Altered proximity-dependent modification enzyme

Abstract

[Problem] A proximity-dependent biotinylated enzyme for an analysis method for interaction between molecules has to be fused with an analysis target substance while maintaining a non-specific biotin labeling activity. Therefore, such an analysis method has been limited in terms of analysis target substances that can be used. [Solution] The present invention was accomplished as a result of confirming that it is possible to use low-molecular weight compounds and natural organic substances as analysis target substances, in an analysis method for interaction between molecules, using an altered proximity-dependent modification enzyme in which lysine on the surface is substituted and in which a tag including lysine is directly or indirectly fused to the N-terminus or the C-terminus if necessary.

Description

Engineered proximity-dependent modification enzymes

[0001] The present disclosure relates to engineered proximity-dependent modification enzymes for use in analyzing interactions between biomolecules and between drugs (small molecules, peptides, nucleic acids, etc.) and proteins. This application claims priority to Japanese Patent Application No. 2023-205732, which is incorporated herein by reference.

[0002] In fields such as disease research and drug discovery, analysis of interactions between biochemicals and chemicals is widely used as an extremely important approach to drug discovery research. In particular, protein-protein interactions are a general term for interactions that occur between proteins in vivo. These interactions are well known to be involved in protein conformational changes and the regulation of fundamental life mechanisms such as signal transduction, transport, and metabolism. Such interactions have an extremely diverse pattern, and are characterized by an enormous variation in the flexibility and breadth of the interaction surface, the length of contact lifetime, and the presence or absence of conformational changes depending on the protein species.

[0003] Many methods for measuring protein-protein interactions have been developed. One of these, immunoprecipitation, uses cell mash extracts, which can lead to false positives and may not detect weak binding events that dissociate during the washing process. To address this issue, BioID, a proximity-dependent biotinylation enzyme, was developed by introducing a mutation (R118G) into Escherichia coli BirA, which releases the intermediate biotinyl-5'-AMP from the active site and randomly biotinylates lysine residues in nearby proteins. Subsequent improvements in activity and labeling nonspecificity led to the development of TurboID and AirID, which the present inventors have also applied to non-denaturing protein array technology (see Patent Document 1 and Non-Patent Document 1). BioID's greatest features are its ability to analyze interactions in living cells and organisms from a variety of organisms, as well as its ability to analyze transient, dynamic, and weak interactions.

[0004] WO / 2022 / 009994

[0005] Proteome Letters 2021;6:9-16

[0006] The analysis of molecular interactions using proximity-dependent biotinylation enzymes is a very useful tool. However, these enzymes must be fused to the target substance while maintaining their nonspecific biotin-labeling activity. For example, it is difficult to fuse these enzymes to low molecular weight compounds or natural organic substances while maintaining their nonspecific biotin-labeling activity. Therefore, the target substances that can be used with this analysis method are limited.

[0007] The present disclosure has been completed based on the finding that a method for analyzing molecular interactions using an engineered proximity-dependent modifying enzyme in which surface lysines have been substituted and in which a lysine-containing tag is fused directly or indirectly to the N-terminus or C-terminus can be used to analyze low molecular weight compounds and natural organic substances.

[0008] 1. A modified proximity-dependent modification enzyme in which a surface lysine has been substituted. 2. The modified proximity-dependent modification enzyme according to item 1 above, wherein the proximity-dependent modification enzyme is a peptide having the following amino acid sequence (SEQ ID NO: 3: AirID): MKDNTVPLTLISILADGEFHSGEQLGEQLGMSRAAINKHIKTLRDWGVDVFRVQGKGYCLPEPIQLLDEEKIRQQLDEGSVTVLPVIDSTNQYLLDRLDELTSGDVCIAEYQQAGRGRRGRKWFSPFGANLYLSMYWRLEQGPAAAMGLSLVIGIVMAETLQKLGADGVRVKWPNDLYLNDRKLAGILVEMTGKTGDAAHIVIGAGINLSMREPETDEVDQSWINLQEAGITIDRNQLAARLIKDLRSALRQFEQQGLAPFLSRWEALDNFINRPVKLIIGDREIHGIARGINEQGALLLEQDGVIKPWIGGEISLRSA. 3. The modified proximity-dependent modification enzyme according to claim 2, wherein the lysine substitution is at one or more of the following positions, and the substituted amino acid is arginine, histidine, glutamic acid, aspartic acid, or serine: 1) K2, 2) K38, 3) K41, 4) K56, 5) K71, 6) K122, 7) K163, 8) K194, 9) K244R, 10) K277, and 11) K307. 4. The modified proximity-dependent modification enzyme according to claim 2, wherein the lysine substitution is at one or more of the following positions: 1) K2R, 2) K38R, 3) K41R, 4) K56R, 5) K71R, 6) K122R, 7) K163R, 8) K194R, 9) K244R, 10) K277R, and 11) K307R. 3. The modified proximity-dependent modifying enzyme according to 2 above, wherein the lysine substitutions are any of the following: 1) K2R, 2) K38R, 3) K41R, 4) K56R, 5) K71R, 6) K122R, 7) K163R, 8) K194R, 9) K244R, 10) K277R, and 11) K307R.6. The modified proximity-dependent modification enzyme according to the preceding paragraph 1, wherein the proximity-dependent modification enzyme is a peptide having the following amino acid sequence (SEQ ID NO: 2: TurboID): MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGDACIAEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAAIGLGPVIGIVMAEALRKLGADKVRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWITLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK. The modified proximity-dependent modifying enzyme according to the preceding item 6, wherein the lysine substitution positions are any one or more of lysines other than the underlined K172 and K183: MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGDACIAEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAAIGLGPVIGIVMAEALRKLGADKVRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWITLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK. 8. The modified proximity-dependent modifying enzyme according to any one of the preceding items 1 to 7, wherein one or more lysine-containing tags are fused directly or indirectly to the N-terminus or C-terminus. 9. The modified proximity-dependent modifying enzyme according to any one of the preceding items 1 to 7, wherein any one of the following tags is fused directly or indirectly to the N-terminus or C-terminus:1) DYKDDDDK (N-terminus) 2) DYKDDDDK (C-terminus) 3) DYKDHDGDYKDHDIDYKDDDDK (N-terminus) 4) GGKKKGKK (C-terminus) 5) KKKDKKDD (N-terminus) 6) DDKKKDKK (C-terminus) 10. A modification proximity-dependent modification enzyme-labeled analyte labeled with the modification proximity-dependent modification enzyme described in any one of the preceding paragraphs 1 to 9. 11. A modification proximity-dependent modification enzyme-labeled analyte labeled with the modification proximity-dependent modification enzyme described in the preceding paragraph 5. 12. The modification proximity-dependent modification enzyme-labeled analyte according to the preceding paragraph 10, wherein the analyte has an N-hydroxysuccinimide ester terminus, an N-hydroxysuccinimide ester-linker azide terminus, or an N-hydroxysuccinimide ester-linker alkyne terminus. 13. 14. A method for evaluating the interaction between an immobilized substance immobilized directly or indirectly on a substrate and a modification proximity-dependent modifying enzyme-labeled analyte, comprising the following steps: (1) adding the modification proximity-dependent modifying enzyme-labeled analyte according to paragraph 12 or paragraph 13 to the immobilized substance immobilized directly or indirectly on a substrate in the presence of a labeling substance; and (2) detecting the labeling substance. 15. The method for evaluation according to paragraph 14, comprising washing the substrate between steps (1) and (2). 16. The method for evaluation according to paragraph 14 or 15, wherein the modification proximity-dependent modifying enzyme-labeled analyte is the modification proximity-dependent modifying enzyme-labeled analyte according to paragraph 12. 17. A method for evaluating a protein, which is an immobilized substance indirectly immobilized to an array via magnetic beads, and a modified proximity-dependent modifying enzyme-labeled analyte, comprising the following steps: (1) adding the modified proximity-dependent modifying enzyme-labeled analyte described in the preceding paragraph 12 or 13 to the immobilized substance indirectly immobilized to an array via magnetic beads in the presence of biotin; and (2) detecting the biotin.18. The evaluation method according to claim 17, comprising a step of washing the array between step (1) and step (2). 19. The evaluation method according to preceding paragraph 17 or 18, wherein the modification proximity-dependent modifying enzyme-labeled analyte is the modification proximity-dependent modifying enzyme-labeled analyte according to preceding paragraph 13. 20. A method for introducing a modification proximity-dependent modifying enzyme-labeled analyte into a cell, comprising introducing the modification proximity-dependent modifying enzyme-labeled analyte according to preceding paragraph 12 or 13 into the cell by electroporation. 21. The evaluation method according to preceding paragraph 14, wherein the analyte maintains water solubility. 22. The evaluation method according to preceding paragraph 17, wherein the analyte maintains water solubility. 23. The evaluation method according to preceding paragraph 14, wherein the analyte has an alkynyl group and the modification proximity-dependent modifying enzyme has an azide group. 24. The evaluation method according to preceding paragraph 14, wherein the analyte has an azide group and the modification proximity-dependent modifying enzyme has an alkynyl group.

