Novel protein targeted degradation technology based on artificially modified NEL structural domain

Abstract

The invention provides a recombinant protein containing an artificially modified NEL structural domain and application of the recombinant protein in targeted degradation of protein. Experimental results show that the recombinant protein can simply and efficiently realize controllable adjustment of the target protein level in cells, and has better stability by reducing the self-ubiquitination level. The invention also provides a construction method of the recombinant protein and an application of the recombinant protein in the process of inhibiting cell autophagy.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular, the present invention relates to the construction and practical application of a recombinant protein containing an artificially modified NEL domain. Background Art

[0002] Traditional drug molecules are mostly small chemical molecules that recognize and regulate the activity of target proteins. These small molecule drugs inhibit the physiological function of target proteins by binding to target proteins and occupying corresponding binding pockets. This strategy has obvious advantages for many enzymes or receptors containing active catalytic cavities or allosteric pockets, but this strategy does not work well for most proteins. Due to the lack of available binding pockets, about 80% of human proteins are currently "untargetable". At present, PROTACs (Proteolysis-Targeting Chimeras) have become a hot spot in the field of new drug research and development due to their targeting function and low-dose effects. Unlike traditional small molecule drugs, PROTACs are bifunctional chimeras that use the natural protein degradation system in cells, namely the ubiquitin-proteasome pathway, to target the target protein at one end and hijack the endogenous E3 ubiquitin ligase at the other end to form a ternary complex, and ubiquitinate the target protein, so that the target protein is recognized and degraded by the proteasome. After the target protein is hydrolyzed, PROTACs can be released and enter the next cycle, so they can work at a lower dose. Given the above advantages of PROTACs, a variety of PROTACs targeting different genes have entered the clinical development stage, with indications including various cancers, inflammation, etc.

[0003] Despite this, the emergence of PROTACs has enabled humans to break through many "undruggable" targets, but it still has off-target effects and the risk of causing side effects. At the same time, most proteins currently lack small molecules that can effectively and specifically target them, which severely limits the range of proteins that PROTACs can target.

[0004] Based on this, there is an urgent need in this field for a new targeted protein degradation technology that can reduce off-target effects. Summary of the invention

[0005] The purpose of the present invention is to provide a construction and application of an artificially modified NEL domain recombinant protein.

[0006] In a first aspect of the present invention, a recombinant protein is provided, wherein the recombinant protein has a structure shown in Formula I or Formula II:

[0007] Z0-Z1-L-Z2(I);

[0008] Z0-Z2-L-Z1(II)

[0009] In the formula,

[0010] Z0 is selected from the group consisting of none, tag, restriction site, or a combination thereof;

[0011] Z1 is the target protein-specific recognition module;

[0012] Z2 is the NEL domain;

[0013] L is none or a link sequence;

[0014] “-” indicates the peptide bond connecting the above sequences;

[0015] Wherein, the Z1 specifically recognizes the target protein, and the Z2 ubiquitinates the target protein.

[0016] In another preferred embodiment, the tag is selected from the following group: Trx, Trx6×his, or HA.

[0017] In another preferred embodiment, the restriction enzyme cleavage site is the HRV 3C restriction enzyme cleavage site.

[0018] In another preferred example, the amino acid sequence of the HRV 3C restriction site is shown in SEQ ID NO:9.

[0019] In another preferred embodiment, the Z1 is selected from the following group: an antibody, a protein motif, or a protein domain.

[0020] In another preferred embodiment, the Z1 contains an artificially introduced mutation.

[0021] In another preferred embodiment, the use of the mutation is selected from the following group:

[0022] (U1) changing the affinity of Z1 for the target protein;

[0023] (U2) reducing the ubiquitination modification of the recombinant protein itself;

[0024] or a combination thereof.

[0025] In another preferred embodiment, the antibody is a nanobody.

[0026] In another preferred example, the amino acid sequence of the Nanobody is as shown in SEQ ID NO:1.

[0027] In another preferred embodiment, the nanobody specifically recognizes green fluorescent protein.

[0028] In another preferred embodiment, the Nanobody contains artificially introduced mutations.

[0029] In another preferred embodiment, the amino acid sequence of the Nanobody into which the mutation is introduced is selected from the following group:

[0030] (1) the amino acid sequence as shown in SEQ ID NO:1 and having the following mutations in SEQ ID NO:1: R35F, F102R and mutation of all lysines in the sequence to arginines;

[0031] (2) the amino acid sequence as shown in SEQ ID NO:1 and having the following mutation in SEQ ID NO:1: R35F;

[0032] (3) The amino acid sequence as shown in SEQ ID NO: 1 and having the following mutations in SEQ ID NO: 1: R35F and F102R.

[0033] (4) the amino acid sequence as shown in SEQ ID NO:1 and having the following mutations in SEQ ID NO:1: N99F, F102R and E103R;

[0034] (5) the amino acid sequence as shown in SEQ ID NO:1 and having the following mutations in SEQ ID NO:1: R35F, N99F, F102R and E103R;

[0035] (6) the amino acid sequence as shown in SEQ ID NO:1 and having the following mutations in SEQ ID NO:1: all lysines in the sequence are mutated to arginines;

[0036] or a combination thereof.

[0037] In another preferred example, the amino acid sequence of the Nanobody with introduced mutations is: the amino acid sequence is as shown in SEQ ID NO: 1 and there are the following mutations in SEQ ID NO: 1: R35F, F102R and all lysines in the sequence are mutated to arginine.

[0038] In another preferred embodiment, the protein motif is the WBS1 motif of ATG16L1.

[0039] In another preferred example, the amino acid sequence of the WBS1 motif of ATG16L1 is shown in SEQ ID NO:2.

[0040] In another preferred example, the WBS1 motif of ATG16L1 specifically recognizes WIPI2b.

[0041] In another preferred embodiment, the WBS1 motif of ATG16L1 contains an artificially introduced mutation.

[0042] In another preferred example, the amino acid sequence of the WBS1 motif of ATG16L1 into which the mutation is introduced comprises: the amino acid sequence as shown in SEQ ID NO: 2 and the following mutation is present in SEQ ID NO: 2: all lysines in the sequence are mutated to arginines.

[0043] In another preferred embodiment, the target protein is selected from the following group: a protein tag, a protein domain, or an amino acid sequence.

[0044] In another preferred embodiment, the protein tag includes green fluorescent protein.

[0045] In another preferred example, the amino acid sequence is the sequence of human WIPI2b.

[0046] In another preferred embodiment, the Z2 contains an artificially introduced mutation.

[0047] In another preferred embodiment, the purpose of the mutation is to reduce the ubiquitination modification of the recombinant protein itself.

[0048] In another preferred embodiment, the Z2 is the 274th to 575th amino acid residues of Shigella flexneri IpaH1.4.

[0049] In another preferred example, the amino acid sequence of amino acid residue 274 to amino acid residue 575 of Shigella flexneri IpaH1.4 is shown in SEQ ID NO:3.

[0050] In another preferred embodiment, the Shigella flexneri IpaH1.4 contains artificially introduced mutations from amino acid residue 274 to amino acid residue 575.

[0051] In another preferred embodiment, the amino acid sequence of amino acid residue 274 to amino acid residue 575 of the Shigella flexneri IpaH1.4 into which the mutation is introduced is selected from the following group:

[0052] (a) the amino acid sequence as shown in SEQ ID NO:3 and having the following mutation in SEQ ID NO:3: all lysines in the sequence are mutated to arginines;

[0053] (b) the amino acid sequence as shown in SEQ ID NO:3 and having the following mutation in SEQ ID NO:3: all serines in the sequence are mutated to alanine;

[0054] (c) the amino acid sequence as shown in SEQ ID NO:3 and having the following mutation in SEQ ID NO:3: all threonine residues in the sequence are mutated to alanine residues;

[0055] (d) the amino acid sequence as shown in SEQ ID NO:3 and having the following mutation in SEQ ID NO:3: cysteine ​​at position 368 in the sequence is mutated to alanine;

[0056] or a combination thereof.

