Protein assembly structure

Abstract

To provide, in one embodiment, a protein assembly structure formed by linking two or more types of modules.SOLUTION: The present disclosure relates, in one embodiment, to a protein assembly structure formed by linking two or more types of modules. The modules are each a homomultimer formed of a fusion protein comprising a protein capable of forming a symmetric homomultimer and a peptide serving as a binding site between modules. The peptide serving as the binding site is capable of reversibly forming a heterodimer, and different modules are linked by the formation of the heterodimer between the peptides.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] The present disclosure relates to a technology for forming two-component protein assemblies formed by linkage using heterodimeric peptides, and more specifically, to a protein assembly structure formed by linkage of two or more types of homomultimers, a method for producing the same, and a kit. [Background technology]

[0002] Biological phenomena are realized by the dynamic (reversible) formation and disassembly of various higher-order assemblies of multiple proteins as needed. Examples of such assemblies include actin filaments and microtubules of the cytoskeleton and capsid proteins that form viruses. These naturally occurring structures possess highly tuned functionality and complexity. Recent advances in computational science and other technologies have made it possible to design artificial protein assemblies comparable to those found in nature. However, most designs to date have focused on the formation of assemblies consisting of a single protein. Although cages, sheets, and three-dimensional crystal structures consisting of two proteins have been proposed (e.g., Non-Patent Documents 1-6), there are still few reported examples. Furthermore, designing protein assemblies that exhibit the flexibility and reversible assembly and disassembly characteristics of naturally occurring higher-order assemblies, such as tubular structures reminiscent of the cytoskeleton, remains an even more challenging task.

[0003] One of the present inventors focused on the symmetry of proteins and succeeded in creating protein units in which cysteines or histidines were placed as connectors at the corners of a tetrameric (C4 symmetry) protein, and linking the protein units by disulfide bonds or coordination to metals to form a two-dimensional protein structure (Non-Patent Document 7). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Fletcher, JM et al. Self-assembling cages from coiled-coil peptide modules. Science 340, 595-599, doi:10.1126 / science.1233936 (2013).

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[0005] The creation of assemblies combining multiple types of protein units remains a major challenge. Although examples of ordered assembly and cooperation of multiple types of proteins are common in nature, designing assemblies with flexibility and reversibility of formation and disassembly closer to those found in nature, as well as higher-order assemblies combining heterogeneous protein units in an ordered manner, remains a major challenge. Among these assemblies, the construction of tubular structures, in particular, has yet to be realized.

[0006] In one aspect, the present disclosure provides a higher-order protein assembly in which proteins are assembled in an ordered manner, a method for producing the same, and a kit. [Means for solving the problem]

[0007] In one aspect, the present disclosure relates to a protein assembly structure formed by linking two or more types of modules, wherein the modules are homomultimers formed by a fusion protein of a protein capable of forming a symmetric homomultimer and a peptide that serves as a binding site between the modules, and the peptide that serves as the binding site is capable of reversibly forming a heterodimer, and different modules are linked by the formation of the heterodimer by the peptide.

[0008] In another aspect, the present disclosure relates to a method for producing a protein assembly structure formed by linking two or more types of modules, the method including: preparing two or more types of modules, wherein the modules are homomultimers formed by fusion proteins of a protein capable of forming a symmetric homomultimer and a peptide serving as a binding site between the modules, the peptide serving as the binding site being capable of reversibly forming a heterodimer; and mixing the prepared modules to drive spontaneous assembly by interaction between the binding sites of the different modules.

[0009] In another aspect, the present disclosure relates to a kit for producing a protein assembly structure, the kit comprising: a first module, which is a homomultimer formed by a fusion protein of a protein capable of forming a symmetric homomultimer and a peptide that serves as a binding site between modules; and a second module, which is a homomultimer formed by a fusion protein of a protein capable of forming a symmetric homomultimer and a peptide that serves as a binding site between modules, wherein the binding site of the first module and the binding site of the second module are capable of forming a reversible heterodimer. [Effects of the Invention]

[0010] According to one aspect of the present disclosure, there can be provided a higher-order protein assembly in which proteins are assembled in an orderly manner, a method for producing the same, and a kit. DETAILED DESCRIPTION OF THE INVENTION

[0011] To create nature-inspired protein assemblies, the present inventors have discovered a unique design method called Nature Inspired Protein Assembly Design (NIPAD), which integrates the design of protein units using a combination of rational design and computational science techniques with the design of assembly conditions based on biophysical knowledge. They then attempted to create a new type of protein assembly that artificially mimics the principles of natural protein assembly formation.

[0012] The NIPAD-based design is based on the rational design philosophy of avoiding going against nature as much as possible. It combines naturally occurring "peptide pairs capable of forming heterodimers (hereinafter also referred to as "heterolinkers")" with "proteins capable of forming homomultimers" to form modules. This prevents spontaneous assembly of each module alone, while enabling the expression and purification of each module. Furthermore, this design allows the specific selectivity and reversibility of formation and disassembly inherent to heterolinkers to be utilized in assembly formation, which we believe will facilitate the creation of higher-order protein assemblies that are closer to those found in nature.

[0013] First, we focused on the complex formation between methyl-cytosine binding domain protein 2 (MBD2), which is involved in epigenetics in humans, and nucleosome remodeling and deacetylation (NuRD). The antiparallel coiled-coil interaction domain (MBD2 / p66α) plays an important role in the formation of the MBD2-NuRD complex, and this MBD2 / p66α can be considered as a heterodimer-forming peptide pair with specific selectivity. In other words, MBD2 / p66α can be a heterolinker in NIPAD. Furthermore, MBD2 / p66α (T m = 65°C) than the denaturation midpoint temperature (T mWe hypothesized that the temperature-dependent reversible aggregation of M3L2 / p66α (T m = 35°C) was used as a heterolinker (Figure 1).

[0014] Next, the inventors selected proteins that could be combined with a heterolinker in the simplest way possible. Specifically, the selection criteria for proteins to be combined with a heterolinker were homomultimers with rotational symmetry, in which the C-termini of the proteins constituting the homomultimer could be positioned at each vertex of the quaternary structure. Non-Patent Document 7 also shows that locating a binding site at the C-terminus of such a protein allows for the simple formation of a desired structure. The C4-symmetric tetrameric protein, PuuE allantoinase (PuuE), was selected as a protein that met these criteria (Figure 2). PuuE is an allantoinase found in Pseudomonas fluorescens. However, we first focused on its pure shape (a square approximately 10 nm square with C-termini positioned at each vertex) rather than its function.

[0015] We then created two types of modules (PuuE-M3L2 and PuuE-p66α) by fusing the "M3L2 / p66α" peptide pair with PuuE, and attempted to combine them. We succeeded for the first time in forming tubular structures consisting of the two components (PuuE-M3L2 and PuuE-p66α). The formed tubular structures possessed flexibility similar to that seen in the cytoskeleton, and structural analysis using cryo-electron microscopy (cryo-EM) revealed that they could take on a variety of morphologies (Figures 14 and 15). These results contribute to the artificial reproduction of the complex properties and functionality of the cytoskeleton, and further deepen our understanding of protein assembly design and mimicking natural structures. Such research marks an important step in expanding the boundaries between life science and materials science, and further exploration in this field is expected to lead to the elucidation of deep secrets of nature and new technological innovations.

[0016] [Protein assembly structure] Thus, in one aspect, the present disclosure relates to a protein assembly structure formed by linking two or more types of modules. In the present disclosure, a "module" refers to a homomultimer formed by a fusion protein of a protein capable of forming a symmetric homomultimer (hereinafter also referred to as a "base protein") and a peptide (heterolinker) capable of forming a reversible heterodimer. In the present disclosure, the heterolinker serves as a binding site between modules, and different modules are linked by the formation of a heterodimer between the heterolinkers of different modules. In the present disclosure, in one or more embodiments, the term "heterolinker" refers to a peptide pair capable of forming a reversible heterodimer, or may refer to one or the other peptide of the peptide pair.

[0017] According to one or more embodiments of the present disclosure, it is possible to provide a structure that can be reversibly controlled (e.g., the formation and disassembly of a structure), and further, it is possible to provide a protein assembly structure that has flexibility similar to that found in the cytoskeleton in addition to this reversibility.

[0018] In the present disclosure, "(a structure) has reversibility" means that the formation of a structure by linking binding sites of modules and the decomposition of the structure by severing the link (dissociation of the modules) can be controlled by a trigger. In one or more embodiments, an example of the trigger is changing the conditions of a solution in which the structure or module of the present disclosure is present. In one or more embodiments, examples of the trigger are not particularly limited and include the salt concentration, temperature, pH, and module concentration in the solution.

[0019] In the present disclosure, the term "flexible structure" refers to a structure having at least one of the characteristics of stretchability, extensibility, and bendability, and preferably refers to a structure having all of stretchability, extensibility, and bendability. In one or more embodiments, the flexible structure is not particularly limited, but examples thereof include structures that can form various structures such as microtubules. In one or more embodiments, examples of the various structures include linear bundles, helical structures, and three-dimensional gel structures.

[0020] In the present disclosure, a "protein assembly structure" refers to a protein assembly formed by linking one or more types of proteins, and can also be referred to as a protein higher-order assembly. In the present disclosure, a "protein assembly structure formed by linking two or more types of modules" refers to a protein assembly structure formed by different modules. In one or more embodiments, "different modules" in the present disclosure include modules having the same base protein but different heterolinkers, and modules having different base proteins and heterolinkers. In one or more embodiments, structures of the present disclosure include protein assembly structures in which modules having the same base protein but different heterolinkers are linked, and protein assembly structures in which modules having different base proteins and heterolinkers are linked. In one or more embodiments, examples of heterolinkers being different include one or the other peptide constituting a peptide pair capable of forming a reversible heterodimer.

[0021] In one or more embodiments, the structures of the present disclosure may be capable of reversible formation and disassembly. In one or more embodiments, the structures of the present disclosure may be in various forms, such as cages, wires, sheets, tubes, and crystalline structures. In one or more embodiments, the structures of the present disclosure may also be in a helical structure, in which two or more tubular structures are twisted together.

[0022] [Module] The modules constituting the structure of the present disclosure are homomultimers formed by fusion proteins of proteins capable of forming symmetric homomultimers with peptides that serve as binding sites between modules, and the peptides that serve as binding sites between modules (heterolinkers) are capable of reversibly forming heterodimers. In one or more embodiments, the heterolinkers spontaneously form heterodimers by interacting with the binding sites of different modules, thereby linking the different modules and forming the protein assembly structure of the present disclosure. In one or more embodiments, the number of subunits (e.g., base protein or fusion protein) constituting a homomultimer in a module of the present disclosure is the same as the number of subunits of a homomultimer that can be formed by the base protein.