[0009] The disclosed method for analyzing intermolecular interactions using a modified proximity-dependent modifying enzyme in which lysines on the surface have been substituted has one or more of the following advantages over conventional methods for analyzing intermolecular interactions using proximity-dependent modifying enzymes: 1) The water solubility of the target substance can be maintained; and 2) The type of target substance can be unrestricted.

[0010] Analysis of the interaction between AirID-modified enzyme-IκBα and FG-RelA 1 Analysis of the interaction between AirID-modified enzyme-IκBα and FG-RelA 2 Structure of surface lysine mutant AirID fused with each tag Yields after synthesis and purification of surface lysine mutant AirID fused with each tag Analysis of surface lysine mutant AirID fused with each tag 1 Geldanamycin-AirID 2 Preparation of C. geldanamycin-AirID 2 Results of interaction between C and immobilized material 1 Geldanamycin-AirID 2 Results of the interaction of C2 geldanamycin with immobilized materials - AirID 2Confirmation of the preservation of C Comparison of solubility of AirID (native) and modified AirID after reaction with the target substance Analysis results using the small molecule thalidomide as the target substance Electrophoresis results of the proteins used 1 Binding reaction between the target substance with an alkynyl group and modified AirID with an azide group (analysis results using JQ1 as the target substance) Electrophoresis results of the proteins used 2 AirID 2 Protein array analysis results using C. Analysis results using surface lysine variant AirID fused with each tag. 2. Binding reaction between analyte with azide group and modified AirID with alkynyl group (analytical results using pomalidomide or thalidomide as analyte). Electrophoresis results of proteins used.

[0011] (Summary of the Present Disclosure) Conventionally, direct binding of an analyte to AirID, a proximity-dependent modifying enzyme, has been achieved by performing room-temperature chemical reactions using the lysine or thiol groups of the target protein as a reactive scaffold. Reactions using cysteine ​​as a scaffold include alkylation with the α-iodo(bromo)acetamide terminal, perfluoroarylation, and maleimide Michael addition. However, cross-reactivity with lysine, which is also a nucleophilic residue, is a concern. In particular, of the two cysteines present in AirID, one is located within the protein folding structure, leaving only one usable cysteine, making these scaffolds insufficient. Another method involves fusing a peptide fragment containing cysteine ​​as a tag to the N- or C-terminus to increase the reactive scaffold. However, cysteine ​​is not preferred because it often alters the properties of proteins. Therefore, in the present invention, we considered it preferable to select lysine, which has an ε-amino group and is highly reactive, as a scaffold. In this case, functional groups with high reactivity with lysine, such as NHS (N-hydroxysuccinimide), isothiocyanate, isocyanate, acyl azide, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imidoester, carbodiimide, anhydride, and fluoroester, can be used for conjugation to lysine. In particular, the reaction between NHS and lysine proceeds under physiological conditions to slightly alkaline conditions (pH 7.2-9), and a wide variety of commercially available reaction reagents are available. In the following example, the 13 lysines in AirID (SEQ ID NO: 3) were converted to arginines to determine which arginines are involved in proximity-dependent biotin enzyme activity. The results confirmed that substitution of K172R and K183R resulted in a loss of proximity-dependent biotin enzyme activity. Substitution of other lysines with arginine did not affect proximity-dependent biotin enzyme activity, although the activity varied slightly. Furthermore, it was confirmed that the number of the remaining 11 lysines can be set to any number, since there is no effect even if all of the remaining 11 lysines are replaced with arginines.This allows the number of binding sites of a test compound to a proximity-dependent biotinylation enzyme to be adjusted according to the physical properties of the test compound, greatly expanding the range of conditions for molecular interaction analysis. As with AirID, we confirmed that converting lysines other than K172 and K183 to arginine did not affect proximity-dependent biotinylation enzyme activity. In both AirID and TourboID, computational predictions using AlphaFold2 (an AI for predicting the three-dimensional structure of protein molecules) also showed that K172 and K183 are located within the folded structure. K172 and K183 are likely lysines required for maintaining the three-dimensional structure. In the following examples, lysine was converted to arginine; however, amino acids with similar properties to lysine, such as glutamic acid, histidine, aspartic acid, and serine, can also be converted. Arginine substitution is preferred because it does not significantly alter the physical properties of the proximity-dependent biotinylation enzyme, such as its surface charge. Furthermore, since there was essentially no change in the proximity-dependent biotinylation enzyme when all lysines were replaced with arginines, by converting all lysines to arginines and then attaching a lysine-containing peptide fragment (reactive scaffold tag) to the N-terminus or C-terminus, the activity of the proximity-dependent biotinylation enzyme can be ensured (maintained) and the analyte can be attached to the scaffold tag. The scaffold tag preferably contains one or more lysines. However, in the following examples, it is sufficient to contain two to five lysines. The present disclosure will be described in detail below, but is not limited to the following description.

[0012] (Proximity-Dependent Modified Enzyme) In the present disclosure, a proximity-dependent labeling enzyme refers to an enzyme that has the ability to bind a detectable molecule (referred to as a "labeling substance" in the present disclosure) to an immobilized substance when an intermolecular interaction occurs between the analyte and an immobilized substance on an array, and the immobilized substance is present in the proximity field of the proximity-dependent labeling enzyme bound to the analyte. Examples of proximity-dependent labeling enzymes include enzymes that have been modified from existing enzymes to weaken their substrate specificity. Examples of such enzymes include transferases, lyases, and ligases. Methods for weakening substrate specificity include amino acid conversion or chemical modification, such as introducing a mutation into the substrate binding site or introducing the sequence of a related enzyme. The bond between the labeling substance and the protein that is the immobilized substance is preferably stronger than the interaction between the analyte and the immobilized substance. However, a covalent bond is desirable in order to prevent loss of the interaction during B / F (B(Bound) / F(Free)) separation. A fusion molecule (proximity-dependent labeling enzyme-labeled analyte) in which a proximity-dependent labeling enzyme is bound to an analyte is contacted with a non-denaturing protein array, whereby the labeling substance is bound to a protein immobilized on the protein array with which the proximity-dependent labeling enzyme-labeled analyte interacts, via covalent or strong binding. The labeling substance is substantially not detached or released from the immobilized substance even after the B / F separation process. Furthermore, after washing, the labeling substance bound to the immobilized substance can be detected and quantified by biochemical techniques (mass spectrometry, electrophoresis, etc.). A preferred proximity-dependent labeling enzyme of the present disclosure is a proximity-dependent biotin ligase in which a portion of the amino acid sequence of the BirA protein, a biotin ligase of Escherichia coli, is modified. The BirA protein recognizes a specific amino acid sequence as a substrate and specifically binds biotin to lysine residues within that amino acid sequence. On the other hand, proximity-dependent biotin ligase loses substrate specificity and functions to bind biotin to lysine residues on the surface of any substance within close range, including immobilized substances.For example, proximity-dependent biotin ligases such as BioID (SEQ ID NO: 1), TurboID (SEQ ID NO: 2), and AirID (SEQ ID NO: 3) have been reported (Choi-Rhee et al., Protein Sci, 2004 (Doi 10.1110 / ps.04911804), Roux, K., et al., JCB, 2012 (Doi 10.1083 / jcb.201112098), Branon, T. C., et al., Nat Biotech, 2018 (Doi: 10.1038 / nbt.4201), Kido, K., et al., eLife, 2020 (Doi 10.7554 / eLife.54983)). The proximity-dependent labeling enzyme used in the present disclosure may be a recombinant or chemically synthesized product. As long as the function is not impaired, derivatives or fragments are acceptable, and modifications, substitutions, deletions, and additions may be performed. An example of a method is to prepare a fusion protein (an AirID-labeled protein) using genetic engineering techniques based on the base sequence of the protein to be analyzed and the base sequence information of the gene encoding AirID. Specifically, a gene linking a gene encoding the substance to be analyzed and a gene encoding AirID is cloned, and the gene is expressed in a cell-free synthesis system to prepare a fusion protein containing the substance to be analyzed and AirID. Alternatively, the protein to be analyzed and AirID may be indirectly bound via a substance that binds to the substance to be analyzed (a support substance for the substance to be analyzed), resulting in an AirID-support substance for the substance to be analyzed. Alternatively, a spacer may be inserted between the AirID and the substance to be analyzed.