[0057] In another preferred example, the amino acid sequence of the 274th to 575th amino acid residues of the Shigella flexneri IpaH1.4 into which the mutation is introduced is: the amino acid sequence as shown in SEQ ID NO:3 and there is the following mutation in SEQ ID NO:3: all lysines in the sequence are mutated to arginines.

[0058] In another preferred embodiment, the L is selected from the following group: a flexible linking sequence or a rigid linking sequence.

[0059] In another preferred embodiment, the L is shown in SEQ ID NO:4.

[0060] In another preferred embodiment, the construction and optimization method of the recombinant protein is selected from the following group:

[0061] (X1) isothermal titration calorimetry to determine the binding strength between the target protein-specific recognition module and the target protein;

[0062] (X2) in vitro ubiquitination assay to determine the ubiquitination level of the recombinant protein;

[0063] (X3) protein structure analysis and mass spectrometry analysis of ubiquitination site modification of the recombinant protein;

[0064] or a combination thereof.

[0065] The second aspect of the present invention provides a polynucleotide, wherein the polynucleotide encodes the recombinant protein described in the first aspect of the present invention.

[0066] In another preferred embodiment, the polynucleotide includes a polynucleotide encoding a target protein-specific recognition module, a polynucleotide encoding a connecting sequence and a polynucleotide encoding a NEL domain.

[0067] In another preferred embodiment, the polynucleotide encoding the target protein-specific recognition module is selected from the following group:

[0068] (A1) a polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1;

[0069] (A2) a polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2;

[0070] or a combination thereof.

[0071] In another preferred embodiment, the polynucleotide encoding the target protein-specific recognition module is selected from the following group:

[0072] (A3) the polynucleotide sequence shown in SEQ ID NO:5;

[0073] (A4) the polynucleotide sequence shown in SEQ ID NO:6;

[0074] or a combination thereof.

[0075] In another preferred embodiment, the polynucleotide encoding the linker sequence is a polynucleotide encoding the amino acid sequence shown in SEQ ID NO:4.

[0076] In another preferred example, the polynucleotide encoding the connection sequence is a polynucleotide sequence as shown in SEQ ID NO:8.

[0077] In another preferred embodiment, the polynucleotide encoding the NEL domain is a polynucleotide encoding the amino acid sequence shown in SEQ ID NO:3.

[0078] In another preferred embodiment, the polynucleotide encoding the NEL domain is a polynucleotide sequence as shown in SEQ ID NO:7.

[0079] The third aspect of the present invention provides an expression vector, wherein the expression vector contains the polynucleotide described in the second aspect of the present invention.

[0080] In another preferred embodiment, the expression vector is selected from the following group: a plasmid vector, a viral vector, or a combination thereof.

[0081] In another preferred embodiment, the plasmid vector is selected from the following group: pRSF-Duet, pLVX-TetOne, pCMV-FLAG-mCherry-P2A, or a combination thereof.

[0082] In another preferred embodiment, the viral vector is selected from the following group: a lentiviral vector, an adenoviral vector, or an adeno-associated virus (AAV) vector, or a combination thereof.

[0083] The fourth aspect of the present invention provides a cell line, wherein the cell line contains the expression vector described in the third aspect of the present invention or the polynucleotide described in the second aspect of the present invention is integrated into its genome.

[0084] In another preferred embodiment, the cell line is selected from the following group: a prokaryotic cell line or a eukaryotic cell line.

[0085] In another preferred embodiment, the prokaryotic cell strain is Escherichia coli.

[0086] In another preferred embodiment, the eukaryotic cell line is the HeLa cell line.

[0087] In another preferred embodiment, the expression vector is selected from the following group: a plasmid vector or a viral vector.

[0088] In another preferred embodiment, the method for preparing the cell line is selected from the following group: plasmid transfection, viral transfection, or liposome transfection.

[0089] In another preferred embodiment, the method for preparing the cell line comprises the following steps:

[0090] (S1) providing a cell line to be transfected and an expression vector;

[0091] (S2) Transfecting the expression vector into the cell line to be transfected, thereby obtaining the cell line.

[0092] In another preferred embodiment, a method for producing the recombinant protein described in the first aspect of the present invention is provided, comprising the following steps: culturing the cell line described in the fourth aspect of the present invention under conditions suitable for expression, thereby expressing the recombinant protein described in the first aspect of the present invention.

[0093] The fifth aspect of the present invention provides a use of the recombinant protein described in the first aspect of the present invention, the polynucleotide described in the second aspect of the present invention, or the expression vector described in the third aspect of the present invention for preparing a drug or a pharmaceutical composition, wherein the drug or pharmaceutical composition is used to degrade the target protein.

[0094] In another preferred embodiment, the drug or drug composition is a targeted drug or drug composition.

[0095] In another preferred embodiment, the targeted drug or drug composition targets a target protein or a target cell.

[0096] In another preferred embodiment, the targeted drug or pharmaceutical composition comprises a carrier selected from the following group: a viral vector, a liposome vector, a virus-like protein vector, an exosome vector, an artificially designed protein vector, or a bacterial molecular injector.

[0097] In another preferred embodiment, the viral vector is selected from the following group: a lentiviral vector, an adenoviral vector, or an adeno-associated viral vector.

[0098] In another preferred embodiment, the liposome carrier is selected from the following group: lipid nanoparticles, lipid complexes, or lipid polymer complexes.

[0099] In another preferred embodiment, the surface of the liposome carrier has a targeting element.

[0100] In another preferred embodiment, the targeting element specifically binds to a marker on the cell surface.

[0101] In another preferred embodiment, the cell surface marker is a cell surface protein.

[0102] In another preferred embodiment, the virus-like protein vector is a human retrovirus-like protein.

[0103] In another preferred embodiment, the human retrovirus-like protein is PEG10.

[0104] In another preferred embodiment, the surface of the exosome carrier has a targeting element.

[0105] In another preferred embodiment, the targeting element is an external targeting peptide.

[0106] In another preferred embodiment, the external targeting peptide specifically binds to a marker on the cell surface.

[0107] In another preferred embodiment, the cell surface marker is a cell surface protein.

[0108] In another preferred embodiment, the artificially designed protein vector is a protein vector designed from scratch based on artificial intelligence.

[0109] In another preferred embodiment, the bacterial molecular injector is an extracellular contractile injection system from bacteria.

[0110] In another preferred embodiment, the extracellular contractile injection system has a recognition domain.

[0111] In another preferred embodiment, the recognition domain has an artificially modified site.

[0112] In another preferred embodiment, the artificially modified site improves the targeting of the bacterial molecular injector.

[0113] In another preferred embodiment, the degradation of the target protein is carried out by ubiquitination of the target protein.

[0114] In another preferred embodiment, the degradation of the target protein regulates the abundance of the target protein.

[0115] In another preferred embodiment, the degradation target protein controls the cellular autophagy process.

[0116] In another preferred embodiment, the target protein is selected from the following group: cancer-related proteins, neurodegenerative disease-related proteins, or other disease-related proteins.

[0117] In another preferred embodiment, the target cells are selected from the following group: tumor cells, neural cells, or other disease-related cells.

[0118] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0119] Figure 1 The results of ITC experiments showing the interaction strength between GN and its related mutants and green fluorescent protein.

