence signal intensity in the phase-change droplets It also increases; when the mixture is completely composed of H3K9me3-KKETPV (that is, when the molar percentage of H3K9me3-KKETPV in the mixture is 1.0), the intensity of the red fluorescence signal in the phase change droplets reaches the highest (Figure 2).

It shows that SGS can be combined with SGP to produce phase-change droplets, which can be marked with the fluorescent signal emitted by GFP. In the system in which mCherry-PDZ exists and Hfq-mCherry-PDZ does not exist, regardless of whether the system contains H3K9me3-KKETPV or H3K9-KKETPV, the red fluorescence signal cannot be enriched in the phase change droplets of the system, and in the absence of mCherry-PDZ In the system with Hfq-mCherry-PDZ, the content of H3K9me3 was positively correlated with the enrichment degree of red fluorescence signal in phase change droplets. It was shown that CD in SGS-CD can recruit H3K9me3-KKETPV into phase-change droplets through the interaction between CD and H3K9me3, and KKETPV in H3K9me3-KKETPV can further recruit Hfq-mCherry-PDZ that emits red fluorescent signal into the phase-change droplets, and then make the phase-change droplets emit an enriched red fluorescence signal; if Hfq-mCherry-PDZ is replaced by mCherry-PDZ, the red fluorescence signal in the phase-change droplets is not obvious, indicating that Hfq- mCherry-PDZ is more suitable for detecting the interaction between CD and H3K9me3 than monovalent mCherry-PDZ after forming a hexavalent state (ie hexamer) through Hfq.

In summary, the set of reagents and multivalent recruitment system based on phase transition of the present invention have the characteristics of amplifying the interaction signal and improving the detection sensitivity of the interaction between H3K9me3 and CD. Provides a new method that is simple and easy to implement.

<110> Tsinghua University

<120> Kit of reagents for detecting interactions between post-translationally modified proteins and their ligands