[0013] (BioID: SEQ ID NO: 1) MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSGDACIAEYQQAGRGGRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRKLGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQGWITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK (TurboID: SEQ ID NO: 2) MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGDACIAEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAAIGLGPVIGIVMAEA LRKLGADKVRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWITLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK (AirID: Sequence number 3)MKDNTVPLTLISILADGEFHSGEQLGEQLGMSRAAINKHIKTLRDWGVDVFRVQGKGYCLPEPIQLLDEEKIRQQLDEGSVTVLPVIDSTNQYLLDRLDELTSGDVCIAEYQQAGRGRRGRKWFSPFGANLYLSMYWRLEQGPAAAMGLSLVIGIVMAETLQKLGADGVRVKWPNDLYLNDRKLAGILVEMTGKTGDAAHIVIGAGINLSMREPETDEVDQSWINLQEAGITIDRNQLAARLIKDLRSALRQFEQQGLAPFLSRWEALDNFINRPVKLIIGDREIHGIARGINEQGALLLEQDGVIKPWIGGEISLRSA

[0014] (Modified Proximity-Dependent Modification Enzyme) The "modified proximity-dependent modification enzyme" of the present disclosure is characterized in that one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, fifteen, twenty, thirty, forty, fifty, or all of the lysines displayed on the surface of the enzyme are substituted. Modified AirID: In the amino acid sequence set forth in SEQ ID NO: 3, the lysine substitutions are at one or more of the following positions, and the substituted amino acids are arginine, glutamic acid, aspartic acid, or serine: 1) K2, 2) K38, 3) K41, 4) K56, 5) K71, 6) K122, 7) K163, 8) K194, 9) K244R, 10) K277, and 11) K307. The lysine substitutions are at one or more of the following positions: 1) K2R, 2) K38R, 3) K41R, 4) K56R, 5) K71R, 6) K122R, 7) K163R, 8) K194R, 9) K244R, 10) K277R, and 11) K307R. The lysine substitutions are as follows: 1) K2R, 2) K38R, 3) K41R, 4) K56R, 5) K71R, 6) K122R, 7) K163R, 8) K194R, 9) K244R, 10) K277R, and 11) K307R. Modified TurboID: In the amino acid sequence set forth in SEQ ID NO: 2, the lysine substitutions are at one or more of the lysines listed below other than the underlined ones, and the substituted amino acid is arginine, histidine, glutamic acid, aspartic acid, or serine. The lysine substitution positions are any one or more of the underlined lysines other than K172 and K183, and the substituted amino acid is arginine. The lysine substitution positions are all of the underlined lysines below, and the substituted amino acid is preferably arginine.MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGDACIAEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAAIGLGPVIGIVMAEALRKLGADKVRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWITLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK (SEQ ID NO: 2) The "engineered proximity-dependent modification enzyme" of the present disclosure also includes variants containing substitutions other than the lysine substitutions set forth above. The mutant does not have mutations at K172R and K183, but may include the following mutations. More specifically, in addition to the lysine substitutions described above, the following may be used: 1) A polypeptide (variant) consisting of an amino acid sequence in which one or two amino acids have been substituted, deleted, inserted, and / or added in the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 3, and which has substantially the same proximity-dependent modification enzyme activity as the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 3; 2) A polypeptide (variant) consisting of an amino acid sequence which has 95% or more identity (particularly, 96% or more, 97% or more, 98% or more, or 99% or more is preferred) to the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 3, and which has substantially the same proximity-dependent modification enzyme activity as the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 3. When introducing a mutation into a peptide, from the viewpoint of not changing the basic properties of the peptide (such as physical properties, functions, physiological activity, or enzymatic activity), for example, mutual substitutions between homologous amino acids (such as polar amino acids, nonpolar amino acids, hydrophobic amino acids, hydrophilic amino acids, positively charged amino acids, negatively charged amino acids, and aromatic amino acids) can easily be envisioned. The term "proximity-dependent modification enzyme activity substantially equivalent to that of the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 3" means that the degree of activity may be stronger or weaker than the proximity-dependent modification enzyme activity of the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 2.For example, the identity can be about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, or about 150% compared to the proximity-dependent modifying enzyme activity of the amino acid sequence set forth in SEQ ID NO: 2 or 3. The identity can also be calculated using BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) or the like (e.g., using default parameters). As used herein, "polypeptide" includes proteins, polypeptides, and oligopeptides, and its minimum size is two amino acids. As used herein, "protein" includes its degradation products, fragmented peptides, and the like.

[0015] The engineered proximity-dependent modifying enzymes of the present disclosure preferably have a tag containing one or more lysines fused directly or indirectly to the N-terminus and / or C-terminus. Preferred examples of tags include: 1) DYKDDDDK (N-terminus: SEQ ID NO: 4) 2) DYKDDDDK (C-terminus: SEQ ID NO: 4) 3) DYKDHDGDYKDHDIDYKDDDDK (N-terminus: SEQ ID NO: 5) 4) GGKKKGKK (C-terminus: SEQ ID NO: 6) 5) KKKDKKDD (N-terminus: SEQ ID NO: 7) 6) DDKKKDKK (C-terminus: SEQ ID NO: 8)

[0016] The modified proximity-dependent modification enzyme of the present invention may be directly or indirectly bound to a target substance by click chemistry. For example, if an azide (alkyne) is directly or indirectly bound to the modified proximity-dependent modification enzyme, then an alkyne (azide) may be directly or indirectly bound to the target substance.

[0017] (Method for evaluating the interaction between an immobilized substance immobilized directly or indirectly to a substrate and an analyte labeled with a modified proximity-dependent modifying enzyme) The present disclosure relates to a method for evaluating the interaction between an immobilized substance immobilized directly or indirectly to a substrate and an analyte labeled with a modified proximity-dependent modifying enzyme (hereinafter, sometimes referred to as the "evaluation method of the present disclosure"), which comprises the following steps: (1) adding the analyte labeled with a modified proximity-dependent modifying enzyme to the immobilized substance immobilized directly or indirectly to a substrate in the presence of a labeling substance; and (2) detecting the labeling substance. Note that evaluating the interaction between the immobilized substance and the analyte includes detecting or quantifying transient or persistent binding between the immobilized substance and the analyte. Note that preferably, the method includes a step of washing the substrate between steps (1) and (2).