[0023] [Base protein] The base protein is capable of forming a homomultimer having symmetry. In one or more embodiments, examples of the homomultimer include a homodimer, a homotrimer, a homotetramer, a homopentamer, a homohexamer, a homoheptamer, and a homooctamer. In one or more embodiments, the homomultimer has rotational symmetry. In one or more embodiments, examples of the homomultimer include a C2-symmetric dimer, a C3-symmetric trimer, a C4-symmetric tetramer, a C5-symmetric pentamer, a C6-symmetric hexamer, a C7-symmetric heptamer, and a C8-symmetric octamer. In one or more embodiments, the protein capable of forming a homomultimer having symmetry has inherent C2, C3, C4, C5, C6, C7, or C8 symmetry.

[0024] When the structure of the present disclosure includes different platform proteins, in one or more embodiments, two or more types of modules containing homomultimers of the same shape, i.e., formed from the same number of subunits (platform proteins), may be combined for use, or two or more types of modules containing homomultimers of different shapes, i.e., formed from different numbers of subunits (platform proteins), may be combined for use. In one or more embodiments, examples of the former combination include a combination of a homotrimer module with a homotrimer module, and a combination of a homotetramer module with a homotetramer module. In one or more embodiments, examples of the latter combination include a combination of a homotrimer module with a homotetramer module, a combination of a homotrimer module with a homopentamer module, and a combination of a homotrimer module with a homotetramer module with a homopentamer module. The module combinations in the present disclosure are not limited to these.

[0025] In one or more embodiments, the base protein is preferably a protein having the above-described symmetry. In one or more embodiments, a naturally occurring protein may be used as the base protein. In one or more embodiments, the protein is not particularly limited, and examples thereof include the following proteins: C2: Rop protein from Escherichia coli (PDB ID: 1ROP), Osmotically Inducible Protein C from Thermus thermophilus HB8(PDB ID: 1UKK), N-terminal domain of Euprosthenops australis Major Ampullate Spidroin 1 from Euprosthenops australis (PDB ID: 6R9D) C3 (triangle): CutA from Pyrococcus horikoshiiOT3 (PDB ID: 1J2V), stereoselective esterase from Pseudomonas putida IFO12996 (PDB ID: 1ZOI), serine protease HTRA1 from Homo sapiens (PDB ID: 3NUM), alkaline exonuclease from Laribacter hongkongensis (PDB ID: 3SZ4), nitrite reductase from Geobacillus thermodenitrificans NG80-2 (PDB ID: 4ZK8), phosphotransfer protein B from Pseudomonas aeruginosa PAO1 (PDB ID: 7C1I) C4 (quadrilateral): PuuE Allantoinase (Allantoinase from Pseudomonas fluorescens (PDB ID: 3CL6), L-aspartate-alpha decarbozylase from Escherichia coli (PDB ID: 1PQH), Peptidoglycan deacetylase from Helicobacter pylori G27 (PDB ID: 3QBU), Polysaccharide deacetylase family protein from Burkholderia pseudomallei (PDB ID: 3SO6), cyclic nucleotide-binding domain of HCN4 channel from Homo sapiens (PDB ID: 4NVP) C5 (pentagon): fructose 1,6-bisphosphate aldolase from Thermoproteus tenax(PDB ID: 1OJX), Sm-like protein of putative cyanophage origin (PDB ID: 3BY7), The SAM domain-containing protein 1 from Homo sapiens (PDB ID: 6LUK) C6 (hexagon): BMC-H shell protein from Haliangium ochraceum DSM 14365 (PDB ID: 5DJB), carboxysome shell protein ccmK4 from Synechocystis sp. PCC6803 (PDB ID: 2A10), Hfq protein from Escherichia coli (PDB ID: 1HK9) C7 (heptagon) GP31 Co-Chaperonin from Tequatrovirus T4 (PDB ID: 1G31), chaperonin-10 from Mycobacterium tuberculosis (PDB ID: 1HX5), Sm protein from Pyrobaculum aerophilum (PDB ID: 1I8F) C8 (octagon) Rim domain of main porin from Mycobacteria smegmatis MC2 1,55 (PDB ID: 2V9U), tyrosine-kinase Wzc from Escherichia coli K12 (PDB ID: 3LA6), human homologous-pairing protein dmc1 from Homo sapiens (PDB ID: 1V5W)

[0026] In the present disclosure, the terms "naturally occurring protein" and "protein occurring in nature" refer to a protein isolated from a natural source, a homologue thereof, or a variant thereof, and in one or more embodiments may include a protein obtained by genetic engineering based on the sequence of a protein isolated from a natural source.

[0027] [Heterolinker] The heterolinker is a peptide that serves as a binding site between modules and is capable of reversibly forming a heterodimer. The heterolinker forms a reversible heterodimer between different modules, thereby linking the base proteins and forming the structure of the present disclosure. Therefore, in one or more embodiments, the heterolinker of the present disclosure is preferably a peptide that can selectively form a reversible heterodimer with the heterolinker of a different module. In one or more embodiments, a naturally occurring peptide that can selectively form a reversible heterodimer may be used as the heterolinker.

[0028] In the present disclosure, a "peptide capable of reversibly forming a heterodimer" refers to a peptide whose dimerization and monomerization (dissociation of the dimer) can be controlled by a trigger. In one or more embodiments, examples of the trigger include, but are not limited to, varying solution conditions such as the salt concentration, temperature, pH, and heterolinker concentration in the solution. In the present disclosure, a "naturally occurring peptide" refers to a peptide isolated from a natural source, a homolog thereof, or a variant thereof, and in one or more embodiments, may include a peptide obtained by genetic engineering based on the sequence of a peptide isolated from a natural source.

[0029] In one or more embodiments, the heterolinker is preferably capable of forming a heterodimer (heterodimeric coiled coil) with a heterolinker of a different module through a coiled coil (double coil) structure, more preferably capable of forming a heterodimeric coiled coil through electrostatic interaction and / or hydrophobic interaction, and even more preferably capable of forming a heterodimeric coiled coil through the cooperation of electrostatic interaction and hydrophobic interaction. The modules in the structure of the present disclosure are linked by heterodimer formation of heterolinkers, and in one or more embodiments, are preferably linked by heterodimeric coiled-coil formation of heterolinkers, more preferably by heterodimeric coiled-coil formation due to electrostatic and / or hydrophobic interactions of the heterolinkers, and even more preferably by heterodimeric coiled-coil formation due to the cooperation of electrostatic and hydrophobic interactions of the heterolinkers.

[0030] In one or more embodiments, the heterolinker may be a known peptide pair capable of forming a reversible heterodimer, or a known peptide pair capable of forming a reversible heterodimeric coiled-coil. In one or more embodiments, the heterolinker may be a naturally occurring peptide pair known to reversibly form a heterodimeric coiled-coil. In one or more embodiments, the coiled-coil structure may be antiparallel or parallel, preferably antiparallel.

[0031] In one or more embodiments, examples of heterolinkers include, but are not limited to, a peptide pair in the coiled-coil region between MBD2 and p66α, a peptide pair in the coiled-coil region between an MBD2 homolog and p66α, and a peptide pair in the coiled-coil region between Jun and Fos. A peptide pair in the coiled-coil region between an MBD2 homolog and p66α is preferred because it makes it easier to control the formation of reversible assemblies. MBD2 and its homologs are known to bind to p66α via an antiparallel coiled-coil structure, and Jun and Fos are known to bind to each other via a parallel coiled-coil structure. In one or more embodiments, examples of MBD2 homologs include MBD3, MBD3L1 (MBD3-like protein 1), and MBD3L2 (MBD3-like protein 2) (Ninad M. Walavalkar et al., J Biol Chem 2013 Feb 1 288(5) 3419-27). In one or more embodiments, a peptide pair in the coiled-coil region of MBD3L2 and p66α is preferred as the heterolinker, as this allows for easier control of the formation of a reversible structure. The above-mentioned documents are incorporated herein by reference. Coiled-coil region of MBD3L2: SGQLVTPADIRRQARRVKKARERLAKALQADRLA (SEQ ID NO: 1) Coiled-coil region of p66α: PEERERMIKQLKEELRLEEAKLVLLKKLRQSQIQ (SEQ ID NO: 2) Coiled-coil region of MBD2: KAFIVTDEDIRKQEERVQQVRKKLEEALMADILS (SEQ ID NO: 3) Coiled-coil region of MBD3: KAFMVTDEDIRKQEELVQQVRKRLEEALMADMLA (SEQ ID NO: 4) Coiled-coil region of MBD3L1: KQFLVTEEDIRKQEGKVKTVRERLAIALIADGLA (SEQ ID NO: 5)

[0032] In one or more embodiments, the heterolinker may be a peptide pair of a peptide having the amino acid sequence shown in SEQ ID NO: 1, 3, 4, or 5 and a peptide having the amino acid sequence shown in SEQ ID NO: 2. In one or more embodiments, the heterolinker may be a peptide pair of a peptide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 1, 3, 4, or 5 and a peptide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 2, which is capable of forming a reversible heterodimer, making it easier to control the reversible linkage of modules. Therefore, a peptide pair capable of forming a reversible heterodimer is preferred, which is a peptide pair consisting of a peptide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 1 and a peptide having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 2, and a peptide pair consisting of a peptide having the amino acid sequence shown in SEQ ID NO: 1 and a peptide having the amino acid sequence shown in SEQ ID NO: 2 is more preferred.

[0033] In the present disclosure, "amino acid sequence identity" can be performed using readily available sequence comparison computer programs, such as the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al. (1984) Nucleic Acids Res. 12: 387), the BLAST package (Ausubel et al. (1999) ibid-Ch. 18), and FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410).

[0034] In one or more embodiments, the heterolinker preferably forms heterodimers more preferentially than homodimers and / or does not substantially form homodimers. In the present disclosure, "does not substantially form homodimers" means that the formation of homodimers does not affect the formation of the protein assembly structure of the present disclosure.

[0035] In order to further improve operability, in one or more embodiments, the heterolinker is fused to the terminus (N-terminus or C-terminus) of the base protein, preferably to the C-terminus of the base protein.

[0036] In one or more embodiments, the heterolinker is fused to the base protein in an orientation such that the base protein forms a homomultimer.

[0037] In one or more embodiments, the heterolinker is preferably fused to the base protein so as to be located at a vertex (corner) or midpoint of the homomultimer formed by the base protein, and from the viewpoint of further improving operability, it is preferably fused to the base protein so as to be located at a vertex of the homomultimer.

