Alpha-helix protein nanofibril

Abstract

The present invention relates to the field of microbial self-assembling protein fibrils as novel bionanomaterials. More specifically, the present invention relates to protein nanofibril materials comprising Bacillus spore appendage (ENA) proteins, which are spontaneously folding helix hairpin subunits having a hydrophobic core and assemble into alpha-helix multimer (A-ENA) fibrils covalently linked by one or more isopeptide bonds, providing nanofibrils with high stability and tensile strength. The present invention further relates to the protein nanofibrils manipulated or modified to provide functionalized bionanomaterials. In particular, the present invention relates to methods for the recombinant production of the self-assembling protein nanofibrils, and their use in modifying bacterial spores and spore activity or pathogenicity.

Description

[Technical Field]

[0001] The present invention relates to the field of microbial self-assembling protein fibrils as novel bionanomaterials. More specifically, the present invention relates to protein nanofibril materials comprising Bacillus spore appendage (ENA) proteins, which are spontaneously folding helix hairpin subunits having a hydrophobic core and assemble into alpha-helix multimer (A-ENA) fibrils covalently linked by one or more isopeptide bonds, providing nanofibrils with high stability and tensile strength. The present invention further relates to the protein nanofibrils manipulated or modified to provide functionalized bionanomaterials. In particular, the present invention relates to methods for the recombinant production of the self-assembling protein nanofibrils, and their use in modifying bacterial spores and spore activity or pathogenicity. [Background technology]

[0002] Nanotechnology facilitates the control of events at the nanoscale and is applied, in particular, to the design of novel functionalized nanomaterials, including nanofibers, nanowires, nanofibrils, and nanoparticles. Nanofibrils are typically constructed from polysaccharides such as cellulose and chitin, and / or proteins such as silk fibroin, thereby providing refined mechanical properties as well as addressing the need for biosustainable and biodegradable alternatives. The most abundant starting material to date is cellulose, which, as a polysaccharide in nanometer form, can be isolated and modified for biomaterial applications. Secondly, protein nanofibrils such as silk, spider silk, and collagen have been characterized in animals and plants and have been described, in particular, as promising nanostructures for the development of bionanomaterials and biomedical applications. Furthermore, starting from biological production, the modification and utilization of these bionanomaterials increases the potential to provide materials with advantageous biological properties. With numerous applications, their value is increasing in an economy transitioning to a more ecological and biosustainable society, and the demand for commercially viable solutions is enormous.

[0003] Bacterial spores are a remarkable example of biological resilience, representing a dormant survival form that can withstand adverse conditions, including physical and chemical hazards such as high salinity, acidity, temperature, and drought, for extended periods (decades). Therefore, the proteinaceous structures constituting the spore coating and its appendages require material properties that can adapt to these harsh conditions. Previously, a fundamentally new class of biological nanofibers was structurally elucidated and identified on the spores of Bacillus cereus sensu lato (sl), and named spore appendages or ENA (Pradhan et al., 2021, the EMBO Journal e106887; Remaut et al., International Publication No. 2022 / 029325). Based on the self-assembling protein fibril structures present in spore samples, the proteins constituting these protein assemblies were classified as S-ENA and L-ENA proteins. These nanofibrils, due to their high flexibility and elasticity, clearly form a new family of components for future bionanomaterials and further functionalization, possessing intriguing properties such as remarkable chemical and physical strength that withstands low pH, protease treatment, pasteurization, drying, and high vacuum. Furthermore, the self-assembly properties of S- and L-ENA fibrils facilitate high-yield recombinant microbial production, and the fibrils' extremely high stability facilitates easy isolation from cell debris, proteins, nucleic acids, and membranes while retaining their native structure and properties. Functionalization by inserting peptides into two different surface loops present on proteins has been demonstrated, at least for S-ENA-based fibrils (Remaut et al., International Publication No. 2022 / 029325). However, heterologous insertion is inherently limited in length and the types of folds possible to manipulate in order to maintain the self-assembly properties of the protein and the mechanical properties of the resulting fibrils, and N-terminal or C-terminal functionalization is undesirable in this type of S- or L-Ena fibril. S-ENA fibrils have been reported to contain intermolecular disulfide covalent bonds to increase their tensile strength, stability, and stiffness.Such rigid covalent interactions are known to occur in other types of protein materials, such as in the widely known ubiquitination reaction, or in the formation of intermolecular isopeptide bonds.

[0004] Bacteria, generally Gram-positive bacteria, possess pyrin, which utilizes enzyme-mediated intermolecular isopeptide bonds to covalently connect subunits and construct long, narrow fibril structures on the cell surface (Hendrickx et al., 2011, Nat. Rev. Micro. 9, 166-176). In addition to these enzyme-dependent processes, intramolecular isopeptide bonds embedded in the hydrophobic environment of β-chain-based immunoglobulins can be formed autocatalytically, playing a role in increasing the protease resistance, thermal, and mechanical stability of pyrin-forming protein domains (e.g., collagen adhesin domain A (CnaA) in FimA, and CnaB in Spy; Kang et al., 2007, Science, 318(5856):1625-8; Echelman et al., 2016, PNAS; 113(9):2490-5). Such autocatalytic isopeptide bonds are formed through proximity-induced reactions, and their self-assembly properties depend primarily on the geometric arrangement of the amino acids involved and the hydrophobic moiety where the isopeptide unit is formed (Kang and Baker, 2011, Trends Biochem Sci. 36(4):229~37). The geometric arrangement of autocatalytic isopeptide bond formation identified in the immunoglobulin-like domain of bacterial pyri has been successfully utilized by biotechnology to covalently bond manipulated derivatives in fragmented domain architectures such as those found in SpyCatcher-SpyTag or SnoopCatcher-SnoopTag technologies (Zakeri et al., 2012, Proc.Nat.Acad.Sci.USA 109:E690~97; Hatlem et al., 2019, Int J Mol Sci 20(9):2129). To date, there is a lack of equivalent isopeptide bond formation geometric arrangements that enable covalent bonding of alpha-helix elements, limiting the stability and application areas of natural and synthetic alpha-helix protein materials. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] International Publication No. 2022 / 029325 [Non-patent literature]

[0006] [Non-Patent Document 1] Pradhan et al., 2021, the EMBO Journal e106887 [Non-Patent Document 2] Hendrickx et al., 2011, Nat. Rev. Micro. 9, 166-176. [Non-Patent Document 3] Kang et al., 2007, Science, 318(5856):1625~8 [Non-Patent Document 4] Echelman et al., 2016, PNAS;113(9):2490~5 [Non-Patent Document 5] Kang and Baker, 2011, Trends Biochem Sci. 36(4):229~37 [Non-Patent Document 6] Zakeri et al., 2012, Proc.Nat.Acad.Sci.USA 109:E690~97 [Non-Patent Document 7] Hatlem et al., 2019, Int J Mol Sci 20(9):2129 [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] To improve and further customize innovations in protein-based materials and nanofibrils, there is a need for novel types of protein fibrils that possess superior characteristics in terms of chemical stability, mechanical resistance, bioengineering potential, and sustainable manufacturing and biodegradability options. [Means for solving the problem]

[0008] (Summary of the invention) This invention discloses a novel class of protein-based nanofibrils derived from Bacillus spores, particularly Bacillus thuringiensis, the fibrils typically appearing on the surface of the spore in association with a parasporal body. The protein nanofibrils of this invention are characterized herein for the first time as being constructed from alpha-helix hairpin proteins, and in agreement with the previously annotated family of spore appendage proteins (see Pradhan et al., 2021), these newly annotated fibril-assembly proteins were referred to as alpha-helix spore appendage proteins or A-ENA proteins. This novel protein family is unique in that it exhibits an ingeniously simplified monomeric or subunit structure of two antiparallel alpha helices linked by short amino acid turns and terminating at the free N-terminus and C-terminus, where the subunits self-assemble into two helically wound protofilaments or protofibrils (see, e.g., Figure 3), resulting in nanofibrils where the side chains of the A-ENA monomers are positioned as a helix wheel, providing an environment that enables crosslinking by isopeptide bonds in an astonishing pattern (see, e.g., Figure 7). The presence of intermolecular covalent interactions between A-ENA subunits within and between protofibrils provides irreversibly organized structural components for forming A-ENA fibrils with high tensile strength. The present invention relates to these protein nanofibrils constituting A-ENA or A-ENA-based protein variants defined by a rational pattern of nucleophilic, electrophilic, and acid / base properties for designing isopeptide bond-forming units for covalently stabilizing helix-helix junctions. Furthermore, through recombinant production in a (microbial) host, nanofibrils can also be produced starting from the expression of recombinant A-ENA or A-ENA-like or A-ENA-based variant proteins from chimeric gene constructs. Self-assembly is conserved for structural preservation of the protein family.Finally, the simple topology of A-ENA-based variants or manipulated A-ENA fibrils allows A-ENA nanofibrils to possess N-terminuses and C-terminuses in addition to (poly)peptide constructs inserted into internal loops or turns, resulting in tunable and high-density fibril functionality.

[0009] Accordingly, a first aspect of the present invention relates to a protein-based fibril, hereafter referred to herein as a protein nanofibril, which is composed of or derived from two protofibrils, each protofibril comprising two or more monomeric protein subunits, and thus the fibril comprising at least a tetramer, each monomeric protein subunit comprising amino acid sequence fragments linked by covalent bonds, the covalent bonds being peptide bonds according to the formula: N-terminal lock (NTL)-helix 1-linker (L)-helix 2-C-terminal tail (CT). The monomeric protein subunit, when expressed or present in aqueous solution, self-assembles into two helices, helix 1 and helix 2, as an alpha-helix antiparallel coiled-coil protein structure, where helix 1 and helix 2 each contain a sequence of at least five heptad (H) elements according to formula H1-1-H1-2-H1-3-H1-4-H1-5 for helix 1 and H2-1-H2-2-H2-3-H2-4-H2-5 for helix 2, and the heptad elements have the following consensus sequence: - Heptad 1-1:XXX-Φ-XX-Φ, - Heptad 1-2:Φ-X-Ψ-Φ-Ψ-Φ-γ, - Heptad 1-3:ε-X-Ψ-Φ-Ψ-X-Φ, - Heptad 1-4:Φ-N-Ψ-γ-Ψ-γ-δ, - Heptad 1-5:Φ-ε-X-Φ-Φ-XX, - Heptad 2-1:Φ-XX-Φ-Φ-X-Φ, - Heptad 2-2: ε-XX-Φ-XXX, - Heptad 2-3: Φ-X-X-Φ-Φ-δ-δ, - Heptad 2-4: ε-Φ-Φ-Φ-ε-X-δ, - Heptad 2-5: Φ-γ-X-Φ-Φ-X-X each comprising 7 amino acid residues designated as "abcdefg", wherein, - Φ is a hydrophobic amino acid selected from the list of M, V, I, L, A, G, H, W, Y, F, preferably a bulky amino acid selected from L, M, I, V, F or W, for at least 70% of the Φ positions shown in the aforementioned heptad elements, - Ψ is a short side chain residue selected from the list of V, C, G, A, P, S, T, N, D, preferably an amino acid selected from A, G, S, T, - γ is a residue functioning as an acid / base catalyst selected from Glu(E) or Asp(D) for at least one or more of the γ positions shown in the aforementioned heptad elements, - δ is a residue capable of functioning as a "donor" or nucleophile for an isopeptide bond selected from lysine for at least one or more of the δ positions shown in the aforementioned heptad elements, - ε is a residue capable of functioning as an "acceptor" or electrophile for an isopeptide bond selected from Glu(E) or Asp(D), Gln(Q) or Asn(N) for at least one or more of the ε positions shown in the aforementioned heptad elements, - γ, δ and ε may be any amino acid at one or more remaining positions not involved in IPB formation, - X may be any type of amino acid, The linker (L) fragment comprises at least 4 amino acids, the N-terminal lock (NTL) and C-terminal tail (CT) fragments each comprise at least one amino acid, and the aforementioned monomeric protein subunits are interconnected via at least one or more isopeptide bonds (IPB).

[0010] Specifically, the protein nanofibril forms at least one IPB to covalently interconnect two protofibrils (f) and (f'). More specifically, the at least one IPB interconnecting the two protofibrils (f) and (f') is positioned between a side chain of an amino acid acting as a nucleophile and a side chain of an amino acid acting as an electrophile: - H2-3f of (i) and H2-4e of (i'), and / or - H2-3f of (i'+1) and H2-4e of (i) In this configuration, it is located between the monomeric protein subunit (i) of protofibril (f) and the monomeric subunit (i' and / or i'+1) of protofibril (f').

[0011] Alternatively, the protein nanofibril may form at least one IPB to covalently interconnect two protofibrils (f') and (f), and the at least one IPB interconnecting the two protofibrils (f') and (f) is positioned between a side chain of an amino acid acting as a nucleophile and a side chain of an amino acid acting as an electrophile: - H2-3f of (i') and H2-4e of (i), and / or - H2-3f of (i+1) and H2-4e of (i') In this configuration, it is located between the monomeric protein subunit (i') of protofibril (f') and the monomeric subunit (i and / or i+1) of protofibril (f).

[0012] In further embodiments, the protein nanofibril has a tertiary structure having a minimum tetrameric entity as shown in Figure 3c, and / or the monomeric protein subunits (i+ / -n) of protofibril(f) and the monomeric subunits (i'+ / -n) of protofibril(f') are covalently linked via at least one IPB, preferably 10 IPBs, where the IPBs are positioned, for example, between the side chains of amino acids acting as nucleophiles and the side chains of amino acids acting as electrophiles, as shown in Figure 9: - (i) NTL and (i-5) or (i-4), (i-3) or (i-2) H1-5b, - H1-4g of (i) and H2-2a of (i-1), - H2-3g of (i) and H2-4a of (i-1), - H2-3f in (i) and H2-4e in (i'), - H2-4g of (i) and H1-3a of (i-1), - H2-4g of (i+1) and H1-3a of (i), - NTL of (i+5) or (i+4), (i+3) or (i+2) and H1-5b of (i), - H1-4g of (i+1) and H2-2a of (i), - H2-3g of (i+1) and H2-4a of (i), and / or - H2-3f of (i'+1) and H2-4e of (i) It is formed in [location].

[0013] Alternatively, the protein nanofibrils have a tertiary structure with a minimum tetrameric entity as shown in Figure 3c, and / or the monomeric protein subunits (i+ / -n) of protofibril(f) and the monomeric subunits (i'+ / -n) of protofibril(f') are covalently linked via at least one IPB, preferably 10 IPBs, where each IPB is positioned between a side chain of an amino acid acting as a nucleophile and a side chain of an amino acid acting as an electrophile: - (i) NTL and (i-5), (i-4), (i-3) or (i-2) H1-5b, - H1-4g of (i) and H2-2a of (i-1), - H2-3g of (i) and H2-4a of (i-1), - H2-3f of (i) and H2-4e of (i'-1), - H2-4g of (i) and H1-3a of (i-1), - H2-4g of (i+1) and H1-3a of (i), - NTL of (i+5), (i+4), (i+3) or (i+2) and H1-5b of (i), - H1-4g of (i+1) and H2-2a of (i), - H2-3g of (i+1) and H2-4a of (i), and / or - H2-3f of (i') and H2-4e of (i) It is formed in [location].

[0014] In a further embodiment, the protein nanofibril has the following consensus sequence: - H1-1:XXX-Φ-XX-Φ, - H1-2:Φ-X-(S / T)-Φ-(A / G)-Φ-(E / Q), - H1-3:EXX-Φ-X-(H / N)-Φ, - H1-4:Φ-N-(A / G / S)-E-(G / A)-EK, - H1-5:Φ-QX-Φ-Φ-XX, - H2-1:Φ-XX-Φ-Φ-X-Φ, - H2-2:(N / Q / D)-XX-Φ-XXX, - H2-3:Φ-XX-Φ-Φ-(K / X)-(K / X), - H2-4:(E / Q)-Φ-Φ-(L)-(Q / X)-XK, - H2-5:Φ-γ-X-Φ-Φ-XX It comprises helical α1 and α2 heptad elements having, where a single-letter amino acid code is applied to a specific residue, and " / " is read as "or", - Φ is a hydrophobic amino acid selected from the list of M, V, I, L, A, G, H, W, Y, F, preferably a bulk amino acid selected from L, M, I, V, F or W. - γ is a residue that functions as an acid / base catalyst selected from Glu(E), Gln(Q), or Asp(D). - X can be any type of amino acid.

[0015] Further specific embodiments relate to protein nanofibrils in which the monomeric protein subunit is based on, derived from, or originates from Bacillus proteins, in particular Bacillus spore appendage (ENA) proteins, and more specifically alpha-helix (A-)ENA proteins, preferably Bacillus thuringiensis.

[0016] Further embodiments provide the protein nanofibril having an N-terminal lock (NTL) amino acid sequence corresponding to M-Ψ-Φ-ZX-Φ-P, where, - Ψ = a short side chain residue selected from the list of V, C, G, A, P, S, T, N, D, preferably A, G, S, or T. - Φ is a hydrophobic amino acid selected from the list of M, V, I, L, A, G, H, W, Y, F, preferably L, M, I, V, F, or W. - X is any amino acid, Z is Ala or proline, and M and P are the single-letter codes for the respective amino acids methionine and proline.

[0017] More specifically, the protein nanofibril may include an NTL having an amino acid sequence corresponding to M-Ψ-Φ-ZX-Φ-P, where Ψ is selected from S, G, or T; Φ is a hydrophobic amino acid selected from the list M, V, I, L, A, G, H, W, Y, F, preferably L, M, I, V, F, or W; X is any amino acid; Z is Ala or proline; and M and P are single-letter codes for the respective amino acids methionine and proline.

[0018] Further specific embodiments relate to a protein fibril in which the monomeric NTL-helix1-L-helix2-CT sequence comprises an A-ENA protein sequence, wherein the A-ENA protein sequence is selected from a list of proteins indicated by their access codes provided in Table 3. In specific embodiments, the A-ENA protein sequence is a modified, manipulated, or functionalized fibril comprising an A-ENA protein selected from the list of SEQ ID NOs: 1-6 or selected from SEQ ID NOs: 1-6, wherein the NTL and / or linker and / or CT are modified.

[0019] In further specific embodiments, the monomeric NTL-helix1-L-helix2-CT sequence is modified with respect to the protein fibrils or modified, manipulated, or functionalized fibrils consisting of the A-ENA(like) protein, defined by hidden Markov model (hmm) profiles for helix1 and helix2 shown in Figure 11 and Tables 1 and 2, respectively, where the NTL and / or linker and / or CT are modified. Those skilled in the art can apply (online) tools and databases to verify whether a protein sequence conforms to an HMM profile representing a protein class or family (e.g., https: / / www.ebi.ac.uk / Tools / hmmer / ), and thus, using the information disclosed herein in Tables 1-3 and Figure 11, it becomes possible to determine whether a protein belongs to the A-Ena family.