[0018] (Substrate) The substrate may be a known substrate for detecting binding between an immobilized substance and an analyte. The substrate may be flat or in the shape of a so-called ELISA plate. Alternatively, the substrate may have micro-dimple-shaped depressions formed on the flat surface, or a porous membrane or nitrocellulose membrane formed on the surface. It is also possible to form pads for loading proteins. Methods for such processing may include molding, lithography, and the like, and may be appropriately selected depending on the substrate material. It is desirable to use a substrate material with low background so as not to affect the luminescence or fluorescence detection used in the subsequent interaction detection. Suitable substrate materials include, for example, non-fluorescent glass, amorphous carbon, quartz, polystyrene, polycarbonate, polymethyl methacrylate, polyolefin, polyethylene terephthalate, and cycloolefin copolymer.

[0019] (Array) The array of the present disclosure is an array of immobilized substances directly or indirectly immobilized on a substrate (preferably on a substrate on which positioning information is specified). The array allows simultaneous evaluation of the interaction of the analyte substance with all of the immobilized substances arranged on the array.

[0020] (Immobilization Substance) The immobilization substance is not particularly limited as long as it can be immobilized directly or indirectly to a substrate, and examples thereof include proteins, antibodies, nucleic acids (including DNA, RNA, etc.), peptides, low molecular weight compounds, medium molecular weight compounds, cell extracts, tissue extracts, sugars, lipids, physiologically active substances, and complexes thereof. The immobilization substance may be a single molecule or a mixture, a natural product, a genetically recombinant product, or a chemically synthesized product, or a derivative or fragment. Modification, substitution, deletion, or addition may be performed.

[0021] (Substance to be Analyzed) The substance to be analyzed is not particularly limited as long as it can be directly or indirectly labeled with a proximity-dependent modifying enzyme, and examples thereof include proteins, antibodies, nucleic acids (including DNA, RNA, etc.), peptides, low molecular weight compounds, medium molecular weight compounds, cell extracts, tissue extracts, sugars, lipids, physiologically active substances, and complexes thereof. As shown in the examples below, low molecular weight compounds that are normally difficult to analyze are preferred. Specific examples of complexes of the substance to be analyzed include those in which protein A and compound B form a complex that enables interaction with immobilized substance C or improves the strength of the interaction. The substance to be analyzed may be a single molecule or a mixture, a natural product, a genetically modified product, or a chemically synthesized product, or a derivative or fragment. Modification, substitution, deletion, or addition may be performed.

[0022] (Direct or Indirect Immobilization of Immobilized Substances on a Substrate) Immobilization of immobilized substances directly or indirectly on a substrate can be performed using known immobilization methods, as long as the immobilized substance is not substantially washed away during the substrate washing step (B / F separation washing step) of the evaluation method of the present disclosure. For example, the immobilized substance must be physically or chemically bound by an appropriate method depending on the material of the substrate. Indirect immobilization on a substrate means immobilizing the immobilized substance to the substrate via some kind of substance (e.g., beads). Immobilization means physically or chemically binding to the substrate. When the immobilized substance is a tag fusion protein fused with a tag, a ligand that specifically binds to the tag, an antibody that recognizes the tag, a metal chelate that binds to the tag, or the like may be formed on the substrate surface. By using a substrate with such a surface and a tag fusion protein, the immobilized substance can be directly or indirectly immobilized on the substrate via tag-ligand binding, tag-antibody binding, or tag-chelate binding. More specifically, examples include His tag and Ni-NTA, GST tag and glutathione, MBP tag and dextrin, biotin and avidin, biotin and streptavidin, biotin and neutravidin, FLAG™ tag and anti-FLAG™ antibody, GST tag and anti-GST antibody, and HA tag and anti-HA antibody. When using inorganic substrates such as glass and using non-tagged proteins as the immobilization substance, it is preferable to treat the substrate surface with a silane coupling agent containing a functional group capable of binding to amino or carboxyl groups (e.g., epoxy group, active ester, amino group, acid anhydride group, isocyanate group, etc.). A solution containing the immobilization substance (e.g., protein) can be spotted onto the treated substrate, and the substance can be covalently immobilized to the substrate surface at the N-terminus or C-terminus of the protein. Silane coupling agents with various chain lengths are commercially available, and any can be used as long as they do not affect the protein structure. It is also possible to adjust the bond distance between the immobilized substance (particularly, a protein) and the substrate using a linker. Other examples include an aminooxy linker having a hydrophobic alkyl and a thiol group, and a hydrazide linker, which are suitable for immobilizing proteins on a metal surface.

[0023] (Proteins as Immobilized Substances) Proteins as immobilized substances immobilized on a substrate or immobilized or mounted on an array are known to be greatly affected by the physical and chemical macro- and micro-environments, leading to denaturation. In particular, at liquid / solid interfaces and liquid / gas interfaces, such denaturation easily and irreversibly progresses, resulting in proteins easily becoming inactive and losing their original functions. In conventional methods, this inactive state makes it difficult to evaluate the interaction between proteins as immobilized substances and proteins as target substances. In other words, it is preferable for proteins as immobilized substances to be maintained in a non-denatured state. It is also known that it is sufficient for even a portion of the proteins as immobilized substances immobilized at each designated position on the array to retain their ability to interact with the target substances. While this depends on the type of protein being immobilized, as described in previous studies on protein-protein interactions (e.g., Song, G. et al., Mol Cell Proteomics. 2019; Al-Mulla, F., et al., Cancer Res., 2011) and nucleic acid-protein interactions (e.g., Hu S et al., Cell, 2009; Liu, L., et al., Nucleic Res., 2019), a protein array can be essentially non-denatured as long as some functionality remains. In other words, for molecular species such as kinases, as long as the kinase substrate protein retains some degree of structure, intermolecular interactions can be evaluated even if the protein as a whole does not retain its native structure. Therefore, the non-denatured state of the immobilized protein means that at least the site that interacts with the target substance maintains its shape or function.

[0024] (Method of Synthesizing Proteins as Analyte Substances and Immobilization Substances) Proteins as analyte substances and immobilization substances can be synthesized by known methods, but it is convenient to use commonly used recombinant proteins. For example, Escherichia coli, Bacillus subtilis, Sf9 insect cells, CHO cells, human cells, yeast, Brevibacillus, filamentous fungi (A. oryzae), tobacco BY-2 cells, or plant transient expression systems such as Nicotiana vesamiana, lettuce, tomato (fruit and leaves), rice, barley, moth orchid, and chili pepper, or cell-free protein synthesis systems can also be used. Suitable examples of cell-free protein synthesis systems include Escherichia coli, E. coli reconstituted systems, wheat, insects, yeast, tobacco, rabbit reticulocytes, and human cells. For the purpose of comprehensively obtaining a wide variety of proteins, wheat cell-free systems are particularly advantageous, offering an extremely high probability of synthesizing proteins in a soluble state and excellent cost advantages. In particular, cell-free protein synthesis using the WEPRO7240 series (Cell-Free Sciences) uses reagents from which GST-like proteins have been removed in advance, allowing for the production of highly purified proteins through simple purification using glutathione beads. This is one of the most preferred methods for preparing a wide variety of purified GST-tagged fusion proteins.