[0038] In one or more embodiments, the fusion protein may contain one or more additional functional groups, additional residues, or the like. In one or more embodiments, the additional residues may be added to the N-terminus and / or C-terminus of the fusion protein or base protein, or may be inserted into the sequence of the base protein to the extent that they do not inhibit homomultimerization of the base protein. Note that additional residues are not included in the determination of sequence identity in the present disclosure. In one or more embodiments, the additional residues include a detectable protein or a fragment thereof (tag). In one or more embodiments, tags include general detectable moieties (e.g., fluorescent proteins and antibody epitope tags), therapeutic agents, purification tags (e.g., His tags), ligands suitable for purification purposes, ligands that determine the localization of the polypeptide, and peptide domains that impart functionality to the polypeptide. In one or more embodiments, the additional functional groups include polypeptide antigens, polypeptide therapeutic agents, enzymes, detectable domains (e.g., fluorescent proteins or fragments thereof), nucleic acid-binding proteins, molecular recognition sites, transcription factors, and nanoparticles. In one or more embodiments, the fusion protein may further comprise a functional polypeptide covalently linked to the N-terminus and / or C-terminus of the fusion protein or base protein, and may be inserted into the sequence of the base protein to the extent that it does not inhibit homomultimerization of the base protein. In one or more embodiments, functional polypeptides include detectable polypeptides such as fluorescent polypeptides or luminescent polypeptides, and receptor-binding domains.

[0039] In one or more embodiments, a reactive site may be introduced into the fusion protein by site-directed mutagenesis (SDM). In one or more embodiments, an example of the reactive site is the introduction of a reactive cysteine ​​residue. In one or more embodiments, the introduction of a reactive cysteine ​​residue allows for chemical modification, functional addition, or the like, using a compound having a reactive site capable of specifically binding to the cysteine ​​residue. In one or more embodiments, examples of the reactive site capable of specifically binding to the cysteine ​​residue include a maleimide group, a halogenated acetic acid group, an aziridine group, an acroyl group, a vinyl sulfone group, a halogenated benzene group, and a pyridyl disulfide group.

[0040] In one or more embodiments, the fusion protein may have at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19, and include a portion corresponding to a protein capable of forming a symmetric homotetramer, and a peptide serving as a binding site capable of forming a reversible heterodimer.

[0041] In one or more embodiments, the module may be a homotetramer composed of four polypeptides having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19, and having binding sites capable of reversibly forming heterodimers.

[0042] In one or more embodiments, the structure of the present disclosure includes a homotetramer (module A) composed of four polypeptides having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 6, 8, 9, 11, 13, 16, 18, or 19, and a homotetramer (module B) composed of four polypeptides having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 7, 10, 12, 14, 15, or 17. and a homotetramer (module B) composed of four polypeptides having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of the target polypeptide, in which modules A and B are alternately linked by forming reversible heterodimers between the binding sites of module A and module B.

[0043] In one or more embodiments, the structure of the present disclosure can be formed into various forms, such as a cage, wire, sheet, tube, or crystal structure, by linking modules that combine a platform protein with a heterolinker (FIG. 3). The protein structures shown as examples of platform proteins in FIG. 3 are a trimer (C3) with 1ZOI (PDB ID), a tetramer (C4) with PuuE (PDB ID: 3CL6) (left) and 1PQH (PDB ID) (right), and a pentamer (C5) with 1OJX (PDB ID).

[0044] When the structure of the present disclosure is tubular, in one or more embodiments, the modules are connected to each other so that the tubular structure has rotational symmetry. In one or more embodiments, the tubular protein assembly structure of the present disclosure has various diameters. Thus, in one or more embodiments, the present disclosure can provide tubular protein assembly structures having various diameters.

[0045] In one or more embodiments, the structure of the present disclosure has a helical structure in which two or more tubular structures are twisted.

[0046] In one or more embodiments, the structures of the present disclosure can be used in a variety of applications. In one or more embodiments, applications include sensing, diagnostics, vaccine development, and drug delivery. In one or more embodiments, other applications include the fabrication of molecular membranes for sieving and filtration, and the fabrication of molecular templates that provide 5-100 nm spatial resolution for patterning and deposition. In one or more embodiments, still other applications include the stabilization of commercially valuable enzymes and proteins, and the fabrication of crystalline molecular scaffolds for macromolecular structure determination by crystallography and electron microscopy.

[0047] [Manufacturing method] In another aspect, the present disclosure relates to a method for producing a protein assembly structure formed by linking two or more types of modules. The production method of the present disclosure includes preparing two or more types of modules, each of which is a homomultimer formed by a fusion protein of a protein capable of forming a symmetric homomultimer and a peptide serving as a binding site between the modules, wherein the peptide serving as the binding site is capable of reversibly forming a heterodimer, and mixing the prepared modules to induce spontaneous assembly due to interactions between the binding sites of the different modules. The production method of the present disclosure makes it possible to produce the structure of the present disclosure.

[0048] The production method of the present disclosure uses modules equipped with binding sites (heterolinkers) that can reversibly form heterodimers, enabling the production of protein-assembly structures by spontaneous assembly (self-organization). By appropriately selecting the types of heterolinkers and base proteins, protein-assembly structures with desired structures and / or sizes can be produced.

[0049] In one or more embodiments, the production method of the present disclosure first includes preparing two or more types of modules. The modules described above can be used as the modules. In one or more embodiments, the two or more types of modules include modules having the same base protein but different heterolinkers, and modules having different base proteins and heterolinkers. In one or more embodiments, the two or more types of modules are prepared in a combination such that the heterolinkers can reversibly form heterodimers.

[0050] In one or more embodiments, the module can be obtained by synthesizing a fusion protein that constitutes the module. In one or more embodiments, the gene for expressing the fusion protein can be synthesized by genetic engineering. In one or more embodiments, the fusion protein can be obtained by transforming a microorganism such as Escherichia coli with a vector into which a gene encoding the fusion protein has been cloned, and then culturing the microorganism to express and purify the fusion protein. In one or more embodiments, the fusion protein may be synthesized using a cell-free protein synthesis system.

[0051] In one or more embodiments, the production method of the present disclosure is not particularly limited and includes preparing two types of modules: a first module and a second module. The first module is a homomultimer formed by a fusion protein of a protein capable of forming a symmetric homomultimer and a peptide that serves as a binding site between modules, and the second module is a homomultimer formed by a fusion protein of a protein capable of forming a symmetric homomultimer and a peptide that serves as a binding site between modules, and the peptide of the first module and the peptide of the second module are capable of reversibly forming a heterodimer. In one or more embodiments, the combination of the first module and the second module is composed of four polypeptides having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 6, 8, 9, 11, 13, 16, 18, or 19, and reversibly forms a heterodimer. and a combination of a homotetramer having a reversible heterodimer-forming binding site and a homotetramer having a reversible heterodimer-forming binding site that is composed of four polypeptides having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7, 10, 12, 14, 15, or 17.

[0052] Next, the resulting two or more types of modules are mixed. Mixing the two or more types of modules induces spontaneous assembly due to interactions between the heterolinkers, and in one or more embodiments, the heterolinkers are linked, the modules are assembled, and a protein assembly structure is formed.

[0053] In one or more embodiments, the modules are mixed in a medium. In one or more embodiments, an aqueous solvent is preferably used as the medium, and an organic solvent such as glycerin may also be used. In one or more embodiments, a mixed medium obtained by mixing an aqueous solvent and an organic solvent may also be used. In one or more embodiments, examples of the aqueous solvent include a buffer and water. In one or more embodiments, examples of the buffer include a Tris buffer such as a Tris-HCl buffer, a phosphate buffer, a HEPES buffer, a citrate-phosphate buffer, and an MES buffer.

[0054] The pH of the buffer can be appropriately determined based on the isoelectric point (pI) of the heterolinker. In one or more embodiments, the pH of the buffer is 6 to 9, and preferably 6 to 8.5.

[0055] In one or more embodiments, the buffer may contain a salt such as NaCl. In one or more embodiments, the salt concentration is, for example, 40 mM to 250 mM, and preferably 50 mM to 200 mM.

[0056] The concentration of each module during mixing can be determined appropriately depending on the type of base protein and heterolinker, and is preferably determined based on the dissociation constant (K d ) In one or more embodiments, the concentration of the module is 100 nM or more, and although the upper limit is not particularly limited, it is 100 μM or less. In one or more embodiments, the concentration of the module is 200 nM or more, 500 nM or more, 750 nM or more, 1 μM or more, 2 μM or more, 3 μM or more, 4 μM or more, 5 μM or more, 6 μM or more, 7 μM or more, 8 μM or more, 9 μM or more, or 10 μM or more. In the present disclosure, the "module concentration" refers to the concentration of the fusion protein in the solution converted into the concentration of the multimer.

[0057] The temperature during mixing can be set appropriately. In one or more embodiments, the temperature may be set to the denaturation midpoint temperature (T m In one or more embodiments, the temperature during mixing is preferably determined based on T (°C) because this can efficiently induce the formation of a thermodynamically stable ordered structure of the heterolinker. m Preferably above -10°C, more preferably T m -8℃~T m +15°C, and more preferably T m -5℃~T m+10°C. In one or more embodiments, when an M3L2 / p66α peptide pair is used as the heterolinker, the temperature during mixing is preferably greater than 25°C, and although there is no particular upper limit, it is preferably 50°C or lower. In one or more embodiments, the temperature during mixing is more preferably 26°C or higher, 27°C or higher, 28°C or higher, 29°C or higher, or 30°C or higher. In one or more embodiments, the temperature during mixing is more preferably 45°C or lower, 44°C or lower, 43°C or lower, 42°C or lower, 41°C or lower, or 40°C or lower.

[0058] In one or more embodiments, the method may include incubating the mixture for a predetermined period of time after mixing. The incubation temperature is the same as described above. The incubation time can be set appropriately, and in one or more embodiments, examples of the incubation time include 30 minutes or more, 1 hour or more, 2 hours or more, 4 hours or more, 6 hours or more, 8 hours or more, 12 hours or more, 24 hours or more, and 72 hours or more.

[0059] In another aspect, the manufacturing method of the present disclosure relates to a method for manufacturing a protein assembly structure, the method including preparing a first module and a second module, mixing the first module and the second module, and driving spontaneous assembly through interaction between a binding site of the first module and a binding site of the second module. According to this aspect, a protein assembly structure in which first modules and second modules are alternately linked can be manufactured.

[0060] [kit] In another aspect, the present disclosure relates to a kit for producing a protein assembly structure. The kit of the present disclosure includes: a first module, which is a homomultimer formed by a fusion protein of a protein capable of forming a symmetric homomultimer and a peptide serving as a binding site between modules; and a second module, which is a homomultimer formed by a fusion protein of a protein capable of forming a symmetric homomultimer and a peptide serving as a binding site between modules, wherein the binding site of the first module and the binding site of the second module can reversibly form a heterodimer. The kit of the present disclosure allows for easy production of the protein assembly structure of the present disclosure. The fusion protein, heterolinker, and module are as described above.