[0120] Figure 2 The results of an in vitro ubiquitination experiment to detect the ubiquitination ability of GN and its mutants on green fluorescent protein fusion protein (GFPuv-HOIL-1L) are shown.

[0121] Figure 3 The results of in vitro ubiquitination experiments of GN and GN K0M2 are shown.

[0122] Figure 4 Shown is an intracellular degradation experiment performed in a HeLa cell line complemented with AcGFP-ATG16L1 after knockdown of ATG16L1.

[0123] Figure 5 Intracellular degradation experiments performed in wild-type HeLa cell lines are shown.

[0124] Figure 6 The schematic diagram shows the product of "self-cleavage" of the GN recombinant protein constructed on the pCMV-FLAG-mCherry-P2A vector. After cleavage, the final expression in the same ORF forms two independent proteins, among which the expression level of the FLAG-mCherry protein is basically consistent with that of the GN recombinant protein. At the same time, since the FLAG-mCherry protein is relatively stable in the cell, it can be used as an internal reference. Finally, the ratio of the protein content of the GN recombinant protein to the protein content of the FLAG-mCherry protein in the cell (GN / FLAG-mCherry) can be used to compare the stability between different mutants under certain conditions.

[0125] Figure 7 Comparison of the abundance of recombinant proteins within cells over time is shown.

[0126] Figure 8 The schematic diagram of the mechanism of action of the recombinant protein in the present invention is shown. The target protein is specifically recognized by the targeting domain of the recombinant protein, and then the target protein is ubiquitinated by the catalytic domain of the recombinant protein. Finally, the target protein modified with the K48-type polyubiquitin chain is transferred to the proteasome for degradation, while the recombinant protein is released and enters the next catalytic cycle.

[0127] Fig. 9 The plasmid map corresponding to the lentiviral packaging vector used in the present invention is shown. DETAILED DESCRIPTION

[0128] Based on the previous extensive and in-depth research on the NEL domain in the E3 effector protein IpaH1.4 of Shigella flexneri, the inventors have developed a class of protein chimeras with novel and unique functions, which have the ability to specifically recognize target substrate proteins and modify them with K48-type polyubiquitin chains; and by performing site-directed mutagenesis on the protein chimeras, their activity and stability are improved. The protein chimeras can target target proteins in cells, and expressing the proteins in cells can simply and efficiently achieve precise regulation of the levels of target proteins in cells. In addition, in the present invention, by integrating the inducible protein chimera gene sequence into the genome of the cell, it can be used to construct a transformed cell line capable of studying related cell physiological processes. On this basis, the present invention is completed.

[0129] Since the interaction between proteins has higher specificity, the targeted protein degradation technology based on targeted protein chimeras in the present invention has lower off-target risk. At the same time, most proteins currently have specific binding motifs or domains, or can be conveniently screened to obtain specific binding nanobodies, or even through artificial intelligence-based protein de novo design and development of targeted binding proteins, so this new technology of targeted protein degradation theoretically has a wider protein targeting range.

[0130] Specifically, the present invention relates to the construction of a class of recombinant proteins and their practical applications. The recombinant protein constructed in the present invention is a bifunctional protein chimera, which is composed of a targeting domain with a targeted substrate and a catalytic domain that catalyzes the ubiquitination modification of the substrate, wherein the targeting domain can be but is not limited to a naturally occurring protein motif, domain that interacts with the substrate protein, or a non-natural binding protein obtained by de novo protein design; the catalytic domain is composed of the NEL domain from the Shigella E3 effector protein IpaH1.4, and its N-terminus or C-terminus has a more flexible connection region. The recombinant protein constructed by the present invention is designed to be able to modify the target protein in the cell with a K48-type polyubiquitin chain and induce its degradation via the ubiquitin-proteasome pathway, thereby achieving simple and efficient controllable regulation of the target protein level in the cell.

[0131] In addition, the present invention provides a structure-based evolutionary strategy that can be used to optimize protein-based targeted protein degradation technology, by introducing the analysis of the strength of mutual binding with the substrate to optimize the ubiquitination ability of the protein chimera to the substrate; by reducing the possible self-ubiquitination sites on the targeted protein chimera itself through mutation to improve its stability. The protein chimera optimized based on these two points shows higher activity and stability in cells, and has better application prospects.

[0132] It should be understood that the specific methods and experimental conditions of the present invention are described below in various degrees of detail to provide a substantial understanding of the present invention. The definitions of certain terms used in this specification are provided below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0133] the term

[0134] As used herein, the terms "target protein-specific recognition module", "targeting domain" or "targeting module" are used interchangeably and refer to a polypeptide region covalently attached to the NEL domain in a recombinant protein, which enables the recombinant protein to have the ability to target a target protein or substrate compared to surrounding locations, cells and / or tissues. The targeting domain in the present invention is derived from a naturally occurring protein motif, protein domain, or antibody that interacts with a potential substrate, or a binding protein obtained by de novo protein design. The protein targeted by the targeting domain may be a conventional protein tag, a domain of an endogenous protein, or a specific amino acid sequence. The design scheme of the binding protein designed from scratch includes but is not limited to computer theoretical calculations based on artificial intelligence and in vitro experimental screening and evolution.

[0135] As used herein, the term "nanoantibody" is derived from the variable region of the heavy chain of camelid antibodies, which has many advantages such as small molecular weight, good stability, and high specificity, and has been widely used in the medical field and biochemical research. The nanoantibody used in the present invention is GFPenhancer, which is a nanoantibody that can efficiently bind to green fluorescent protein GFP, and its typical amino acid sequence is shown in SEQ ID NO: 1, and the typical nucleotide sequence encoding the sequence is shown in SEQ ID NO: 5.

[0136] As used herein, the terms "recombinant protein", "protein chimera", "E3 enzyme chimera", "NEL E3 enzyme chimera" and "chimera" are used interchangeably and refer to a protein having a structure shown in Formula I or Formula II, that is, a recombinant protein comprising a target protein-specific recognition module and a NEL domain.

[0137] As used herein, the term "target protein" or "substrate" refers to a protein that directly or indirectly interacts with the targeting domain in the recombinant protein, which can be covalently linked to a K48-type ubiquitin chain and thus recognized and degraded by the proteasome.

[0138] As used herein, the term "degradation" or "hydrolysis" or "ubiquitin-proteasome pathway" refers to the main pathway for intracellular protein degradation. This pathway can be summarized as three steps: free ubiquitin is activated by ubiquitin activating enzyme E1, ubiquitin transfer mediated by ubiquitin conjugating enzyme E2, and ligation mediated by ubiquitin ligase E3. Finally, it is modified to the protein to be degraded and forms a K48-type ubiquitin chain tag, which is then recognized and hydrolyzed by the proteasome.

[0139] As used herein, the term "E3 ubiquitin ligase" or "E3 enzyme" or "E3 ligase" or "E3" refers to an E3 ubiquitin ligase with ubiquitin ligation activity in the ubiquitin-proteasome pathway, which can link ubiquitin to a target protein or substrate to form a K48-type ubiquitin chain, thereby allowing the target protein or substrate modified with the K48-type ubiquitin chain to be recognized and degraded by the proteasome.

[0140] As used herein, the terms "ubiquitination" and "autoubiquitination" refer to the process of attaching ubiquitin protein to lysine residues of substrate proteins. Ubiquitination of substrate proteins such as peptides or proteins can serve as a signal for their targeting to the proteasome complex and ultimately lead to their degradation in the cell. The process in which a protein or protein composition with E3 ligase activity attaches itself to ubiquitin protein is autoubiquitination.

[0141] As used herein, the terms "Trx" and "HA" are the names of tag proteins, both of which are commonly used tag proteins. "Trx6×his" means that Trx tag is followed by 6 histidines, which is used for protein purification, i.e., purification tag.