[0025] (Evaluation Method of the Present Disclosure) An example of the evaluation method of the present disclosure is not particularly limited as long as it includes the steps of (1) adding an analyte labeled with a modified proximity-dependent modification enzyme to an immobilized substance immobilized directly or indirectly on a substrate in the presence of a labeling substance, and (2) detecting the labeling substance. A method using a non-denatured protein array is exemplified below. An immobilized substance is placed and immobilized on a substrate. In this description, a non-denatured protein array using a non-denatured protein as the immobilized substance is used as a representative example. To maintain the immobilized protein in a non-denatured state, the array or the inside of the array wells is always filled with buffer. During buffer exchange, it is preferable to slowly add and remove buffer from the protein array to prevent the proteins immobilized on the magnetic beads from migrating to adjacent wells. In particular, when adding buffer, it is desirable to use a syringe or the like to inject it toward the wall. During reaction and washing of the protein array, a shaking speed of approximately one back-and-forth per second is desirable to prevent the magnetic beads from moving. The storage buffer in the protein array is removed, and the fusion protein of the modified AirID and the analyte, diluted with the reaction buffer, is added to the protein array in the presence of the labeling substance biotin. Note that "addition" can be by any method that allows contact between the immobilized substance and the analyte. Furthermore, "in the presence of the labeling substance (biotin)" can be by any method that allows contact between the immobilized substance and the labeling substance (biotin). For example, the labeling substance may be added to the array before, simultaneously with, or after the addition of the analyte to the array. The storage buffer refers to, but is not limited to, a near-neutral buffer suitable for biological reactions, containing glycerol or the like to prevent protein aggregation or stabilize the structure. The reaction buffer refers to, but is not limited to, a near-neutral buffer suitable for biological reactions, containing a blocking agent to prevent nonspecific adsorption of the analyte to the substrate or immobilized substance, as well as the labeling substance (biotin) and activation energy source (ATP) required for the reaction.The wash buffer refers to a near-neutral buffer suitable for biological reactions, containing salts and surfactants, used to remove analytes free in solution or bound to the substrate or immobilized substance, but is not limited thereto. In the presence of biotin (and, if necessary, ATP), all proteins immobilized on the array interact with the modified AirID fusion protein, which is the analyte. If the immobilized substance and the analyte bind, the modified AirID labels the lysine residue of the immobilized substance within close range with biotin. If the fusion protein does not contain a lysine residue, the protein may be modified to contain a lysine residue, as necessary. The interaction evaluation method disclosed herein detects biotin bound to the protein immobilized on the array. Therefore, interactions can be detected even if specific but weakly interacting analytes are removed by the washing procedure. The biotin labeled on the protein immobilized on the array after washing is detected using a substance that specifically recognizes and binds to biotin, and the interaction analysis results are obtained as a measurement image. Substances that specifically recognize and bind to biotin and are used to detect interactions include anti-biotin antibodies and streptavidin. These substances are preferably HRP-, AP-, or fluorescently labeled. Anti-biotin antibodies or streptavidin are preferably diluted in reaction buffer and placed in the protein array for the biotin binding reaction. After the reaction, free anti-biotin antibodies or streptavidin must be removed by washing. After washing, if an HRP / AP-labeled substance is used, a chemiluminescent reagent is added, and the luminescence produced by the reaction between the chemiluminescent reagent and HRP / AP is measured using a luminescence detection device. An example of a luminescence detection device is the LAS (GE). If a fluorescent label is used, measurements are performed using a fluorescence detection device. An example of a fluorescence detection device is the Typhoon (GE). The presence or absence of an interaction between the immobilized protein on each array and the target protein is determined from the measurement image.To determine whether an interaction exists, it is desirable to digitize the signal of each spot on the measurement image and determine that an interaction exists if the signal is above a certain value. It is desirable to use analysis software such as Array Pro Analyzer to digitize the measurement image. This allows the strength of interaction (binding strength) between multiple immobilized substances and the substance being analyzed to be measured at once.

[0026] Preferred examples of the modified proximity-dependent modifying enzyme, the N-terminus of the modified proximity-dependent modifying enzyme, the C-terminus of the modified proximity-dependent modifying enzyme, the target substance, and the terminus of the target substance of the present disclosure are as follows, but are not particularly limited thereto. (1) Modified proximity-dependent modification enzymes: an enzyme in which all of K2, K38, K41, K56, K71, K122, K163, K194, K244, K277, and K307 are substituted (AirID_KR11), an enzyme in which only K2 is substituted, an enzyme in which only K38 is substituted, an enzyme in which only K41 is substituted, an enzyme in which only K56 is substituted, an enzyme in which only K71 is substituted, an enzyme in which only K122 is substituted, an enzyme in which only K163 is substituted, an enzyme in which only K194 is substituted, an enzyme in which only K244 is substituted, an enzyme in which only K277 is substituted, an enzyme in which only K307 is substituted, an enzyme having an azide group in these enzymes, and an enzyme having an alkynyl group in these enzymes. (2) N-terminus of modified proximity-dependent modification enzymes: DYKDDDDK, DYKDHDGDYKDHDIDYKDDDDK, or KKKDKKDD. (3) C-terminus of modified proximity-dependent modification enzymes: DYKDDDDK, GGKKKGKK, or DDKKKDKK (C-terminus) (4) Analyte: low molecular weight compound, analyte having an azide group, analyte having an alkynyl group (5) Terminus of Analyte: N-hydroxysuccinimide ester terminus, N-hydroxysuccinimide ester-linker azide terminus, or N-hydroxysuccinimide ester-linker alkyne terminus In the present disclosure, the examples described above in (1) to (5) can be combined. Hereinafter, the present disclosure will be described in more detail using examples, but the following examples should be considered as an aid in gaining a concrete understanding of the present disclosure, and the scope of the present disclosure is not limited by the following examples in any way.

[0027] (Confirmation of Enzyme Activity by Lysine Modification) In this example, it was confirmed whether the enzyme activity was maintained by modifying the lysine of AirID.

[0028] (AirID) The interaction between the AirID-modified enzyme-IκBα and FG-RelA was analyzed using an in-tube GSH magnetic bead assay. The results are shown in Figures 1 and 2. AF analysis confirmed that mutation of either of the internally located lysines (Lys172, Lys183) abolished biotinylation activity. Furthermore, mutation of other lysines (surface lysines) to arginine did not affect activity. Therefore, in the following examples, we used a modified AirID in which lysines other than the internally located lysines (Lys172, Lys183) were mutated to arginine.

[0029] (TurboID) As with the AirID described above, the enzymatic activity of lysine-modified TurboID was confirmed. It was confirmed that modifying lysines other than the two underlined ones (K172 and K183) to arginine did not affect activity. MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGDACIAEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAAIGLGPVIGIVMAEALRKLGADKVRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWITLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK

[0030] (Preparation and Functionality Confirmation of Surface Lysine-Mutated AirIDs Fused with Each Tag) In this example, surface lysine-mutated AirIDs fused with each tag shown in Figure 3 were prepared. Next, the yields after synthesis and purification were confirmed for each tag. Furthermore, the detection sensitivity for each tag was confirmed.

[0031] The yields of the surface lysine-mutated AirIDs fused with each tag after synthesis and purification are shown in Figure 4. The yields after synthesis and purification were confirmed to be equivalent to those of the wild-type.

[0032] The analytical results using surface lysine-mutated AirIDs fused with each tag are shown in Figures 5 and 16. Specifically, 1 mM thalidomide-O-PEG4-NHS ester was bound to each lysine-tagged surface lysine-mutated AirID, and biotinylation upon interaction with CRBN was analyzed. Surface lysine-mutated AirID (1) showed less CRBN biotinylation than WT. Surface lysine-mutated AirIDs (4–7) fused with tags containing 4–5 lysines showed more CRBN biotinylation than WT. This is likely dependent on the amount of thalidomide bound to the lysines. C-terminal tag (3) showed more CRBN biotinylation than N-terminal tag (2). Furthermore, surface lysine-mutated AirIDs (5 and 7) fused with tags containing five lysines showed stronger activity.

[0033] (Analysis using geldanamycin-surface lysine mutation AirID) In ​​this example, geldanamycin-surface lysine mutation AirID_KR11 (geldanamycin-AirID 2 C) was prepared. Furthermore, the surface lysine mutation AirID was evaluated using HSP90AB1, which specifically binds to geldanamycin.