[0061] In one or more embodiments, the combination of heterolinkers in the first module and the second module includes a peptide pair that can form a reversible heterodimer, which is a peptide pair having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 1, 3, 4, or 5, and a peptide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 2, and the reversible heterodimer of the modules can be formed. Peptide pairs capable of forming reversible heterodimers are preferred, because this makes it easier to control the linkage between the two peptides. The peptide pair is made up of a peptide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 1 and a peptide having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 2, and the peptide pair is more preferred, because this makes it easier to control the linkage between the two peptides.

[0062] In one or more embodiments, the combination of the first module and the second module is composed of four polypeptides having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 6, 8, 9, 11, 13, 16, 18, or 19, and reversibly forms a heterodimer. and a combination of a homotetramer having a reversible heterodimer-forming binding site and a homotetramer having a reversible heterodimer-forming binding site that is composed of four polypeptides having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7, 10, 12, 14, 15, or 17.

[0063] In another aspect, the kit of the present disclosure includes: a fusion protein A formed from a protein capable of forming a symmetric homomultimer and a peptide that serves as a binding site between modules; and a fusion protein B formed from a protein capable of forming a symmetric homomultimer and a peptide that serves as a binding site between modules, wherein the binding site of fusion protein A and the binding site of fusion protein B are capable of reversibly forming a heterodimer.

[0064] The fusion protein is as described above. In one or more embodiments, the combination of fusion protein A and fusion protein B has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 6, 8, 9, 11, 13, 16, 18, or 19, and comprises a portion corresponding to a protein capable of forming a symmetric homotetramer and a binding site capable of forming a reversible heterodimer. and a fusion protein B having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence shown in SEQ ID NO: 7, 10, 12, 14, 15, or 17, and including a portion corresponding to a protein capable of forming a symmetric homotetramer and a peptide serving as a binding site capable of forming a reversible heterodimer. Fusion proteins A and B are each capable of forming a symmetric homotetramer, and when the homotetramer formed by fusion protein A (module A) and the homotetramer formed by fusion protein B (module B) are mixed, the mutual binding sites form a reversible heterodimer, thereby alternately linking module A and module B and forming a protein assembly structure.

[0065] In one or more embodiments, the kit of the present disclosure includes an instruction manual that describes a method for producing a protein-assembled structure.

[0066] In another aspect, the present disclosure relates to a vector for introducing the gene for expressing the fusion protein into a microorganism. In one or more embodiments, the vector of the present disclosure can be used to create the above-mentioned module and the structure of the present disclosure. The vector of the present disclosure is not particularly limited and may be a plasmid, etc.

[0067] That is, the present disclosure may relate to one or more of the following embodiments: [1] A protein assembly structure formed by linking two or more types of modules, the module is a homomultimer formed by a fusion protein of a protein capable of forming a symmetric homomultimer and a peptide serving as a binding site between modules, the peptide serving as the binding site is capable of reversibly forming a heterodimer; A protein assembly structure in which different modules are linked by the peptides forming heterodimers. [2] The structure according to [1], wherein the homomultimer has rotational symmetry. [3] The structure according to [1] or [2], wherein the linkage between the different modules is reversible. [4] The module includes a first module and a second module; The structure according to any one of [1] to [3], wherein the first modules and the second modules are alternately connected. [5] The binding site of the first module is a peptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, 3, 4, or 5; the binding site of the second module is a peptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2; The structure according to [4], wherein the binding site of the first module and the binding site of the second module are capable of forming a reversible heterodimer. [6] The structure according to [4] or [5], wherein the proteins capable of forming homomultimers in the first module and the second module are the same or different proteins. [7] The structure according to any one of [1] to [6], wherein the peptide that serves as the binding site between the modules is fused to the end of the protein capable of forming the homomultimer. [8] The structure according to any one of [1] to [7], which has at least one form selected from the group consisting of a cage, a wire, a sheet, a tube, and a crystal structure. [9] A method for producing a protein assembly structure formed by linking two or more types of modules, comprising: Prepare two or more types of modules. wherein the module is a homomultimer formed by a fusion protein of a protein capable of forming a symmetric homomultimer and a peptide serving as a binding site between modules, and the peptide serving as the binding site is capable of reversibly forming a heterodimer; and mixing the prepared modules and driving spontaneous assembly through interactions between binding sites of different modules; method.

[10] The two or more types of modules include a first module and a second module; the protein assembly structure is a protein assembly structure in which the first modules and the second modules are alternately linked, [9] The method described in [9], comprising mixing the first module and the second module and driving spontaneous assembly by interaction between the binding site of the first module and the binding site of the second module.

[11] The method according to [9] or

[10] , wherein the protein assembly structure is a structure according to any one of [1] to [8].

[12] A kit for producing a protein-assembled structure, comprising: a first module, which is a homomultimer formed by a fusion protein of a protein capable of forming a symmetric homomultimer and a peptide serving as a binding site between modules; a second module that is a homomultimer formed by a fusion protein of a protein capable of forming a symmetric homomultimer and a peptide that serves as a binding site between modules; The binding site of the first module and the binding site of the second module are capable of reversibly forming a heterodimer. [Brief explanation of the drawings]

[0068] [Figure 1] Figure 1 shows the predicted structure of the heterodimeric peptide pair M3L2 and p66α using AlphaFold2 (AF2). [Figure 2] Figure 2 shows the crystal structure (front, side, and back) of PuuE (PDB ID: 3CL6). The circle in the figure indicates the position of the C-terminus. [Figure 3] FIG. 3 is a schematic diagram illustrating an example of a structure of the present disclosure, showing examples of possible shapes of a base protein, heterolinker, module, and structure. [Figure 4] Figure 4 shows a schematic diagram of the PuuE-M (PuuE-M3L2) and PuuE-p (PuuE-p66α) fusion protein, the predicted structure of the PuuE-M and PuuE-p tetramers using AF2, and a model of the tube structure predicted based on the predicted structure. A simple diagram illustrating the number of module pairs (n) that form one circumference of the tube is shown in the lower right. [Figure 5] Figure 5 shows the predicted tetramer structures (front and side views) of PuuE-M and PuuE-p using AF2, with 50 predicted models superimposed. The square frames in the figure represent M3L2 or p66α. [Figure 6] Figure 6 shows negative-stain transmission electron microscopy (nsTEM) images of tube structures constructed by incubating PuuE-M and PuuE-p at final concentrations of 12.5 μM each in NaCl(+) buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.5 mM EDTA) at 40°C for 24 hours. [Figure 7] Figure 7 shows an example of the protein concentration dependence of the structure formation using PuuE-M and PuuE-p. The images show nsTEM images of tube structures formed by incubating PuuE-M and PuuE-p at various final concentrations (250 nM, 2.5 μM, and 12.5 μM) in NaCl(+) buffer at 40 °C for 24 h. [Figure 8]Figure 8 shows an example of the dynamics of tube formation over time using PuuE-M and PuuE-p. PuuE-M and PuuE-p (final concentrations of 12.5 μM each) were incubated in NaCl(+) buffer at 40°C for the indicated times. nsTEM images are shown at 0, 0.5, 1, 2, 4, 6, 8, 24, 48, and 72 hours after the start of incubation. [Figure 9] FIG. 9 shows the analysis results of the length of the tube structure formed in the experiment of FIG. [Figure 10] Figure 10 shows nsTEM images demonstrating the stability of the tube structures formed using PuuE-M and PuuE-p. The tube structures were obtained by incubating PuuE-M and PuuE-p at final concentrations of 12.5 μM in NaCl(+) buffer at 40°C for 24 hours, and then left at room temperature for 2 weeks or 1 month. [Figure 11] Figure 11 shows an example of the salt concentration dependence of the structure formation using PuuE-M and PuuE-p. The images show nsTEM images of structures formed by incubating PuuE-M and PuuE-p at final concentrations of 12.5 μM each in NaCl(-) buffer (0 mM NaCl), NaCl(-) buffer with various NaCl concentrations (50 mM and 200 mM), and NaCl(+) buffer (100 mM NaCl) at 40°C for 24 hours. [Figure 12] FIG. 12 shows nsTEM images showing the reversibility of the tube structures formed using PuuE-M and PuuE-p. [Figure 13] Figure 13 shows the results of tube length analysis of the nsTEM images in Figure 12. For tube length analysis, tubes were picked up from the nsTEM images at 5k magnification at each step, and the tube lengths were calculated. 150 tubes with the longest tube lengths were used for each data point. *** p<0.001 (Welch's t-test). [Figure 14]Figure 14 shows representative cryo-EM images of tube structures obtained by incubating PuuE-M and PuuE-p at a final concentration of 12.5 μM each in NaCl(+) buffer at 40°C for 24 hours. The right image shows an example of tube extraction for two-dimensional classification. [Figure 15] Figure 15 illustrates the characteristics of the tube structures formed using PuuE-M and PuuE-p. (a) is a representative cryo-EM image of the tube structure. (b) is a two-dimensional average image of the tube structure. Each structure was determined from a total of 206,658 tube segments. (c) is a representative three-dimensional reconstruction model of a C4, C5, and C6 symmetric tube structure constructed based on the two-dimensional average image. [Figure 16] Figure 16 shows a more detailed structure of the three-dimensional reconstructed model shown in Figure 15. Tube structures of different diameters were formed symmetrically at C4, C5, and C6, suggesting that the obtained tube structures have contractility. [Figure 17] Figure 17 shows real-time images (time lapse) of the changes in the tube structure monitored by total internal reflection fluorescence microscopy (TIRFM). The upper row shows snapshots at the start (0 seconds) and 4 seconds later, while the lower row shows the dynamic flexibility of the tube structure from 0 to 4 seconds (0.4 seconds per image). [Figure 18] FIG. 18 shows a schematic diagram of the PuuE-Dloop-M fusion protein in which Dloop has been grafted onto PuuE-M, and the predicted structure of the tetramer of PuuE-Dloop-M using AF2. [Figure 19] Figure 19 shows nsTEM images of tube structures with helical structures formed using PuuE-Dloop-M and PuuE-p. A helical structure with two twisted tubes (center) or three twisted tubes (right) was formed. [Figure 20] FIG. 20 shows nsTEM images showing the reversibility of the tube structure having a helical structure. [Figure 21]Figure 21 shows the predicted tetramer structures of 1PQH-M and 1PQH-p by AF2, as well as nsTEM images of the structures obtained by combining them. [Figure 22] FIG. 22 shows nsTEM images of the predicted tetramer structures of PuuE-M, 3QBU-p, 3QBU-M and PuuE-p by AF2, and the structures obtained by actually combining them. [Figure 23] Figure 23 shows the predicted tetramer structure of PuuE-M and 1PQH-p by AF2, a model diagram of the structure formed by PuuE-M and 1PQH-p, and an nsTEM image of the structure obtained by actually fabricating and combining them. [Figure 24] Figure 24 shows the predicted tetramer structures of PuuE-M, PuuE-p, PuuE-Rigid-M, and PuuE-Flexible-p by AF2, as well as nsTEM images of the structures obtained using these structures. [Figure 25] Figure 25 shows the predicted tetramer structures of PuuE-p and PuuE-Rigid-p by AF2, as well as nsTEM images of the structures obtained using PuuE-M and PuuE-p (top right) and the structures obtained using PuuE-M and PuuE-Rigid-p (bottom right). [Figure 26] Figure 26 shows nsTEM and fluorescence microscope images of a tube structure obtained by combining PuuE-M with PuuE-K46C-p, which is PuuE-p with a cysteine ​​introduced therein, modified with Alexa488-maleimide. [Figure 27] Figure 27 shows the predicted structures of the tetramers by AF2 of PuuE-DogTag-M, in which DogTag has been introduced into PuuE-M, and G11-PuuE-M, in which G11 has been introduced into PuuE-M, as well as nsTEM images of the tube structures formed using PuuE-DogTag-M and PuuE-p, and G11-PuuE-M and PuuE-p. [Example]

[0069] The present disclosure will be described in more detail below with reference to examples, but these are merely illustrative and the present disclosure is not limited to these examples. All documents cited in this disclosure are incorporated herein by reference.