<160> 11

<170> PatentIn version 3.5

<210> 1

<211> 446

<212> PRT

<213> Artificial sequences

<400> 1

Met Lys His His His His His His His Glu Asn Leu Tyr Phe Gln Gly Gly

1 5 10 15

Met Ser Glu Ser Ser Asp Ile Ser Ala Met Gln Pro Val Asn Pro Lys

20 25 30

Pro Phe Leu Lys Gly Leu Val Asn His Arg Val Gly Val Lys Leu Lys

35 40 45

Phe Asn Ser Thr Glu Tyr Arg Gly Thr Leu Val Ser Thr Asp Asn Tyr

50 55 60

Phe Asn Leu Gln Leu Asn Glu Ala Glu Glu Phe Val Ala Gly Val Ser

65 70 75 80

His Gly Thr Leu Gly Glu Ile Phe Ile Arg Ser Asn Asn Val Leu Tyr

85 90 95

Ile Arg Glu Leu Pro Asn Gly Gly Ser Gly Gly Ser Met Lys Val Ser

100 105 110

Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu

115 120 125

Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Arg Gly Glu Gly Glu

130 135 140

Gly Asp Ala Thr Asn Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr

145 150 155 160

Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr

165 170 175

Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp Tyr Met Lys Gln His Asp

180 185 190

Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile

195 200 205

Ser Phe Lys Asp Asp Gly Thr Tyr Lys Thr Arg Ala Glu Val Lys Phe

210 215 220

Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe

225 230 235 240

Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Phe Asn

245 250 255

Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys

260 265 270

Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu

275 280 285

Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu

290 295 300

Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Lys Leu Ser Lys Asp

305 310 315 320

Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala

325 330 335

Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Thr Met Lys Gly

340 345 350

Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Met Ser Gly His Met

355 360 365

Asp Leu Asn Met Pro Ala Tyr Val Lys Phe Asn Tyr Met Ala Glu Arg

370 375 380

Glu Asp Glu Leu Ser Leu Ile Lys Gly Thr Lys Val Ile Val Met Glu

385 390 395 400

Lys Ser Ser Asp Gly Trp Trp Arg Gly Ser Tyr Asn Gly Gln Val Gly

405 410 415

Trp Phe Pro Ser Asn Tyr Val Thr Glu Glu Gly Asp Ser Pro Leu Gly

420 425 430

Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Ser Met Gly

435 440 445

<210> 2

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<212> DNA

<213> Artificial sequences

<400> 2

aaggagatat accatgaaac atcatcatca tcatcacgaa aacctgtatt ttcagggcgg 60

catgagcgaa agcagcgata ttagcgcgat gcagccggtg aacccgaaac cgtttctgaa 120

aggcctggtg aaccatcgcg tgggcgtgaa actgaaattt aacagcaccg aatatcgcgg 180

caccctggtg agcaccgata actattttaa cctgcaactg aacgaagcgg aagaatttgt 240

ggcgggcgtg agccacggca ccctgggcga aattttatt cgcagcaaca acgtgctgta 300

tattcgcgaa ctgccgaacg gcggttccgg cggttccatg aaagtgagca agggcgagga 360

gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa 420

gttcagcgtg cgcggcgagg gcgagggcga tgccaccaac ggcaagctga ccctgaagtt 480

catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta 540

cggcgtgcag tgcttcagcc gctaccccga ctacatgaag cagcacgact tcttcaagtc 600

cgccatgccc gaaggctacg tccaggagcg caccatctcc ttcaaggacg acggcaccta 660

caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa 720

gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaacttcaa 780

cagccacaac gtctatatca cggccgacaa gcagaagaac ggcatcaagg cgaacttcaa 840

gatccgccac aacgtcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac 900

ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccaa 960

gctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc 1020

cgccgggatc actctcggca tggacgagct gtacaagacc atgaaaggcg gtagcggtgg 1080

cagcggtggt agcggcggct ccatgagcgg ccatatggac ctcaacatgc ccgcttatgt 1140

gaaatttaac tacatggctg agagagga tgaattatca ttgataaagg ggacaaaggt 1200

gatcgtcatg gagaaaagca gtgatgggtg gtggcgtggt agctacaatg gacaagttgg 1260

atggttccct tcaaactatg taactgaaga aggtgacagt cctttgggtg gcagtggcgg 1320

tagcggtggc agcggtggca gctccatggg ctaactcgag tctggtaaag aaac 1374

<210> 3

<211> 65

<212> PRT

<213> Artificial sequences

<400> 3

Glu Glu Tyr Val Val Glu Lys Val Leu Asp Arg Arg Val Val Lys Gly

1 5 10 15

Lys Val Glu Tyr Leu Leu Lys Trp Lys Gly Phe Ser Asp Glu Asp Asn

20 25 30

Thr Trp Glu Pro Glu Glu Asn Leu Asp Cys Pro Asp Leu Ile Ala Glu

35 40 45

Phe Leu Gln Ser Gln Lys Thr Ala His Glu Thr Asp Lys Ser Glu Gly

50 55 60

Gly

65

<210> 4

<211> 226

<212> DNA

<213> Artificial sequences

<400> 4

ccatggggga ggaatatgtg gtggaaaaag ttctcgaccg tcgagtggta aagggcaaag 60

tggagtacct cctaaagtgg aagggattct cagatgagga caacacatgg gagccagaag 120

agaacctgga ttgccccgac ctcattgctg agtttctgca gtcacagaaa acagcacatg 180

agacagataa atcagaggga ggctaactcg agtctggtaa agaaac 226

<210> 5

<211> 380

<212> PRT

<213> Artificial sequences

<400> 5

Met Lys His His His His His His His Glu Asn Leu Tyr Phe Gln Gly Gly

1 5 10 15

Met Ser Glu Ser Ser Asp Ile Ser Ala Met Gln Pro Val Asn Pro Lys

20 25 30

Pro Phe Leu Lys Gly Leu Val Asn His Arg Val Gly Val Lys Leu Lys

35 40 45

Phe Asn Ser Thr Glu Tyr Arg Gly Thr Leu Val Ser Thr Asp Asn Tyr

50 55 60

Phe Asn Leu Gln Leu Asn Glu Ala Glu Glu Phe Val Ala Gly Val Ser

65 70 75 80

His Gly Thr Leu Gly Glu Ile Phe Ile Arg Ser Asn Asn Val Leu Tyr

85 90 95

Ile Arg Glu Leu Pro Asn Gly Gly Ser Gly Gly Ser Met Lys Val Ser

100 105 110

Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu

115 120 125

Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Arg Gly Glu Gly Glu

130 135 140

Gly Asp Ala Thr Asn Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr

145 150 155 160

Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr

165 170 175

Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp Tyr Met Lys Gln His Asp

180 185 190

Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile

195 200 205

Ser Phe Lys Asp Asp Gly Thr Tyr Lys Thr Arg Ala Glu Val Lys Phe

210 215 220

Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe

225 230 235 240

Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Phe Asn

245 250 255

Ser His Asn Val Tyr Ile Thr Ala Asp Lys Gln Lys Asn Gly Ile Lys

260 265 270

Ala Asn Phe Lys Ile Arg His Asn Val Glu Asp Gly Ser Val Gln Leu

275 280 285

Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu

290 295 300

Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Lys Leu Ser Lys Asp

305 310 315 320

Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala

325 330 335

Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Thr Met Lys Gly

340 345 350

Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Met Ser Ser Lys Lys

355 360 365

Thr Pro Pro Pro Val Pro Pro Arg Thr Thr Ser Lys

370 375 380

<210> 6

<211> 1183

<212> DNA

<213> Artificial sequences

<400> 6

aaggagatat accatgaaac atcatcatca tcatcacgaa aacctgtatt ttcagggcgg 60

catgagcgaa agcagcgata ttagcgcgat gcagccggtg aacccgaaac cgtttctgaa 120