[0142] As used herein, the representative GN XX or AN XX involved in the present invention has the following protein structure: GN XX or AN XX, wherein G represents the nanobody GFP enhancer, N represents the NEL domain of IpaH1.4, A represents the WBS1 motif of ATG16L1 (which can specifically recognize WIPI2), "-" represents a peptide chain connecting the NEL structure and the LRR domain on IpaH1.4 (with a more flexible spatial structure), and XX represents the code for none or a mutant.

[0143] The mutant code for GN XX can be M1, M2, M3, M4, K0, K0M2, K0M2K0, wherein M1 represents the mutation of R35F on GFP enhancer; M2 represents the mutation of R35F and F102R on GFP enhancer; M3 represents the mutation of N99F, F102R and E103R on GFP enhancer; M4 represents the mutation of R35F, N99F, F102R and E103R on GFP enhancer; K0 represents the mutation of replacing all lysine with arginine in GFP enhancer; K0M2 includes all mutations of K0 and M2; K0M2K0 further replaces all lysine residues on the NEL domain with arginine on the basis of including all mutations of K0 and M2. The mutant code for AN XX may be K0, where K0 represents a mutation in which all lysines are replaced by arginines in the WBS1 motif of ATG16L1.

[0144] The targeting module of the recombinant protein contains artificially introduced mutations, whose functions include but are not limited to changing the affinity for the target protein and reducing the ubiquitination modification of the recombinant protein itself.

[0145] As mentioned above, the mutants are all rationally designed based on the structure. The verification methods for the design include computer calculation verification and biochemical experimental verification and other means. It can be further verified or optimized by methods including but not limited to single-point saturation mutation.

[0146] The present invention includes methods for constructing and optimizing recombinant proteins, including but not limited to isothermal titration calorimetry to determine the binding strength between the substrate and the targeting domain, in vitro ubiquitination experiments to determine the ubiquitination level of the recombinant protein on the substrate, and amino acid sequence modification of ubiquitination sites based on protein structure and mass spectrometry analysis.

[0147] The present invention relates to a recombinant protein that can degrade endogenous proteins in cells. For example, based on known protein interactions, the above-mentioned construction and optimization of recombinant proteins are adopted to finally evolve a recombinant protein AN K0 that can be stably expressed in cells. The targeting binding domain of the recombinant protein comes from the amino acid residues 207 to 236 of the protein ATG16L1, which is a motif that has been reported to bind to the autophagy-related protein WIPI2b. The expression of this recombinant protein in cells can achieve specific degradation of endogenous WIPI2b.

[0148] The present invention relates to a polynucleotide sequence or coding sequence capable of expressing the recombinant protein, which includes but is not limited to the polynucleotide sequence or coding sequence shown in SEQ ID NO: 5-8, and a polynucleotide sequence or coding sequence formed by changing one or more nucleotides in the sequence shown in SEQ ID NO: 5-8, wherein the changed sequence can still express the amino acid sequence shown in SEQ ID NO: 1-4. Preferably, the change includes a synonymous mutation, which refers to a gene mutation that does not change the amino acid sequence of the protein.

[0149] The present invention relates to a system for controllable regulation of cell physiological processes and a cell line integrated into the system, including but not limited to an inducible recombinant protein expression system based on the Tet-on system and a cell line containing such a system in the genome. For example, a recombinant protein GN K0M2 containing a nanobody that can specifically bind to green fluorescent protein is constructed and optimized using the above-mentioned method for constructing and optimizing recombinant proteins. The antibody is cloned into a Tet-on system plasmid and integrated into a HeLa cell line that can stably express AcGFP-ATG16L1 through a lentiviral vector. Effective control of the cellular macroautophagy process can be achieved by additionally applying doxycycline.

[0150] The present invention includes a method and detection means for optimizing the stability of recombinant proteins, such as reducing the self-ubiquitination modification of recombinant proteins by mutating lysine residues that do not have substrate targeting function or are not related to catalytic activity into arginine residues on the recombinant proteins. The principle is to avoid the ubiquitination modification of the highly active E3 ubiquitin ligase activity of the recombinant proteins on their own lysine residues, thereby reducing the degradation of the recombinant proteins in cells. The detection means corresponding to the optimization method include in vitro self-ubiquitination level detection and intracellular protein level detection, including but not limited to detection means such as immunoblotting.

[0151] The techniques used in the present invention to construct relevant recombinant proteins include various conventional techniques in molecular biology, structural biology, cell biology and genetic engineering.

[0152] The present invention can be used to regulate various cellular processes within cells, including but not limited to the cellular autophagy process.

[0153] The present invention is applicable to eukaryotic cells containing the ubiquitin-proteasome system, including but not limited to human cervical cancer cells.

[0154] The present invention rationally designs and experimentally evaluates the above-mentioned recombinant protein based on in vitro ubiquitination experimental results, mass spectrometry data results, isothermal calorimetric titration and other in vitro biochemical data. The recombinant protein can be understood as an artificial protein obtained by directed evolution with multiple functions such as basic research and practical application research.

[0155] NEL domain

[0156] As used herein, the NEL domain of the recombinant protein involved in the present invention is the 274th amino acid residue to the 575th amino acid residue of the effector protein IpaH1.4 of Shigella flexneri. Artificial mutations can be introduced into the NEL domain, including but not limited to mutating lysine to arginine or mutating serine / threonine to alanine to reduce the self-ubiquitination of the recombinant protein. The NEL domain contains a natural flexible region at its N-terminus and C-terminus, and the way it is connected to the targeting domain can be either through the N-terminus or through the C-terminus. The cysteine ​​residue at position 368 of the NEL domain is a catalytic active site, and an inactivating mutation can be performed on it for experiments to study functions that are not dependent on its E3 enzyme activity. The mutation includes but is not limited to mutation to alanine.

[0157] Tet-on system

[0158] The Tet-on system is a tetracycline-induced gene on system. In this system, tetracycline or its derivative doxycycline controls the transcription activator rtTA to bind to the Tet response element TRE, thereby regulating the expression of downstream genes. When there is no inducer, rtTA does not bind to TRE, blocking the expression of downstream genes; when there is an inducer such as doxycycline, the combination of doxycycline and rtTA binds to TRE, thereby starting the expression of downstream genes.

[0159] The present invention utilizes the Tet-on system as a protein induction expression system, and the technology used is well known in the art, and the specific process is as follows: the recombinant protein is constructed on the pLVX-TetOne plasmid by means of molecular cloning, and an HA tag is fused to the N segment of the recombinant protein for detecting the expression of the recombinant protein; the virus packaging plasmid is co-transfected into HEK293T cells, and the collected virus particles are used to infect the cell line to be studied, and the cells in which the recombinant protein is integrated into the genome are screened out by resistance tags; the expression of the recombinant protein is induced by adding different concentrations of doxycycline to the culture medium, and the experimental results are observed by means of immunoblotting or cell fluorescence imaging. The pLVX-TetOne plasmid is a commonly used lentiviral vector. The HEK293T cells are derived from human embryonic kidney cells and are a cell line commonly used in experiments. The Tet-on system is a type of protein induction expression system, and other suitable protein expression systems can also be used to regulate the recombinant protein described in the present invention.

[0160] Carrier

[0161] As used herein, the terms "viral vector", "liposome vector", "virus-like protein vector", "exosome vector", "artificially designed protein vector", and "bacterial molecular injector" are several common or promising delivery systems for genetic materials or proteins.