[0020] Further specific embodiments relate to any one of the protein fibrils in which the NTL is cleaved in such a way that it comprises at least one but fewer than four amino acids.

[0021] Another embodiment relates to the protein-based fibril, wherein the monomeric protein subunit comprises at least four amino acid NTLs, and the monomeric protein is a mutant variant that is modified on its surface or compared to the consensus motif of fragment NTL-Helix 1-L-Helix 2-CT as defined herein, having at least one amino acid substitution at the following positions in the heptad of the helix 1 or helix 2 region: H1-1a, H1-1b, H1-1c, H1-3f, H1-4f, H1-5b, H1-5f, H2-1b, H2-1c and / or H2-5b, and having different amino acids from the list of amino acids provided for the consensus sequence described herein for the heptad positions.

[0022] In alternative embodiments, the protein-based fibril comprises a monomeric protein subunit containing at least one but fewer than four amino acid NTLs, wherein the monomeric protein is a mutant variant modified on its surface or compared to the consensus motif of the fragment NTL-Helix 1-L-Helix 2-CT as defined herein, having at least one amino acid substitution at the following positions in the heptad of the helix 1 or helix 2 region: H1-1a, H1-1b, H1-1c, H1-1f, H1-2f, H1-3b, H1-3f, H1-4b, H1-4f, H1-5b, H1-5f, H2-1b, H2-1c, H2-2b, H2-2f, H2-3f, H2-4f, H2-5b, H2-5c and / or H2-5f, and having different amino acids from the list of amino acids provided for the consensus sequence described herein for the heptad positions.

[0023] Further embodiments relate to the protein nanofibrils, wherein a monomeric protein containing a consensus sequence corresponding to the formula NTL-Helix 1-L-Helix 2-CT as defined herein is further ligated to a tag or protein domain or conjugated within the N-terminus, C-terminus, or linker region of the monomer to form a functionalized fibril. The fusion or ligation may occur directly or in conjunction with a (flexible and / or optimized) linker sequence. Thus, the self-assembled protein fibril comprises one or more monomers to which a functional fusion to a further portion exists, the further portion may be a folded protein, tag, label, or functional portion fused or conjugated directly or via a linker to the A-ENA-based monomer. In certain embodiments, the monomeric protein of the protein fibril ligated to a further portion to form a functionalized protein nanofibril is obtained from (recombinant) expression from a gene fusion of monomers operably ligated to the further functional portion. More specifically, the gene fusion of the monomer may be present as its N-terminus or C-terminus, or as an insertion in the linker region. In another particular embodiment, the monomer protein of the protein nanofibril is functionalized or modified after fibril formation, and the modified or functionalized protein nanofibril is obtained from the orthogonal conjugation of one or more monomer protein subunits of the protein nanofibril after the self-assembly of the fibril. In preferred embodiments, the further orthogonal conjugation comprises a covalent bond, more preferably an isopeptide bond, and even more preferably, the functionalization comprises a modification comprising a peptide tag / binding partner pair derived from an isopeptide bond-forming chain-fimbriae subunit folding complement, more specifically a (Spy) tag / collagen adhesin domain B (CnaB2) catcher type (e.g., SpyCatcher) or a (Snoop) tag / D4 domain (RrgA) catcher type (e.g., SnoopCatcher) binding partner type protein or fusion protein pair (Hatlem et al., 2019, Int J Mol Sci 20(9):2129).

[0024] Further embodiments provide the protein nanofibrils comprising the monomeric proteins described herein, wherein at least one monomer, preferably A-ENA or an A-ENA-based variant monomer, further comprises fusion with a heterogeneous protein domain for surface presentation on the protein nanofibrils. Certain embodiments relate to the protein nanofibrils comprising at least one monomer described herein further fused to a heterogeneous protein domain, wherein the fusion is made at the N-terminus or C-terminus of the monomer or inserted at the L- or linker region of the monomer. In certain further embodiments, the heterogeneous protein domain fused to the at least one protein monomer comprises a VHH for nanobody surface presentation or a peptide tag / binding partner pair counterpart, preferably a covalent interaction bond such as an isopeptide bond, more specifically, a tag / catcher binding pair such as SpyTag / SpyCatcher or SnoopTag / SnoopCatcher, or further proteins used for surface presentation by being present on the surface of the protein nanofibrils. In a further specific embodiment, the heterologous protein comprises a sequence selected from SEQ ID NOs: 7-11, 13-15, or 67.

[0025] In one particular embodiment, the protein nanofibril comprises monomers fused to heterogeneous protein domains, each monomer being identical, and forming a homopolymer fibril by self-assembly. In a further alternative embodiment, the protein nanofibril comprises one or more monomeric protein subunits as described herein, each monomer further comprising a heterogeneous protein domain, which comprises a flexible (optimized) linker between the monomer and the heterogeneous protein domain as described herein, and the protein domain has a hydrodynamic radius of less than 11 nm, which is suitable for surface presentation when the heterogeneous protein domain is on a homopolymer fibril.

[0026] Another specific embodiment relates to the nanofibrils, wherein the monomer subunits described herein are fused to heterologous protein domains in at least one monomer to form protein nanofibrils, and after self-assembly of the monomers into protofibrils and entanglement with the nanofibrils, at least two different sequences of monomer proteins appear as part of the heteropolymer fibrils.

[0027] Further specific embodiments provide protein nanofibrils that are protein nanofibrils isolated "as is" from a bacterial or spore environment and / or that do not contain further biological materials such as spores or parasporial attachment components. In further specific embodiments, the protein nanofibrils described herein are isolated from a recombinantly produced bacterial cell culture, preferably from Bacillus or from a Bacillus cell culture or spore-forming culture, preferably from Bacillus thuringiensis, wherein the bacterial cell culture comprises heterologous A-ENA or A-ENA base or modified A-ENA monomer protein subunits for self-assembly into the protein nanofibrils described herein. Alternatively, the protein nanofibrils described herein are recombinantly produced protein nanofibrils, such as protein fibrils expressed in the cytoplasm of a host cell and isolated from the host cell, wherein the host cell may be a prokaryotic or eukaryotic host cell.

[0028] In a second aspect, the present invention relates to a modified bacterial spore, preferably a Bacillus spore, compared to a wild-type bacterial spore, preferably a Bacillus spore, in that the spore comprises and / or presents a modified A-ENA monomer protein subunit or a functionalized protein nanofibril as described herein.

[0029] More specifically, the modified bacterial spore, preferably a Bacillus spore, may lack an endogenous A-ENA monomer protein conforming to the consensus sequence of the formula NTL-helix1-L-helix2-CT as described herein, and the bacterial strain, preferably a Bacillus strain, may contain an exogenously introduced A-ENA monomer protein comprising the consensus sequence in the form of the formula NTL-helix1-L-helix2-CT as described herein, or may contain the protein nanofibrils presenting the heterologous or exogenously introduced modified A-ENA monomer protein or monomer mutant variant or monomer fusion protein previously described herein.

[0030] Further embodiments relate to the use of the modified bacterial spores, preferably Bacillus spores, to increase the activity of the bacterial spores, and more preferably to improve or increase Bt pathogenicity.

[0031] An alternative embodiment relates to the application of the protein nanofibrils described herein in combination with bacterial spores or bacterial spore-forming suspensions, preferably Bacillus spores or Bacillus suspensions, to enhance the toxicity of the bacteria or spores, preferably Bacillus or Bt spores, so as to enhance the pesticide activity, preferably insecticidal activity, of the spores or suspensions.

[0032] Further aspects of the present invention relate to nucleic acid molecules, vectors, host cells, or compositions for providing A-ENA monomers in modified mutant variants or fusion subunits for the spontaneous assembly of protein nanofibrils.

[0033] The final aspect of the present invention is a method for producing the A-ENA protein nanofibrils or A-ENA-based variant protein nanofibrils described herein, comprising the following steps: - The steps of introducing a nucleic acid molecule encoding an A-ENA monomer, an A-ENA-based monomer, or a modified A-ENA monomer as described herein into a cell, or recombinantly expressing the monomer protein described herein in a host cell, incubating the cell to allow the monomer protein to self-assemble into a protofibril and consequently into a protein nanofibril, or alternatively, culturing a host cell containing the monomer protein subunit described herein or assembling the protein nanofibril as described herein. - A step of obtaining self-assembled monomers and / or protein nanofibrils from host cells and / or cell suspensions, preferably by cell lysis, and - A step of purifying the self-assembled protein nanofibrils, preferably by resuspension from an insoluble fraction and / or further purification from a cell lysate. This includes methods.

[0034] Specific embodiments further relate to a method for producing the recombinant A-ENA protein nanofibrils, wherein one or more protein monomers are modified mutant variants or fusion proteins described herein, also referred to herein as functionalized A-ENA protein nanofibrils. Alternatively, the isolation and / or purification of the protein fibrils in the final step may be carried out by alternative methods known to those skilled in the art to remove undesirable cell debris, DNA, or host cell proteins or compounds for further application of the protein nanofibrils or functionalized protein nanofibrils obtained from the method.

[0035] Drawings are only schematic and not limiting. In drawings, the sizes of some elements may be exaggerated for illustrative purposes and may not be drawn to scale. [Brief explanation of the drawing]