aggcctggtg aaccatcgcg tgggcgtgaa actgaaattt aacagcaccg aatatcgcgg 180

caccctggtg agcaccgata actattttaa cctgcaactg aacgaagcgg aagaatttgt 240

ggcgggcgtg agccacggca ccctgggcga aattttatt cgcagcaaca acgtgctgta 300

tattcgcgaa ctgccgaacg gcggttccgg cggttccatg aaagtgagca agggcgagga 360

gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa 420

gttcagcgtg cgcggcgagg gcgagggcga tgccaccaac ggcaagctga ccctgaagtt 480

catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta 540

cggcgtgcag tgcttcagcc gctaccccga ctacatgaag cagcacgact tcttcaagtc 600

cgccatgccc gaaggctacg tccaggagcg caccatctcc ttcaaggacg acggcaccta 660

caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa 720

gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaacttcaa 780

cagccacaac gtctatatca cggccgacaa gcagaagaac ggcatcaagg cgaacttcaa 840

gatccgccac aacgtcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac 900

ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccaa 960

gctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc 1020

cgccgggatc actctcggca tggacgagct gtacaagacc atgaaaggcg gtagcggtgg 1080

cagcggtggt agcggcggct ccatgagcag caaaaaaacc ccgccgccgg tgccgccgcg 1140

caccaccagc aaataactcg agtctggtaa agaaaccgct gct 1183

<210> 7

<211> 465

<212> PRT

<213> Artificial sequences

<400> 7

Met Lys His His His His His His His Glu Asn Leu Tyr Phe Gln Gly Gly

1 5 10 15

Gly Pro Leu Gly Ser Met Lys Pro Ile Asn Ile Gln Asp Gln Phe Leu

20 25 30

Asn Gln Ile Arg Lys Glu Asn Thr Tyr Val Thr Val Phe Leu Leu Asn

35 40 45

Gly Phe Gln Leu Arg Gly Gln Val Lys Gly Phe Asp Asn Phe Thr Val

50 55 60

Leu Leu Glu Ser Glu Gly Lys Gln Gln Leu Ile Tyr Lys His Ala Ile

65 70 75 80

Ser Thr Phe Ala Pro Gln Lys Asn Val Gln Leu Glu Leu Glu Gly Gly

85 90 95

Ser Gly Gly Ser Met Lys Val Ser Lys Gly Glu Glu Asp Asn Met Ala

100 105 110

Ile Ile Lys Glu Phe Met Arg Phe Lys Val His Met Glu Gly Ser Val

115 120 125

Asn Gly His Glu Phe Glu Ile Glu Gly Glu Gly Glu Gly Arg Pro Tyr

130 135 140

Glu Gly Thr Gln Thr Ala Lys Leu Lys Val Thr Lys Gly Gly Pro Leu

145 150 155 160

Pro Phe Ala Trp Asp Ile Leu Ser Pro Gln Phe Met Tyr Gly Ser Lys

165 170 175

Ala Tyr Val Lys His Pro Ala Asp Ile Pro Asp Tyr Leu Lys Leu Ser

180 185 190

Phe Pro Glu Gly Phe Lys Trp Glu Arg Val Met Asn Phe Glu Asp Gly

195 200 205

Gly Val Val Thr Val Thr Gln Asp Ser Ser Leu Gln Asp Gly Glu Phe

210 215 220

Ile Tyr Lys Val Lys Leu Arg Gly Thr Asn Phe Pro Ser Asp Gly Pro

225 230 235 240

Val Met Gln Lys Lys Thr Met Gly Trp Glu Ala Ser Ser Glu Arg Met

245 250 255

Tyr Pro Glu Asp Gly Ala Leu Lys Gly Glu Ile Lys Gln Arg Leu Lys

260 265 270

Leu Lys Asp Gly Gly His Tyr Asp Ala Glu Val Lys Thr Thr Tyr Lys

275 280 285

Ala Lys Lys Pro Val Gln Leu Pro Gly Ala Tyr Asn Val Asn Ile Lys

290 295 300

Leu Asp Ile Thr Ser His Asn Glu Asp Tyr Thr Ile Val Glu Gln Tyr

305 310 315 320

Glu Arg Ala Glu Gly Arg His Ser Thr Gly Gly Met Asp Glu Leu Tyr

325 330 335

Lys Thr Met Lys Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser

340 345 350

Met Gly Gly Ser Gly Gly Ser Gly Ser Pro Glu Phe Leu Gly Glu Glu

355 360 365

Asp Ile Pro Arg Glu Pro Arg Arg Ile Val Ile His Arg Gly Ser Thr

370 375 380

Gly Leu Gly Phe Asn Ile Val Gly Gly Glu Asp Gly Glu Gly Ile Phe

385 390 395 400

Ile Ser Phe Ile Leu Ala Gly Gly Pro Ala Asp Leu Ser Gly Glu Leu

405 410 415

Arg Lys Gly Asp Gln Ile Leu Ser Val Asn Gly Val Asp Leu Arg Asn

420 425 430