[0034] (geldanamycin-AirID 2 C) Preparation of Geldanamycin-AirID 2 The results of preparation of C are shown in Figure 6. The geldanamycin-AirID prepared 2 C was confirmed to be soluble.

[0035] (geldanamycin-AirID 2 The results of the interaction between geldanamycin-AirID2C and immobilized substances are shown in Figures 7 and 8. From the results in Figure 7, it was found that GM-AirID2C prepared under the conditions of 20 μM AirID_KR11 + 100 μM GM-NHS 2It was confirmed that GM-AirID C specifically interacts with and biotinylates HSP90AB1. The results in Figure 8 show that adding unmodified geldanamycin as a competitor reduced the activity of GM-AirID C. 2 This confirmed the specific interaction by inhibiting the reaction between HSP90AB1 and biotin-modified GM. The interaction between biotin-modified GM and HSP90AB1 without AirID could not be detected by this assay. This confirms that the evaluation method of the present disclosure is superior because it is specific and highly sensitive.

[0036] (geldanamycin-AirID 2 Storage stability of geldanamycin-AirID stored at -80°C 2 The results of using geldanamycin-AirID stored at -80°C are shown in Figure 9. 2 It was confirmed that C has a function.

[0037] (Comparison of solubility of AirID (native) and AirID modified bodies after reaction with the target substance) In this example, the solubility of AirID (native) and AirID modified bodies after reaction with the target substance was compared. When reacted with a high concentration (5000 μM) of (S,R,S)-AHPC-PEG4-NHS ester (VHL ligand), a difference in solubility was confirmed between AirID and the AirID modified body (AirID_KR11) (see Figure 10). In addition, a "drug-binding specialized modified AirID = AirID" that maintains stability even when bound to a drug was confirmed. 2 I was able to get a C.

[0038] (Analysis 1 Using a Low-Molecular-Weight Analyte) In this example, thalidomide, a low-molecular-weight compound, was used as the analyte.

[0039] (Analysis of the intermolecular interaction between CRBN and thalidomide-bound AirID variants prepared by NHS ester reaction) In this example, the interaction between thalidomide and CRBN, which are known to bind to each other, was analyzed. The details are as follows. (Protein synthesis of immobilized substances) BRD2, BRD3, BRD4, CRBN, and CRBN YW / AA were used as immobilized substances (target proteins) using FLAG TM Template DNA fused to tag proteins and GST proteins was synthesized. Each template DNA was used to express FLAG in a wheat cell-free expression system. TM FLAG-GST fusion immobilized material was synthesized. (Binding of immobilized material to magnetic beads and purification) TMThe -GST fusion immobilized material was bound to glutathione magnetic beads used in non-denaturing protein arrays and purified. The purified magnetic beads were dispensed into tubes and immobilized by magnetic force, allowing for solution exchange during washing. (Protein Synthesis and Purification of AirID Modified Proteins) Template DNA was synthesized by fusing the AirID modified protein (AirID_KR11) with a lysine-containing tag (containing multiple lysine residues) and a His-tagged protein. Specifically, tags containing one or more lysines at the N-terminus and / or C-terminus (see paragraph "0015") were used. The inventors have confirmed that the effects of this example can be achieved with any tag containing one or more lysines. Using the synthesized template DNA and a wheat cell-free expression system, lysine-containing tag- and His-tag-fused AirID modified proteins were synthesized. The synthesized AirID modified proteins were bound to Ni resin and purified. The purified AirID modified proteins were eluted from the Ni resin using imidazole. (Binding Reaction between the Analyte and AirID Modified Proteins) After purification, the eluted AirID modified protein and the analyte containing an NHS ester were mixed in solution and allowed to stand at 4–37°C for 1–6 hours to carry out the binding reaction. The reaction temperature and time could be varied within a range that did not affect the protein properties. The AirID modified protein was tested at a concentration of approximately 5–100 μM, but lower and higher concentrations were also confirmed to be possible. The concentration of the analyte containing an NHS ester was tested at approximately 100–1000 μM, which was higher than the concentration of the AirID modified protein, but lower and higher concentrations were also confirmed to be possible. Phosphate buffer adjusted to pH 7.4 was used as the reaction solvent, but it was confirmed that the pH could be adjusted between 7.2–8.5, and other buffers could also be used. AirID modified proteins bound to the analyte were prepared. Unreacted analyte containing an NHS ester was removed by solution exchange via dialysis. (Biotin labeling of immobilized substance in the presence of the analyte) The analyte (thalidomide)-bound AirID variant diluted in a reaction buffer containing biotin and ATP was immobilized on the FLAG marker on magnetic beads. TM React with FLAG-GST fusion immobilized material TMThe immobilized α-GST fusion material was biotinylated. (Removal of the target substance) To remove the target substance-bound AirID variants that were free in the reaction buffer or adsorbed on the magnetic beads, the reaction buffer was removed and then washed multiple times with washing buffer. (Biotin detection with anti-biotin antibody) The washing buffer was removed, and anti-biotin antibody diluted in reaction buffer was added to detect the FLAG on the magnetic beads. TM The antibody was reacted with the thalidomide-bound AirID modified substance and GST fusion immobilized material. To remove any free anti-biotin antibody, the reaction buffer was removed, followed by multiple washes with the wash buffer. After removing the wash buffer, a chemiluminescent reagent was added and allowed to react. The resulting luminescence was detected using an LAS4000 (GE) and a measurement image was obtained. (Biotin detection results) The results are shown in Figure 11. Luminescence was observed between the thalidomide-bound AirID modified substance and CRBN. Luminescence was not observed between other proteins. For details, see the thalidomide-bound AirID 2 In C, only CRBN was biotinylated. Pomalidomide, added as a competitor, inhibited the biotinylation reaction of CRBN, indicating that the biotinylated AirID 2 It was confirmed that C specifically bound to CRBN. The electrophoresis results for the proteins used are shown in Figure 12. From the above, this example confirmed that the analytical method of the present disclosure can analyze the specific interaction between an immobilized substance and a low-molecular-weight analyte.

[0040] (Analysis 2 Using a Small Molecular Weight Analyte) In this example, the small molecular weight compound JQ1 was used as the analyte. In this example, we analyzed the interactions between JQ1 and the BRD family, or between thalidomide and CRBN, which are known to bind to each other. Details are as follows.