[0070] [Experimental Method] Plasmids and cloning Primers and synthetic genes for PuuE-M3L2 (PuuE-M: SEQ ID NO: 6) and PuuE-p66α (PuuE-p: SEQ ID NO: 7) were purchased from Eurofins Genomics. PCR was performed using PrimeSTAR Max DNA polymerase (Takara Bio) according to the manufacturer's protocol, and the size of the PCR product was confirmed by standard agarose gel electrophoresis. Cloning was performed using In-Fusion Snap Assembly (Takara Bio) according to the manufacturer's protocol, as a standard method. Each amplified gene fragment was ligated between the NdeI and BamHI multiple cloning sites of the pET11a expression vector (Novagen). PuuE-M MHHHHHHENLYFQGVDYPRDLIGYGSNPPHPHWPGKARIALSFVLNYEEGGERNILHGDKESEAFLSEMVSAQPLQGERNMSMESLYEYGSRAGVWRILKLFKAFDIPLTIFAVAMAAQRHPDVIRAMVAAGHEICSHGYRWIDYQYMDEAQEREHMLEAIRILTELTGERPLGWYTGRTGPNTRRLVMEEGGFLYDCDTYDDDLPYWEPNNPTGKPHLVIPYTLDTNDMRFTQVQGFNKGDDFFEYLKDAFDVLYAEGAEAPKMLSIGLHCRLIGRPARLAALQRFIEYAKSHEQVWFTRRVDIARHWHATHPYTSGQLVTPADIRRQARRVKKARERLAKALQADRLA (SEQ ID NO: 6) PuuE-p MHHHHHHENLYFQGVDYPRDLIGYGSNPPHPHWPGKARIALSFVLNYEEGGERNILHGDKESEAFLSEMVSAQPLQGERNMSMESLYEYGSRAGVWRILKLFKAFDIPLTIFAVAMAAQRHPDVIRAMVAAGHEICSHGYRWIDYQYMDEAQEREHMLEAIRILTELTGERPLGWYTGRTGPNTRRLVMEEGGFLYDCDTYDDDLPYWEPNNPTGKPHLVIPYTLDTNDMRFTQVQGFNKGDDFFEYLKDAFDVLYAEGAEAPKMLSIGLHCRLIGRPARLAALQRFIEYAKSHEQVWFTRRVDIARHWHATHPYTPEERERMIKQLKEELRLEEAKLVLLKKLRQSQIQ (SEQ ID NO: 7) The cloning primers for PuuE-Dloop-M3L2 (PuuE-Dloop-M: SEQ ID NO: 8) and the synthetic DNA fragment of Dloop were purchased from Eurofins Genomics. The PuuE-Dloop-M plasmid was constructed using the PuuE-M plasmid as a base in the same manner as above. The underlined portion in the PuuE-Dloop-M sequence below corresponds to the sequence of Dloop. PuuE-Dloop-M MHHHHHHENLYFQGVDYPRDLIGYGSNPPHPHWPGKARIALSFVLNYEEGGERNILHGDKESEAFLSEMVSAQPLQ QGVMVGMGQK GERNMSMESLYEYGSRAGVWRILKLFKAFDIPLTIFAVAMAAQRHPDVIRAMVAAGHEICSHGYRWIDYQYMDEAQEREHMLEAIRILTELTGERPLGWYTGRTGPNTRRLVMEEGGFLYDCDTYDDDLPYWEPNNPTGKPHLVIPYTLDTNDMRFTQVQGFNKGDDFFEYLKDAFDVLYAEGAEAPKMLSIGLHCRLIGRPARLAALQRFIEYAKSHEQVWFTRRVDIARHWHATHPYTSGQLVTPADIRRQARRVKKARERLAKALQADRLA (SEQ ID NO: 8) All plasmids were amplified using Escherichia coli strain DH5α (NIPPON GENE) and extracted using NucleoSpin Plasmid EasyPure (MACHEREY-NAGEL) according to the manufacturer's protocol. The DNA sequences of the final products were confirmed by the sequencing service of Eurofins Genomics.

[0071] Protein expression and purification The recombinant protein was expressed using Escherichia coli strain BL21(DE3) (NIPPON GENE) cotransformed with the pGro7 chaperone plasmid (Takara Bio) and purified as follows. After transformation with the plasmid DNA, colonies were grown overnight at 37°C on LB agar plates supplemented with 100 μg / mL ampicillin (Amp) and 20 μg / mL chloramphenicol (Crm). The resulting colonies were then inoculated into 5 mL of LB-Amp-Crm and cultured overnight at 37°C and 200 rpm. The overnight culture was diluted with 1 L of LB-Amp-Crm supplemented with 0.5 mg / mL L-arabinose and cultured at 37°C and 200 rpm until the optical density at 600 nm reached 0.6–0.8. Protein synthesis was then induced by adding 0.1 mM isopropyl-β-D-thiogalactopyranoside, and the culture was continued for 16–20 hours at 16°C. After incubation, cells were collected by centrifugation at 10,000 rpm at 4°C for 5 minutes and stored at -80°C as a cell pellet. Then, the cells were thawed at room temperature, resuspended in 60 mL of ice-cold purification buffer (20 mM Tris-HCl, pH 8.0, containing 300 mM NaCl), and disrupted on ice using sonication (1:2 on / off cycle, 70% amplitude for 9 minutes; SFX250, Branson). Cell debris was removed by centrifugation at 10,000 rpm at 4°C for 30 minutes. The supernatant was filtered through a 0.45 μm membrane filter (Merck) and applied to a HisTrap FF Crude column (Cytiva) pre-equilibrated with buffer (20 mM Tris-HCl, 300 mM NaCl, pH 8.0). The column was washed with 5 volumes of 2% elution buffer (20 mM Tris-HCl, 300 mM NaCl, 20 mM imidazole, pH 8.0). The 6xHis-tagged protein was eluted with 10 column volumes of elution buffer containing a linear gradient of 20–400 mM imidazole.Fractions containing proteins confirmed by UV absorption and SDS-PAGE were collected and dialyzed twice at 4°C against 50 volumes of NaCl(+) buffer (50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.5 mM EDTA) or NaCl(-) buffer (50 mM Tris-HCl, pH 8.0, 0 mM NaCl, 0.5 mM EDTA). Each purified protein was concentrated using an Amicon Ultra centrifugal filter unit (Merck) with the appropriate molecular weight cutoff and then filtered through a 0.45 μm pore-size membrane filter (Merck). Protein concentration was determined by absorbance measurement at 280 nm using a NanoDrop OneC spectrophotometer (Thermo Scientific). The molar extinction coefficient at 280 nm of the protein was calculated based on its amino acid composition. The concentrated proteins were frozen in liquid nitrogen and stored at -80°C before experiments.

[0072] Sample preparation All proteins were thawed immediately on ice immediately before the tube formation experiment. Each sample was prepared in a 1.5 mL microtube at room temperature to a volume of 200 μL using the appropriate buffer for each concentration described below. NaCl(+) buffer was used except for the NaCl concentration-dependent experiment, in which NaCl(-) buffer was used. Samples were incubated at the temperatures described below using a ThermoMixer C (Eppendorf) or MATRIX Orbital Delta Plus (IKA) with shaking at 300 rpm. For disassembly and reassembly experiments, buffer exchange was performed four times at each step using Microcon50 centrifugal filter units (Merck) according to the manufacturer's protocol, using NaCl(-) and NaCl(+) buffers, respectively.

[0073] Negative stain transmission electron microscopy (nsTEM) A G600TT copper grid (Nissin EM) was carbon-coated using a VE-2030 vacuum device. After glow-discharging the grid using a PIB-10 vacuum device, 5 μL of sample solution was placed on the grid for 1 minute. The remaining solution was removed with filter paper (No. 2, Advantec), followed by rinsing three times with 5 μL of Milli-Q water. After wiping off the moisture with filter paper, the grid was briefly stained three times with 3 μL of 2% (w / v) uranyl acetate solution. The remaining solution was removed with filter paper, and the grid was allowed to dry. nsTEM observations were performed using a transmission electron microscope (HT-7700) (Hitachi) at an accelerating voltage of 80 kV. Images were recorded using the HT-7700 control software (Hitachi).

[0074] Tube Length Analysis Several hundred distinguishable tubes were manually selected from nsTEM images at 5k magnification. Tube length was analyzed using ImageJ (Fiji) and calculated as half the circumference. Plots were performed using Igor Pro 9 (WaveMetrics) to select 150 long tubes. Analysis of disassembly and reformation was performed using Welch's t-test.