Ala Ser His Glu Gln Ala Ala Ile Ala Leu Lys Asn Ala Gly Gln Thr

435 440 445

Val Thr Ile Ile Ala Gln Tyr Lys Pro Glu Glu Tyr Ser Arg Phe Glu

450 455 460

Ala

465

<210> 8

<211> 1406

<212> DNA

<213> Artificial sequences

<400> 8

ccatgaaaca tcatcatcat catcacgaaa acctgtattt tcagggcggc ggcccgctgg 60

gtagcatgaa accgattaac attcaggatc agtttctgaa ccagattcgc aaagaaaaca 120

cctatgtgac cgtgtttctg ctgaacggct ttcagctgcg cggccaggtg aaaggctttg 180

ataactttac cgtgctgctg gaaagcgaag gcaaacagca gctgattat aaacatgcga 240

ttagcacctt tgcgccgcag aaaaacgtgc agctggaact ggaaggcggt tccggcggtt 300

ccatgaaagt gagcaagggc gaggaggata acatggccat catcaaggag ttcatgcgct 360

tcaaggtgca catggagggc tccgtgaacg gccacgagtt cgagatcgag ggcgagggcg 420

agggccgccc ctacgagggc accccagaccg ccaagctgaa ggtgaccaag ggtggccccc 480

tgcccttcgc ctgggacatc ctgtcccctc agttcatgta cggctccaag gcctacgtga 540

agcaccccgc cgacatcccc gactacttga agctgtcctt ccccgagggc ttcaagtggg 600

agcgcgtgat gaacttcgag gacggcggcg tggtgaccgt gacccaggac tcctccctcc 660

aggacggcga gttcatctac aaggtgaagc tgcgtggcac caacttcccc tccgacggcc 720

ccgtaatgca gaagaagaca atgggctggg aggcctcctc cgagcggatg taccccgagg 780

acggcgccct gaagggcgag atcaagcaga ggctgaagct gaaggacggc ggccactacg 840

acgctgaggt caagaccacc tacaaggcca agaagcccgt gcagctgccc ggcgcctaca 900

acgtcaacat caagttggac atcacctccc acaacgagga cctacaccatc gtggaacagt 960

acgaacgcgc cgagggccgc cactccaccg gcggcatgga cgagctgtac aagaccatga 1020

aaggcggtag cggtggcagc ggtggtagcg gcggctccat gggtggcagc ggcggtagcg 1080

gatccccgga attcctgggg gaggaagaca ttccccggga accaaggcgg atcgtgatcc 1140

atcggggctc caccggcctg ggcttcaaca ttgtgggcgg cgaggatggt gaaggcatct 1200

tcatctcctt catccttgct gggggtccag ccgacctcag tggggagcta cggaaggggg 1260

accagatcct gtcggtcaat ggtgttgacc tccgcaatgc cagtcacgaa caggctgcca 1320

ttgccctgaa gaatgcgggt cagacggtca cgatcatcgc tcagtataaa ccagaagagt 1380

atagtcgatt cgaggcgtaa ctcgag 1406

<210> 9

<211> 374

<212> PRT

<213> Artificial sequences

<400> 9

Met Lys His His His His His His His Glu Asn Leu Tyr Phe Gln Gly Gly

1 5 10 15

Met Lys Val Ser Lys Gly Glu Glu Asp Asn Met Ala Ile Ile Lys Glu

20 25 30

Phe Met Arg Phe Lys Val His Met Glu Gly Ser Val Asn Gly His Glu

35 40 45

Phe Glu Ile Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln

50 55 60

Thr Ala Lys Leu Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp

65 70 75 80

Asp Ile Leu Ser Pro Gln Phe Met Tyr Gly Ser Lys Ala Tyr Val Lys

85 90 95

His Pro Ala Asp Ile Pro Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly

100 105 110

Phe Lys Trp Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val Val Thr

115 120 125

Val Thr Gln Asp Ser Ser Leu Gln Asp Gly Glu Phe Ile Tyr Lys Val

130 135 140

Lys Leu Arg Gly Thr Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys

145 150 155 160

Lys Thr Met Gly Trp Glu Ala Ser Ser Glu Arg Met Tyr Pro Glu Asp

165 170 175

Gly Ala Leu Lys Gly Glu Ile Lys Gln Arg Leu Lys Leu Lys Asp Gly

180 185 190

Gly His Tyr Asp Ala Glu Val Lys Thr Thr Tyr Lys Ala Lys Lys Pro

195 200 205

Val Gln Leu Pro Gly Ala Tyr Asn Val Asn Ile Lys Leu Asp Ile Thr

210 215 220

Ser His Asn Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu

225 230 235 240

Gly Arg His Ser Thr Gly Gly Met Asp Glu Leu Tyr Lys Thr Met Lys

245 250 255

Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Met Gly Pro Glu

260 265 270

Phe Leu Gly Glu Glu Asp Ile Pro Arg Glu Pro Arg Arg Ile Val Ile