[0162] As used herein, the term "viral vector" includes retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated virus vectors. Among them, retroviruses and lentiviruses are both retroviruses, with single-stranded RNA as the genome, reverse transcribed into cDNA and then integrated into the host cell genome. Compared with general retroviruses, there are regulatory genes in lentiviruses, which can effectively infect cells in the division phase and the resting phase, and have the advantages of long-term stable expression of exogenous genes and higher biosafety, and are often used as delivery vectors. Adenovirus is a double-stranded DNA virus with a wide range of infection and large exogenous gene loading capacity, and is also commonly used as a delivery vector. Adeno-associated virus is a type of single-stranded linear DNA virus. Only with the assistance of helper viruses such as adenovirus or herpes virus can the host produce infectious adeno-associated virus. Compared with adenovirus, adeno-associated virus has low immunogenicity and a long expression time, which is suitable for in vivo animal experiments.

[0163] As used herein, the term "liposome carrier" includes lipid nanoparticles, lipid complexes, or lipid polymer complexes. The liposome is an artificial membrane, which is a spherical particle with a double layer of lipid molecules. Hydrophilic drugs can be enclosed in the hydrophilic internal region of the liposome, and hydrophobic drugs can be encapsulated in the hydrocarbon chain region of the lipid bilayer. Lipid nanoparticles do not have a hydrophilic cavity structure, but form a multilayer core structure inside the lipid layer through the electrostatic complexation between cationic phospholipids and the negatively charged nucleic acid material encapsulated.

[0164] As used herein, the term "virus-like protein vector" is expressed by a virus-like genetic element "retrotransposon". The protein expressed by this type of retrotransposon can form a virus-like structure and participate in the transfer of RNA between cells. By modifying the virus-like protein expressed by the relevant retrotransposon, such as PEG10, it can selectively package RNA and target specific cells, so that it can fuse with the cells and complete the delivery process.

[0165] As used herein, the term "exosome carrier" refers to a membrane vesicle secreted by cells. Compared with artificial membrane carriers such as liposomes, it has a more complex lipid bilayer, contains a variety of lipids, proteins and carbohydrates, can carry a variety of components, has low immunogenicity, high physical and chemical stability, and high tissue penetration ability, and can be used as a delivery carrier. By modifying the exosomes with external targeting peptides, it can be targeted to specific cells, thereby releasing the recombinant protein described in the present invention.

[0166] As used herein, the term "artificially designed protein carrier" refers to the position of each amino acid residue designed based on biophysical and biochemical principles to obtain a protein carrier with a target structure and function. This process involves protein design. There are currently three protein design strategies: one is structure prediction, that is, the amino acid sequence is known, and the structure is predicted through backbone sampling and side chain sampling; the second is fixed backbone design, that is, the backbone structure is known, and the sequence is designed through backbone sampling and side chain sampling; the third is de novo design, that is, designing proteins when both the amino acid sequence and the backbone structure are unknown. At present, the de novo design of proteins is usually designed using artificial intelligence such as AlphaFold. Artificially designed protein carriers can be designed with targeting domains, etc. according to needs, so as to target target proteins or cells.

[0167] As used herein, the term "contractible injection system" is a type of cell puncturing nanodevice that can transfer substances such as exogenous nucleic acids or proteins into other cells. The bacterial molecular injector is composed of an outer tube (Sheath) and a baseplate complex (Baseplate), wherein the retractable outer tube uses the baseplate complex to form a fulcrum, and the rigid inner tube (Tube) carrying the effector is punctured into the receptor cell through a contraction and rotation action. Common bacterial molecular injectors include a contractile injection system anchored on the cell membrane represented by the type VI secretion system (T6SS), and an extracellular contractile injection system. The bacterial molecular injector from the contractile injection system can be artificially modified to target the target protein or target cell, thereby releasing the loaded recombinant protein in the present invention to exert its protein degradation effect in a directional manner.

[0168] The main advantages of the present invention include:

[0169] (1) The present invention provides a recombinant protein comprising an artificially modified NEL domain and its application in targeted protein degradation. Based on the structural analysis of related proteins, by rationally mutating specific amino acids in the NEL domain or the targeting domain, a recombinant protein with significantly improved ubiquitination ability and enhanced activity, as well as reduced self-ubiquitination level and enhanced stability is unexpectedly obtained.

[0170] (2) The recombinant protein of the present invention can specifically target the target protein and ubiquitinate it, thereby efficiently achieving controllable regulation of the level of the target protein in the cell.

[0171] (3) The recombinant protein in the present invention can effectively inhibit the cell autophagy process.

[0172] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0173] Example 1: Construction of Targeting Protein Chimera Sequence

[0174] This embodiment demonstrates the construction of a targeted protein chimera sequence. The present invention involves two different plasmid systems that can be expressed in prokaryotic cells (such as Escherichia coli) or eukaryotic cells (such as HeLa cells), and the protein fragments contained in the translation products in the open reading frame (ORF) can be basically divided into three parts, namely Trx6×his-G / AN expressed in prokaryotic cells or HA-G / AN used for expression in eukaryotic cells. Among them, Trx and HA are commonly used tag proteins, 6×his represents the 6 histidines following the Trx tag (used for protein purification, i.e., purification tags), G / A represents the WBS1 motif of GFPenhancer and ATG16L1 and their related mutants, N represents the NEL domain of the E3 effector protein IpaH1.4 of Shigella flexneri, and "-" is a segment of artificial or natural amino acid sequences used to connect various protein fragments. The relevant construction background information is as follows:

[0175] (1) Shigella flexneri is a type of Gram-negative bacteria that mainly uses humans and other primates as hosts. After invading the human intestine, it can cause severe bacillary dysentery. IpaH1.4 is an effector protein secreted by Shigella flexneri through the T3SS secretion system. Its structural characteristics are that the N-terminus has an LRR domain that can bind to the substrate, and the C-terminus has a NEL domain that can mediate ubiquitination modification and is a bacterial E3 ligase. In the present invention, the inventors constructed a series of targeting protein chimeras with novel and unique functions, wherein the amino acid sequence of the wild-type NEL domain is as follows: LADAVTAWFPENKQSDVSQIWHAFEHEEHANTFSAFLDRLSDTVSARNTSGFREQVAAWLEKLSASAELRQQSFAVAADATESCEDRVALTWNNLRKTLLVHQASEGLFDNDTGALLSLGREMFRLEILEDIARDKVRTLHFVDEIEVYLAFQTMLAEKLQLSTAVKEMRFYGVSGVTANDLRTAEAMVRSREENEFTDWFSLWGPWHAVLKRTEADRWAQAEEQKYEMLENEYSQRVADRLKASGLSGDADAEREAGAQVMRETEQQIYRQLTDEVLALRLSENGSNHIA (SEQ ID NO: 3)

[0176] The corresponding nucleotide sequence is (including other nucleotide sequences that can be used to translate into the above amino acid sequence):