[0041] (Protein synthesis of immobilized substances) BRD2, BRD3, BRD4, CRBN, and CRBN YW / AA were used as immobilized substances (target proteins) using FLAG. TMTemplate DNA fused to tag proteins and GST proteins was synthesized. Each template DNA was used to express FLAG in a wheat cell-free expression system. TM FLAG-GST fusion immobilized material was synthesized. (Binding of immobilized material to magnetic beads and purification) TMThe -GST fusion immobilized material was bound to glutathione magnetic beads used in non-denaturing protein arrays and purified. The purified magnetic beads were dispensed into tubes and immobilized by magnetic force, allowing for solution exchange during washing. (Protein Synthesis and Purification of AirID Modified Proteins) Template DNA was synthesized by fusing the AirID modified protein (AirID_KR11) with a lysine-containing tag (containing multiple lysine residues) and a His-tagged protein. Specifically, tags containing one or more lysines at the N-terminus and / or C-terminus (see paragraph "0015") were used. The inventors have confirmed that the effects of this example can be achieved with any tag containing one or more lysines. Using the synthesized template DNA and a wheat cell-free expression system, lysine-containing tag- and His-tag-fused AirID modified proteins were synthesized. The synthesized AirID modified proteins were bound to Ni resin and purified. The purified AirID modified proteins were eluted from the Ni resin using imidazole. (Conjugation reaction of azide and AirID modified compounds) After purification, the eluted AirID modified compounds were mixed with Azido-PEG4-NHS ester in a solution and allowed to stand at 4-37°C for 1-6 hours to carry out the conjugation reaction. The reaction temperature and time could be varied within a range that did not affect the protein properties. The AirID modified compounds were used at concentrations of approximately 5-100 μM, but lower and higher concentrations were also confirmed to be possible. The Azido-PEG4-NHS ester was used at concentrations of approximately 100-1000 μM, which was higher than the concentration of the AirID modified compounds, but lower and higher concentrations were also confirmed to be possible. Phosphate buffer adjusted to pH 7.4 was used as the reaction solvent, but it was confirmed that the pH could be adjusted between 7.2 and 8.5, and other buffers could also be used. Azide-conjugated AirID modified compounds were prepared. Unreacted Azido-PEG4-NHS ester was removed by solution exchange via dialysis. (Binding reaction between the target substance and the azide-bound AirID modified substance) The prepared azide-bound (having an azide group) AirID modified substance and the target substance having an alkyne (alkynyl group) were mixed, and a binding reaction was carried out by click chemistry reaction.For the click chemistry reaction, the analyte containing an alkyne (alkynyl group) was added to the azide-conjugated AirID modified solution in a mixture of 250 μM THPTA, 50 μM CuSO4, 2.5 mM sodium ascorbate, and 1 mM aminoguanidine. The analyte concentration was 100 μM, but it was confirmed that this was not a problem as long as it exceeded the number of azides conjugated to the AirID modified solution. The analyte-conjugated AirID modified solution was prepared. Unreacted alkyne (alkynyl group) analyte was removed by dialysis. (Biotin labeling of immobilized substance in the presence of analyte) The analyte (JQ1 or thalidomide)-conjugated AirID modified solution diluted in reaction buffer containing biotin and ATP was attached to the FLAG on magnetic beads. TM React with FLAG-GST fusion immobilized material TM The immobilized α-GST fusion material was biotinylated. (Removal of the target substance) To remove the target substance-bound AirID variants that were free in the reaction buffer or adsorbed on the magnetic beads, the reaction buffer was removed and then washed multiple times with washing buffer. (Biotin detection with anti-biotin antibody) The washing buffer was removed, and anti-biotin antibody diluted in reaction buffer was added to detect the FLAG on the magnetic beads. TM The antibody was reacted with the thalidomide-GST fusion immobilized substance. To remove any free anti-biotin antibody, the reaction buffer was removed and the antibody was washed multiple times with the washing buffer. After removing the washing buffer, a chemiluminescent reagent was added and reacted. The resulting luminescence was detected using an LAS4000 (GE) and a measurement image was obtained. (Biotin detection results) The results are shown in Figure 13. Luminescence was observed between the JQ1-bound AirID variant and the BRD family, as well as between the thalidomide-bound AirID variant and CRBN. Luminescence was not observed between other compounds and proteins. For details, see JQ1-bound AirID. 2 C only modified the BRD family with biotin. JQ1, added as a competitor, inhibited the biotin modification reaction of the BRD family, indicating that JQ1-bound AirID2 It was confirmed that C specifically binds to the BRD family. The electrophoresis results for the proteins used are shown in Figure 14. This example confirmed that the analytical method of the present disclosure can analyze the specific interaction between the immobilized substance and the target substance.

[0042] (AirID 2 Protein array analysis using AirID C) Using an E3 ligase array (loaded with approximately 570 proteins), we searched for proteins that interact with drug X. Drug X, added as a competitor, inhibited the biotin modification reaction of the positive protein (1). 2 C_Drug X can search for E3 ligases that specifically bind to it (see Figure 15). As described above, the engineered proximity-dependent modification enzymes of the present disclosure are useful in protein array analysis.

[0043] (Analysis of intermolecular interaction between pomalidomide- or thalidomide-bound AirID variants and CRBN) In this example, an analysis of the interaction between pomalidomide or thalidomide, which are known to bind, and CRBN was performed. AirID variants bound to azide and alkyne attached to pomalidomide, AirID variants bound to alkyne and azide attached to thalidomide, and AirID variants bound to the substance to be analyzed, each bound in different combinations by click chemistry reaction, were used. Details are as follows. (Protein synthesis of immobilized substance) CRBN, CRBN YW / AA, and BRD4 were used as immobilized substances (target proteins) using FLAG TM Template DNA fused to tag proteins and GST proteins was synthesized. Each template DNA was used to express FLAG in a wheat cell-free expression system. TM FLAG-GST fusion immobilized material was synthesized. (Binding of immobilized material to magnetic beads and purification) TMThe -GST fusion immobilized material was bound to glutathione magnetic beads used in non-denaturing protein arrays and purified. The purified magnetic beads were dispensed into tubes and immobilized by magnetic force, allowing for solution exchange during washing. (Protein Synthesis and Purification of AirID Modified Proteins) Template DNA was synthesized by fusing AirID modified proteins with lysine-containing tags (containing multiple lysine residues) and His-tagged proteins. Specifically, tags containing one or more lysines at the N-terminus and / or C-terminus (see paragraph "0015") were used. The inventors have confirmed that the effects of this example can be achieved with tags containing one or more lysines. Lysine-containing tag and His-tag fused AirID modified proteins were synthesized using the synthesized template DNA in a wheat cell-free expression system. The synthesized AirID modified proteins were bound to Ni resin and purified. The AirID modified proteins were eluted from the purified Ni resin using imidazole. (Conjugation reaction of alkyne or azide with AirID modified proteins) After purification, the eluted AirID modified proteins were mixed with propargyl-PEG4-NHS ester or azido-PEG4-NHS ester in a solution and incubated at 4-37°C for 1-6 hours to carry out the conjugation reaction. The reaction temperature and time could be varied within a range that did not affect the protein properties. The AirID modified protein concentrations were approximately 5-100 μM, but lower and higher concentrations were also confirmed to be feasible. The propargyl-PEG4-NHS ester or azido-PEG4-NHS ester concentrations were approximately 100-1000 μM, which was higher than the concentration of the AirID modified protein. However, lower and higher concentrations were also confirmed to be feasible. Phosphate buffer adjusted to pH 7.4 was used as the reaction solvent, but the pH could be adjusted between 7.2 and 8.5, and other buffers could also be used. Alkyne- or azide-conjugated AirID modified proteins were prepared. Unreacted Propargyl-PEG4-NHS ester or Azido-PEG4-NHS ester was removed by solution exchange via dialysis.(Conjugation reaction between the analyte and alkyne- or azide-conjugated AirID modified molecules) The prepared alkyne- or azide-conjugated AirID modified molecules were mixed with the analyte containing an alkyne group and conjugated via click chemistry. For the click chemistry reaction, the analyte containing an azide or alkynyl group was added to the alkyne- or azide-conjugated AirID modified molecules in a mixture containing 250 μM THPTA, 50 μM CuSO4, 2.5 mM sodium ascorbate, and 1 mM aminoguanidine. The analyte containing an azide or alkynyl group was added at a concentration of 100 μM, although it was confirmed that this was not a problem as long as the concentration exceeded the number of alkynes or azides conjugated to the AirID modified molecules. The analyte was conjugated to the AirID modified molecules. Unreacted azide or alkyne-conjugated analyte was removed by solution exchange via dialysis. (Biotin labeling of immobilized substance in the presence of the analyte) The analyte (pomalidomide or thalidomide)-bound AirID variant diluted with a reaction buffer containing biotin and ATP was attached to the FLAG on magnetic beads. TM React with FLAG-GST fusion immobilized material TM The immobilized α-GST fusion material was biotinylated. (Removal of the target substance) To remove the target substance-bound AirID variants that were free in the reaction buffer or adsorbed on the magnetic beads, the reaction buffer was removed and then washed multiple times with washing buffer. (Biotin detection with anti-biotin antibody) The washing buffer was removed, and anti-biotin antibody diluted in reaction buffer was added to detect the FLAG on the magnetic beads. TMThe membrane was reacted with the α-GST fusion immobilized material. To remove free anti-biotin antibodies, the reaction buffer was removed, followed by multiple washes with wash buffer. After removing the wash buffer, a chemiluminescent reagent was added and reacted. The resulting luminescence was detected using an LAS4000 (GE) and images were obtained. (Biotin detection results) The results are shown in Figure 17. Luminescence was observed between the pomalidomide- or thalidomide-conjugated AirID variants and CRBN. Luminescence was not observed between other proteins. Specifically, with pomalidomide- or thalidomide-conjugated AirID2C, only CRBN was biotinylated. Pomalidomide, added as a competitor, inhibited the biotinylation reaction of CRBN, confirming that pomalidomide- or thalidomide-conjugated AirID2C specifically bound to CRBN. Figure 18 shows the electrophoresis results of the proteins used. Alkynyl group modification of the AirID variants was confirmed. This Example and Example 6 confirmed that the analytical method of the present disclosure can analyze the specific interaction between an immobilized substance and an analyte (especially a low molecular weight compound) using an analyte having an alkynyl group (azide group) and a modified proximity-dependent modifying enzyme having an azide group (alkynyl group).