275 280 285

His Arg Gly Ser Thr Gly Leu Gly Phe Asn Ile Val Gly Gly Glu Asp

290 295 300

Gly Glu Gly Ile Phe Ile Ser Phe Ile Leu Ala Gly Gly Pro Ala Asp

305 310 315 320

Leu Ser Gly Glu Leu Arg Lys Gly Asp Gln Ile Leu Ser Val Asn Gly

325 330 335

Val Asp Leu Arg Asn Ala Ser His Glu Gln Ala Ala Ile Ala Leu Lys

340 345 350

Asn Ala Gly Gln Thr Val Thr Ile Ile Ala Gln Tyr Lys Pro Glu Glu

355 360 365

Tyr Ser Arg Phe Glu Ala

370

<210> 10

<211> 1158

<212> DNA

<213> Artificial sequences

<400> 10

aggagatata ccatgaaaca tcatcatcat catcacgaaa acctgtattt tcagggcggc 60

atgaaagtga gcaagggcga ggaggataac atggccatca tcaaggagtt catgcgcttc 120

aaggtgcaca tggagggctc cgtgaacggc cacgagttcg agatcgaggg cgagggcgag 180

ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg tggccccctg 240

cccttcgcct gggacatcct gtcccctcag ttcatgtacg gctccaaggc ctacgtgaag 300

caccccgccg acatccccga ctacttgaag ctgtccttcc ccgaggggctt caagtggggag 360

cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga cccaggactc ctccctccag 420

gacggcgagt tcatctacaa ggtgaagctg cgtggcacca acttcccctc cgacggcccc 480

gtaatgcaga agaagacaat gggctgggag gcctcctccg agcggatgta ccccgaggac 540

ggcgccctga agggcgagat caagcagagg ctgaagctga aggacggcgg ccactacgac 600

gctgaggtca agaccaccta caaggccaag aagcccgtgc agctgcccgg cgcctacaac 660

gtcaacatca agttggacat cacctcccac aacgaggact aacaccatcgt ggaacagtac 720

gaacgcgccg agggccgcca ctccaccggc ggcatggacg agctgtacaa gaccatgaaa 780

ggcggtagcg gtggcagcgg tggtagcggc ggctccatgg gcccggaatt cctgggggag 840

gaagacattc cccgggaacc aaggcggatc gtgatccatc ggggctccac cggcctgggc 900

ttcaacattg tgggcggcga ggatggtgaa ggcatcttca tctccttcat ccttgctggg 960

ggtccagccg acctcagtgg ggagctacgg aagggggacc agatcctgtc ggtcaatggt 1020

gttgacctcc gcaatgccag tcacgaacag gctgccattg ccctgaagaa tgcgggtcag 1080

acggtcacga tcatcgctca gtataaacca gaagagtata gtcgattcga ggcgtaactc 1140

gagtctggta aagaaacc 1158

<210> 11

<211> 29

<212> PRT

<213> Artificial sequences

<400> 11

Ala Arg Thr Lys Gln Thr Ala Arg Gly Gly Ser Gly Gly Ser Gly Gly

1 5 10 15

Ser Trp Gly Gly Ser Lys Lys Glu Thr Pro Val Ala Val

20 25

Claims (23)

1. For detecting proteins named X and proteins named X _L The kit of reagents with modified proteins having or not having an interaction, consisting of four reagents named a, B, C and D, respectively;

the A is formed by connecting a biomolecule with the name of R and the X;

said B contains a biomolecule designated L;

the R and the L are the same or different and have interaction, and the R and the L have phase change after interaction;

the C is a polymer formed by C monomers, and the C monomers are the following cl) or C2):

cl) from a monomer named mc, a reporter group named nail, and a reporter group named Y _C The biomolecule of (a) is linked to form a molecule, and more than or equal to two of said mc are capable of forming a multimer;

c2 A molecule obtained by attaching a tag to cl);

said D is defined by said X _L And the name is Y _D Are connected to form the biological molecules;

said Y _C And said Y _D Have interaction between them; said Y is _C And said Y _D Are all proteins;

the R is protein, and the L is protein;

determining whether X and X contain the nail by determining whether the nail is contained in a phase-change liquid drop generated by the phase change of the interaction of R in A and L in B _L Whether or not there is an interaction between them.