[0177] CTGGCTGATGCCGTGACAGCATGGTTCCCGGAAAACAAACAATCTGATGTATCACAGATATGGCATGCTTTTGAACATGAAGAGCATGCCAACACCTTTTCCGCGTTCCTTGACCGCCTTTCCGATACCGTCTCTGCACGCAATACCTCCGGATTCCGTGAACAGGTCGCTGCATGGCTGGAAAAACTCAGTGCCTCTGCGGAGCTTCGACAGCAGTCTTTCGCTGTTGCTGCTGATGCCACTGAGAGCTGTGAGGACCGTGTCGCGCTCACATGGAACAATCTCCGGAAAACCCTCCTGGTCCATCAGGCATCAGAAGGCCTTTTCGATAATGATACCGGCGCTCTGCTCTCCCTGGGCAGGGAAATGTTCCGCCTCGAAATTCTGGAGGACATTGCCCGGGATAAAGTCAGAACTCTCCATTTTGTGGATGAGATAGAAGTCTACCTGGCCTTCCAGACCATGCTCGCAGAGAAACTTCAGCTCTCCACTGCCGTGAAGGAAATGCGTTTCTATGGCGTGTCGGGAGTGACAGCAAATGACCTCCGCACTGCCGAAGCTATGGTCAGAAGCCGTGAAGAGAATGAATTTACGGACTGGTTCTCCCTCTGGGGACCATGGCATGCTGTACTGAAGCGTACGGAAGCTGACCGCTGGGCGCAGGCAGAAGAGCAGAAGTATGAGATGCTGGAGAATGAGTACTCTCAGAGGGTGGCTGACCGGCTGAAAGCATCAGGTCTGAGCGGTGATGCGGATGCGGAGAGGGAAGCCGGTGCACAGGTGATGCGTGAGACTGAACAGCAGATTTACCGTCAGTTGACTGACGAGGTACTGGCCCTGCGATTGTCTGAAAACGGCTCAAATCATATCGCA(SEQ ID NO:7)

[0178] For the linker segment between G / A and N, in the present invention, the natural protein linker segment between the LRR domain and the NEL domain of IpaH1.4 is usually selected, and its amino acid sequence is as follows:

[0179] NSATISAPEHS (SEQ ID NO:4)

[0180] The corresponding nucleotide sequence is (including other nucleotide sequences that can be used to translate into the above amino acid sequence):

[0181] AATTCTGCCACAATTTCCGTCCAGAACACTCC(SEQ ID NO:8)

[0182] (2) GFPenhancer is a nanobody that can efficiently bind to green fluorescent protein GFP. Nanobodies are derived from the variable region of the heavy chain of camelid antibodies and have many advantages such as small molecular weight, good stability, and high specificity. They have been widely used in the medical field and biochemical research. In the present invention, the wild-type amino acid sequence of the nanobody we used is as follows:

[0183] HMAQVQLVESGGALVQPGGSLRLSCAASGFPVNRYSMRWYRQAPGKEREWVAG MSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKPEDTAVYYCNVNVGFEYWGQ GTQVTVS(SEQ ID NO:1)

[0184] The corresponding nucleotide sequence is (including other nucleotide sequences that can be used to translate into the above amino acid sequence):

[0185] CATATGGCCCAAGTTCAGCTGGTTGAGAGTGGTGGTGCGCTGGTTCAGCCGGGTGGTAGTCTGCGTCTCAGTTGCGCCGCGAGCGGTTTCCCGGTGAATCGCTACAGTATGCGCTGGTACCGTCAAGCCCCGGGCAAAGAACGCGAATGGGTTGCCGGTATGAGTAGTGCCGGTGAT CGCAGCAGCTATGAAGACAGCGTGAAAGGCCGCTTTACCATCAGCCGCGATGATGCGCGCAATACCGTGTACCTCCAGATGAACAGTCTGAAGCCAGAGGATACCGCCGTGTACTACTGCAACGTGAACGTGGGCTTCGAATATTGGGGTCAAGGCACGCAAGTTACCGTTAGT(SEQ ID NO:5)

[0186] (3) The WBS1 motif of ATG16L1 is the WIPI2b binding site 1. In the initial stage of cellular autophagy, this motif is involved in the interaction between ATG16L1 and WIPI2b. In the present invention, the wild-type amino acid sequence of the WBS1 motif used by us is as follows:

[0187] AENEKDSRRRQARLQKELAEAAKEPLPVEQ(SEQ ID NO:2)

[0188] The corresponding nucleotide sequence is (including other nucleotide sequences that can be used to translate into the above amino acid sequence) (SEQ ID NO: 6):

[0189] GCAGAGAATGAAAAAGACTCCAGGAGGCGGCAAGCCCGGCTGCAGAAAGAGC TTGCAGAAGCAGCAAAGGAACCTCTACCAGTCGAACAG (SEQ ID NO: 6)

[0190] (4) For prokaryotically expressed Trx6×his-G / AN, the linker between Trx6×his and G / A contains a HRV 3C restriction site, which can be treated with the corresponding HRV 3C enzyme and then the Trx6×his portion can be removed. The sequence of the HRV 3C site is a commonly used amino acid sequence:

[0191] LEVLFQGP (SEQ ID NO: 9) (cleavage site is located between Q and G)

[0192] Example 2: Construction of prokaryotic plasmids and protein expression and purification

[0193] This example relates to the construction of prokaryotic plasmids and protein expression and purification. For prokaryotic expression of GN, AN or their mutants, they were constructed on the pRSF-Duet vector, and connected to the first multiple cloning site after restriction endonuclease treatment. All plasmids were sequenced after construction to confirm that the nucleotide sequence was correct.

[0194] (1) The mutation was constructed by circular PCR, that is, upstream and downstream primers containing the sequence after mutation were designed at the position of the point mutation, and the product containing the point mutation was obtained by PCR amplification. Subsequently, the wild-type plasmid used as a template was digested with DpnI. The final product can be directly used for subsequent plasmid transformation.

[0195] (2) The DH5α strain was used for amplification of related plasmids, and the BL21 (DE3) strain was used for expression of related proteins.

[0196] (3) For the BL21 (DE3) strain expressing the protein, add it to LB medium containing kanamycin one day before expression and culture it at 37°C, 200 rpm overnight; on the day of expression, take 10 ml of the overnight culture solution and add it to 1 L LB medium containing kanamycin, and culture it at 37°C, 200 rpm until the OD 600 When the value reached about 0.8, the culture temperature was lowered to 16°C, and IPTG with a final concentration of 100mM was added to induce expression overnight; the next morning, the bacteria expressing the target protein were harvested by centrifuge, crushed under high pressure, and purified using Ni column affinity chromatography; all proteins were further purified using size exclusion chromatography after Trx6×his was removed by HRV 3C treatment, and the protein purity was finally confirmed by SDS PAGE experiment.

[0197] Example 3: Construction of eukaryotic plasmids and cell lines

[0198] This example relates to the construction of eukaryotic plasmids and cell lines. For eukaryotic expression of GN, AN or their mutants, they were constructed on pCMV vectors, and the genes used were all from the enzyme digestion products of prokaryotic plasmids, and then connected to the corresponding restriction endonuclease positions on the multiple cloning site. After construction, all plasmids were sequenced to confirm that the nucleotide sequences were correct.

[0199] (1) The vectors of eukaryotic plasmids with specific functions are different. For the inducible expression of GN K0M2 and ANK0, we constructed them on the pLVX-TetOne vector; in the experiment to detect the stability of the targeted protein chimera, the corresponding GN and its mutants were constructed on the pCMV-FLAG-mCherry-P2A vector, where FLAG is a commonly used protein tag, mCherry is a commonly used red fluorescent protein, and P2A is a self-cleaving peptide. The targeted protein chimera located behind P2A will be separated from the front P2A during the intracellular translation process, thereby achieving two independent protein products in one ORF.

[0200] (2) All experiments were conducted using the HeLa cell line and its corresponding stable cell lines.

[0201] (3) The plasmid was transfected by liposome transfection. The cells were plated at a cell density of 25% one day before transfection. On the day of transfection, the culture medium was aspirated and the culture dish was rinsed once with PBS to reduce FBS residue. Then, lipo2000 was mixed with the plasmid and the cells were transfected in serum-free culture medium. After 4 hours, the serum-free culture was replaced with serum-containing culture medium and cultured for another 24 hours. The cells can be processed according to the purpose of the experiment.