[0044] The evaluation method of the present invention can analyze low molecular weight compounds, which has been difficult to do with conventional evaluation methods.

Claims

1. Engineered proximity-dependent modifying enzymes in which surface lysines have been substituted.

2. The modified proximity dependent modifying enzyme of claim 1, wherein the proximity dependent modifying enzyme is a peptide having the following amino acid sequence (SEQ ID NO:3: AirID): MKDNTVPLTLISILADGEFHSGEQLGEQLGMSRAAINKHIKTLRDWGVDVFRVQGKGYCLPEPIQLLDEEKIRQQLDEGSVTVLPVIDSTNQYLLDRLDELTSGDVCIAEYQQAGRGRRGRKWFSPFGANLYLSMYWRLEQGPAAAMGLSLVIGIVMAETLQKLGADGVRVKWPNDLYLNDRKLAGILVEMTGKTGDAAHIVIGAGINLSMREPETDEVDQSWINLQEAGITIDRNQLAARLIKDLRSALRQFEQQGLAPFLSRWEALDNFINRPVKLIIGDREIHGIARGINEQGALLLEQDGVIKPWIGGEISLRSA.

3. The engineered proximity-dependent modification enzyme of claim 2, wherein the lysine substitution position is any one or more of the following and the substituted amino acid is arginine, histidine, glutamic acid, or aspartic acid: 1) K2, 2) K38, 3) K41, 4) K56, 5) K71, 6) K122, 7) K163, 8) K194, 9) K244R, 10) K277, and 11) K307.

4. The engineered proximity-dependent modification enzyme of claim 2, wherein the lysine substitutions are any one or more of the following: 1) K2R, 2) K38R, 3) K41R, 4) K56R, 5) K71R, 6) K122R, 7) K163R, 8) K194R, 9) K244R, 10) K277R, and 11) K307R.

5. The engineered proximity dependent modification enzyme of claim 2, wherein the lysine substitutions are: 1) K2R, 2) K38R, 3) K41R, 4) K56R, 5) K71R, 6) K122R, 7) K163R, 8) K194R, 9) K244R, 10) K277R, and 11) K307R.

6. The modified proximity dependent modifying enzyme of claim 1, wherein the proximity dependent modifying enzyme is a peptide having the following amino acid sequence (SEQ ID NO:2: TurboID): MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGDACIAEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAAIGLGPVIGIVMAEALRKLGADKVRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWITLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK.

7. The modified proximity dependent modifying enzyme of claim 6, wherein the lysine substitution positions are any one or more of the lysines other than K172 and K183 as underlined: MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIPLLNAKQILGQLDGGSVAVLPVVDSTNQYLLDRIGELKSGDACIAEYQQAGRGSRGRKWFSPFGANLYLSMFWRLKRGPAAAIGLGPVIGIVMAEALRKLGADKVRVKWPNDLYLQDRKLAGILVELAGITGDAAQIVIGAGINVAMRRVEESVVNQGWITLQEAGINLDRNTLAATLIRELRAALELFEQEGLAPYLPRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGVIKPWMGGEISLRSAEK.

8. An engineered proximity-dependent modifying enzyme according to any one of claims 1 to 7, wherein one or more lysine-containing tags are fused directly or indirectly to the N-terminus or C-terminus.

9. The modified proximity-dependent modification enzyme of any one of claims 1 to 7, wherein any one of the following tags is fused directly or indirectly to the N-terminus or C-terminus: 1) DYKDDDDK (N-terminus: SEQ ID NO: 4) 2) DYKDDDDK (C-terminus: SEQ ID NO: 4) 3) DYKDHDGDYKDHDIDYKDDDDK (N-terminus: SEQ ID NO: 5) 4) GGKKKGKK (C-terminus: SEQ ID NO: 6) 5) KKKDKKDD (N-terminus: SEQ ID NO: 7) 6) DDKKKDKK (C-terminus: SEQ ID NO: 8).

10. A modified proximity-dependent modifying enzyme-labeled analyte, which is labeled with the modified proximity-dependent modifying enzyme according to any one of claims 1 to 9.

11. A modification proximity-dependent modifying enzyme-labeled analyte, which is labeled with the modification proximity-dependent modifying enzyme described in claim 5.

12. The modified proximity-dependent modifying enzyme labeled analyte of claim 10, wherein the analyte has an N-hydroxysuccinimide ester terminus, an N-hydroxysuccinimide ester-linker azide terminus, or an N-hydroxysuccinimide ester-linker alkyne terminus.

13. The modified proximity-dependent modified enzyme labeled analyte of claim 11, wherein the analyte has an N-hydroxysuccinimide ester terminus, an N-hydroxysuccinimide ester-linker azide terminus, or an N-hydroxysuccinimide ester-linker alkyne terminus.

14. A method for evaluating the interaction between an immobilized substance immobilized directly or indirectly on a substrate and a modification proximity-dependent modification enzyme-labeled analysis target substance, comprising the following steps: (1) a step of adding the modification proximity-dependent modification enzyme-labeled analysis target substance described in claim 12 or claim 13 to the immobilized substance immobilized directly or indirectly on a substrate in the presence of a labeled substance; (2) a step of detecting the labeled substance.

15. The evaluation method according to claim 14, further comprising a step of cleaning the substrate between the steps (1) and (2).

16. The evaluation method described in claim 14 or 15, wherein the modification proximity-dependent modification enzyme-labeled analysis target substance is the modification proximity-dependent modification enzyme-labeled analysis target substance described in claim 12.

17. A method for evaluating a protein, which is an immobilized substance indirectly immobilized to an array via magnetic beads, and a modified proximity-dependent modifying enzyme-labeled analysis target substance, comprising the following steps: (1) a step of adding the modified proximity-dependent modifying enzyme-labeled analysis target substance described in claim 12 or claim 13 to the immobilized substance indirectly immobilized to an array via magnetic beads in the presence of biotin; and (2) a step of detecting the biotin.

18. The evaluation method according to claim 17, further comprising a step of washing the array between steps (1) and (2).

19. The evaluation method described in claim 17 or 18, wherein the modification proximity-dependent modification enzyme-labeled analysis target substance is the modification proximity-dependent modification enzyme-labeled analysis target substance described in claim 13.

20. A method for introducing a modified proximity-dependent modification enzyme-labeled analyte into a cell, comprising introducing the modified proximity-dependent modification enzyme-labeled analyte described in claim 12 or claim 13 into the cell by electroporation.

21. The evaluation method according to claim 14, wherein the substance to be analyzed maintains water solubility.

22. The evaluation method according to claim 17, wherein the substance to be analyzed maintains water solubility.

23. The evaluation method according to claim 14, wherein the target substance to be analyzed has an alkynyl group and the modified proximity-dependent modifying enzyme has an azide group.

24. The evaluation method according to claim 14, wherein the analyte has an azide group and the modified proximity-dependent modifying enzyme has an alkynyl group.

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