2. The kit of claim 1, wherein: said Y is _C Is Y11) or Y13):

y11) is a protein represented by the 362-465 th amino acid sequence of SEQ ID No. 7;

y13) fusion protein obtained by connecting a label to the N terminal or/and the C terminal of Y11);

and/or, said Y _D Is Y21) or Y23):

y21) is a protein represented by the 22 nd to 29 th positions of the sequence 11;

y23) is a fusion protein obtained by attaching a tag to the N-terminus or/and C-terminus of Y21).

3. The kit of claim 1 or 2, wherein: said R contains a binding domain designated binding domain 1; said L contains a binding domain designated binding domain 2; the interaction between the R and the L is performed 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.

4. The kit of claim 1 or 2, wherein: a is also connected with a report group named B;

and/or, a reporter group named propane is also connected in the B.

5. The kit of claim 4, wherein: said B and said C are the same or different;

and/or said a is different from said b and said c.

6. The kit of claim 5, wherein: the A, the B and the C are all fluorescent reporter groups.

7. The kit of claim 6, wherein: the fluorescent reporter group is a fluorescent protein.

8. The kit according to any one of claims 1 or 2, characterized in that: the number ratio of the X to the R in the A is an integer of 1 or more.

9. The kit according to any one of claims 1 or 2, characterized in that: the R is a polymer formed by R monomers, the R monomers all contain monomers with the name of mr, and more than or equal to two mr can form the polymer;

and/or, said L is a multimer formed from L monomers, said L monomers each comprising a monomer named ml, greater than or equal to two of said ml being capable of forming a multimer;

said mc, said mr and said ml are identical or at least two are identical or are different from each other.

10. The kit of claim 9, wherein:

at least one monomer in the R contains a binding region 1;

and/or at least one monomer in L comprises a binding region 2;

the interaction between said R and said L is via said binding domain 1 and said binding domain 2.

11. The kit of claim 9, wherein: the R monomers contain the mr and a binding region 1;

and/or, said L monomers each contain said ml and binding region 2;

the interaction between said R and said L is via said binding domain 1 and said binding domain 2.

12. The kit of claim 11, wherein: in the R monomer, mr is connected with the binding region 1 or the biomolecule containing the binding region 1 through a connecting region or a chemical bond;

and/or, in the L monomer, the ml is connected with the binding region 2 or the biological molecule containing the binding region 2 through a connecting region or a chemical bond.

13. The kit of claim 11 or 12, characterized in that: the R monomers also contain a reporter group named B;

and/or, the L monomers each further contain a reporter group designated as c.

14. The kit of claim 13, wherein: in the R monomer, mr and B are connected with the binding region 1 or a biomolecule containing the binding region 1 through a connecting region or a chemical bond;

and/or, in the L monomer, the ml, the propyl and the binding region 2 or the biological molecule containing the binding region 2 are connected through a connecting region or a chemical bond.

15. The kit of claim 14, wherein: the R monomers are the same, the L monomers are the same, and the C monomers are the same;

and/or, the mr and the ml are yeast protein SmF; and/or mc is Bacillus subtilis protein Hfq;

and/or, the binding domain 1 is a region that binds to PRMH shown at positions 366 to 380 of sequence 5 in SH3 shown at positions 364 to 431 of sequence 1; the binding region 2 is a region that binds to SH3 at positions 364 through 431 of sequence 1 in PRMH at positions 366 through 380 of sequence 5;

and/or the connecting region is (Gly-Gly-Ser) _n Or contains (Gly-Gly-Ser) _n N is a natural number of 2 or more;

and/or, the formazan is a red fluorescent protein;

and/or both the B and the C are green fluorescent proteins.