[0202] (4) For the construction of inducible expression of GN K0M2 and AN K0 cell lines, the gene was integrated into the cell genome by lentiviral packaging vectors. The specific operation steps are as follows: before infection, 293T cells were used to assemble virus particles, and the virus titer was increased to the required concentration using a virus concentration reagent; the cell density was 5% on the day before virus infection. For infection experiments with only one type of virus particles, five wells in a six-well plate were required, of which three were used to test the working concentration required for screening antibiotics (test wells), one well was used for virus infection (experimental well), and the other was reserved as a negative control that was not infected (control well); on the day of transfection, the virus particles were diluted with an appropriate volume of serum-containing culture medium and 6 μg / mL of polybrene was added to the experimental wells. After 24 hours of infection, the virus-containing culture medium was replaced with a normal serum-containing culture medium; referring to the concentration in the test well that just caused the cells to die after 24 hours of puromycin treatment, an appropriate concentration of puromycin was used to screen positive clones (mixed clones); using Western Blot ... Blot was used to detect the protein expression after doxycycline induction.

[0203] Example 4: Mutant design and screening

[0204] This embodiment relates to the design and screening of recombinant protein mutants. The present invention designs mutants of targeted protein chimeras based on two main goals, namely, improving the ability of targeted protein chimeras to ubiquitinate substrates and reducing the ability of targeted protein chimeras to ubiquitinate themselves.

[0205] (1) Previous studies have shown that there is a certain relationship between the ubiquitination ability of E3 ligases on substrates and the strength of their binding to the substrates. Based on this, the present invention focuses on the binding interface between GN and the target protein GFP. Combining existing crystallographic data and analysis of the protein-protein interaction interface, mutations were specifically designed for several key amino acid residues on the nanobody GFP enhancer located on the interaction interface, which successfully weakened the binding strength of the nanobody with the substrate. The interaction strength of the mutants with GFP ranges from nM level, μM level to no obvious interaction.

[0206] (2) The interaction strength between GN and its mutants and green fluorescent protein was verified by isothermal titration calorimetry (ITC). The specific steps are as follows: the corresponding mutant proteins and green fluorescent protein were purified by the above two protein purification methods; the protein sample concentration for ITC experiments is generally required to be 300 μM for needle sample protein and 30 μM for pool protein. In the present invention, green fluorescent protein was used as the needle sample, GN and its mutants were used as pool samples, and green fluorescent protein was used to titrate the protein buffer for background subtraction; the protein buffer composition was 20 mM Tris-HCl (pH = 7.5), 100 mM NaCl and 1 mM DTT; data processing and analysis used the relevant analysis software provided by Malvern.

[0207] (3) The ubiquitination ability of GN and its mutants on green fluorescent protein fusion protein was characterized by an in vitro ubiquitination experiment. The specific steps are as follows: the relevant proteins were purified using the above two protein purification methods; the complete in vitro ubiquitination reaction system contained a final concentration of 1 μM GN or its mutants, 10 mM ATP, 10 mM MgCl2, 200 nM ubiquitin activating enzyme UBA1, 400 nM ubiquitin conjugating enzyme UBE2D1 and 100 μM ubiquitin monomer, and the protein buffer composition was 20 mM Tris-HCl (pH = 7.5), 100 mM NaCl and 1 mM DTT; the reactions were all carried out at 37°C, and the loading buffer (containing SDS and DTT) was added to terminate the reaction after sampling at the corresponding time points; the reaction results were detected by SDS PAGE experiment to detect the reduction of ubiquitin, and the ubiquitination level of green fluorescent protein fusion protein was detected by Western Blot experiment (using GFP antibody).

[0208] (4) Since it was accidentally discovered during the research process that GN and AN exhibited self-ubiquitination in the in vitro ubiquitination reaction, in order to weaken or eliminate this phenomenon, based on the existing crystal structure data, the present invention constructed GN K0 and AN K0 mutants. The specific principle is to determine that all lysine residues on the GFP enhancer part of GN and the WBS1 sequence of AN do not participate in the interaction with the substrate. Therefore, in theory, as long as all lysine residues of GN and AN are mutated to arginine residues, their self-ubiquitination levels can be effectively reduced. The self-ubiquitination effects of the early mutants were detected by in vitro ubiquitination experiments. Further intracellular detection was performed by plasmid transient transfection. GN and AN with HA tags and their K0 mutants were overexpressed in HeLa cells. After 24 hours of expression in a cell culture incubator, the intracellular protein level was detected by Western Blot (using HA antibody).

[0209] Example 5: Detection of the interaction intensity between GN and its related mutants and green fluorescent protein

[0210] This example involves using isothermal titration calorimetry to determine the binding strength between a substrate and a targeting domain. The specific steps of the isothermal titration calorimetry are shown in Example 4.

[0211] The results are as follows Figure 1 As shown, GFPuv is used as a needle sample, and GN and its mutants are used as pool samples. By increasing the number of point mutations on GFPenhancer, it can be clearly observed that the value of the protein dissociation equilibrium constant gradually increases, which indicates that the interaction between GFP protein and GFPenhancer gradually weakens. In addition, considering that there are many GFP protein variants in practical applications, further testing is necessary for the compatibility of GFP variants. The GFPenhancer used in the present invention also has a high binding strength for the commonly used AcGFP.

[0212] Example 6: In vitro ubiquitination detection of GN and its mutants on the ubiquitination ability of green fluorescent protein fusion protein (GFPuv-HOIL-1L)

[0213] This example involves using an in vitro ubiquitination assay to detect the ubiquitination level of a green fluorescent protein fusion protein. The specific steps of the in vitro ubiquitination assay are shown in Example 4.

[0214] The results are as follows Figure 2 As shown, the lighter the substrate band and the darker the ubiquitinated substrate band above, the stronger the ubiquitination ability of GN or its mutants to the substrate. In the present invention, the ubiquitination ability of GN M2 to the substrate is significantly improved compared with GN.

[0215] Example 7: In vitro ubiquitination detection of the ubiquitination ability of GN and mutant GN K0M2

[0216] This example relates to the use of an in vitro ubiquitination assay to detect the in vitro ubiquitination results of GN and GN K0M2. The specific steps of the in vitro ubiquitination assay are shown in Example 4.

[0217] The results are as follows Figure 3 As shown, the introduction of K0 mutation significantly weakened the self-ubiquitination ability of GN K0M2. The GN K0M2 band at 150 minutes was significantly denser than the GN band, indicating that it itself was not significantly ubiquitinated. In addition, the introduction of M2 mutation further improved its ubiquitination ability for substrates. In summary, the activity and stability of the GN K0M2 mutant constructed by the present invention were significantly improved compared with GN.

[0218] Example 8: Mutant GN K0M2 can inhibit macroautophagy by degrading AcGFP-ATG16L1

[0219] This embodiment involves constructing a HeLa cell line with ATG16L1 knockout and detecting the degradation level of AcGFP-ATG16L1 in the cell line using an in vitro ubiquitination experiment. The preparation method of the HeLa cell line with ATG16L1 knockout: using the CRISPR-Cas9 system to base edit the exons in the region where ATG16L1 is located in the genome, so that it produces a frameshift mutation to silence the expression of ATG16L1; Gong Xinyu, a postdoctoral fellow in the research group of Pan Lifeng of the Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, provided the HeLa cell line with ATG16L1 knockout. The specific steps of the in vitro ubiquitination experiment are shown in Example 4.

[0220] The results are as follows Figure 4 As shown, in the intracellular degradation experiment conducted in the HeLa cell line in which AcGFP-ATG16L1 was complemented after ATG16L1 was knocked out, the degradation of the intracellularly expressed AcGFP-ATG16L1 protein can be achieved within 24 hours by inducing the expression of the recombinant protein GN K0M2 using doxycycline (Dox). After the cells were treated with amino acid-deficient culture medium and the autophagy inhibitor Bafilomycin A1 (Baf-A1), it was found that the recombinant protein GN K0M2 caused the degradation of AcGFP-ATG16L1, thereby preventing the macroautophagy process of the cells from proceeding normally.