[0036] [Figure 1] Identification of type A spore appendages (A-ENA) as a novel family of protein fibrils produced on the spores of Bacillus thuringiensis Sv. Israelensis (Bti). Transmission electron microscopy (TEM) characterization of Bti spore biofilms reveals the presence of a nanoscale fibril network. (a) Unstained TEM micrograph of resuspended Bti spore biofilm, composed of spores and parasporial bodies (PSBs), grown on LB agar and deposited on a Formvar / Carbon grid; (b) High-magnification image of the same grid used in (a), revealing A-ENA filaments penetrating the biofilm; (c) Negative-stained TEM (nsTEM) micrograph of Bti spores, revealing A-ENA filaments emanating from the spore surface; (d-f) Examples of A-ENA-mediated binding between spores and PSBs, between spores and PSBs, and between spores; (g) nsTEM micrograph of a cluster of parasporial bodies modified with A-ENA filaments emanating from the PSB surface; (h) nsTEM micrograph of a wild-type A-ENA fibril with a corresponding 2D class mean (box size 384 × 384 Å); (i) cryoEM micrograph of a vitrified wild-type A-ENA fibril with a corresponding 2D class mean (box size 230 × 230 Å). [Figure 2]Recombinant production of self-assembled A-ENA fibrils. (a) nsTEM micrograph of recombinant A-ENA (UniprotKB Q8KNV8; SEQ ID NO: 1) expressed in the cytoplasm of E. coli. A-ENA self-assembles into protein nanofibrils isolated from the culture by resuspending and incubating the cell pellet in EDTA-lysozyme, incubation in warm 1% SDS, subsequent centrifugation, and a series of washing steps in water (resuspending and centrifugation) to remove the dissolved lysates (all procedures in Example 2). (b) The obtained suspension of A-ENA fibrils was deposited on a Formvar / Carbon-supported copper grid and stained with 2% (w / v) uranyl acetate solution for observation by nsTEM (scale bar: 100 nm; inset: corresponding 2D class mean with box size 384 × 384 Å); (b) Reconstructed cryoEM volume derived from recombinant A-ENA fibrils vitrified in deionized water (resolution: 2.59 Å according to 0.143 FSC standard); (c) Corresponding Fourier shell correlation function of the volume shown in panel b; (d) Cartoon representation of atomic model constructed based on electrostatic potential map shown in (b). [Figure 3]Visualization of the architecture and structure of type A spore appendages (A-ENA). (a, b) Ribbon and space-filling models of A-ENA fibrils viewed (a) perpendicular to and (b) along the long axis of the fibril. An A-ENA fibril consists of two protofibrils (colored white and dark gray and labeled f and f') that intertwine into a two-start helix superstructure (i.e., a parallel double helix) with twists and rises of approximately 12° and 10.8 Å, respectively. (c) Top: Ribbon and surface representation of a pair of juxtaposed A-ENA monomers labeled i and i'. An A-ENA subunit consists of an alpha-helix hairpin with an N-terminal extension projecting downward along the fibril axis. The A-ENA helix hairpin forms an angle of approximately 68° with respect to the long axis of the fibril. Within a single vertical plane of the fibril, two A-ENA monomers, one of each protofibril, interact laterally to form a dimer entity. Center: The A-ENA pair stacks axially, thereby elongating each protofibril with a relative twist of approximately 12° and an elevation of 10.8 Å, forming a tetramer complex that can be considered a minimal fibril entity or fibril nucleus. Bottom: As a result, the elongation of the fibril is achieved by axially stacking additional dimer units at either pole of the A-ENA tetramer, with a relative twist and elevation of approximately ±12° and 10.8 Å, respectively. The A-ENA fibril is depicted with the C-terminus of the subunit pointing upward (referred to as the C-terminus or pointed fibril end, i.e., the C-pole) and the N-terminal extension of the subunit pointing downward (referred to as the N-terminus or spiny fibril end, i.e., the N-pole). The A-ENA monomers are called i and i' in the f and f' protofibrils, respectively, and each protomer stacked toward the C and N poles is assigned an increment (i.e., i+1, i+2, i+3, etc.) or decay (i.e., i-1, i-2, i-3, etc.). [Figure 4-1]Structure and protomer interactions in Bacillus thuringiensis Sv. islaerensis (Bti) A-ENA fibrils. (a) Cartoon representation of a single Bti A-ENA monomer (UniprotKB Q8KNV8; SEQ ID NO: 1) identified in the cryoEM structure of recombinant A-ENA fibrils. The A-ENA monomer shown here consists of alpha-helix hairpins (α1 and α2) having a 13-residue N-terminal extension (NTL; i.e., res2-14 in SEQ ID NO: 1). (b) The residues indicated by the stick symbol (residue numbers indicated by bold underlines) are involved in the formation of intermolecular isopeptide bonds (IPBs) with adjacent A-ENA monomers (the receiving residue of the corresponding A-ENA partner subunit is shown next to each highlighted residue, with arrows drawn from nucleophilic residues to electrophilic residues); (b) Front view of Bti A-ENA (SEQ ID NO: 1; rotated 90° relative to panel a) with residues related to intermolecular isopeptide bonds highlighted and indicated by the stick symbol: Interprotofibril contact of monomer i is made with monomer i' (i.e., coplanar within protofibril f') and monomer i'+1 (shifted 1 unit upward in protofibril f'); intraprotofibril contact is made with the A-ENA monomer directly above (i+1) or directly below (i-1) subunit i, and with monomer i-5 via residue 2 in the N-terminal connector. Thus, each subunit i is involved in as many as 10 IPBs, bridging seven different protomers within and across the protofibril; (c) Top view (C pole) and bottom view (N pole) of a single A-ENA monomer in surface representation: both surfaces represent the terminal poles of the A-ENA fibril, forming contact sites for the upper and lower subunits, respectively.These contact surfaces are mostly hydrophobic, except for the residues involved in IPB formation, which are highlighted and annotated with arrows; (d) Ultrastructure of the A-ENA fibril, with both protofibrils (f' and f) shown in light gray and gray, and a single A-ENA monomer highlighted in cartoon representation; (e) Magnified view of the framed region of panel d, with subunits (i+1~i-5) within protofibril f shown in cartoon representation, and residues involved in intraprotofibril IPB between i / i+1, i / i-1 and i / i-5 shown as sticks; (f) Cartoon representation of the interprotofibril IPB between i / i' and i / i'+1. [Figure 4-2]Structure and protomer interactions in Bacillus thuringiensis Sv. islaerensis (Bti) A-ENA fibrils. (a) Cartoon representation of a single Bti A-ENA monomer (UniprotKB Q8KNV8; SEQ ID NO: 1) identified in the cryoEM structure of recombinant A-ENA fibrils. The A-ENA monomer shown here consists of alpha-helix hairpins (α1 and α2) having a 13-residue N-terminal extension (NTL; i.e., res2-14 in SEQ ID NO: 1). (b) The residues indicated by the stick symbol (residue numbers indicated by bold underlines) are involved in the formation of intermolecular isopeptide bonds (IPBs) with adjacent A-ENA monomers (the receiving residue of the corresponding A-ENA partner subunit is shown next to each highlighted residue, with arrows drawn from nucleophilic residues to electrophilic residues); (b) Front view of Bti A-ENA (SEQ ID NO: 1; rotated 90° relative to panel a) with residues related to intermolecular isopeptide bonds highlighted and indicated by the stick symbol: Interprotofibril contact of monomer i is made with monomer i' (i.e., coplanar within protofibril f') and monomer i'+1 (shifted 1 unit upward in protofibril f'); intraprotofibril contact is made with the A-ENA monomer directly above (i+1) or directly below (i-1) subunit i, and with monomer i-5 via residue 2 in the N-terminal connector. Thus, each subunit i is involved in as many as 10 IPBs, bridging seven different protomers within and across the protofibril; (c) Top view (C pole) and bottom view (N pole) of a single A-ENA monomer in surface representation: both surfaces represent the terminal poles of the A-ENA fibril, forming contact sites for the upper and lower subunits, respectively.These contact surfaces are mostly hydrophobic, except for the residues involved in IPB formation, which are highlighted and annotated with arrows; (d) Ultrastructure of the A-ENA fibril, with both protofibrils (f' and f) shown in light gray and gray, and a single A-ENA monomer highlighted in cartoon representation; (e) Magnified view of the framed region of panel d, with subunits (i+1~i-5) within protofibril f shown in cartoon representation, and residues involved in intraprotofibril IPB between i / i+1, i / i-1 and i / i-5 shown as sticks; (f) Cartoon representation of the interprotofibril IPB between i / i' and i / i'+1. [Figure 5] Helix crosslinking by autocatalytic units for isopeptide bond formation. A-ENA forms 10 isopeptide bond interactions with adjacent A-ENA subunits. These isopeptide bonds are formed by autocatalytic units consisting of an acid-base catalyst (preferably Glu), a nucleophilic residue (preferably Lys or an N-terminal amine), and an electrophilic residue (preferably Glu, Gln, Asn, or Asp). Autocatalytic isopeptide bond formation depends on the proximity and defined relative arrangement of the acid-base catalysts, thereby activating the nucleophilic subunit for attack and nucleophilic substitution (i.e., dehydration) of the electrophilic residue. The defined arrangement of nucleophilic and electrophilic residues on adjacent helices results in spontaneous and autocatalytic crosslinking of A-ENA subunits under physiological conditions. (a) Outline of subunit "i" and its adjacent subunits, including the isopeptide bond units mediating crosslinking within and between fibrils of the helix hairpin; (b) Enlarged view of the five IPB units (IPB-U1~IPB-U5) involved in subunit crosslinking. [Figure 6]Sequence conservation in A-ENA(like) proteins. The A-ENA consensus weblogo is derived from a multi-sequence alignment of the top 250 blast hits using Q8KNV8 (SEQ ID NO: 1) as the query sequence, filtered to a maximum of 95% sequence identity redundancy; residues involved in IPB formation (derived from the cryoEM structure of recombinant Q8KNV8 fibril) are highlighted with asterisks. Catalytic acid / base residues (mainly Glu) are highlighted with plus signs. The residue numbers presented here do not correspond to the residue number frame used for Q8KNV8 (shifted by +4). This discrepancy is due to the multi-sequence alignment that served as the source for generating the consensus logo. [Figure 7-1]Helix wheel representation of the A-ENA interaction network. The helix secondary structure results in a defined arrangement of side chains and defined knob-in-hole interactions at helix-helix junctions. When viewed along the long axis of the helix, the positions of the side chains can be depicted on the helix wheel. Uniform helix elevation and twist of 1.5 Å and 100°, respectively, in the alpha helix results in a residue arrangement such that every eighth residue is placed in equivalent positions on the helix wheel and employs a 10.5 Å translation along the helix. Thus, a helix can be defined as a successive heptamer unit or "heptad" (H), with side chains named "a" through "g". The protrusions of side chains on the alpha helix create ridges and valleys called "knobs" and "holes" placed in systematically defined positions, resulting in defined knob-in-hole interactions when two adjacent alpha helices interact. Within the A-ENA subunit, alpha helices α1 and α2 each consist of five heptads (H1-1 to H1-5 and H2-1 to H2-5, respectively) and form a helix hairpin by joining to antiparallel packing contacts via hydrophobic knob-in-hole interactions along the length of the helix (i.e., positions "d" on the helix wheel as defined above). During the stacking of A-ENA subunits (i.e., i / i+1 and i / i-1 interactions), alpha helices α1 and α2 within the consecutive subunits enter pairwise parallel packings via primarily hydrophobic knob-in-hole interactions along the length of the helix, including heptad positions "a" and "e" on the C-pole side and "g" and "c" on the N-pole side of the A-ENA hairpin. Exceptions in hydrophobic contacts are formed by residues that enter into isopeptide bond formation (highlighted by solid arrows, pointing from nucleophile to electrophile) and / or residues that form acid-base catalysts within the IPB unit (highlighted by red circles and labeled).The packing of f and f' protofibrils in A-ENA is mediated by the "b," "e," and "f" positions of the α2 heptads H2-3 and H2-4 in subunit i, and by knob-in-hole and IPB interactions with i' and i'+1. See Figure 7 below: Alignment of A-ENA orthologue with A-ENA (UniprotKB Q8KNV8; Sequence ID No. 1) with A-ENA, which has a low sequence identity of 24%. The A-ENA subunit employs a common structure consisting of an α-helix "α1" consisting of a variable N-terminal lock ("NTL") followed by five heptad repeats (H1-1 to H1-5), a variable-length linker sequence ("L") followed by a second α-helix "α2", and terminated by a variable-length C-terminal tail ("CT"). The alignment of A-ENA orthologues according to heptad repeats demonstrates the strong conservation of the positions of hydrophobic residues involved in intrahelical and interhelical knob-in-hole interactions (labeled φ and Ψ for hydrophobic and short-chain residues, respectively), as well as the formation of interhelical isopeptide bonding units (labeled γ, δ, and ε for acid / base catalysts, nucleophilic and electrophilic residues, respectively). [Figure 7-2]Helix wheel representation of the A-ENA interaction network. The helix secondary structure results in a defined arrangement of side chains and defined knob-in-hole interactions at helix-helix junctions. When viewed along the long axis of the helix, the positions of the side chains can be depicted on the helix wheel. Uniform helix elevation and twist of 1.5 Å and 100°, respectively, in the alpha helix results in a residue arrangement such that every eighth residue is placed in equivalent positions on the helix wheel and employs a 10.5 Å translation along the helix. Thus, a helix can be defined as a successive heptamer unit or "heptad" (H), with side chains named "a" through "g". The protrusions of side chains on the alpha helix create ridges and valleys called "knobs" and "holes" placed in systematically defined positions, resulting in defined knob-in-hole interactions when two adjacent alpha helices interact. Within the A-ENA subunit, alpha helices α1 and α2 each consist of five heptads (H1-1 to H1-5 and H2-1 to H2-5, respectively) and form a helix hairpin by joining to antiparallel packing contacts via hydrophobic knob-in-hole interactions along the length of the helix (i.e., positions "d" on the helix wheel as defined above). During the stacking of A-ENA subunits (i.e., i / i+1 and i / i-1 interactions), alpha helices α1 and α2 within the consecutive subunits enter pairwise parallel packings via primarily hydrophobic knob-in-hole interactions along the length of the helix, including heptad positions "a" and "e" on the C-pole side and "g" and "c" on the N-pole side of the A-ENA hairpin. Exceptions in hydrophobic contacts are formed by residues that enter into isopeptide bond formation (highlighted by solid arrows, pointing from nucleophile to electrophile) and / or residues that form acid-base catalysts within the IPB unit (highlighted by red circles and labeled).The packing of f and f' protofibrils in A-ENA is mediated by the "b," "e," and "f" positions of the α2 heptads H2-3 and H2-4 in subunit i, and by knob-in-hole and IPB interactions with i' and i'+1. See Figure 7 below: Alignment of A-ENA orthologue with A-ENA (UniprotKB Q8KNV8; Sequence ID No. 1) with A-ENA, which has a low sequence identity of 24%. The A-ENA subunit employs a common structure consisting of an α-helix "α1" consisting of a variable N-terminal lock ("NTL") followed by five heptad repeats (H1-1 to H1-5), a variable-length linker sequence ("L") followed by a second α-helix "α2", and terminated by a variable-length C-terminal tail ("CT"). The alignment of A-ENA orthologues according to heptad repeats demonstrates the strong conservation of the positions of hydrophobic residues involved in intrahelical and interhelical knob-in-hole interactions (labeled φ and Ψ for hydrophobic and short-chain residues, respectively), as well as the formation of interhelical isopeptide bonding units (labeled γ, δ, and ε for acid / base catalysts, nucleophilic and electrophilic residues, respectively). [Figure 7-3]Helix wheel representation of the A-ENA interaction network. The helix secondary structure results in a defined arrangement of side chains and defined knob-in-hole interactions at helix-helix junctions. When viewed along the long axis of the helix, the positions of the side chains can be depicted on the helix wheel. Uniform helix elevation and twist of 1.5 Å and 100°, respectively, in the alpha helix results in a residue arrangement such that every eighth residue is placed in equivalent positions on the helix wheel and employs a 10.5 Å translation along the helix. Thus, a helix can be defined as a successive heptamer unit or "heptad" (H), with side chains named "a" through "g". The protrusions of side chains on the alpha helix create ridges and valleys called "knobs" and "holes" placed in systematically defined positions, resulting in defined knob-in-hole interactions when two adjacent alpha helices interact. Within the A-ENA subunit, alpha helices α1 and α2 each consist of five heptads (H1-1 to H1-5 and H2-1 to H2-5, respectively) and form a helix hairpin by joining to antiparallel packing contacts via hydrophobic knob-in-hole interactions along the length of the helix (i.e., positions "d" on the helix wheel as defined above). During the stacking of A-ENA subunits (i.e., i / i+1 and i / i-1 interactions), alpha helices α1 and α2 within the consecutive subunits enter pairwise parallel packings via primarily hydrophobic knob-in-hole interactions along the length of the helix, including heptad positions "a" and "e" on the C-pole side and "g" and "c" on the N-pole side of the A-ENA hairpin. Exceptions in hydrophobic contacts are formed by residues that enter into isopeptide bond formation (highlighted by solid arrows, pointing from nucleophile to electrophile) and / or residues that form acid-base catalysts within the IPB unit (highlighted by red circles and labeled).The packing of f and f' protofibrils in A-ENA is mediated by the "b," "e," and "f" positions of the α2 heptads H2-3 and H2-4 in subunit i, and by knob-in-hole and IPB interactions with i' and i'+1. See Figure 7 below: Alignment of A-ENA orthologue with A-ENA (UniprotKB Q8KNV8; Sequence ID No. 1) with A-ENA, which has a low sequence identity of 24%. The A-ENA subunit employs a common structure consisting of an α-helix "α1" consisting of a variable N-terminal lock ("NTL") followed by five heptad repeats (H1-1 to H1-5), a variable-length linker sequence ("L") followed by a second α-helix "α2", and terminated by a variable-length C-terminal tail ("CT"). The alignment of A-ENA orthologues according to heptad repeats demonstrates the strong conservation of the positions of hydrophobic residues involved in intrahelical and interhelical knob-in-hole interactions (labeled φ and Ψ for hydrophobic and short-chain residues, respectively), as well as the formation of interhelical isopeptide bonding units (labeled γ, δ, and ε for acid / base catalysts, nucleophilic and electrophilic residues, respectively). [Figure 7-4]Helix wheel representation of the A-ENA interaction network. The helix secondary structure results in a defined arrangement of side chains and defined knob-in-hole interactions at helix-helix junctions. When viewed along the long axis of the helix, the positions of the side chains can be depicted on the helix wheel. Uniform helix elevation and twist of 1.5 Å and 100°, respectively, in the alpha helix results in a residue arrangement such that every eighth residue is placed in equivalent positions on the helix wheel and employs a 10.5 Å translation along the helix. Thus, a helix can be defined as a successive heptamer unit or "heptad" (H), with side chains named "a" through "g". The protrusions of side chains on the alpha helix create ridges and valleys called "knobs" and "holes" placed in systematically defined positions, resulting in defined knob-in-hole interactions when two adjacent alpha helices interact. Within the A-ENA subunit, alpha helices α1 and α2 each consist of five heptads (H1-1 to H1-5 and H2-1 to H2-5, respectively) and form a helix hairpin by joining to antiparallel packing contacts via hydrophobic knob-in-hole interactions along the length of the helix (i.e., positions "d" on the helix wheel as defined above). During the stacking of A-ENA subunits (i.e., i / i+1 and i / i-1 interactions), alpha helices α1 and α2 within the consecutive subunits enter pairwise parallel packings via primarily hydrophobic knob-in-hole interactions along the length of the helix, including heptad positions "a" and "e" on the C-pole side and "g" and "c" on the N-pole side of the A-ENA hairpin. Exceptions in hydrophobic contacts are formed by residues that enter into isopeptide bond formation (highlighted by solid arrows, pointing from nucleophile to electrophile) and / or residues that form acid-base catalysts within the IPB unit (highlighted by red circles and labeled).The packing of f and f' protofibrils in A-ENA is mediated by the "b," "e," and "f" positions of the α2 heptads H2-3 and H2-4 in subunit i, and by knob-in-hole and IPB interactions with i' and i'+1. See Figure 7 below: Alignment of A-ENA orthologue with A-ENA (UniprotKB Q8KNV8; Sequence ID No. 1) with A-ENA, which has a low sequence identity of 24%. The A-ENA subunit employs a common structure consisting of an α-helix "α1" consisting of a variable N-terminal lock ("NTL") followed by five heptad repeats (H1-1 to H1-5), a variable-length linker sequence ("L") followed by a second α-helix "α2", and terminated by a variable-length C-terminal tail ("CT"). The alignment of A-ENA orthologues according to heptad repeats demonstrates the strong conservation of the positions of hydrophobic residues involved in intrahelical and interhelical knob-in-hole interactions (labeled φ and Ψ for hydrophobic and short-chain residues, respectively), as well as the formation of interhelical isopeptide bonding units (labeled γ, δ, and ε for acid / base catalysts, nucleophilic and electrophilic residues, respectively). [Figure 8-1]Recombinant production and fibril formation of A-ENA orthologs. (a) Uniprot accession codes (SEQ ID NOs. 3-5) of putative A-ENA orthologs and corresponding sequence identity to Bti A-ENA (Uniprot: Q8KNV8, SEQ ID NO. 1); (b) nsTEM micrographs of autoassembled fibrils formed by recombinant expression of the corresponding A-ENA ortholog in the cytoplasm of E. coli. Fibrils were purified from the cytoplasm of E. coli C43 (DE3) by enzymatic digestion in 1 mg / mL-1 lysozyme, 5 mM ethylenediaminetetraacetic acid (EDTA), 50 mM Tris pH 6.8, and 50 mM NaCl at 37°C for 18 hours, followed by surfactant extraction in 1% (w / v) sodium dodecyl sulfate (SDS) at 100°C for 30 minutes (see Example 2 for further details). The fibrils obtained for the three orthologues tested have diameters consistent with those observed for recombinant Q8KNV8 and recombinant Q8KNV7. The nsTEM images, obtained from samples derived from the insoluble fraction after SDS / thermal extraction, demonstrate the extremely high physicochemical robustness of the obtained fibrils. The latter shows the autocatalytic formation of one or more IPBs; (c) AlphaFold2 multimer v1.3 model of the hexameric sequences of each A-ENA orthologue shown in (a); (d) Multiple sequence alignment of the A-ENA orthologue sequences tested, as well as Q8KNV8 (A-ENA; SEQ ID NO: 1) and Q8KNV7 (A-ENA-1 SEQ ID NO: 2): Residues involved in IPB formation and catalytic acidic glutamic acid residues are highlighted with black asterisks and red plus signs, respectively. [Figure 8-2]Recombinant production and fibril formation of A-ENA orthologs. (a) Uniprot accession codes (SEQ ID NOs. 3-5) of putative A-ENA orthologs and corresponding sequence identity to Bti A-ENA (Uniprot: Q8KNV8, SEQ ID NO. 1); (b) nsTEM micrographs of autoassembled fibrils formed by recombinant expression of the corresponding A-ENA ortholog in the cytoplasm of E. coli. Fibrils were purified from the cytoplasm of E. coli C43 (DE3) by enzymatic digestion in 1 mg / mL-1 lysozyme, 5 mM ethylenediaminetetraacetic acid (EDTA), 50 mM Tris pH 6.8, and 50 mM NaCl at 37°C for 18 hours, followed by surfactant extraction in 1% (w / v) sodium dodecyl sulfate (SDS) at 100°C for 30 minutes (see Example 2 for further details). The fibrils obtained for the three orthologues tested have diameters consistent with those observed for recombinant Q8KNV8 and recombinant Q8KNV7. The nsTEM images, obtained from samples derived from the insoluble fraction after SDS / thermal extraction, demonstrate the extremely high physicochemical robustness of the obtained fibrils. The latter shows the autocatalytic formation of one or more IPBs; (c) AlphaFold2 multimer v1.3 model of the hexameric sequences of each A-ENA orthologue shown in (a); (d) Multiple sequence alignment of the A-ENA orthologue sequences tested, as well as Q8KNV8 (A-ENA; SEQ ID NO: 1) and Q8KNV7 (A-ENA-1 SEQ ID NO: 2): Residues involved in IPB formation and catalytic acidic glutamic acid residues are highlighted with black asterisks and red plus signs, respectively. [Figure 9-1]Conserved architecture and isopeptide bonding patterns in A-ENA-like protein nanofibrils. (a, b) The A-ENA-like fibril assembly unit employs a general structure comprising, in order, a variable-length and sequenced N-terminal lock (NTL), followed by an α-helix (α1) containing preferentially five helix heptads (H1-1 to H1-5), a variable-length linker region (L; frequently a turn of 4-5 residues, but not limited thereto), an α-helix (α2) containing preferentially five helix heptads (H2-1 to H2-5), and a variable-length C-terminal tail (CT). In the fibril assembly unit, the α1 and α2 heptads retain consensus sequence motifs of hydrophobic residues and short side-chain residues, as shown in Figure 10, thereby promoting the adoption of antiparallel helix hairpins within the A-ENA-like unit and facilitating the stacking of pairs of fibril assembly units (i / i+1 and i / i-1) into helixic protofibrils (labeled f) with a twist of approximately 12° and an elevation of 10.8 Å. In addition, the conserved sequence motifs of hydrophilic residues in the α1 helix heptads H1-2, H1-3, H1-4 and H1-5, and in α2 H2-2, H2-3, H2-4 and H2-5 (see Figure 10) form isopeptide bond-forming units consisting of an acid-base catalyst (preferably Glu or Asp), a nucleophilic residue (preferably Lys or an N-terminal amine), and an electrophilic residue (preferably Glu, Gln, Asp, or Asn). Isopeptide bond pairs are highlighted within the frame, showing conserved heptad positions in different subunits of protofibril