16. The kit of claim 12, wherein: the mr and the ml are both yeast SmF shown in 17 th to 102 th positions of a sequence 1;

and/or mc is Hfq shown in 17 th to 94 th positions of the sequence 7;

and/or the biological molecule containing the binding domain 1 is SH3 shown in the 364 th to 431 th positions of the sequence 1;

and/or the biomolecule containing the binding area 2 is PRMH shown in 366 th to 380 th positions of a sequence 5.

17. The kit of claim 9, wherein: the R monomer is H1) or H3):

h1 ) the amino acid sequence is a protein represented by the 17 th to 431 th positions of the sequence 1;

h3 A fusion protein obtained by connecting a label to the N terminal or/and the C terminal of H1);

and/or the L monomer is I1) or I3):

i1 ) the amino acid sequence is a protein shown in the 17 th to 380 th positions of the sequence 5;

i3 A fusion protein obtained by connecting labels to the N terminal or/and the C terminal of I1);

and/or, the C monomer is J1) or J3):

j1 ) the amino acid sequence is a protein shown in the 17 th to 465 th positions of the sequence 7;

j3 A fusion protein obtained by attaching a tag to the N-terminus or/and C-terminus of J1).

18. The kit of claim 1 or 2, wherein: the modification is a post-translational modification of the protein or a de-modification of the post-translational modification of the protein.

19. The kit of claim 18, wherein the protein post-translational modification is a methylation, acetylation, phosphorylation, ubiquitination, or glycosylation modification;

the post-translational modification of the protein is demethylation, deacetylation, dephosphorylation, deubiquitination or deglycosylation.

20. Method for detecting the presence of interactions between biomolecules, named X and X respectively _L Two biomolecules of (2), said X _L Is a protein with a modification, wherein X is a protein, the method comprising:

mixing the solution A, the solution B, the solution C and the solution D to obtain a solution to be detected, wherein the solution A is a solution containing the A in any one of claims 1 to 16; the solution B is a solution containing the B according to any one of claims 1 to 16; the solution C is a solution containing C according to any one of claims 1 to 16; the solution D is a solution containing D according to any one of claims 1 to 16; the interaction of R in the solution A and L in the solution B to be detected is subjected to phase change to generate phase change liquid drops; determining the X and the X according to the signal whether the phase change liquid drop in the liquid to be detected contains the nail _L Whether or not there is an interaction between them.

21. The method of claim 20, wherein: the modification is a post-translational modification of the protein or a de-modification of the post-translational modification of the protein.

22. The method of claim 21, wherein,

the protein post-translational modification is methylation, acetylation, phosphorylation, ubiquitination or glycosylation modification;

the post-translational modification of the protein is demethylation, deacetylation, dephosphorylation, deubiquitination or deglycosylation.

23. Use of any one of the following kits of any one of claims 1 to 19:

z1) the use for detecting or aiding in the detection of interactions between proteins with modifications and other biomolecules;

z2) in screening the protein with modification and other biomolecule interaction regulation factors;

z3) in the identification or auxiliary identification of protein with modification and other biomolecule interaction regulating factors;

z4) in detecting the effect of the substance on the interaction between the protein with the modification and other biomolecules;

z5) in detecting whether the protein has enzyme activity participating in post-translational modification of the protein;

z6) in the preparation of products for detecting whether the protein with the modification and other biomolecules have interaction or not;

z7) in the preparation of the protein with modification and other biological molecular interaction regulation factor products of screening;

z8) in the preparation of products for identifying the interaction regulatory factors between the protein with the modification and other biomolecules;

z9) in the preparation of products for detecting whether proteins have enzyme activity participating in protein posttranslational modification.

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