[0075] Assembly formation scheme 1 We used genetic engineering to construct PuuE, fusing M3L2 (SEQ ID NO: 1) or p66α (SEQ ID NO: 2) to the C-terminus of PuuE. These constructs, "PuuE-M: SEQ ID NO: 6" and "PuuE-p: SEQ ID NO: 7," were constructed (Figure 4). Furthermore, a 6xHis tag was introduced to the N-terminus of PuuE to simplify the purification of each module. Based on structural prediction using AF2, we predicted that PuuE-M would have a relatively flexible orientation of M3L2, whereas PuuE-p would have a rigid structure with a significantly restricted orientation of p66α (Figure 5). Therefore, we predicted that the ligation of these two modules would form a tubular structure (Figure 4). When PuuE-M and PuuE-p were expressed in E. coli, both modules were found in the soluble fraction and could be purified. Neither PuuE-M nor PuuE-p forms aggregates by themselves (PuuE-M does not link to PuuE-M, and PuuE-p does not link to PuuE-p). However, mixing the two in NaCl(+) buffer (final concentration of each 12.5 μM tetramer-equivalent) resulted in the formation of a characteristic checkerboard-patterned tube structure (PuuE-M / PuuE-p tube) as confirmed by nsTEM observation (Figure 6). While cage, sheet, and three-dimensional crystalline structures have previously been achieved with the two types of modules, this is the first time we have successfully formed a tube structure. Note that the concentrations of the fusion proteins in the following examples refer to the tetramer-equivalent concentration (module concentration).

[0076] Protein concentration dependence Here, it is known that conditions such as protein concentration, temperature, time, and salt concentration have a significant effect on the formation of actin filaments, microtubules, amyloid fibrils, or artificially designed higher-order protein assemblies. Therefore, the design of the assembly formation conditions in NIPAD was based on these biophysical findings. First, we investigated the protein concentration dependence of PuuE-M / PuuE-p tube formation, and found that tubes formed after 24 hours of incubation at 40°C even when mixed at 250 nM tetramer equivalents (Figure 7). This is due to the K dThis is consistent with the fact that the concentrations of the proteins are of the same order of magnitude (~268 nM). When the mixing concentrations were increased above 2.5 μM, the number and length of the formed tubes clearly increased, and when the concentrations of each module (PuuE-M and PuuE-p) were increased to 12.5 μM, the tube formation ability significantly increased (Fig. 7). This result suggests that the formation of PuuE-M / PuuE-p tubes also depends on the protein concentration.

[0077] Temperature dependence Next, we investigated the temperature dependence of tube formation by PuuE-M and PuuE-p at 12.5 μM each (NaCl(+) buffer). m Although the temperature was around 35°C, almost no tube formation was observed even after 24 hours of incubation at temperatures well below 20-25°C. m At temperatures around 30-40°C, tube formation was evident. m At temperatures well below T, the driving force for the association of PuuE-M and PuuE-p is too strong, and the aggregate remains in a kinetically trapped state. m It is thought that the driving force for association becomes weaker around 45°C, and the dynamic rearrangement of M3L2 / p66α bonds due to thermal perturbation efficiently induces the formation of thermodynamically stable ordered structures. This concept is well known as a general theory for crystal formation, and has also been reported for the formation of ordered nanoparticle structures, naturally occurring protein assemblies, and artificially designed protein assemblies. Above 45°C, thermal denaturation and aggregation of PuuE-M and PuuE-p became dominant, significantly reducing their ability to form tubes.

[0078] Kinetics (time dependence) Next, we investigated the kinetics of PuuE-M / PuuE-p tube formation. When a sample containing a mixture of PuuE-M and PuuE-p at 12.5 μM (NaCl(+) buffer) was incubated at 40°C, tiny tube-like structures formed within 30 minutes, and within 1–2 hours, clearly distinguishable tubes measuring several hundred nanometers to 1 μm were observed. The tubes continued to elongate over time, reaching lengths of several μm after 24 hours and reaching tubes of approximately 5 μm after 48 hours (Figures 8 and 9). Furthermore, tubes formed by incubation at 40°C for 24 hours were left at room temperature for one month. No changes in the structure of the formed tubes were observed even after two weeks and one month, suggesting their stability (Figure 10).

[0079] Salt concentration dependence (reversibility) Finally, we investigated the salt concentration dependence of PuuE-M / PuuE-p tube formation. Samples were prepared by mixing 12.5 μM of each protein in the presence of 0–400 mM NaCl, and the results were compared after 24 h of incubation at 40°C. Tube formation was observed at 50–200 mM NaCl, but not at other NaCl concentrations (Figure 11). Because PuuE-M (pI = 6.44) and PuuE-p (pI = 6.02) both carry the same charge at pH 8.0, low salt concentrations likely inhibited tube formation due to electrostatic repulsion. In the 50–200 mM NaCl range, moderate electrostatic shielding appears to provide favorable conditions for tube formation. At higher NaCl concentrations, either excessive electrostatic shielding resulted in excessive driving force for association, or aggregation due to salting out inhibited tube formation. It is known that in protein crystal formation using electrostatic interactions, electrostatic shielding inhibits crystal formation at NaCl concentrations of several tens of mM or higher. In contrast, protein crystals formed through the combined action of electrostatic and hydrophobic interactions remain stable up to NaCl concentrations of approximately 200 mM. The binding of M3L2 / p66α is consistent with the latter example, as both electrostatic and hydrophobic interactions contribute to the binding.

[0080] The salt-dependent PuuE-M / PuuE-p tube formation and the T m We hypothesized that reversible tube disassembly and reformation by changing the NaCl concentration would be possible by exploiting the dynamic recombination of the bond between PuuE-M and PuuE-p near the nucleus. Indeed, when a sample formed tubes in 100 mM NaCl was solvent-exchanged with a 0 mM NaCl buffer (NaCl(-) buffer) and then incubated at 40°C for 24 hours, the tubes significantly shortened (Figures 12 and 13). When this sample was solvent-exchanged with a 100 mM NaCl buffer (NaCl(+) buffer) and then incubated in the same manner, the tubes grew significantly. Thus, although we were unable to achieve the reversible control of tube formation by temperature as originally envisioned, we instead achieved artificial control by salt concentration. Recent studies have demonstrated the disassembly of artificially prepared protein assemblies at sites (methods) different from those used for assembly, and the reversible disassembly and reformation under extreme conditions, such as the addition of denaturants. On the other hand, the salt-concentration-dependent reversibility observed in tube formation is realized under mild conditions, and the properties are similar to those of actin filaments. Therefore, NIPAD can be said to be the first design to successfully incorporate behavior similar to the reversibility exhibited by biomolecular assemblies into the formation of artificial protein higher-order assemblies.

[0081] structural analysis Based on these results, we designed the optimal conditions for the formation of PuuE-M / PuuE-p tubes (12.5 μM PuuE-M and PuuE-p, 100 mM NaCl, 40°C for 24 hours). To clarify the structural features of PuuE-M / PuuE-p tubes formed under these optimal conditions, we performed cryo-EM structural analysis (Fig. 14). Average images obtained by 2D classification revealed a diversity of diameters (i.e., symmetry) among the formed tubes (Fig. 15). This diversity in tube structure is reminiscent of microtubules. From these 2D average images, we then successfully constructed 3D reconstructed models of tube structures with C4, C5, and C6 symmetries (Fig. 15). Based on the known PuuE structure, the back face of PuuE has a characteristic central depression, revealing that the face and back of PuuE are arranged alternately in all 3D reconstructed models. However, the resolution was not sufficient to distinguish between PuuE-M and PuuE-p. Furthermore, the 3D reconstructions of tubes with larger diameters had lower resolution. We speculate that these poor resolutions result from the flexibility of the binding sites that form the tube structure. Indeed, bent and collapsed tubes were frequently observed in nsTEM images. Many previous designs have employed point or edge (face) connections to the platform protein itself to create relatively uniform protein assemblies. In contrast, designs such as NIPAD, in which "independent" linkers are fused to the platform protein with a certain degree of flexibility, have been reported to result in morphological diversity even among assemblies formed from the same units. Therefore, we speculate that the diversity of the PuuE-M / PuuE-p tubes, like those formed by microtubules, is manifested in the form of various diameters. Furthermore, cryo-electron microscopy analysis provided data suggesting that the flexibility of the connecting sites allows the entire tube structure to contract (Figure 16).

[0082] To confirm the flexibility of PuuE-M / PuuE-p tubes in more detail, we fluorescently labeled tubes formed under the optimal conditions described above and attempted real-time observation by TIRFM. Time-lapse observations in the same field of view successfully captured tubes bending in solution due to Brownian motion (Figure 17). Figure 17 shows real-time images (time-lapse) of the changes in tube structure monitored by TIRFM. The top row shows snapshots taken at the start (0 s) and 4 s later, while the bottom row shows the dynamic flexibility of the tube structure over the period from 0 to 4 s (0.4 s per image). Fluorescent labeling The tube structures were fabricated under the optimized conditions described above and then fluorescently labeled using the following reaction. Alexa Fluor 488 succinimidyl ester dissolved in dimethyl sulfoxide (DMSO) was added to the tube solution at a final concentration of 0.7 mM and incubated at 25°C for 1 hour with gentle shaking in the dark. Excess dye was removed using a Microcon 300 centrifugal filter unit (Merck) by four NaCl(+) buffer exchanges according to the protocol. The labeled tubes were stored at 25°C in the dark until further experiments. Total internal reflection fluorescence microscope (TIRFM) observation Silicone-coated coverslip (24 x 36 mm) 2 Place two pieces of double-sided tape (approximately 100 μm thick) on top of the glass (18 × 18 mm thick; Matsunami Glass Industry Co., Ltd.), and then place another cover slip (18 × 18 mm thick) on top of the tape. 2The observation chamber was assembled with a 10 mg / mL Pluronic F-127 (Sigma-Aldrich) solution (thickness: 1; Matsunami Glass Industry Co., Ltd.). To prevent nonspecific protein adhesion, the chamber was filled with 10 mg / mL Pluronic F-127 (Sigma-Aldrich) dissolved in distilled water over 10 minutes at room temperature. After washing the Pluronic F-127 solution with five chamber volumes of NaCl(+) buffer, the chamber was filled with TIRFM buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 0.5 mM EDTA, 0.2% (w / v) methylcellulose (1500 cP, Wako), 1 mM DTT, 2 mM Trolox). Next, the fluorescently labeled tubing solution was diluted 1 / 10 with the NaCl(+) solution and further diluted 1 / 10 with TIRFM buffer (final dilution: 1 / 100). The diluted tubing solution was then perfused into the observation chamber, which was then sealed with a Valap to prevent flow. Fluorescence images of the tubular structures were acquired at 40 ms intervals using an inverted microscope (IX-71, Olympus) equipped with a 60x objective lens (PlanApo NA 1.45 oil, Olympus), an EMCCD camera (iXon3, Andor Technology), and a 488 nm excitation laser (OBIS 488-60-LS, Coherent). All observations were performed at 25 ± 1°C.