f(i, i+1, i-1, i-5) and protofibril f'(i' and i'+1) (e.g., H1-3a in subunit "i"), with arrows pointing from nucleophilic to electrophilic residues. The schematic diagram shows the positions best suited for maximum isopeptide bond pairing ability. Individual IPB pairs can be considered conditional and can be replaced by hydrophobic knob-in-hole interactions. Removal of all IPB pairs overall results in insufficient A-ENA-like self-assembly (see Figure 12) and insufficient fibril stability.The N-terminal amine within the NTL may be involved in IPB formation with non-adjacent subunits, i.e., residues H1-5b of subunit i-5 in the case of Bti A-ENA (SEQ ID NO: 1). The exact subunit "ix" involved in the IPB can be altered by elongating or shortening the NTL; (c, d) Side and top views (C pole) of a cartoon representation of a 16-mer fragment of an A-ENA-like nanofibril showing the helical stacking of A-ENA-like units (i, i+1, i-1, etc.) and the association of two A-ENA-like protofibrils (f and f'). The fibrils are elongated by the addition of new A-ENA-like monomer units at the C or N pole of the fibril. Shown in the spherical representation are the residues involved in intrafibril and interfibril isopeptide bonds that bridge the A-ENA-like subunits according to the schematic diagrams in panels a and b. [Figure 9-2]Conserved architecture and isopeptide bonding patterns in A-ENA-like protein nanofibrils. (a, b) The A-ENA-like fibril assembly unit employs a general structure comprising, in order, a variable-length and sequenced N-terminal lock (NTL), followed by an α-helix (α1) containing preferentially five helix heptads (H1-1 to H1-5), a variable-length linker region (L; frequently a turn of 4-5 residues, but not limited thereto), an α-helix (α2) containing preferentially five helix heptads (H2-1 to H2-5), and a variable-length C-terminal tail (CT). In the fibril assembly unit, the α1 and α2 heptads retain consensus sequence motifs of hydrophobic residues and short side-chain residues, as shown in Figure 10, thereby promoting the adoption of antiparallel helix hairpins within the A-ENA-like unit and facilitating the stacking of pairs of fibril assembly units (i / i+1 and i / i-1) into helixic protofibrils (labeled f) with a twist of approximately 12° and an elevation of 10.8 Å. In addition, the conserved sequence motifs of hydrophilic residues in the α1 helix heptads H1-2, H1-3, H1-4 and H1-5, and in α2 H2-2, H2-3, H2-4 and H2-5 (see Figure 10) form isopeptide bond-forming units consisting of an acid-base catalyst (preferably Glu or Asp), a nucleophilic residue (preferably Lys or an N-terminal amine), and an electrophilic residue (preferably Glu, Gln, Asp, or Asn). Isopeptide bond pairs are highlighted within the frame, showing conserved heptad positions in different subunits of protofibril f(i, i+1, i-1, i-5) and protofibril f'(i' and i'+1) (e.g., H1-3a in subunit "i"), with arrows pointing from nucleophilic to electrophilic residues. The schematic diagram shows the positions best suited for maximum isopeptide bond pairing ability. Individual IPB pairs can be considered conditional and can be replaced by hydrophobic knob-in-hole interactions. Removal of all IPB pairs overall results in insufficient A-ENA-like self-assembly (see Figure 12) and insufficient fibril stability.The N-terminal amine within the NTL may be involved in IPB formation with non-adjacent subunits, i.e., residues H1-5b of subunit i-5 in the case of Bti A-ENA (SEQ ID NO: 1). The exact subunit "ix" involved in the IPB can be altered by elongating or shortening the NTL; (c, d) Side and top views (C pole) of a cartoon representation of a 16-mer fragment of an A-ENA-like nanofibril showing the helical stacking of A-ENA-like units (i, i+1, i-1, etc.) and the association of two A-ENA-like protofibrils (f and f'). The fibrils are elongated by the addition of new A-ENA-like monomer units at the C or N pole of the fibril. Shown in the spherical representation are the residues involved in intrafibril and interfibril isopeptide bonds that bridge the A-ENA-like subunits according to the schematic diagrams in panels a and b. [Figure 10-1]Design principles and sequence motifs of helix hairpin self-assembly and self-crosslinking protein nanofibrils. (a, b) The A-ENA-like fibril assembly unit employs a general structure that, in order, includes a variable-length and sequenced N-terminal lock (NTL), followed by an α-helix (α1) containing preferentially five helix heptads (H1-1 to H1-5), a variable-length linker region (L), an α-helix (α2) containing preferentially five helix heptads (H2-1 to H2-5), and a variable-length C-terminal tail (CT). In the A-ENA-like fibril assembly unit, the α1 and α2 heptads maintain consensus sequence motifs of hydrophobic residues and short side-chain residues, thereby promoting the adoption of antiparallel helix hairpins within the A-ENA-like unit and facilitating the stacking of pairs of fibril assembly units (i / i+1 and i / i-1) into helix protofibrils (see Figure 7). In addition, the conserved sequence motifs of hydrophilic residues in the α1 helix heptads H1-2, H1-3, H1-4 and H1-5 and the α2 helix heptads H2-2, H2-3, H2-4 and H2-5 form an isopeptide bond-forming unit consisting of an acid-base catalyst (γ; preferably Glu or Asp), a nucleophilic residue (δ; preferably Lys or an N-terminal amine), and an electrophilic residue (ε; preferably Glu, Gln, Asp, or Asn). The isopeptide bond-forming units (shown in Figures 5, 7, and 9) can be individually replaced with hydrophobic residues for hydrophobic knob-in-hole interactions. The NTL, linker (L), and C-tail elements are variable in length and sequence. Preferred consensus sequences of the NTL in non-limited embodiments are shown in panel c. [Figure 10-2]Design principles and sequence motifs of helix hairpin self-assembly and self-crosslinking protein nanofibrils. (a, b) The A-ENA-like fibril assembly unit employs a general structure that, in order, includes a variable-length and sequenced N-terminal lock (NTL), followed by an α-helix (α1) containing preferentially five helix heptads (H1-1 to H1-5), a variable-length linker region (L), an α-helix (α2) containing preferentially five helix heptads (H2-1 to H2-5), and a variable-length C-terminal tail (CT). In the A-ENA-like fibril assembly unit, the α1 and α2 heptads maintain consensus sequence motifs of hydrophobic residues and short side-chain residues, thereby promoting the adoption of antiparallel helix hairpins within the A-ENA-like unit and facilitating the stacking of pairs of fibril assembly units (i / i+1 and i / i-1) into helix protofibrils (see Figure 7). In addition, the conserved sequence motifs of hydrophilic residues in the α1 helix heptads H1-2, H1-3, H1-4 and H1-5 and the α2 helix heptads H2-2, H2-3, H2-4 and H2-5 form an isopeptide bond-forming unit consisting of an acid-base catalyst (γ; preferably Glu or Asp), a nucleophilic residue (δ; preferably Lys or an N-terminal amine), and an electrophilic residue (ε; preferably Glu, Gln, Asp, or Asn). The isopeptide bond-forming units (shown in Figures 5, 7, and 9) can be individually replaced with hydrophobic residues for hydrophobic knob-in-hole interactions. The NTL, linker (L), and C-tail elements are variable in length and sequence. Preferred consensus sequences of the NTL in non-limited embodiments are shown in panel c. [Figure 11]HMM profiles of helix 1 and helix 2. Sequence logos of 35-residue length hmm profiles of helix 1 and helix 2 constructed based on a set of 593 A-ENA(like) sequences with the accession numbers listed in Table 3. Although the pairwise sequence identity in this list of proteins is low, at 20-30%, strong conservation exists at key positions in helix 1 and helix 2, as reflected in the formulas provided herein for the heptad elements of each helix. [Figure 12] Isopeptide bond formation is required for the self-assembly of stable A-ENA-like nanofibrils. (a) SDS-PAGE of purified A-ENA mutant (i.e., A-ENA E29A Q44A N64A E78A Q82A; SEQ ID NO: 15) in which residues E29, Q44, N64, E78, and Q82 are mutated to alanine residues; (b) and (c) nsTEM micrographs of 1 mg / mL-1 of A-ENA in 1×PBS. Scale bars represent 1 μm and 0.1 μm, respectively. [Figure 13-1] Overview of A-ENA manipulation sites: (a) Sites for insertion of a single amino acid, peptide, or complete domain into the N-terminus, C-terminus, or loop connecting helices α1 and α2 of wild-type A-ENA; (b) Sites for insertion of a single amino acid, peptide, or complete domain into the N-terminus, C-terminus, or loop connecting helices α1 and α2 of A-ENA ΔNTL (e.g., SEQ ID NO: 12); (c) and (e) Sites for site-directed mutagenesis of surface-exposed residues on wild-type A-ENA fibrils: indicated by sticks in the cartoon representation of A-ENA (c) and in bold in the five heptads of helices α1 and α2, respectively; (d) and (f) Sites for site-directed mutagenesis of surface-exposed residues on ΔNTL A-ENA fibrils: indicated by sticks in the cartoon representation of A-ENA (c) and in bold in the five heptads of helices α1 and α2, respectively. [Figure 13-2]Overview of A-ENA manipulation sites: (a) Sites for insertion of a single amino acid, peptide, or complete domain into the N-terminus, C-terminus, or loop connecting helices α1 and α2 of wild-type A-ENA; (b) Sites for insertion of a single amino acid, peptide, or complete domain into the N-terminus, C-terminus, or loop connecting helices α1 and α2 of A-ENA ΔNTL (e.g., SEQ ID NO: 12); (c) and (e) Sites for site-directed mutagenesis of surface-exposed residues on wild-type A-ENA fibrils: indicated by sticks in the cartoon representation of A-ENA (c) and in bold in the five heptads of helices α1 and α2, respectively; (d) and (f) Sites for site-directed mutagenesis of surface-exposed residues on ΔNTL A-ENA fibrils: indicated by sticks in the cartoon representation of A-ENA (c) and in bold in the five heptads of helices α1 and α2, respectively. [Figure 14] Post-polymerization and functionalization of A-ENA fibrils using the SpyTag / SpyCatcher system. (a) Required genetic components: A-ENA with a C-terminal SpyTag and a SpyCatcher with a C-terminal fusion protein (here: Superfolder green fluorescent protein (sfGFP); Pedelacq et al., 2006. Nat Biotechnol. 24(1):79~88), as well as a molecular model of a SpyTagged A-ENA fibril having a bound SpyCatcher-sfGFP unit. (b) nsTEM micrograph of purified homopolymer A-ENA-SpyTag fibrils; (c) SDS-PAGE analysis of sfGFP with and without prior incubation with A-ENA-(1)-SpyTag fibrils, showing the presence of the fluorescence signal of Spycatch-sfGFP bound to A-ENA fibrils in a stacked gel of lanes (2-3) (high MW - no separation); (d) fluorescence image of a centrifugal suspension of A-ENA-SpyTag fibrils incubated with SpyCatcher-sfGFP: the fluorescent insoluble phase consists of A-ENA fibrils. [Figure 15-1] Optimized linker design strategy for surface presentation of heterogeneous domains on A-ENA scaffolds. (a) Molecular model of homopolymer A-ENA fibrils presenting fusion domains on the fibril surface: In this strategy, the domain of interest is fused to A-ENA (either at the N-terminus, C-terminus, or within the α1-α2 loop linker region). Upon recombination, homopolymer A-ENA fibrils are produced in which the ratio of A-ENA to the fusion domain is 1:1. Crucially, a flexible linker is required to avoid steric hindrance between the presented domains. This approach allows for the efficient presentation of fusion domains with a hydrodynamic radius of less than 150% of the pitch of the A-ENA fibril (i.e., 10.8 nm); (b) Examples of surface presentation of different heterogeneous domains bound to the C-terminus of A-ENA: nsTEM micrographs of A-ENA fibrils presenting p66α (SEQ ID NO: 8), MBD2 (SEQ ID NO: 9), and rubredoxin (SEQ ID NO: 10), respectively; (c) AlphaFold2 models and molecular weights of the corresponding domains discussed in (b); (d) Eppendorf inversion tests performed to investigate hydrogelation; (e) Melting curves of wild-type A-ENA and A-ENA-rubredoxin (RR) fusion proteins. [Figure 15-2]Optimized linker design strategy for surface presentation of heterogeneous domains on A-ENA scaffolds. (a) Molecular model of homopolymer A-ENA fibrils presenting fusion domains on the fibril surface: In this strategy, the domain of interest is fused to A-ENA (either at the N-terminus, C-terminus, or within the α1-α2 loop linker region). Upon recombination, homopolymer A-ENA fibrils are produced in which the ratio of A-ENA to the fusion domain is 1:1. Crucially, a flexible linker is required to avoid steric hindrance between the presented domains. This approach allows for the efficient presentation of fusion domains with a hydrodynamic radius of less than 150% of the pitch of the A-ENA fibril (i.e., 10.8 nm); (b) Examples of surface presentation of different heterogeneous domains bound to the C-terminus of A-ENA: nsTEM micrographs of A-ENA fibrils presenting p66α (SEQ ID NO: 8), MBD2 (SEQ ID NO: 9), and rubredoxin (SEQ ID NO: 10), respectively; (c) AlphaFold2 models and molecular weights of the corresponding domains discussed in (b); (d) Eppendorf inversion tests performed to investigate hydrogelation; (e) Melting curves of wild-type A-ENA and A-ENA-rubredoxin (RR) fusion proteins. [Figure 16]Block copolymer design strategy for surface presentation of heterogeneous domains on A-ENA scaffolds. (a) Molecular model of heteropolymer A-ENA fibrils presenting fusion domains on the fibril surface: In this strategy, the domain of interest is genetically fused to A-ENA (either at the N-terminus or C-terminus within the α1-α2 loop linker region). When expressed by recombination in a background strain also expressing wild-type A-ENA, heteropolymer A-ENA fibrils are produced in which the ratio of A-ENA to fusion domains is greater than 1. Using this approach, fusion domains with a hydrodynamic radius of more than 150% of the pitch of the A-ENA fibril (i.e., 10.8 nm) can be efficiently presented; (b) nsTEM micrograph of block copolymer A-ENA fibrils consisting of wild-type A-ENA (block A) and nanobody-TEV-A-ENA (SEQ ID NO: 11) units; (c) Alphafold2 model of the presented nanobody. [Figure 17] Functional presentation of camelid antibodies. Schematic diagram of A-ENA-like nanofibrils having SpyCatcher (left) or SpyTag (right) sequences selectively fused to the NTL or CT of A-ENA monomers (only single fusions are shown for clarity). SpyCatcher and SpyTag A-ENA fusions form fibrils that can be used to capture SpyTagged and SpyCatcher constructs, respectively. Shown here are SpyTagged (left) or SpyCatcher fusion (right) Nb conjugated to modified A-ENA nanofibrils. [Figure 18-1]The A-ENA sporesilk network associates spores of Bacillus thuringiensis Sv. islaerensis with parasporial bodies. (a) Unstained TEM image of A-ENA biofilm in Bti, grown on LB agar, scraped from plate, and resuspended in water. The sample was then deposited on a Formvar / Carbon grid. The observed fibrils emerging from the spores correspond to A-ENA based on their diameter (approximately 10 nm); (b) Unstained TEM image of the Bti ΔA-ENA strain, grown on LB agar, scraped from plate, resuspended in water, and finally deposited on a Formvar / Carbon grid. This strain shows a complete lack of A-ENA fibrils; (c, d) Unstained TEM images of spores isolated from Bti WT and Bti ΔA-ENA strains. To isolate spores, 200 μl of spore suspension was added to 1 ml of 50% histodenz, and the sample was centrifuged at 16000 × g for 40 minutes. Before deposition on a Formvar / Carbon grid, the supernatant was removed and the pellet was washed three times with water (at 16000 × g for 5 minutes); (e) The Bti ΔA-ENA strain was supplemented with a plasmid containing the ORF of the A-ENA gene under its native promoter. An A-ENA biofilm surrounding the spores and PSBs was observed; (f) Based on TEM images, spores and PSBs were manually counted, and the PSB / spore ratio is shown in a plot comparing the spore suspension and spore isolate. The experiment was repeated four times, and each time more than 100 entities (spores or PSBs) were counted for each group. Panels (a) to (e) are representative images for each group. [Figure 18-2]The A-ENA sporesilk network associates spores of Bacillus thuringiensis Sv. islaerensis with parasporial bodies. (a) Unstained TEM image of A-ENA biofilm in Bti, grown on LB agar, scraped from plate, and resuspended in water. The sample was then deposited on a Formvar / Carbon grid. The observed fibrils emerging from the spores correspond to A-ENA based on their diameter (approximately 10 nm); (b) Unstained TEM image of the Bti ΔA-ENA strain, grown on LB agar, scraped from plate, resuspended in water, and finally deposited on a Formvar / Carbon grid. This strain shows a complete lack of A-ENA fibrils; (c, d) Unstained TEM images of spores isolated from Bti WT and Bti ΔA-ENA strains. To isolate spores, 200 μl of spore suspension was added to 1 ml of 50% histodenz, and the sample was centrifuged at 16000 × g for 40 minutes. Before deposition on a Formvar / Carbon grid, the supernatant was removed and the pellet was washed three times with water (at 16000 × g for 5 minutes); (e) The Bti ΔA-ENA strain was supplemented with a plasmid containing the ORF of the A-ENA gene under its native promoter. An A-ENA biofilm surrounding the spores and PSBs was observed; (f) Based on TEM images, spores and PSBs were manually counted, and the PSB / spore ratio is shown in a plot comparing the spore suspension and spore isolate. The experiment was repeated four times, and each time more than 100 entities (spores or PSBs) were counted for each group. Panels (a) to (e) are representative images for each group. [Figure 19]Exogenous A-ENA forms a spore silk network in Bacillus thuringiensis Sv. Kurstaki (Btk) that associates spores and parasporal toxin crystals. (a) Unstained TEM image of WT Btk strain grown on LB agar, scraped from plate, and resuspended in water. PSB crystals are free in the medium and lack direct attachment to spores. (b) Unstained TEM image of Btk strain expressing the A-ENA gene derived from Bti under a native promoter. The image clearly shows that spores are linked to PSB by A-ENA filaments and that A-ENA forms a biofilm as observed in Bti. (c, d) Isolated spores of WT Btk and +A-ENA Btk. Spores were isolated by sucrose cushion. Three different sucrose concentrations (80%, 70%, and 60%) were added to 1.5 ml tubes, and 200 μl of spore suspension was added on top. The samples were centrifuged at 16000 × g for 20 minutes. Free PSBs were found at the interface between 70% and 80% sucrose, while spores were found in the pellet. Before deposition on a Formvar / Carbon grid, the supernatant was completely removed and the pellet was washed three times with water (at 16000 × g for 5 minutes). (e) Detailed PSBs from Btk connected by A-ENA bundles. (f) Based on TEM images, spores and PSBs were manually counted, and the PSB / spore ratio is shown in a plot comparing the spore suspension and spore isolate. The experiment was repeated four times, and each time more than 100 entities (spores or PSBs) were counted for each group. Panels (a) to (d) are representative images of each group. [Figure 20]A-ENA-mediated association of spores and parasporal toxins enhances the entomopathogenic activity of Bacillus thuringiensis. (a) The survival curve of Chironomus aprilinus in the presence of isolated spores was determined, as shown in Figure 18; (b) Similarly, the survival curve of Chironomus aprilinus in the presence of spore suspension was evaluated. For both experiments a) and b), each group consisted of a minimum of eight larvae. The viability of all larvae was confirmed before starting the experiment. Larvae of various sizes representing different maturation stages were included. Isolated spores and spore suspension were administered at OD600 with a final 0.02. Phosphate-buffered saline (PBS) was used as a negative control. Larval survival was monitored daily for one week. The experiment was conducted in triplicate under room temperature conditions. In both plots, the three curves are statistically significant according to the log-rank (Mantel-Cox) test (p<0.0001); (c) Images showing examples of both viable and dead larvae from the Tyronomus aprilinus cohort used in the experimental setup. [Figure 21]In vivo functionalization of the A-ENA spore silk network in Bacillus thuringiensis. Bacillus thuringiensis islaerensis (Bti) and Bacillus thuringiensis krustaki (Btk) strains were subjected to electroporation using a pA180 plasmid containing A-ENA (SEQ ID NO: 7) fused to the C-terminus of a SpyTag under the control of native promoters and terminators. After isolating spores as described above, the spores were incubated with 100 μM purified sfGFP fused to the N-terminus of a SpyCatcher for 10 minutes at room temperature with continuous stirring. Subsequently, the spores were centrifuged at 10000 × g for 5 minutes and washed with PSB to remove any unbound sfGFP:SpyCatcher. After repeating this washing step three more times, the spores were imaged. Fluorescence microscopy revealed that spores transformed with A-ENA:SpyTag showed strong fluorescence signals from spore clusters and PSBs in both Bti and Btk strains. Wild-type (WT) Bti and Btk strains were included as negative controls to account for potential autofluorescence originating from the spores. Scale bars in the images represent lengths of 2.5 μm. [Figure 22] Stability analysis of recombinant A-ENA. The panel in the figure shows A-ENA fibrils subjected to the following treatments: (a) incubation in 2% (w / v) SDS at 99°C for 1 hour, (b) incubation in 8M urea at room temperature for 1 hour, (c) high-temperature drying; i.e., A-ENA fibers suspended in miliQ water were dried at 200°C for 15 minutes and rehydrated in miliQ water for imaging, (d) incubation in 2M NaOH at room temperature for 1 hour, (e) autoclaving at 121°C for 20 minutes, (f) incubation in 100% (v / v) formic acid at room temperature for 30 minutes. [Figure 23]Enhanced entomopathogenic activity of Bacillus thuringiensis against Trichoderma ni (nettle moth) larvae by purified recombinant A-ENA fibrils. a. Representative ns-EM images of a wild-type Btk spore suspension incubated with purified recombinant A-ENA fibrils (4.18 mg / mL, produced in E. coli) show that the exogenous addition of fibrils to the spore preparation results in the clustering of spores and Cry toxin crystals within the A-ENA fibril network. b. Representative photographs of dead and living Trichoderma ni (nettle moth) larvae after feeding with medium supplemented with Btk spore suspension. c. Different formulations: Survival curves of Trichoderma ni larvae fed with spore suspensions of wild-type B. thuringiensis sv. krustaki ("BTK WT"), recombinant B. thuringiensis sv. krustaki expressing A-ENA ("BTK+A-ENA"), or wild-type B. thuringiensis sv. islaerensis ("BTI"), and culture media supplemented with phosphate-buffered saline ("PBS") as a negative control. d. Spore suspension of wild-type B. thuringiensis sv. krustaki ("BTK WT"); spore suspension of wild-type B. thuringiensis sv. krustaki ("BTK WT + A-ENA fibril") incubated with purified recombinant A-ENA (4.18 mg / ml, produced in E. coli); and survival curves of T. 2 larvae fed with medium supplemented with purified recombinant A-ENA fibril (4.18 mg / ml) to eliminate any toxic effects as a negative control. [Figure 24]Functionalized A-ENA fibrils manipulated by insertion into the linker region. a.-c. Schematic diagram of an A-ENA linker insertion mutant (A-ENA_LI). d. Negative stained EM image of a cell suspension of E. coli DH5α expressing A-ENA_LI-R4.5-2RFD (SEQ ID NO: 67); scale bar = 100 nm. The image shows the presence of assembled A-ENA_LI-R4.5-2RFD fibrils in the cell suspension. e. nsEM image of purified A-ENA_LI-R4.5-2RFD showing the presence of abundant micrometer-length A-ENA-like nanofibrils with a thickness of approximately 10 nm, confirming that the linker insertion mutant retains the ability to self-assemble into SDS-stable protein nanofibrils; scale bar = 200 nm (left) and 100 nm (right). [Figure 25-1]A mutant A_ENA_E28Q_E39Q_E41Q has a single IPB for A-ENA fibril self-assembly. A-ENA was mutated with Gln(Q) at residues E28, E39 and E41, thereby removing the acid-base catalysts IPB1, 2, 4 and 5 normally formed in wild-type A-ENA fibrils, and thus allowing only IPB3 as the interconnection between A-ENA monomer subunits, which was shown to be sufficient for fibril assembly (a, b). Ribbon diagram (a) and schematic diagram (b) of an A-ENA fibril having residues (K76->Q82; SEQ ID NO: 68) forming an IPB3 isopeptide bond, shown as a sphere (a) or as an arrow (b). IPB3 cross-links the f and f' protofibrils in the manner of i->i'-1. Therefore, a single isopeptide bond across the f-f' protofibril interface results in a nanofibril in which all protomers are crosslinked to form a single covalent unit. To generate an A-ENA variant with a single IPB3, the acid-base catalyst (Figure 5) in A-ENA (SEQ ID NO: 1), which is required for the formation of IPB1 (E41), IPB2 (E39), and IPB4 and 5 (E28), was mutated to Gln, a similar but non-catalyzable residue, resulting in "A_ENA_E28Q_E39Q_E41Q" (SEQ ID NO: 68). (c) Alphafold3 prediction and associated PAE plot of the protofibril of A_ENA_E28Q_E39Q_E41Q. (d) Negative staining EM image of purified SDS-resistant mutant A-ENA fibrils formed by A_ENA_E28Q_E39Q_E41Q, demonstrating that a single crossover fibril IPB (i.e., IPB3) is sufficient for the formation of robust protein nanofibrils. Scale bar = 100 nm. [Figure 25-2]A mutant A_ENA_E28Q_E39Q_E41Q has a single IPB for A-ENA fibril self-assembly. A-ENA was mutated with Gln(Q) at residues E28, E39 and E41, thereby removing the acid-base catalysts IPB1, 2, 4 and 5 normally formed in wild-type A-ENA fibrils, and thus allowing only IPB3 as the interconnection between A-ENA monomer subunits, which was shown to be sufficient for fibril assembly (a, b). Ribbon diagram (a) and schematic diagram (b) of an A-ENA fibril having residues (K76->Q82; SEQ ID NO: 68) forming an IPB3 isopeptide bond, shown as a sphere (a) or as an arrow (b). IPB3 cross-links the f and f' protofibrils in the manner of i->i'-1. Therefore, a single isopeptide bond across the f-f' protofibril interface results in a nanofibril in which all protomers are crosslinked to form a single covalent unit. To generate an A-ENA variant with a single IPB3, the acid-base catalyst (Figure 5) in A-ENA (SEQ ID NO: 1), which is required for the formation of IPB1 (E41), IPB2 (E39), and IPB4 and 5 (E28), was mutated to Gln, a similar but non-catalyzable residue, resulting in "A_ENA_E28Q_E39Q_E41Q" (SEQ ID NO: 68). (c) Alphafold3 prediction and associated PAE plot of the protofibril of A_ENA_E28Q_E39Q_E41Q. (d) Negative staining EM image of purified SDS-resistant mutant A-ENA fibrils formed by A_ENA_E28Q_E39Q_E41Q, demonstrating that a single crossover fibril IPB (i.e., IPB3) is sufficient for the formation of robust protein nanofibrils. Scale bar = 100 nm. [Figure 26] A general overview of the A-ENA production and processing pipeline. [Modes for carrying out the invention]