[0221] Example 9: Mutant AN K0 can regulate the abundance of endogenous WIPI2b protein in cells

[0222] This embodiment involves using an intracellular degradation experiment to detect the protein abundance of WIPI2b in cells within 24 hours. The specific steps of the intracellular degradation experiment are to treat a HeLa cell line that can be induced to express AN KO with different concentrations of doxycycline (Dox), and the construction method of the cell line is shown in Example 3; then, after 24 hours, Western Blot is used to detect the content of WIPI2b in the cell (using WIPI2b antibody).

[0223] The results are as follows Figure 5 As shown, in the intracellular degradation experiment carried out in the wild-type HeLa cell line, the abundance of endogenous WIPI2b protein in the cell can be regulated within 24 hours by inducing the expression of the recombinant protein AN K0 using different concentrations of doxycycline.

[0224] Example 10: Detection and comparison of autoubiquitination levels of mutant GN K0M2K0

[0225] This example involves using an intracellular degradation experiment to detect the protein abundance of GN K0M2K0 and GO K0M2, thereby indirectly reflecting their self-ubiquitination levels. The specific steps of the intracellular degradation experiment are shown in Example 3.

[0226] The results are as follows Figure 7 As shown, over time, the protein abundance of GN K0M2K0 was higher than that of GN K0M2. The above results indicate that the recombinant protein GN K0M2K0 obtained by mutating all lysine residues in the catalytic domain of GN K0M2 to arginine residues has a higher protein abundance, which indicates that it has a lower level of self-ubiquitination and can exist more stably in cells.

[0227] discuss

[0228] At present, the research in this field is in its infancy, and there are few related work reports. It has broad development and application prospects. The subsequent development plans for this technology are as follows:

[0229] 1. The first is to broaden the types of targetable substrate proteins, that is, to obtain more targeted binding proteins that can specifically target the target protein. The designed solutions include but are not limited to the following points:

[0230] (1) Based on artificial intelligence, a series of binding proteins that can target proteins related to major human diseases (such as cancer and neurodegenerative diseases) are designed, their interactions are verified through in vitro experiments, and the optimal de novo designed proteins are obtained through high-throughput screening technology and directed evolution technology.

[0231] (2) Through traditional nanoantibody screening and preparation methods, nanoantibodies with high affinity for pathogenic proteins are obtained.

[0232] (3) By analyzing natural protein-protein interactions, a series of motifs or domains that can specifically bind to the target protein are optimized with the help of relevant in vitro biochemical experiments and directed evolution technology.

[0233] 2. The second step is to develop relevant delivery strategies, that is, to explore the practicability of the invention in organisms. It is expected that the solutions that can be tried include but are not limited to the following:

[0234] (1) DNA delivery is used to deliver genes expressing recombinant proteins using viral vectors (such as adeno-associated virus (AAV)) to form infectious viral particles in vitro and deliver them to specific organs or tissues in the body. The corresponding recombinant proteins are then transcribed and translated into target cells.

[0235] (2) The messenger RNA (mRNA) is delivered by lipid nanoparticle (LNP) delivery system or human retrovirus-like protein (PEG10 and other proteins) to deliver the prepared mRNA encoding the recombinant protein, and then the corresponding recombinant protein is directly translated into the target cells.

[0236] (3) Develop new protein delivery strategies, such as secretion systems similar to those used by pathogenic bacteria to secrete effector proteins, or exosome carriers modified with targeting peptides or artificially designed protein carriers to directly deliver high-purity recombinant proteins into specific target cells.

[0237] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A recombinant protein, characterized in that The recombinant protein has a structure shown in Formula I or Formula II: Z0-Z1-L-Z2(I); Z0-Z2-L-Z1(II); In the formula, Z0 is selected from the group consisting of none, tag, restriction site, or a combination thereof; Z1 is the target protein-specific recognition module; Z2 is the NEL domain; L is none or a link sequence; "-" indicates the peptide bond connecting the above sequences; Wherein, the Z1 specifically recognizes the target protein, and the Z2 ubiquitinates the target protein.

2. The recombinant protein according to claim 1, characterized in that The Z1 is selected from the group consisting of an antibody, a protein motif, a protein domain, or a combination thereof.

3. The recombinant protein according to claim 2, characterized in that The antibody is a nanobody, and the nanobody is an unmutated or mutated nanobody; the amino acid sequence of the mutated nanobody is selected from the following group: (1) the amino acid sequence as shown in SEQ ID NO:1 and having the following mutations in SEQ ID NO:1: R35F, F102R and mutation of all lysines in the sequence to arginines; (2) the amino acid sequence as shown in SEQ ID NO:1 and having the following mutation in SEQ ID NO:1: R35F; (3) the amino acid sequence as shown in SEQ ID NO:1 and having the following mutations in SEQ ID NO:1: R35F and F102R; (4) the amino acid sequence as shown in SEQ ID NO:1 and having the following mutations in SEQ ID NO:1: N99F, F102R and E103R; (5) the amino acid sequence as shown in SEQ ID NO:1 and having the following mutations in SEQ ID NO:1: R35F, N99F, F102R and E103R; (6) the amino acid sequence as shown in SEQ ID NO:1 and having the following mutations in SEQ ID NO:1: all lysines in the sequence are mutated to arginines; or a combination thereof.

4. The recombinant protein according to claim 2, characterized in that The protein motif is an unmutated or mutated WBS1 motif of ATG16L1; the amino acid sequence of the mutated WBS1 motif of ATG16L1 includes: the amino acid sequence shown in SEQ ID NO: 2 and the following mutation in SEQ ID NO: 2: all lysines in the sequence are mutated to arginines.

5. The recombinant protein according to claim 1, characterized in that The Z2 is the amino acid residues 274 to 575 of the unmutated or mutated Shigella flexneri IpaH1.4; the amino acid sequence of the amino acid residues 274 to 575 of the mutated Shigella flexneri IpaH1.4 is selected from the following group: (a) the amino acid sequence as shown in SEQ ID NO:3 and having the following mutation in SEQ ID NO:3: all lysines in the sequence are mutated to arginines; (b) the amino acid sequence as shown in SEQ ID NO:3 and having the following mutation in SEQ ID NO:3: all serines in the sequence are mutated to alanine; (c) the amino acid sequence as shown in SEQ ID NO:3 and having the following mutation in SEQ ID NO:3: all threonine residues in the sequence are mutated to alanine residues; (d) the amino acid sequence as shown in SEQ ID NO:3 and having the following mutation in SEQ ID NO:3: cysteine ​​at position 368 in the sequence is mutated to alanine; or a combination thereof.

6. A polynucleotide, characterized in that The polynucleotide encodes the recombinant protein according to claim 1.

7. An expression vector, characterized in that: The expression vector contains the polynucleotide according to claim 6.

8. The expression vector according to claim 7, characterized in that The expression vector is selected from the following group: a plasmid vector, a viral vector, or a combination thereof, wherein the viral vector is selected from the following group: a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or a combination thereof.

9. A cell line, characterized in that The cell line contains the expression vector according to claim 7 or the polynucleotide according to claim 6 is integrated into its genome.

10. Use of the recombinant protein according to claim 1, the polynucleotide according to claim 6 or the expression vector according to claim 7, characterized in that: Used to prepare a drug or a pharmaceutical composition, wherein the drug or the pharmaceutical composition is used to degrade the target protein.

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