[0083] Porting Drop Inspired by natural structures, we attempted to significantly alter the morphology of the PuuE-M / PuuE-p tube. Specifically, we hypothesized that transplanting the D-loop of actin into the PuuE-M structure would produce a helically twisted tube similar to actin filaments. It has been suggested that the D-loop plays an important role in actin filament formation through its interaction with hydrophobic pockets. The characteristic depression on the back side of PuuE also has a high hydrophobicity, leading to this idea. We selected the loop structure on the back side of PuuE-M as the target for D-loop transplantation and constructed a fusion construct, "PuuE-Dloop-M: SEQ ID NO: 8," by genetic engineering (Figure 18). When PuuE-Dloop-M was expressed in E. coli, the target product was found in the soluble fraction and could be purified. PuuE-Dloop-M does not form aggregates on its own, but when mixed with PuuE-p, nsTEM observations confirmed that not only did it form the same tube structure as before, but also new helical structures (Figure 19, center) in which two (Figure 19, center) or three tubes were twisted (Figure 19, right) (these will be referred to as PuuE-Dloop-M / PuuE-p tubes) (Figure 19).

[0084] The helical structure due to the twisting of the tubes, as shown in Figure 19 (center and right), is clearly the result of the transplantation of D-loop, since it is not observed at all in the combination of PuuE-M and PuuE-p. This contributes to elucidating, from a bottom-up perspective, the importance of D-loop for the formation of twisted helical actin filaments. The twisted helical structure of PuuE-Dloop-M / PuuE-p tubes is thought to be generated by hydrophobic interactions. It is known that hydrophobic interactions are temperature-dependent and weaken at low temperatures. Therefore, we predicted that the disassembly and reformation of the twisted structure of PuuE-Dloop-M / PuuE-p tubes could be reversibly controlled by changing the temperature. Surprisingly, when a sample of PuuE-Dloop-M / PuuE-p tubes formed at 30°C was incubated at 0°C for 1 hour, we obtained results suggesting not only dissociation of the twist but also the decomposition of the tube itself (Figure 20). When this decomposed sample was again incubated at 30°C for 24 hours, the long twisted tubes reformed. In other words, by transplanting the D-loop, not only did the tubes form a twisted helical structure (i.e., PuuE-D-loop-M / PuuE-p tube), but the disassembly and reformation of the tube itself also acquired a new temperature-dependent reversibility in addition to the salt-dependent reversibility. This temperature sensitivity is reminiscent of microtubules, and this result further reinforces the idea that NIPAD is a design that can realize behavior similar to that of biomolecular assemblies in artificial protein higher-order assemblies.

[0085] Assembly formation scheme 2 Genes for expressing the modules "1PQH-M3L2 (1PQH-M): SEQ ID NO: 9" and "1PQH-p66α (1PQH-p): SEQ ID NO: 10," in which M3L2 or p66α was fused to the C-terminus of N-terminal 6xHis-tagged 1PQH, were constructed as described above, expressed in E. coli, and purified. 1PQH is a protein that forms a homotetramer with rotational symmetry, and each C-terminus is located at each vertex of the quaternary structure (each vertex of a square). Therefore, M3L2 and p66α fused to the C-terminus are both located at each vertex of the module and can be linked to each other. 1PQH-M and 1PQH-p were mixed in NaCl(+) buffer to a final concentration of 12.5 μM each and incubated at 40°C for 24 hours. As a result, a wire structure was formed. The structural model obtained from the tetramer structure prediction using AF2 and the observed image by nsTEM are shown in Figure 21. 1PQH-M MHHHHHHENLYFQGMIRTMLQGKLHRVKVTHADLHYEGTCAIDQDFLDAAGILENEAIDIWNVTNGKRFSTYAIAAERGSRIISVNGAAAHCASVGDIVIIASFVTMPDEEARTWRPNVAYFEGDNEMKRSGQLVTPADIRRQARRVKKARERLAKALQADRLA (SEQ ID NO: 9) 1PQH-p MHHHHHHENLYFQGMIRTMLQGKLHRVKVTHADLHYEGTCAIDQDFLDAAGILENEAIDIWNVTNGKRFSTYAIAAERGSRIISVNGAAAHCASVGDIVIIASFVTMPDEEARTWRPNVAYFEGDNEMKRGGGGSPEERERMIKQLKEELRLEEAKLVLLKKLRQSQIQ (SEQ ID NO: 10)

[0086] Aggregation Scheme 3 Genes for expressing the units "3QBU-M3L2 (3QBU-M): SEQ ID NO: 11" and "3QBU-p66α (3QBU-p): SEQ ID NO: 12," in which M3L2 or p66α was fused to the C-terminus of N-terminal 6xHis-tagged 3QBU, were constructed as described above, expressed in E. coli, and purified. 3QBU is a protein that forms a homotetramer with rotational symmetry, and each C-terminus is located at each vertex of the quaternary structure (each vertex of a square). Therefore, M3L2 and p66α fused to the C-terminus are both located at each vertex of the unit and can be linked to each other. PuuE-M and 3QBU-p, and 3QBU-M and PuuE-p were mixed in NaCl(+) buffer to a final concentration of 6.25 μM each and incubated at 40°C for 24 hours. As a result, tube structures were formed in both cases. The structural model obtained from the tetramer structure prediction using AF2 and the observed image by nsTEM are shown in Figure 22. This shows that proteins could be assembled to form tube structures even when modules based on different proteins were combined. 3QBU-M MHHHHHHGKPIPNPLLGLDSTENLYFQGIDPFTMAKEILVAYGVDIDAVAGWLGSYGGEDSPDDISRGLFAGEVGIPRLLKLFKKYHLPATWFVPGHSIETFPEQMKMIVDAGHEVGAHGYSHENPIAMSTKQEEDVLLKSVELIKDLTGKAPTGYVAPWWEFSNITNELLLKHGFKYDHSLMHNDFTPYYVRVGDSWSKIDYSLEAKDWMKPLIRGVETNLVEIPANWYLDDLPPMMFIKKSPNSFGFVSPRDIGQMWIDQFDWVYREMDYAVFSMTIHPDVSARPQVLLMHEKIIEHINKHEGVRWVTFNEIADDFLKRNPRKKGSGSGSGQLVTPADIRRQARRVKKARERLAKALQADRLA (SEQ ID NO: 11) 3QBU-p MHHHHHHGKPIPNPLLGLDSTENLYFQGIDPFTMAKEILVAYGVDIDAVAGWLGSYGGEDSPDDISRGLFAGEVGIPRLLKLFKKYHLPATWFVPGHSIETFPEQMKMIVDAGHEVGAHGYSHENPIAMSTKQEEDVLLKSVELIKDLTGKAPTGYVAPWWEFSNITNELLLKHGFKYDHSLMHNDFTPYYVRVGDSWSKIDYSLEAKDWMKPLIRGVETNLVEIPANWYLDDLPPMMFIKKSPNSFGFVSPRDIGQMWIDQFDWVYREMDYAVFSMTIHPDVSARPQVLLMHEKIIEHINKHEGVRWVTFNEIADDFLKRNPRKKGSGSGPEERERMIKQLKEELRLEEAKLVLLKKLRQSQIQ (SEQ ID NO: 12)

[0087] Aggregation Scheme 4 PuuE-M and 1PQH-p were mixed in NaCl(+) buffer to a final concentration of 12.5 μM each and incubated at 40°C for 24 hours. The results are shown in Figure 23. As shown in Figure 23, a sheet structure was formed. This indicates that proteins could be assembled to form a sheet structure even when modules based on different proteins were combined.

[0088] Aggregation Scheme 5 As described in the assembly formation scheme 1, it is presumed that the orientation of M3L2 in PuuE-M is relatively flexible, whereas the orientation of p66α in PuuE-p is highly restricted, resulting in a rigid structure. Therefore, we attempted to control the structure of the formed assembly by changing the movement or orientation of the heterolinkers M3L2 and p66α. The movement or orientation of M3L2 and p66α was controlled by introducing amino acid mutations between the base protein and the heterolinker. (1) Regulation of M3L2 and p66α activity To restrict the orientation of M3L2 to a rigid structure, a site-directed mutagenesis kit (KOD-Plus-Mutagenesis Kit, TOYOBO) was used to replace S at position 317 in the amino acid sequence of PuuE-M (SEQ ID NO: 6) with P and delete G at position 318 to construct PuuE-Rigid-M3L2 (PuuE-Rigid-M: SEQ ID NO: 13). This construct was then expressed in E. coli and purified as described above. To make p66α more flexible, PuuE-Flexible-p66α (PuuE-Flexible-p: SEQ ID NO: 14) was constructed by replacing PYTP at positions 314 to 317 in the amino acid sequence of PuuE-p (SEQ ID NO: 7) with GGGGS. This construct was then expressed in E. coli and purified as described above. The underlined parts in the following sequences indicate the substituted portions. Combinations of PuuE-M and PuuE-Flexible-p, PuuE-Rigid-M and PuuE-Flexible-p, and PuuE-Rigid-M and PuuE-p were mixed in NaCl(+) buffer to a final concentration of 12.5 μM and incubated at 40°C for 24 hours. The results are shown in Figure 24. As shown in Figure 24, mixing PuuE-M and PuuE-Flexible-p resulted in the formation of a cage structure. Mixing PuuE-Rigid-M and PuuE-Flexible-p resulted in the formation of a tube structure, similar to that of PuuE-M and PuuE-p (see Figure 6). Mixing PuuE-Rigid-M and PuuE-p resulted in the formation of a sheet structure. PuuE-Rigid-M MHHHHHHENLYFQGVDYPRDLIGYGSNPPHPHWPGKARIALSFVLNYEEGGERNILHGDKESEAFLSEMVSAQPLQGERNMSMESLYEYGSRAGVWRILKLFKAFDIPLTIFAVAMAAQRHPDVIRAMVAAGHEICSHGYRWIDYQYMDEAQEREHML EAIRILTELTGERPLGWYTGRTGPNTRRLVMEEGGFLYDCDTYDDDLPYWEPNNPTGKPHLVIPYTLDTNDMRFTQVQGFNKGDDFFEYLKDAFDVLYAEGAEAPKMLSIGLHCRLIGRPARLAALQRFIEYAKSHEQVWFTRRVDIARHWHATHPYT P QLVTPADIRRQARRVKKARERLAKALQADRLA (SEQ ID NO: 13) PuuE-Flexible-p MHHHHHHENLYFQGVDYPRDLIGYGSNPPHPHWPGKARIALSFVLNYEEGGERNILHGDKESEAFLSEMVSAQPLQGERNMSMESLYEYGSRAGVWRILKLFKAFDIPLTIFAVAMAAQRHPDVIRAMVAAGHEICSHGYRWIDYQYMDEAQEREH MLEAIRILTELTGERPLGWYTGRTGPNTRRLVMEEGGFLYDCDTYDDDLPYWEPNNPTGKPHLVIPYTLDTNDMRFTQVQGFNKGDDFFEYLKDAFDVLYAEGAEAPKMLSIGLHCRLIGRPARLAALQRFIEYAKSHEQVWFTRRVDIARHWHATH GGGGS EERERMIKQLKEELRLEEAKLVLLKKLRQSQIQ (SEQ ID NO: 14) (2) Regulation of the orientation of M3L2 and p66α PuuE-Rigid-p66α (PuuE-Rigid-p: SEQ ID NO: 15) was constructed by deleting the 316th T in the amino acid sequence of PuuE-p (SEQ ID NO: 7) so that the orientation of p66α in PuuE-p was the same (plane) as that of M3L2 in PuuE-M. PuuE-Rigid-p was then expressed in E. coli and purified as described above. PuuE-M and PuuE-Rigid-p were mixed in NaCl(+) buffer to a final concentration of 12.5 μM each and incubated at 40°C for 24 hours. The results are shown in Figure 25. As shown in Figure 25 (bottom right), a tape-like structure was formed. PuuE-Rigid-p MHHHHHHENLYFQGVDYPRDLIGYGSNPPHPHWPGKARIALSFVLNYEEGGERNILHGDKESEAFLSEMVSAQPLQGERNMSMESLYEYGSRAGVWRILKLFKAFDIPLTIFAVAMAAQRHPDVIRAMVAAGHEICSHGYRWIDYQYMDEAQEREHM LEAIRILTELTGERPLGWYTGRTGPNTRRLVMEEGGFLYDCDTYDDDLPYWEPNNPTGKPHLVIPYTLDTNDMRFTQVQGFNKGDDFFEYLKDAFDVLYAEGAEAPKMLSIGLHCRLIGRPARLAALQRFIEYAKSHEQVWFTRRVDIARHWHATHP YP EERERMIKQLKEELRLEEAKLVLLKKLRQSQIQ (SEQ ID NO: 15) These results indicate that by controlling the movement and orientation of the heterolinker, structures with various structures (morphologies) can be formed.