[0037] The present invention is described with reference to certain drawings in relation to specific embodiments, but the present invention is not limited thereto and is limited only by the claims. No reference numerals in the claims shall be considered limiting. Naturally, it shall be understood that not all aspects or advantages may not necessarily be achieved by following any particular embodiment of the present invention. Accordingly, for example, a person skilled in the art will recognize that the present invention may be implemented or performed in a manner that achieves or optimizes one advantage or group of advantages taught herein, without necessarily achieving other aspects or advantages that may be taught or suggested herein. The present invention, both in terms of organization and operation, is best understood by referring to the following detailed description when read in conjunction with the accompanying drawings, along with its features and advantages. The aspects and advantages of the present invention become apparent from and are explained with reference to the embodiments described below herein. Throughout this specification, any reference to “one embodiment” or “a certain embodiment” means that a particular feature, structure or characteristic described in relation to an embodiment is included in at least one embodiment of the present invention. Therefore, throughout this specification, the appearance of the phrases "in one embodiment" or "in a particular embodiment" in various places does not necessarily all refer to the same embodiment, although there may be such cases.

[0038] definition When referring to a singular noun, if an indefinite or definite article is used, e.g., “a” or “an,” or “that,” this includes the plural form of the noun unless something else is specifically stated. Where the term “includes” is used in this description and claims, it does not exclude other elements or steps. Where the terms “essentially consist of,” “essentially made from,” or “substantially contain” are used in this specification with respect to a chemical substance or compound such as a protein, it means that certain further components or substances may be present, i.e., those that do not substantially affect the essential characteristics of the chemical substance. Where specifically referred to in this specification, “substantially contain,” “essentially consist of,” or “essentially made from” refers to the majority or bulk of a chemical substance such as a polypeptide, thereby defining its functional outcome, but may include further substances such as further amino acids. Furthermore, in this description and claims, terms such as first, second, third, etc., are used to identify similar elements, but are not necessarily used to describe a sequential or chronological order. The terms used herein are interchangeable under appropriate circumstances, and it is understood that the embodiments of the invention described herein may be operated in an order other than that described or illustrated herein. The following terms or definitions are provided solely to aid in the understanding of the invention. Unless otherwise specifically defined herein, all terms used herein have the same meanings as they have to those skilled in the art of the invention. The practitioner refers to the definitions and terms of the art in particular to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th edition, Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Suppl. 114), John Wiley & Sons, New York (2016).Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art (e.g., molecular biology, biochemistry, structural biology and / or mathematical biology).

[0039] As used herein, the terms “nucleic acid sequence,” “DNA sequence,” or “nucleic acid molecule” refer to polymeric forms of nucleotides of any length, which are either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of a molecule. Therefore, this term includes double-stranded and single-stranded DNA and RNA. This term also includes known types of modifications of naturally occurring nucleotides by analogs, such as methylation and “cap” substitution. “Nucleic acid construct” means a nucleic acid molecule constructed to contain one or more functional units that are not found together in nature. Examples include circular DNA molecules, linear DNA molecules, double-stranded DNA molecules, extrachromosomal DNA molecules (plasmids), cosmids (plasmids containing COS sequences derived from lambda phages), and viral genomes containing non-natural nucleic acid sequences. “Coded sequence” is a nucleotide sequence that, under the control of appropriate regulatory sequences, is transcribed into mRNA and / or translated into polypeptides. The boundaries of a coding sequence are determined by a translation start codon at the 5' end and a translation stop codon at the 3' end. The coding sequence may include, but is not limited to, mRNA, cDNA, recombinant nucleotide sequences, or genomic DNA, while introns may also be present under certain circumstances. As used herein, “promoter region of a gene” or “regulatory element” refers to a functional DNA sequence unit that is operably ligated to a coding sequence and is possibly sufficient to promote the transcription of the coding sequence when placed under appropriate induction conditions. “Operatably ligated” refers to a juxtaposition in which the components thus described are related in a way that enables them to function in their intended form. A promoter sequence “operatably ligated” to a nucleic acid molecule that is a coding sequence is ligated in such a way that the expression of the coding sequence is achieved under conditions of compatibility with the promoter sequence. As used herein, “gene” includes both the promoter region and the coding sequence of a gene. This refers to both genomic sequences (including possible introns) and cDNA derived from spliced ​​messengers that are operatably ligated to a promoter sequence.The terms “terminator” or “transcription termination signal” encompass regulatory sequences, which are DNA sequences at the end of a transcription unit that transmit signals for 3' processing, polyadenylation of the primary transcript, and transcription termination. Terminators may originate from native genes or from various other (bacterial) genes. A “chimeric gene” or “chimeric construct” or “chimeric gene construct” means a recombinant nucleic acid sequence molecule in which a promoter sequence or regulatory nucleic acid sequence is operatively ligated to or associated with a nucleic acid sequence encoding mRNA, so that the promoter sequence or regulatory nucleic acid sequence can regulate the transcription or expression of the associated nucleic acid coding sequence. The regulatory nucleic acid sequence of a chimeric gene may not be operatively ligated to the associated nucleic acid sequence found in nature and may be heterologous to the coding nucleic acid sequence molecule, meaning that the sequence does not exist in the same configuration as it is in nature within the chimeric construct. More generally, in this specification, the term “heterologous” is defined as a sequence or molecule of different origin.

[0040] In this specification, the terms “protein,” “polypeptide,” and “protein domain” are used interchangeably to refer to polymers of amino acid residues, as well as their variants and synthetic analogues. A monomer or protomer is defined as a single polypeptide chain extending from the amino terminus (also referred herein as the N-terminus or N-terminal terminus) to the carboxyl terminus (also referred herein as the C-terminus or C-terminal terminus). As used herein, a “protein subunit” refers to a monomer or protomer that can form part of a complex or assembly of a multimeric protein. As used herein, a “protein domain” refers to a folded protein or folded portion of a protein as a distinctly different functional and / or structural unit within a protein. Typically, a protein domain contributes to a particular function or interaction that plays a role in the overall function of the protein. Domains can exist in a variety of biological contexts where similar domains may be found within proteins with different functions. Protein secondary structural elements typically form spontaneously as intermediates before the protein folds into its three-dimensional tertiary structure. The two most common secondary structural elements of proteins are the alpha-helix and the beta (β) sheet, although β-turns and omega-loops also occur. A beta sheet consists of β-chains (also called β-chains) connected horizontally by at least two or three skeletal hydrogen bonds, and generally forms a twisted, pleated sheet. A β-chain is a sequence of polypeptide chains, typically 3-10 amino acids long, with a backbone in the elongation conformation. A β-turn is a type of irregular secondary structure in proteins that causes a change in the orientation of polypeptide chains. Beta-turns (β-turns, β-turns, β-bends, tight turns, reverse turns) are extremely common motifs within proteins and polypeptides, primarily used to connect β-chains.

[0041] A "coiled coil" is a tertiary protein structure in which alpha helices wrap around each other to form a superhelix bundle, typically consisting of two or three helices oriented parallel or antiparallel. Coiled coils are usually formed by multimeric proteins of either the same (homo) or different (hetero) chains. Consecutive helices originating from the same polypeptide chain can also fold into a coiled coil structure, typically in which case the helices of the same monomer are oriented antiparallel. The helices constituting a coiled coil interact via a knob-in-hole geometric arrangement of amino acid side chains at their interface (Crick FHC (1952) Nature 170:882~883), where a residue (knob) from one helix packs into a space (hole) surrounded by the four side chains of the opposing helix. This regular packing also necessitates heptad repeats, defined as positions where side chains repeat precisely every seven residues along the helix interface. Therefore, the heptad regularity of the side-chain positions of coiled coils is indicated by a sequence repeat of seven residues, the positions of which are labeled a-g, typically the core-forming positions (a and d) are usually occupied by hydrophobic residues, while the remaining solvent-exposed positions (b, c, e, f, and g) are occupied by hydrophilic residues. Through this rigorous patterning, coiled coils can be predicted based on their amino acid sequences to a level of detail that allows for the assignment of individual residues to the positions of the heptad repeats. Predictive tools are known to those skilled in the art, for example, as described by Lupas et al. (2017; The Structure and Topology of α-Helical Coiled Coils. Subcell Biochem; 82:95-129).

[0042] In the context of this invention, “self-assembly” refers to the spontaneous organization of molecules within a supramolecular ordered structure as a result of their mutual non-covalent interactions, without external control or templates. The chemical structure and conformational structure of individual molecules hold instructions on how they are assembled. The same or different molecules may constitute the building blocks of a molecular self-assembly system. Generally, interactions are established under low-order states such as solutions, random coils, or disordered aggregates, leading to a final ordered state, which may be crystals or folded macromolecules, or further assemblies of macromolecules. The association of small molecules or proteins into a sufficiently ordered structure is driven by thermodynamic principles and therefore based on energy minimization. Interactions involved in molecular assembly processes include electrostatic interactions, hydrophobic interactions, hydrogen bonding, van der Waals interactions, aromatic stacking, and / or metallic coordination. Although non-covalent and individually weak forces, these forces can generate highly stable assemblies and control the shape and function of the final assembly (Lombardi et al., 2019; Pharmaceuticals, 11, 166). The self-assembling protein subunits described herein and referred herein as A-ENA monomeric proteins are capable of self-assembling into monomers and can further multimerize, fold, and engage with protein fibrils as described herein. Fiber assemblies can be obtained from existing components, more specifically isolated self-assembling A-ENA monomeric proteins as described herein, referred to as constituent units or subunits.

[0043] The terms “chimeric polypeptide,” “chimeric protein,” “kaimer,” “fusion polypeptide,” “fusion protein,” or “heterofusion” are used interchangeably herein and refer to a protein comprising at least two distinct and significantly different polypeptide components, which may or may not originate from the same protein. The term also refers to a molecule that does not exist in nature, meaning that it is artificial. When referring to a chimeric polypeptide or fusion polypeptide (as defined herein), the term “fused to” and other grammatical equivalents such as “covalently linked,” “connected,” “joined,” “ligated,” “conjugated,” and, as used specifically herein, “inserted to,” refer to any chemical or recombination mechanism for linking two or more polypeptide components. The fusion of two or more polypeptide components may be a direct fusion of sequences or an indirect fusion, for example, with intervening amino acid sequences or linker sequences or chemical linkers. The fusion of an amino acid residue or (poly)peptide to an Ena protein, or its insertion into the Ena protein or another protein of interest as described herein, may be a covalent peptide bond or may also refer to a fusion obtained by chemical linkage. Where used herein, and where used interchangeably with “conjugated to,” “ligated to,” or “connected to,” the term “fused to” refers in particular to “genetic fusion,” such as by recombinant DNA technology, as well as “chemical conjugation and / or enzymatic conjugation,” which result in a stable covalent linkage.

[0044] As used herein, the terms “protein complex,” “protein assembly,” or “multimer” refer to a group of two or more associated macromolecules in which at least one of the macromolecules is a protein. As used herein, a protein complex or protein assembly typically refers to the binding or association of macromolecules that can be formed under physiological conditions. Individual members of a protein complex, such as protein subunits or protomers, are linked by non-covalent or covalent interactions. “Binding to” means any interaction, whether direct or indirect. A direct interaction implies contact between binding partners. An indirect interaction means any interaction in which interacting partners interact within a complex of two or more molecules. Interactions may be entirely indirect with the assistance of one or more crosslinking molecules, or they may be partially indirect, with direct contact between partners still present, stabilized by further interactions of one or more molecules. The bonding or association may be non-covalent (in which case the juxtaposition is energetically preferred, for example, by hydrogen bonding, van der Waals interactions, or electrostatic interactions) or it may be covalent, for example, by peptide bonds, disulfide bonds, or isopeptide bonds.

[0045] It is understood that protein complexes can be multimers. Protein complex assemblies can result in the formation of homo-multimerized or hetero-multimerized complexes. Furthermore, interactions can be stable or transient. The terms “multimer,” “multimerized complex,” or “multimerized protein or multimerized assembly” include multiple identical or heterogeneous polypeptide monomers. Polypeptides may be able to self-assemble into multimerized assemblies (i.e., dimers, trimers, pentamers, hexamers, heptamers, octamers, etc.) formed from the self-assemblies of multiple single polypeptide monomers (i.e., “homo-multimerized assemblies”) or from the self-assemblies of multiple different polypeptide monomers (i.e., “hetero-multimerized assemblies”). As used herein, “multiple” means two or more. Multimerized assemblies include 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or preferably more polypeptide monomers. Multimerized assemblies can be used for any purpose and can provide a means for developing a wide range of protein “nanomaterials.” In addition to finite, cage-like or shell-like protein assemblies, multimer assemblies can also be designed by selecting a target architecture with appropriate symmetry. The monomers or protomers and / or multimer assemblies of the present invention can be used in the design of higher-order assemblies such as protofibrils, fibril assemblies, or fibrils and further fibers, which have the advantages of hierarchical assembly. The resulting multimers or fiber assemblies are higher-order materials with excellent rigidity and monodispersity, and may function as the multimer or fiber structure itself or form custom-designed molecular machines with high-performance materials and a wide range of applications, such as modified surfaces containing the multimer or fiber structure. More specifically, as used herein, a multimer refers to a homo- or hetero-multimer, or homo- or heteropolymer protein complex, that associates with each other to form a structure organized by non-covalent and / or covalent interactions, and / or is further modified to grow or develop into self-assembly or induced formation of nanofibrils.The aforementioned multimer assembly may contain other proteins that associate with the aforementioned ENA protein base or particularly A-ENA base multimer, referred to as an engineered ENA protein, such as a monomer ENA fusion protein or ENA protein variant, mutant, and / or an ENA protein as exemplified and described herein, and the aforementioned ENA protein base or particularly A-ENA base multimer, thereby extending the multimer to further modifications required for a particular application.

[0046] "Recombinant polypeptide" means a polypeptide produced using recombinant techniques, i.e., through the expression of recombinant or synthetic polynucleotides, which may be obtained in vitro and / or intracellular contexts. When a chimeric polypeptide or fusion polypeptide or its bioactive (i.e., functional) portion is produced by recombinant, it is also preferably concentrated, purified or prepared substantially without culture medium, i.e., impurities represent less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation. "Isolated" or "purified" usually means material that is substantially or essentially free of its associated components in its natural state.

[0047] The terms "homogram" and "multiple homologs" of a protein include peptides, oligopeptides, polypeptides, proteins, and enzymes that have amino acid substitutions, deletions, and / or insertions compared to the unmodified or wild-type protein in question, and that possess similar biological and functional activity to the unmodified protein from which they are derived. As used herein, the term "amino acid identity" refers to the degree to which sequences are identical on a one-to-one correspondence basis of amino acids across a comparison window. Therefore, the "percentage of sequence identity" is calculated by comparing two optimally aligned sequences across a comparison window, determining the number of positions in both sequences where the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met, as shown herein and in conventionally known single-letter codes) occurs, obtaining the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the size of the window), and multiplying the result by 100 to obtain the percentage of sequence identity. As used herein, a "substitution" or "mutation" results from a substitution by one or more amino acids or nucleotides that differs from the amino acid or nucleotide sequence of the parent protein or fragment thereof. It is understood that a protein or fragment thereof may have conserved amino acid substitutions that have no substantial effect on the protein's activity. The amino acid identity percentages presented herein are preferably percentages relative to the full length of the natural or wild-type protein or to a comparison window corresponding to the specific amino acid sequence mentioned.

[0048] The term "wild-type" refers to a gene or gene product isolated from a naturally occurring source or contained in a cell, cell line, or organism. Since wild-type genes or gene products are the most frequently observed genes in a population, they are arbitrarily referred to as the "normal" or "wild-type" form of a gene or gene product observed in nature. In contrast, the terms "modified," "engineered," "mutant," or "variant" refer to a gene or gene product that exhibits modifications to its sequence, post-translational modifications, and / or functional properties (i.e., alterations in features) compared to a wild-type or naturally occurring gene or gene product. A knockout refers to a non-functional gene product and / or a modified gene, mutant gene, or deletion gene that results in a functional gene. It should be noted that naturally occurring mutants or variants can also be isolated, and these are identified by the fact that they exhibit altered features compared to a wild-type gene or gene product and have a different sequence compared to a reference gene or reference protein. By the term “functional homolog” or “functional variant,” the functional homolog or functional variant, as used herein, refers to a modified, manipulated, or mutated gene product that has retained functionality compared to the A-ENA wild-type or original sequence on which the modification or introduction of mutation is based. “Functionality,” as used herein, refers to the self-assembly characteristic of monomeric protein subunits and the further spontaneous assembly of such monomers into nanofibril structures as described herein.

[0049] As used herein, the terms “vector,” “vector construct,” or “recombinant vector” may be double-stranded or single-stranded in any conformation, including but not limited to linear, cyclic, coiled, supercoiled, twisted, or nicked, and may be DNA, RNA, or DNA / RNA hybrid molecules. These vectors of the present invention include, but are not limited to, plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as adenovirus, AAV, or baculovirus vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or P1 artificial chromosomes (PAC), all of which are well known and can be purchased from commercial sources. Any vector can be used to construct and express the fusion molecules used in the present invention. A general class of vectors for specific purposes includes prokaryotic and / or eukaryotic cell cloning vectors, expression vectors, fusion vectors, phage and yeast presentation vectors, shuttle vectors for use in different hosts, mutagenic vectors, transcription vectors, and vectors for accepting large inserts. Most of the necessary methodologies can be found in Ausubel et al., 2007. Vector constructs prepared for introduction into prokaryotic or eukaryotic cell hosts typically include a host-recognized replication system containing a DNA fragment intended to encode the nucleic acid molecule of the present invention, and preferably also including transcription and translation initiation regulatory sequences operably linked to the molecular coding segment. The expression system may include, for example, an origin of replication or self-replication sequence (ARS) and expression regulatory sequences, promoters, enhancers, and necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, transcription terminator sequences, and mRNA stabilization sequences. Optionally, signal peptides derived from secretory polypeptides of the same or related species may also be included, thereby enabling the protein to cross and / or remain on the cell membrane, or to be secreted from the cell. Appropriate promoters and other necessary vector sequences are selected to be functional within the host.Other vectors may be incorporated into the host cell's genome upon introduction into the host cell, thereby replicating alongside the host genome. Examples of viable combinations of cell lines and expression vectors are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th edition, Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Suppl. 114), John Wiley & Sons, New York (2016). Many vectors useful for expression in bacteria, yeast, fungi, mammals, insects, plants, or other cells are well known in the art.

[0050] The “host cell” may be either a prokaryotic or eukaryotic cell. The cell may be transfected transiently or stably. Such transfection of expression vectors into prokaryotic and eukaryotic cells may be achieved by any technique known in the art, including but not limited to standard bacterial transformation, calcium phosphate coprecipitation, electroporation or liposome-mediated transfection, DEAE dextran-mediated transfection, polycation-mediated transfection or virus-mediated transfection. For all standard techniques, see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th edition, Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Suppl. 114), John Wiley & Sons, New York (2016). In this context, recombinant host cell is a host cell genetically modified to contain the isolated DNA molecule, nucleic acid molecule or expression construct or vector of the present invention. DNA can be introduced by any means known in the art that are appropriate for specific types of cells, including, but not limited to, transformation, lipofection, electroporation, or virus-mediated transduction. DNA constructs enabling the expression of the chimeric protein of the present invention can be readily prepared by techniques known in the art, such as cloning, hybridization screening, and polymerase chain reaction (PCR). Standard techniques for cloning, DNA isolation, amplification, and purification, as well as standard techniques for enzymatic reactions and various separation techniques involving DNA ligases, DNA polymerases, restriction endonucleases, etc., are known to those skilled in the art and are commonly used. Numerous standard techniques are described in Sambrook et al. (2012), Wu (ed.) (1993), and Ausubel et al. (2016). Representative host cells that may be used in the present invention include, but are not limited to, bacterial cells, yeast cells, plant cells, and animal cells.Bacterial host cells suitable for use according to the present invention include Escherichia spp. species cells, Bacillus species cells, Pasteuria species cells, Streptomyces spp. species cells, Erwinia spp. species cells, Klebsiella spp. species cells, Serratia spp. species cells, Pseudomonas spp. species cells, Lactococcus spp. species cells, Lactobacillus spp. species cells, and Salmonella spp. species cells. Animal host cells suitable for use according to the present invention include insect cells and mammalian cells (most specifically derived from Chinese hamster (e.g., CHO) and human cell lines such as HeLa). Yeast host cells suitable for use according to the present invention include species from the genera Saccharomyces, Schizosaccharomyces, Kluyveromyces, Pichia (e.g., Pichia pastoris), Hansenula (e.g., Hansenula polymorpha), Yarowia, Schwaniomyces, Schizosaccharomyces, and Zygosaccharomyces. Saccharomyces cerevisiae, S. carlsbergensis, and K. lactis are the most commonly used yeast hosts and are convenient fungal hosts. Host cells can be prepared in suspension, flask cultures, tissue cultures, organ cultures, etc. Alternatively, host cells can also be transgenic animals or plants, or parts or materials derived therefrom.

[0051] As used herein, the terms “medicine” or “drug” refer to a substance / composition used in treatment, i.e., a substance / composition used in the prevention or treatment of an object that may be suffering from a disease or disorder. The terms “disease” or “disorder” refer to any pathological condition, and in particular to a disease or disorder as defined herein.

[0052] The terms “subject,” “individual,” or “patient,” as used interchangeably herein, relate to any organism, such as any mammal, including both vertebrates, particularly humans and other mammals, such as rodents, rabbits, cattle, sheep, horses, dogs, cats, llamas, pigs, or non-human primates (e.g., monkeys), for which diagnosis, treatment, or prevention is desired. Rodents may be mice, rats, hamsters, guinea pigs, or chinchillas. In one embodiment, the subject is a human, a rat, or a non-human primate. Preferably, the subject is a human. In one embodiment, the subject is a subject who has or is suspected of having a disease or disorder, or a subject who is expected to be at high risk of developing a disease or disorder, particularly one of the diseases or disorders disclosed herein, and is also referred to herein as “patient.”

[0053] The terms “treatment,” “treating,” “to treat,” or “method for treating” are interchangeable and are defined by therapeutic interventions that slow, interrupt, block, control, halt, reduce or reverse the progression or severity of signs, symptoms, disorders, conditions, or diseases, but not necessarily involve the complete elimination of all signs, symptoms, conditions, or disorders associated with a disease. Thus, therapeutic measures are designed to treat a disease or improve the health of a human or animal, rather than prevent the disease. Treatment may also refer to preventive measures relating to drugs or treatments designed and used to prevent the onset of a disease, and are referred to herein as “prevention.”

[0054] "Composition" refers to one or more active molecules, preferably a combination comprising one or more protein nanofibrils of the present invention, and may further include a pH buffer, a buffer solution such as water, physiological saline, physiological saline solution, glycerol, a preservative, and / or a solute, whose suitability for obtaining optimal performance is recognized by those skilled in the art. Suitable conditions as used herein may also refer to conditions for using, applying or administering the protein nanofibrils described herein, or, if the protein nanofibrils are produced in and / or isolated from spore-forming bacteria, may include bacterial cultures or spore materials or residues derived from bacterial cultures.

[0055] A “pharmaceutical composition” is a therapeutic active agent or therapeutic active composition provided by the present invention, and optionally comprises a carrier, diluent, or excipient. A “carrier” or “adjuvant,” in particular a “pharmaceutically acceptable carrier” or “pharmaceutically acceptable adjuvant,” is any suitable excipient, diluent, carrier, and / or adjuvant that does not induce the production of antibodies harmful to the individual receiving the composition, nor does it cause any protection. “pharmaceutically acceptable” means a material that is not biologically or otherwise undesirable, i.e., a material that can be administered to an individual together with a compound without causing any undesirable biological effects or interacting in a harmful manner with any of the other components of the pharmaceutical composition it contains. A pharmaceutically acceptable carrier is preferably one that is relatively non-toxic and harmless to the patient at concentrations consistent with the effective activity of the active ingredient, so that any side effects attributable to the carrier do not impair the beneficial effects of the active ingredient. Preferably, a pharmaceutically acceptable carrier or adjuvant enhances the immune response induced by the antigen. A suitable carrier or adjuvant typically comprises one or more compounds from the following non-exclusive list: large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, and inactive virus particles. As used herein, the term “excipient” is intended to include all substances that may be present in a pharmaceutical composition but are not active ingredients, e.g., salts, binders (e.g., lactose, dextrose, sucrose, trehalose, sorbitol, mannitol), lubricants, thickeners, surfactants, preservatives, emulsifiers, buffers, stabilizers, flavorings, or colorants. “Diluent” includes vehicles such as water, physiological saline, saline solution, glycerol, and ethanol. Auxiliary substances such as wetting agents or emulsifiers, pH buffers, or preservatives may be included in such vehicles. A pharmaceutically effective amount of the polypeptide or conjugate of the present invention and a pharmaceutically acceptable carrier is preferably an amount that produces or affects a particular condition being treated. For therapeutic purposes, the pharmaceutical composition of the present invention can be administered to any patient according to standard techniques.Administration can be by any appropriate mode, including oral, parenteral, topical, nasal, intraocular, intrathecal, intraventricular, sublingual, rectal, and vaginal. Further formulation techniques such as nanotechnology and aerosols and inhalants are also within the scope of this invention. The dosage and frequency of administration depend on the patient's age, sex and condition, concomitant administration of other drugs, contraindications, and other parameters to be considered by the clinician. The pharmaceutical compositions of this invention may be lyophilized for storage and reconstituted in a suitable carrier before use. When prepared as a lyophilized product or liquid, physiologically acceptable carriers, excipients, and stabilizers must be added to the pharmaceutical compositions of this invention (Remington's Pharmaceutical Sciences, 22nd edition, Ed. Allen, Loyd V, Jr. (2012)). The dosage and concentration of carriers, excipients, and stabilizers should be safe for the target organism (humans, mice, and other mammals), including buffers such as phosphates, citrates, and other organic acids; antioxidants such as vitamin C; proteins such as small polypeptides, serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as PVP; amino acids such as aminoacetates, glutamates, asparagine, arginine, and lysine; glucose such as glucose, mannose, or dextrin; disaccharides and other carbohydrates; chelating agents such as EDTA; sugar alcohols such as mannitol and sorbitol; counterions such as Na+ and / or surfactants such as TWEEN®, PLURONICS®, or PEG.

[0056] Detailed explanation The present invention is based on a novel type of spore appendage (ENA) protein, particularly the alpha-helix spore appendage protein or A-ENA, which has been found to function as a self-assembling protein that forms multimeric fibrous structures associated with Bacillus thuringiensis (Bt) spores and parasporial bodies, contributing, for example, to the pathogenicity of bacterial spores such as insecticides. These fibrous structures appearing in Bt spore cultures have been elucidated herein for the first time and revealed to be proteinaceous components constituting the A-ENA protein, which appears as highly organized, forced protein fibril structures that spontaneously fold to provide extremely high stability and rigidity for strong association of spores with parasporial bodies. Investigating the biological function of these fiber assemblies through the application of Bt A-ENA knockout strains and complementarity with spore-forming bacteria lacking endogenous A-ENA protein, it was revealed that the association of parasporial bodies containing biconical crystals with toxic proteins for killing lepidopteran insects is indeed associated with Bt spores via A-ENA-based fibrils. The rigidity, thermal and mechanical stability, and tensile strength of these A-ENA-based protein fibrils appear to stem from the presence of several isopeptide covalent interactions within the fibrils, resulting in a biological structural material with enormous potential in different application areas. However, these naturally occurring A-ENA-based fibrils appearing in Bt spore cultures always strongly associate with parasporial bodies among the potential further components of the spore-forming culture, thereby playing a major role in the toxicity of Bt spores to insects. However, their isolation and structural analysis have been difficult, making ex vivo isolation of A-ENA fibrils isolated from parasporial bodies virtually impossible.Ultimately, through more advanced technological advancements in the Cryo-EM field, structural analysis of Bacillus thuringiensis islaerensis (Bti) spore cultures made it possible to identify these Bti fibrils as A-ENA protein-based fibrils, represented in the genome of this Bacillus species by two A-ENA genes encoding two proteins (described herein as A-ENA and A-ENA-1, corresponding to Sequence IDs 1 and 2, respectively). Based on further comparative analysis of homologous bacterial protein sequences and novel structural insights, it became possible to define the structural features of this newly annotated "A-ENA" protein family.

[0057] Alpha-helix spore appendage (A-ENA) protein A-ENA proteins are defined herein as protein monomers that appear as self-assembled alpha-helix (α-helix) antiparallel coiled-coil monomers that provide a helix pattern in their structure as determined by their amino acid sequence, as is known for coiled-coil structured proteins, consisting of a consensus sequence determined by the fragments NTL-Helix 1, L, Helix 2, CT, as further defined herein, where each helix is ​​defined by the sequence of at least five heptad elements (H1-5), each heptad element containing seven amino acids whose side chain is defined as "abcdefg", as further defined herein. Furthermore, in addition to the structural features of their sequence-based alpha-helix coiled coils, A-ENA proteins are characterized by the presence of numerous amino acid residues in geometric positions that promote the autocatalytic formation of one or more isopeptide covalent bonds within or with other A-ENA monomers within the self-assembled antiparallel coiled coil hydrophobic environment, resulting in a “functional” A-ENA protein capable of spontaneously forming fibril assemblies as described herein.

[0058] Accordingly, the present invention provides a newly annotated family of bacterial proteins, defined as “A-ENA proteins,” that are functional in the spontaneous formation of protein fibrils, which are intermolecularly linked via isopeptide bonds, as shown herein, to be responsible for the irreversible nature of the strongly assembled fibrous structures. Further elucidation of fibrils produced by recombination using the exogenous introduction of Bti A-ENA into E. coli has led to the matriculate structural design of fibril constructions required to obtain the protein nanofibrils described herein, based on their heptad-repeat elements and coiled-coil monomeric structure defined by the consensus formula described herein, thereby providing the necessary rigidity through the autocatalytic association of at least one isopeptide (IPB) bond during their self-assembly. The wild-type A-ENA, identified herein as Sequence ID No. 1, which assembles these Bti protein fibril structures connecting spores and parasporial bodies, contains multiple IPBs. Interestingly, however, it was found that the nanofibrils still assemble spontaneously if at least one IPB can be formed between the A-ENA protein monomer subunits of the two protofibrils due to all mutations except for one of the conserved acid / base autocatalytic residues. This need for at least one (cross-fibril) IPB supports the further finding that A-ENA structural homologs with low sequence identity still allow self-assembly to nanofibrils to generate at least one covalent IPB linkage to establish the necessary stability of the assembled protein nanofibrils, provided that they conform to the sequence motif provided for the heptad element and include at least one of the IPB acid / base catalyst (γ), donor (δ), and acceptor (ε) positions in the heptad, conforming to E / D (γ), K as the donor, and E / Q / D / N as the acceptor amino acid identity, respectively.

[0059] Accordingly, a first aspect of the present invention relates to a protein fibril constructed from or derived from two protofibrils, each protofibril comprising two or more monomeric protein subunits based on, derived from, or derived from an A-ENA protein subunit, which spontaneously fold as an alpha-helix antiparallel coiled-coil structured monomer and having an amino acid sequence consisting of protein fragments according to the formula:NTL-helix1(h1)-L-helix2(h2)-CT, where each fragment NTL, L, and CT comprises at least one, one, and four amino acids, respectively, and fragments h1 and h2 (also referred herein as helix α1 and helix α2) comprises at least five heptad elements to enable the formation of a helix hairpin, the amino acids being defined as further detailed below, and the monomeric protein subunits are interconnected via at least one isopeptide bond (IPB). The self-assembling monomeric protein subunit is an A-ENA protein as defined herein, or a protein derived from an A-ENA-based protein or A-ENA protein subunit as further defined herein, and the protein fibril constitutes a basic A-ENA protein structural scaffold as defined herein, and when A-ENA-based or derived protein monomer sequences are applied to form a fibril, they are functional when assembled into a fibril as wild-type A-ENA proteins, but may include modifications such as insertions, mutations, fusions or conjugations to the A-ENA monomer (hence referred to as "A-ENA-based" monomers or "A-ENA-derived" monomer sequences, i.e., functional homologs or functional variants or manipulated A-ENA proteins), which are provided by manipulating A-ENA monomer sequences or proteins as further described herein.