[0089] Aggregation Scheme 6 PuuE-K46C-M3L2 (PuuE-K46C-M: SEQ ID NO: 16) and PuuE-K46C-p66α (PuuE-K46C-p: SEQ ID NO: 17) were constructed by substituting K at position 46 of PuuE in PuuE-M (SEQ ID NO: 6) and PuuE-p (SEQ ID NO: 7) and were expressed in E. coli and purified as described above. In SEQ ID NOs: 16 and 17 below, the substituted C at position 46 is underlined. Then, PuuE-K46C-p and PuuE-M modified with Alexa488-Maleimide were mixed in NaCl(+) buffer to a final concentration of 12.5 μM each and incubated at 40°C for 24 hours. The results are shown in Figure 26. The formation of tubular structures was confirmed as shown in Figure 26 (left), and the image observed by fluorescence microscopy is shown in Figure 26 (right). Fluorescent labeling PuuE-K46C-p was thawed on ice immediately prior to reduction and fluorescent labeling. For the reduction reaction, a 12.5 μM PuuE-K46C-p solution was prepared on ice using NaCl(+) buffer. Tris(2-carboxyethyl)phosphine (TCEP) was added to a final concentration of 0.5 mM, and the mixture was incubated at 4°C for several hours with gentle shaking. For the labeling reaction, Alexa Fluor 488 C5 maleimide dissolved in DMSO was added to the reduced PuuE-K46C-p solution at a final concentration of 0.5 mM, and the mixture was incubated overnight at 4°C with gentle shaking, protected from light. Excess dye was removed using a Microcon 50 centrifugal filter unit (Merck) by four NaCl(+) buffer exchanges according to the protocol. The labeled PuuE-K46C-p was frozen in liquid nitrogen and stored at -80°C protected from light until further experiments. PuuE-K46C-M MHHHHHHENLYFQGVDYPRDLIGYGSNPPHPHWPGKARIALSFVLNYEEGGERNILHGD C ESEAFLSEMVSAQPLQGERNMSMESLYEYGSRAGVWRILKLFKAFDIPLTIFAVAMAAQRHPDVIRAMVAAGHEICSHGYRWIDYQYMDEAQEREHMLEAIRILTELTGERPLGWYTGRTGPNTRRLVMEEGGFLYDCDTYDDDLPYWEPNNPTGKPHLVIPYTLDTNDMRFTQVQGFNKGDDFFEYLKDAFDVLYAEGAEAPKMLSIGLHCRLIGRPARLAALQRFIEYAKSHEQVWFTRRVDIARHWHATHPYTGSGSGSGQLVTPADIRRQARRVKKARERLAKALQADRLA (SEQ ID NO: 16) PuuE-K46C-p MHHHHHHENLYFQGVDYPRDLIGYGSNPPHPHWPGKARIALSFVLNYEEGGERNILHGD C ESEAFLSEMVSAQPLQGERNMSMESLYEYGSRAGVWRILKLFKAFDIPLTIFAVAMAAQRHPDVIRAMVAAGHEICSHGYRWIDYQYMDEAQEREHMLEAIRILTELTGERPLGWYTGRTGPNTRRLVMEEGGFLYDCDTYDDDLPYWEPNNPTGKPHLVIPYTLDTNDMRFTQVQGFNKGDDFFEYLKDAFDVLYAEGAEAPKMLSIGLHCRLIGRPARLAALQRFIEYAKSHEQVWFTRRVDIARHWHATHPYTPEERERMIKQLKEELRLEEAKLVLLKKLRQSQIQ (SEQ ID NO: 17)

[0090] Aggregation Scheme 7 We attempted to form tubular structures loaded with functions (DogTag or SplitGFP). A gene for expressing PuuE-DogTag-M3L2 (PuuE-DogTag-M: SEQ ID NO: 18) was constructed by inserting DogTag into the loop structure located on the back side of PuuE-M (between Q at position 76 and G at position 77 in PuuE-M (SEQ ID NO: 6)). This gene was expressed in E. coli and purified as described above. Furthermore, a gene for expressing G11-PuuE-M3L2 (G11-PuuE-M: SEQ ID NO: 19) was constructed by inserting GFP11 (Split GFP) into the N-terminus of PuuE-M. This gene was expressed in E. coli and purified as described above. The underlined portion in the PuuE-DogTag-M sequence is the DogTag sequence, and the underlined portion in the G11-PuuE-M sequence is the GFP11 sequence. PuuE-DogTag-M and PuuE-p, and G11-PuuE-M and PuuE-p were mixed in NaCl(+) buffer to a final concentration of 12.5 μM, and incubated at 30°C for 24 hours. The results are shown in Figure 27. As shown in Figure 27, tube structures were formed in both cases. PuuE-DogTag-M MHHHHHHENLYFQGVDYPRDLIGYGSNPPHPHWPGKARIALSFVLNYEEGGERNILHGDKESEAFLSEMVSAQPLQ DIPATYEFTDGKHYITNEPIPPK GERNMSMESLYEYGSRAGVWRILKLFKAFDIPLTIFAVAMAAQRHPDVIRAMVAAGHEICSHGYRWIDYQYMDEAQEREHMLEAIRILTELTGERPLGWYTGRTGPNTRRLVMEEGGFLYDCDTYDDDLPYWEPNNPTGKPHLVIPYTLDTNDMRFTQVQGFNKGDDFFEYLKDAFDVLYAEGAEAPKMLSIGLHCRLIGRPARLAALQRFIEYAKSHEQVWFTRRVDIARHWHATHPYTSGQLVTPADIRRQARRVKKARERLAKALQADRLA(SEQ ID NO: 18) G11-PuuE-M M RDHMVLHEYVNAAGIT HHHHHHENLYFQGVDYPRDLIGYGSNPPHPHWPGKARIALSFVLNYEEGGERNILHGDKESEAFLSEMVSAQPLQGERNMSMESLYEYGSRAGVWRILKLFKAFDIPLTIFAVAMAAQRHPDVIRAMVAAGHEICSHGYRWIDYQYMDEAQEREHMLEAIRILTELTGERPLGWYTGRTGPNTRRLVMEEGGFLYDCDTYDDDLPYWEPNNPTGKPHLVIPYTLDTNDMRFTQVQGFNKGDDFFEYLKDAFDVLYAEGAEAPKMLSIGLHCRLIGRPARLAALQRFIEYAKSHEQVWFTRRVDIARHWHATHPYTSGQLVTPADIRRQARRVKKARERLAKALQADRLA(SEQ ID NO: 19)

Claims

1. A protein assembly structure formed by linking two or more types of modules, the module is a homomultimer formed by a fusion protein of a protein capable of forming a symmetric homomultimer and a peptide serving as a binding site between modules, the peptide serving as the binding site is capable of reversibly forming a heterodimer; A protein assembly structure in which different modules are linked by the peptides forming heterodimers.

2. The structure of claim 1 , wherein the homomultimer has rotational symmetry.

3. The structure of claim 1 , wherein the connections between the different modules are reversible.

4. the modules include a first module and a second module; The structure of claim 1 , wherein the first modules and the second modules are interlocked.

5. the binding site of the first module is a peptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, 3, 4 or 5; the binding site of the second module is a peptide having at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 2; The structure of claim 4 , wherein the binding site of the first module and the binding site of the second module are capable of forming a reversible heterodimer.

6. The structure according to claim 4 , wherein the proteins capable of forming homomultimers in the first module and the second module are the same or different proteins.

7. The structure according to claim 1 , wherein the peptide that serves as a binding site between the modules is fused to the end of the protein capable of forming a homomultimer.

8. The structure of claim 1 , having at least one morphology selected from the group consisting of a cage, a wire, a sheet, a tube, and a crystalline structure.

9. A method for producing a protein assembly structure formed by linking two or more types of modules, comprising the steps of: Prepare two or more types of modules. wherein the module is a homomultimer formed by a fusion protein of a protein capable of forming a symmetric homomultimer and a peptide serving as a binding site between modules, and the peptide serving as the binding site is capable of reversibly forming a heterodimer; and mixing the prepared modules and driving spontaneous assembly through interactions between binding sites of different modules; method.

10. the two or more types of modules include a first module and a second module; the protein assembly structure is a protein assembly structure in which the first modules and the second modules are alternately linked, 10. The method of claim 9, comprising mixing the first module and the second module and driving spontaneous assembly by interaction between a binding site of the first module and a binding site of the second module.

11. The method according to claim 9, wherein the protein assembly structure is a structure according to any one of claims 1 to 8.

12. A kit for producing a protein-assembled structure, comprising: a first module, which is a homomultimer formed by a fusion protein of a protein capable of forming a symmetric homomultimer and a peptide serving as a binding site between modules; a second module which is a homomultimer formed by a fusion protein of a protein capable of forming a symmetric homomultimer and a peptide which serves as a binding site between modules; The binding site of the first module and the binding site of the second module are capable of reversibly forming a heterodimer.

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