[0060] Certain embodiments may also relate to a protein fibril constructed from two protofibrils, each protofibril comprising two or more monomeric protein subunits based on, derived from, or derived from an A-ENA protein subunit, and the protein nanofibril is an "A-ENA-based protein nanofibril" in that, in addition to the A-ENA protein subunit and / or derived from or variants thereof, the fibril may also contain further components derived from a production host, an attached spore-forming culture, or further components intentionally attached to or in contact with the protein nanofibril. The components may be synthetic materials such as chemicals, plastics, and surfaces, or natural materials such as other proteins, carbohydrates, lipids, LPS, metabolites, and bacterial components.

[0061] To clearly define the fundamental structural features of the A-ENA protein monomer subunit, features considered necessary to enable functionally folded proteins in spontaneous fibrous assembly were inferred based on the structural information disclosed herein for Bti A-ENA nanofibrils and verified by structural modeling of potential A-ENA orthologues, followed by their recombination production and self-assembly into fibrils. Indeed, the alignment of A-ENA orthologues based on heptad repeat elements demonstrated strong conservation of the positions of hydrophobic residues (labeled φ and Ψ for hydrophobic and short-chain residues, respectively) involved in intra-helix and inter-helix knob-in-hole interactions, necessary to obtain coiled-coil structures, as well as residues involved in inter-helix isopeptide bonding units (labeled γ, δ, and ε for acid / base catalysts, nucleophilic and electrophilic residues, respectively).

[0062] The protein nanofibrils of the present invention comprise or consist of at least two protofibrils, each comprising at least two monomeric A-ENA or A-ENA-based protein subunits, each comprising amino acid sequence fragments linked by covalent bonds according to the formula NTL-helix1(h1)-L-helix2(h2)-CT, wherein the monomeric protein subunits spontaneously fold in aqueous solution into two alpha helices named helix1(h1) and helix2(h2) as an alpha-helix antiparallel coiled-coil structure as defined herein. Helices h1 and h2, or alternatively referred to as alpha-helices α1 and α2, are defined herein as consecutive heptameric units or "heptads," "heptad repeats," or "heptad elements" (H), comprising amino acid residues having side chains designated a-g, and therefore "abcdefg." Each of the two helices comprises a contiguous sequence of at least five heptad (H) elements, defined according to formula H1-1-H1-2-H1-3-H1-4-H1-5 for helix 1 and H2-1-H2-2-H2-3-H2-4-H2-5 for helix 2, wherein each heptad element comprises seven amino acid residues designated "abcdefg", and each heptad element has a consensus sequence defined herein below, such that it forms a helix hairpin by joining to an antiparallel packing junction via hydrophobic knob-in-hole interactions, a pattern necessary to provide an antiparallel coiled-coil structure, and creates a hydrophobic environment with residues arranged to autocatalyze the formation of an intermolecular IPB in the presence of further monomers.

[0063] Therefore, within the A-ENA subunit, the primary amino acid sequences of the h1 and h2 helices in the formula NTL-h1-L-h2-CT determine a 3D structure along the length of the helix to obtain the helix wheel in order to assemble into a coiled-coil structure, and IPB formation is induced in the presence of further A-ENA or A-ENA-based monomers.

[0064] Coiled coils are conventionally known to form helices that fold from heptad elements present in the amino acid sequence and pack as knob-in-hole structures, due to the systematic appearance of side chains of the heptad element residue patterns a-g that form ridges and valleys. Therefore, the spontaneous folding of the coiled coil structure of a monomer is predictable by those skilled in the art who possess amino acid sequences, for example, by applying a predictive tool described in Lupas et al. (2017; The Structure and Topology of α-Helical Coiled Coils. Subcell Biochem; 82:95-129). Thus, the coiled coil structural features of a polypeptide are determined by the conserved motifs in each of the subsequently defined heptad elements of the monomer amino acid sequence, and using tools available in the art, those skilled in the art can infer the coiled coil structural features of a polypeptide. In the present invention, the helix hairpin is formed by monomers as a result of the presence of an intramolecular antiparallel coiled-coil structure induced by the sequences of h1 and h2 in the NTL-h1-L-h2-CT consensus sequence of the A-ENA protein as defined herein.

[0065] The A-ENA monomer protein subunits contained in the protofibrils and nanofibrils of the present invention are based on an initial characterization of Bti fibrous assemblies structurally analyzed from spore cultures, thereby revealing that the main components, as disclosed herein, are subunits provided by A-ENA protein sequences (SEQ ID NOs: 1 and 2), which form fibrils on spore junctions that firmly associate spores with parasporial bodies. Based on structural elucidation and protein sequences, monomer protein sequence consensus proteins for providing the basis for the coiled coil of the self-assembling protein nanofibril structure of the present invention are presented to constitute an NTL-h1-L-h2-CT fragment, wherein the fragments NTL, h1, L, h2, and CT are linked to one another to form a single polypeptide chain, and thus via peptide bonds, with helix 1 and helix 2 comprising at least five heptad elements (H), and the consensus sequence for each of the heptad elements is provided herein to result in monomer protein subunits of the A-ENA protein family as defined herein, which can be further assembled into a protofibril, which then entangle to form an antiparallel coiled coil, resulting in the protein nanofibril of the present invention. Furthermore, the heptad elements as defined herein include provisions necessary to enable spontaneous intermolecular isopeptide interactions that irreversibly link the monomers within the protein nanofibril.

[0066] The monomeric protein subunits of protein nanofibrils provided herein are called alpha-helix spore appendage (A-ENA) polypeptides, a family of proteins originating from bacteria, particularly spore-forming bacteria, most specifically the genus Bacillus, and are defined herein as spontaneously folding monomeric protein subunits that form two alpha helices that assemble into an antiparallel coiled-coil structure packed as knob-in-holes, and have an amino acid sequence comprising a fragment following the formula NTL-h1-L-h2-CT, which is covalently linked via peptide bonds to form a single polypeptide chain, in which, - NTL refers to an N-terminal lock or N-terminal amino acid sequence, which includes at least one amino acid preceding the sequence constituting helix 1, and provides a variable fragment. - L refers to a linker fragment that forms a loop or turn in the monomer's 3D structure, comprising at least four amino acid residues located between helix 1 and helix 2, providing a covalent connection (via peptide bonds) to enable an alpha-helix intramolecular coiled coil, and typically the linker is variable in its sequence. - CT refers to the C-terminal tail or C-terminal end of the A-ENA monomer polypeptide, and includes at least one amino acid residue following the sequence constituting helix 2, providing a variable fragment. - h1 and h2 refer to helix 1 and helix 2, also known as the α1 helix or α2 helix of the coiled coil, respectively, which are further defined by the presence of (at least) five heptad elements (shown as H1-5) in each of helix 1 and helix 2, and are defined for the A-ENA protein family as follows: Helix 1 contains or consists of H1-1-H1-2-H1-3-H1-4-H1-5, where the heptad elements are defined by the residues "abcdefg", connected in this specific order from the N-terminus to the C-terminus of the helix, and the consensus amino acid sequence "abcdefg" of each heptad element (H1-1 to H1-5) is, respectively, - Regarding Heptad 1-1, XXX-Φ-XX-Φ, - Regarding the heptad 1-2, Φ-X-Ψ-Φ-Ψ-Φ-γ, - Regarding heptads 1-3, ε-X-Ψ-Φ-Ψ-X-Φ, - Regarding heptads 1-4, Φ-N-Ψ-γ-Ψ-γ-δ, - Regarding heptads 1-5, Φ-ε-X-Φ-Φ-XX is or corresponds to this, Helix 2 contains or consists of H2-1-H2-2-H2-3-H2-4-H2-5, the heptad elements are defined by the residues "abcdefg", and are connected in this specific order from the N-terminus to the C-terminus of the helix, and the consensus amino acid sequence "abcdefg" of each heptad element is, - Regarding Heptad 2-1, Φ-XX-Φ-Φ-X-Φ, - Regarding heptad 2-2, ε-XX-Φ-XXX, - Regarding heptad 2-3, Φ-XX-Φ-Φ-δ-δ, - Regarding heptad 2-4, ε-Φ-Φ-Φ-ε-X-δ, - Regarding heptad 2-5, Φ-γ-X-Φ-Φ-XX is or corresponds to this, The aforementioned consensus amino acid sequence residues are defined as follows: "Φ" represents hydrophobic amino acid residues selected from the list of M, V, I, L, A, G, H, W, Y, F, preferably from the list of L, M, I, V, F, or W, for at least 70% of the Φ positions shown in the heptad element. "Ψ" represents a short side-chain amino acid residue selected from the list of V, C, G, A, P, S, T, N, D, preferably from the list of A, G, S, and T. "γ" is an amino acid residue for isopeptide interactions that functions as an acid / base catalyst, selected from E or D, at least one of the γ positions shown in the heptad element, and preferably the amino acid representing γ is E. "δ" is an amino acid residue for isopeptide interaction that functions as a "donor" or nucleophile, preferably a lysine residue (K), at least one of the δ positions shown in the heptad element. "ε" is an amino acid residue for isopeptide interactions that functions as an "acceptor" or electrophile selected from the list of E, Q, D, and N, preferably from the list of E, Q, and N, for at least one of the ε positions shown in the heptad element, X represents an amino acid that may be any type of amino acid residue known in the art, and its identity does not limit the functionality of the A-ENA protein.

[0067] Therefore, as used herein, amino acid "X" or "any type of amino acid residue" may be defined as any one of the 20 naturally occurring amino acid residues listed herein (below), as well as their different isomers or enantiomers and their synthetic analogs or variants. Amino acids are presented herein by three-letter or one-letter nomenclature, as defined and provided in the IUPAC-IUB Joint Commission on Biochemical Nomenclature (Nomenclature and Symbolism for Amino Acids and Peptides. Eur.J.Biochem.138:9~37(1984)), as follows: alanine (A or Ala), cysteine ​​(C or Cys), aspartic acid (D or Asp), glutamic acid (E or Glu), phenylalanine (F or Phe), glycine (G or Gly), histamine Din (H or His), isoleucine (I or Ile), lysine (K or Lys), leucine (L or Leu), methionine (M or Met), asparagine (N or Asn), proline (P or Pro), glutamine (Q or Gln), arginine (R or Arg), serine (S or Ser), threonine (T or Thr), valine (V or Val), tryptophan (W or Trp), and tyrosine (Y or Tyr).

[0068] In a further specific embodiment, "Φ" represents a hydrophobic amino acid residue selected from the list of M, V, I, L, A, G, H, W, Y, F for at least 75%, 80%, 85%, 90%, 95%, or 99% or 100% of the Φ positions shown in the heptad element.

[0069] In certain embodiments in which the monomeric protein subunits are interconnected in the protein nanofibrils described herein via a single IPB per monomer, the positions in the monomer defined as "γ", "δ", and "ε" provide acid / base catalyst, donor, and acceptor residues, respectively, at H2-5 "γ" which is at least E or D, at H2-3 "δ" which provides at least lysine, and at H2-4 "ε" which provides at least E, Q, D, or N acceptors, thereby enabling the covalent IPB formation of the cross-protofibril (see Figure 25). The positions of the residues defined as "γ", "δ", and "ε" elsewhere in the heptad motif may, in this case, be any amino acid (defined herein as "X" above), preferably an amino acid that does not sterically hinder folding or fibril formation, such as Q instead of E or D for the acid / base catalyst.

[0070] In a more specific embodiment, the monomeric protein subunit of the protein nanofibril described herein is covalently interconnected with further monomers via multiple IPBs per monomer, and the monomer sequence is defined by the "γ", "δ", and "ε" residues in the heptad element to provide, respectively, acid / base catalyst, donor, and acceptor residues required to form the IPB (also shown in Figure 7) between one or more specific side chain residues: - (i) NTL and (i-5), (i-4), (i-3) or (i-2) H1-5b, - H1-4g of (i) and H2-2a of (i-1), - H2-3g of (i) and H2-4a of (i-1), - H2-3f in (i) and H2-4e in (i'), - H2-4g of (i) and H1-3a of (i-1), - H2-4g of (i+1) and H1-3a of (i), - NTL of (i+5), (i+4), (i+3) or (i+2) and H1-5b of (i), - H1-4g of (i+1) and H2-2a of (i), - H2-3g of (i+1) and H2-4a of (i), and / or - H2-3f of (i'+1) and H2-4e of (i).

[0071] Therefore, as shown in Figure 7, the "γ", "δ", and "ε" residues at these positions required to form the IPB between some or all of these IPB links are present in these monomers as E or D as catalyst, lysine as donor, and E, Q, D, or N as acceptor. Thus, for monomers in which not all of the "γ", "δ", and "ε" residues in the heptad element are involved in the IPB linkage, the positions may also include alternative amino acids that do not function as catalysts (e.g., Gln), donors, or acceptors, i.e., any type of amino acid, preferably an amino acid whose side chain is structurally similar to the limitations of the amino acid side chain that functions as a catalyst, donor, or acceptor.

[0072] In further embodiments, the protein nanofibrils described comprise or consist of two protofibrils, each containing at least two A-ENA monomers as defined herein above, wherein helix 1 and helix 2 of the monomer protein subunits are given by the following formula: - Helix 1 containing XXX-Φ-XX-Φ-Φ-X-Ψ-Φ-Ψ-Φ-γ-ε-X-Ψ-Φ-Ψ-X-Φ-Φ-N-Ψ-γ-Ψ-γ-δ-Φ-ε-X-Φ-Φ-XX and - Helix 2 containing Φ-XX-Φ-Φ-X-Φ-ε-XX-Φ-XXX-Φ-XX-Φ-Φ-δ-δ-ε-Φ-Φ-Φ-ε-X-δ-Φ-γ-X-Φ-Φ-XX A consensus array of heptad elements that follows, or is derived from or defined by, The aforementioned consensus amino acid sequence residues are defined as follows: - Φ is a hydrophobic amino acid residue selected from the list M, V, I, L, A, G, H, W, Y, F, preferably L, M, I, V, F, or W. - Ψ is a short side-chain amino acid residue selected from the list of V, C, G, A, P, S, T, N, D, preferably A, G, S, or T. - At least one of the γ positions is an amino acid residue for isopeptide interactions that function as an acid / base catalyst, selected from E or D, preferably E, and the remaining γ residues may be any type of amino acid residue. - At least one of the δ-positions is an amino acid residue selected from K for isopeptide interactions that function as a "donor" or nucleophile, and the remaining δ-residues may be any type of amino acid residue. - At least one of the ε-positions is an amino acid residue for isopeptide interactions that functions as an "acceptor" or electrophile, selected from E, Q, D, or N, preferably E, Q, or N, and the remaining ε-residues may be any type of amino acid residue. - X may be any type of amino acid residue.

[0073] In certain embodiments, the A-ENA protein monomer initially characterized as described herein is applied to form the isolated protein nanofibrils of the present invention. Thus, the isolated protein nanofibrils can be obtained from a host, the A-ENA protein is produced by recombination by the expression of a nucleic acid molecule encoding the A-ENA protein, and / or the A-ENA protein monomer is provided as a monomeric subunit selected from a list of A-ENA proteins that follow a protein sequence represented by any one of the accession numbers listed in Table 3.

[0074] This list of 593 accession numbers, representing 593 different proteins classified herein as A-ENA(like) proteins or A-ENA protein orthologues, is comprised of analysis of structurally identified Bti A-ENA and is consistent with findings regarding potential orthologues tested in Example 7, also exemplified herein as Sequence IDs 3-5, and is based on proposed consensus sequence motifs for the NTL-h1-L-h2-CT fragments and heptad elements described herein to provide a self-assembling protein unit that spontaneously forms irreversibly crosslinked fibrils via the presence of one or more autocatalyzed IPBs, preferably cross-protofibrils.

[0075] Furthermore, alignment of the A-ENA protein family, including members whose accession numbers are provided in Table 3, yielded a low pairwise sequence identity of 20-30% for the entire sequence (NTL-h1-L-h2-CT). Despite this low sequence conservation, the consensus motifs necessary to enable structural self-assembly and IPB formation are conserved as discussed herein, and in particular, the residues present as heptad elements in helix 1 and helix 2 have made it possible to establish a methodology for determining whether a protein sequence can be classified as A-ENA or an A-ENA-like protein. An A-ENA-like protein is defined herein as an A-ENA protein conforming to the NTL-h1-L-h2-CT formula, in which the heptad elements contain the scaffold motif as defined herein, but with potential variability in the length properties of NTL, L, and CT, as well as potential modifications or additions to the protein that do not affect the A-ENA scaffold or self-assembly structural properties. In fact, the data in Tables 1 and 2 include established Hidden Markov Model (HMM) profiles for helix 1 and helix 2 of the A-ENA monomer composed of the formula NTL-h1-L-h2-CT as defined herein. The HMM calculations are based on 593 A-ENA(like) sequences, yielding sequence logos of 35-residue HMM profiles for helix 1 and helix 2, respectively, which should be interpreted as Wheeler et al. (2014): "The Hidden Markov Model is shown by drawing a stack of characters for each position, where the height of the stack corresponds to conservation at that position, and the height of each character in the stack depends on the frequency of that character at that position."

[0076] In certain embodiments, the A-ENA protein monomer subunit is defined by an amino acid sequence following the formula NTL-h1-L-h2-CT, where NTL, CT, and L each comprise at least one, one, or four amino acids, and h1 and h2 provide helix 1 and helix 2, each comprising or essentially comprising five heptad elements H1, H2, H3, H4, and H5, according to the consensus motif provided below. A heptad element, composed of residues abcdefg, may be more specifically defined as follows: - Regarding heptad 1-1 (H1-1), XXX-Φ-XX-Φ, - For H1-2, Φ-X-(S or T)-Φ-(A or G)-Φ-(E or Q), - For H1-3, EXX-Φ-X-(H or N)-Φ, - For H1-4, Φ-N-(A or G or S)-E-(G or A)-EK, - Regarding H1-5, Φ-QX-Φ-Φ-XX, - Regarding H2-1, Φ-XX-Φ-Φ-X-Φ, - Regarding H2-2, (N or Q or D)-XX-Φ-XXX, - For H2-3, Φ-XX-Φ-Φ-(K or X)-(K or X), - For H2-4, (E or Q)-Φ-Φ-(L)-(Q or X)-XK, - Regarding H2-5, Φ-γ-X-Φ-Φ-XX, The consensus amino acid sequence residues of the heptad element are defined as follows: - Φ is a hydrophobic amino acid residue selected from the list M, V, I, L, A, G, H, W, Y, F, preferably L, M, I, V, F, or W. - Ψ is a short side-chain amino acid residue selected from the list of V, C, G, A, P, S, T, N, D, preferably A, G, S, or T. - γ is an amino acid residue for isopeptide interactions that function as an acid / base catalyst, selected from E or D, preferably E. - ε is an amino acid residue for isopeptide interactions that functions as an "acceptor" or electrophile, selected from E, Q, D, or N, preferably E, Q, or N. - X may be any type of amino acid residue. - The single-letter code for the amino acid residue as shown, - and the amino acids in parentheses provide alternatives (indicated by "or") to the "abcdegf" residues at their respective positions.

[0077] In further specific embodiments, the A-ENA protein monomer subunit is defined by the formula of the NTL-h1-L-h2-CT consensus as defined herein, and is further specified by the following h1 and h2 polypeptide sequences, such as polypeptide sequences containing or essentially derived from the heptad elements H1-1 to H1-5 and H2-1 to H2-5, corresponding to the following sequences: H1-1 is DQAINII, H1-2 is LASIGLE, H1-3 is ELGLAHV, H1-4 is INAEGEK, H1-5 is VQAVVAG, H2-1 is FDQLLAT, H2-2 is NESVTQT, H2-3 is LKTVIKK, H2-4 is EMLLQFK, H2-5 is LEEAKSL (corresponding to sequence numbers 24-33 respectively), or -H1-1 is EQAINII, H1-2 is LASIGLE, H1-3 is ELGLAHV, H1-4 is INAEGEK, H1-5 is VQAVVTE, H2-1 is LDQLLAT, H2-2 is NESTVDT, H2-3 is LKTVIKK, H2-4 is EMLLQLK, and H2-5 is LEETKSI (corresponding to sequence numbers 34-43 respectively). These are the heptad element sequences "abcdefg" corresponding to the heptad elements present in the A-ENA protein of Bacillus thuringiensis Sv israerensis strain ATCC 35646 (defined by UniprotKB Q8KNV8-SEQ ID NO: 1 and UniprotKB Q8KNV7-SEQ ID NO: 2, respectively), The monomer spontaneously folds into an alpha-helix antiparallel coiled-coil structure, and further assembly into a fibrous structure enables autocatalytically induced isopeptide bond formation.

[0078] In this specification, the fragments referred to as NTL, CT, and L are used to define the monomer polypeptide sequences of the N-terminal NTL preceding helix 1, the C-terminal CT following helix 2, and the internal linker sequence L connecting helix 1 and helix 2, respectively. The minimum number of amino acid residues constituting the fragments is defined in this specification as one amino acid for the NTL and CT fragments, and a minimum of four amino acids for the linker. The maximum number of amino acids is not limited herein, and the sole requirement is that the A-ENA monomer protein spontaneously folds into an alpha-helix antiparallel coiled-coil structure, enabling further self-assembly into a protofibril and ultimately a nanofibril.

[0079] NTLs have been observed to play a role in the covalent linkage of protein nanofibrils and thus function as a "lock" for the fibril structure, and therefore, depending on their sequence, this fragment can be applied to modulate the fibril structure. In certain embodiments, the length of an NTL sequence is at least one and at most (or maximum) three amino acids, or at least one and at most four amino acids, or at least one and at most five amino acids, or at least one and at most six amino acids, or at least one and at most seven amino acids, or at least one and at most eight amino acids, or at least one and at most nine amino acids, or at least one and at most ten amino acids, or at least one and at most eleven amino acids, or at least one and at most twelve amino acids, or at least one and at most thirteen amino acids, or at least one and at most fourteen amino acids. The NTL is an amino acid, or at least one and up to 15 amino acids, or at least one and up to 16 amino acids, or at least one and up to 17 amino acids, or at least one and up to 18 amino acids, or at least one and up to 19 amino acids, or at least one and up to 20 amino acids, or at least one and up to 21 amino acids, or at least one and up to 22 amino acids, or at least one and up to 23 amino acids, or at least one and up to 24 or more amino acids, or at least 2, 3, 4, 5, 6, or 7 amino acids and up to 3, 4, 5, 6, 7, or 8 or more amino acids. The total length or number of amino acids in the NTL may or may not include an additional tag, protein domain, or peptide amino acid sequence attached to the A-ENA NTL fragment to provide a modified A-ENA or A-ENA-based monomeric protein.

[0080] The C-terminal tail (CT) provides a structurally exposed fragment on the surface of the monomer or fibril, and is therefore suitable for attachment, connection, or fusion to further parts such as protein domains, tags, or other molecules. Thus, in another particular embodiment, the length of the C-terminal tail sequence is at least one and up to (or maximum) two amino acids, or at least one and up to three or four amino acids, or at least one and up to five amino acids, or at least one and up to six amino acids, or at least one and up to seven amino acids, or at least one and up to eight amino acids, or at least one and up to nine amino acids, or at least one and up to ten amino acids, or at least one and up to eleven amino acids, or at least one and up to twelve amino acids, or at least one and up to thirteen amino acids, or at least one and up to ten. The CT has a maximum of 14 amino acids, or at least 1 and a maximum of 15 amino acids, or at least 1 and a maximum of 16 amino acids, or at least 1 and a maximum of 17 amino acids, or at least 1 and a maximum of 18 amino acids, or at least 1 and a maximum of 19 amino acids, or at least 1 and a maximum of 20 amino acids, or at least 1 and a maximum of 21 amino acids, or at least 1 and a maximum of 22 amino acids, or at least 1 and a maximum of 23 amino acids, or at least 1 and a maximum of 24 or more amino acids, or at least 2, 3, 4, 5, 6, or 7 amino acids and a maximum of 3, 4, 5, 6, 7, or 8 or more amino acids. The total length or number of amino acids in the CT may or may not include the amino acid sequences of additional tags, protein domains, or peptides attached to the A-ENA CT fragment to provide a modified A-ENA or A-ENA-based monomeric protein.

[0081] The linker(L) sequence appears in the structure as a loop or turn, providing a covalent link between the two helices of the hairpin, and in parallel, allowing for the insertion or conjugate of further proteins, molecules, or tags onto the A-ENA protein. Thus, in certain embodiments, the length of the linker sequence located between the sequence of helix 1 and the sequence of helix 2 is at least 4 and at most (or maximum) the number of amino acids that result in a 3D structure that prevents helices 1 and 2 from forming a coiled coil. Therefore, preferably, the linker fragment is at least 4 and at most 30 amino acids, or at least 4 and at most 25 amino acids, or at least 4 and at most 20 amino acids, or at least 4 and at most 15 amino acids, or at least 4 and at most 10 amino acids, or at least 5 and at most 9 amino acids, or at least 6 and at most 8 amino acids, or at least 7 amino acids. The total length or number of amino acids of the linker may or may not include the amino acid sequences of additional tags, protein domains, or peptides inserted into the A-ENA linker fragment to provide a modified A-ENA or A-ENA-based monomeric protein.

[0082] The A-ENA-based monomeric protein subunits forming the protein nanofibrils of the present invention as described herein provide an A-ENA protein having a sequence based on or derived from the original bacterial A-ENA protein, as annotated herein, but a portion of the consensus sequence of the fragments may be adapted, modified, or altered by mutation, substitution, addition, or deletion of further amino acids, peptides, or proteins to provide a functionalized A-ENA protein or its fibrous structure. In certain embodiments, heterologous amino acid sequences or proteins or tags may be fused, added, or inserted into any one of the NTL, CT, or L fragments, resulting in an A-ENA-based protein monomer or a modified or manipulated A-ENA, ultimately providing a functionalized protein that self-assembles into a functionalized protein fibril and / or fibril having a modified surface.

[0083] Specifically, the N-terminal lock or N-terminus of A-ENA or an A-ENA-based monomeric protein subunit may further be defined as an amino acid sequence of at least one amino acid, preferably at least seven amino acids, and the NTL fragment comprises or consists of a consensus sequence defined as M-Ψ-Φ-ZX-Φ-P, where, - Ψ is a short side-chain amino acid residue selected from the list of V, C, G, A, P, S, T, N, D, preferably A, G, S, or T. - Φ is a hydrophobic amino acid residue selected from the list of M, V, I, L, A, G, H, W, Y, F, preferably from the list of L, M, I, V, F, or W. - X = any amino acid residue, - Z is Ala or proline, and M and P are single-letter codes for the respective amino acids methionine and proline.

[0084] In a further specific embodiment, the NTL fragment includes or comprises a consensus sequence defined as M-Ψ-Φ-ZX-Φ-P, where, - Ψ is selected from S, G, or T, - Φ is a hydrophobic amino acid residue selected from the list of M, V, I, L, A, G, H, W, Y, F. - X = any amino acid residue, - Z is Ala or proline, and M and P are single-letter codes for the respective amino acids methionine and proline.

[0085] A-ENA proteins having NTLs defined by the aforementioned consensus motif offer options for forming an IPB, where the donor or nucleophilic residue is provided by the N-terminal free amino (NH2-) group present in the peptide backbone. The NTL consensus motif forms structural elements that help pack the NTL onto the fibril surface, thereby helping to position the N-terminal amine at the optimal orientation and distance for isopeptide bond formation.

[0086] In certain embodiments, the NTL comprises or consists of MG-(M or I)-P-(T or N)-IP, where a single-letter code is applied to each amino acid, and the amino acid in parentheses provides an alternative (indicated by "or") at the position, and the NTL may also comprise further amino acids, such as the NTL fragments of SEQ ID NO: 1 and SEQ ID NO: 2, which further include EGLDIT (SEQ ID NO: 44) as part of the NTL.

[0087] In certain embodiments, an A-ENA monomeric protein subunit comprising or composed of an amino acid sequence corresponding to the formula NTL-h1-L-h2-CT described herein includes further amino acid residues in the h1 and / or h2 helix sequences located at the N-terminus and / or C-terminus from the heptad elements H1-H5, even though these are not part of the NTL, CT, or linker region, and therefore helix 1 and helix 2 are, for helix 1, formula:[X] n1 -H1-1-H1-2-H1-3-H1-4-H1-5-[X] n2 , and / or Helix II: [X] n1-H2-1-H2-2-H2-3-H2-4-H2-5-[X] n2 Corresponding to this, the heptad element is defined by the residues "abcdefg" as defined herein, and is covalently linked in the order described above, [X] n X represents an additional amino acid present as part of the helix structure h1 and / or h2, where X may be any amino acid, and n1 and n2 refer to the number of different amino acids in the helix preceding or following the heptad element, respectively, where each of the n amino acids X may be the same or different such that the polypeptide chain of helix 1 or helix 2 forms the alpha(α)-helix, respectively.

[0088] In a particular embodiment, the [X] with respect to h1 n1 is a single arginine (R), leucine (L), asparagine (N), or lysine (K) residue, and / or the aforementioned [X] of helix 1. n2 This includes or consists of three amino acids such as FEK or FKK, and / or the aforementioned [X] of helix 2. n2 It contains or consists of three amino acids, such as IQS or LKL.

[0089] A-ENA protein fibril The present invention relates to protein fibrils, preferably nanofibrils, and “nanofibrill” is defined herein as a fibrous assembly having a nanoscale diameter and a significant length extending to several micrometers. Where used interchangeably herein, the terms “fibril,” “filament,” “fibrous assembly,” or “fibrous structure” refer to structured biochemical compounds such as protein assemblies or protein-based assemblies, preferably composed of protein material, and forming ordered structures of long shapes having a diameter of up to 100 nanometers, potentially as part of a larger hierarchical structure. Taking into account the terminology used herein to define (nano)fibrils, the inventors herein refer herein to a plurality of potential nanofibrils as “fibers,” and fibers are generally considered to represent structures with considerably larger diameters (microscale to milliscale) compared to fibrils, and therefore “fibers” preferably provide a higher-order hierarchical structure of the plurality of fibrils. In certain embodiments, such fibers comprise a plurality of A-ENA nanofibrils, each fibril further undergoing lateral association with another fibril to provide a structured fiber. A further example of a fiber is a woven assembly of protein nanofibrils.

[0090] Nanofibril structures offer unique and intriguing properties that make them subject to investigation in several fields of research and application, such as microbiology, biomechanics, and materials science. The present invention provides protein nanofibrils obtained by the spontaneous entanglement of two protofibril structures, where the term “protofibril” refers to a fibrous self-assembly of a polymer or multimer, which is fundamentally composed of monomeric protein subunits described herein, called A-ENA proteins or A-ENA-based proteins, comprising protein fragments following the formula defined herein as NTL-helix 1(h1)-L-helix 2(h2)-CT, interconnected via at least one isopeptide bond, as further detailed herein. In particular, the protofibrils are composed of functional A-ENA proteins or their orthologues, where “functional” refers to their ability to self-assemble as coiled-coil structures into entangled protofibril structures that form the nanofibrils described herein.

[0091] Previously, self-assembling S-ENA and L-ENA proteins have been reported based on the elucidation of Bacillus cereus spore appendages (Pradhan et al., 2021; Remaut et al., International Publication No. 2022 / 029325), and these also provide self-assembling proteins that multimerize into protein fibrils. These S-ENA and L-ENA protein-based fibrous structures also provide nanofibril structures in the sense of the present invention, where S-ENA and L-ENA fibrils are composed of multiple polypeptide chains that appear as helix multimers or rather disks, stacking into helix (S-ENA) or ladder-type (L-ENA) multimer assemblies or fibrils, and the multimers of the fibrils are interconnected by disulfide crosslinks as covalent intermolecular rigidity links. Pradhan et al. (2021) and Remaut et al. (International Publication No. 2022 / 029325) refer to S-ENA or L-ENA “fibers,” but the inventors have chosen to redefine these structures herein as nanofibrils or “fibrils” in order to better align with the conventional use of the term fiber in some fields, which refers to multiple fibrils.

[0092] In this invention, the alpha-helix spore appendage protein, referred to as the A-ENA protein, first annotated herein in Bacillus thuringiensis, has a significantly different protein structure compared to the previously discovered S-ENA and L-ENA proteins. For the A-ENA protein as well, the structural appearance of the resulting assembled fibrils is determined or driven by the amino acid sequence of the monomeric protein, which not only self-assembles into monomers to form protofibrils, but also connects intermolecularly via one or more isopeptide bonds. The resulting A-ENA multimer assembly has a (proto)fibrous structure, while the S-ENA and L-ENA multimer assemblies form helix arcs or disks, respectively, which can be visually distinguished from the fibrous structure (under a microscope). As a result of the aforementioned structural differences, nanofibrils composed of S-ENA protein or L-ENA protein are each considered to be multiple stacked helix arcs or disks, which further extend into fibrous assemblies covalently interconnected by disulfide crosslinks between different polymer assemblies, whereas nanofibrils composed of A-ENA protein contain at least two entangled alpha-helix protofibrils covalently linked by isopeptide bonds.

[0093] One advantage of the aforementioned fibrous material for at least some biotechnology applications is that isopeptide bonds are formed irreversibly, thus providing the extremely high tensile strength of the assembled fibrils, as well as the remarkable mechanical and thermal stability of the protein nanofibrils. While S-ENA and L-ENA-based fibrils are also considered rigid and stable bionanomaterials, the properties of isopeptide covalent linkages are stronger compared to disulfide linkages.

[0094] Therefore, from a phenotypic standpoint, A-ENA fibrils are readily distinguishable from any other fibrils in bacterial or more specifically Bacillus spore samples simply by analyzing them under a microscope. Indeed, examination of 2D images of microscopic analysis, for example, in Figure 2, as shown herein, revealed that A-ENA nanofibrils exhibit a uniquely highly ordered fibril structure compared to the fibrous structures described for, for example, S-ENA and L-ENA proteins (see Pradhan et al., 2021 or Remaut et al., International Publication No. 2022 / 029325).

[0095] In one embodiment described herein, a protein nanofibril comprises or consists of two protofibrils, each protofibril comprising or consisting of at least two monomeric protein subunits, each monomeric protein subunit comprising fragments interconnected by covalent bonds according to the formula NTL-h1-L-h2-CT, which spontaneously fold in aqueous solution into two helices, h1 and h2, as an alpha-helix antiparallel coiled-coil structure, each helix comprising five or more heptad elements according to the formulas H1- to H5 previously described herein, according to the consensus sequence provided for their “abcdefg” side chain format, where L, NTL, and CT are defined as described herein, and the monomeric protein subunits interact via at least one isopeptide covalent bond. In certain embodiments, the nanofibril is therefore composed of A-ENA monomers, the A-ENA monomers as defined herein and / or the A-ENA monomers may also be A-ENA-based monomers defined as A-ENA proteins, which include one or more modifications necessary to functionalize the (nano)fibril, but the ability to self-assemble into protein nanofibrils is thus retained.

[0096] Therefore, the fibrils of the present invention are preferably formed from two protofibrils of at least two A-ENA monomers or A-ENA-based monomers, preferably at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400 or more A-ENA monomers or A-ENA-based monomers. Thus, the basic structure of the protein nanofibrils described herein is a tetrameric structure, comprising two protofibrils of at least two monomers each. In embodiments in which the protein nanofibrils comprise two protofibrils of at least three monomers each, a hexameric structure is provided, and so on. The A-ENA monomer or A-ENA-based monomer of the protofibril may be the same or different as further described herein.

[0097] From the viewpoint of visualizing the 3D structure of the protein nanofibrils, the A-ENA monomers of the fibrils are referred to as i and i', respectively, in the f and f' protofibrils that form entangled helix fibrils, and the protomers further stacked toward the C and N poles of the fibrils are numbered incrementally (i.e., i+1, i+2, i+3, etc., or i'+1, i'+2, i'+3, etc.) or decaying (i.e., i-1, i-2, i-3, etc., or i'-1, i'-2, i'-3, etc.). Throughout this specification and this application, the A-ENA fibrils are oriented in the drawings such that the C-terminus of their subunits faces upward (referred to as the C-terminus or pointed fibril end, i.e., the C-pole) and the N-terminal extension or end of their subunits faces downward (referred to as the N-terminus or spiny fibril end, i.e., the N-pole).

[0098] In further specific embodiments, the protein nanofibrils are isolated protein nanofibrils, where “isolated” means that the nanofibrils do not contain other compounds such as material from bacterial spore cultures or material from bacterial components such as parasporial bodies, spores, or other bacterial-derived materials. In practice, ex vivo extraction or isolation of A-ENA fibrils from naturally occurring bacterial spores has been impossible. In retrospect, this is thought to be due to their specific structure and the presence of isopeptide bonds. Since A-ENA nanofibrils cannot be obtained “as is” from bacterial spores, the protein nanofibrils of the present invention, which are fibrils isolated from any further bacterial compound (e.g., without attached parasporial bodies or spores), can therefore be isolated or obtained from other means, for example from recombinantly produced fibrils, without the presence of spore formation and associated bacterial compounds. Therefore, in certain embodiments, the protein nanofibrils of the present invention, also referred to as A-ENA protein nanofibrils or A-ENA-based protein nanofibrils, are recombinantly produced A-ENA nanofibrils or A-ENA-based nanofibrils.

[0099] Iso-peptide bond (IPB) The present invention relates to a protein nanofibril described herein that has extremely high strength and stability, which is the result of the presence of spontaneously formed covalent linkages of each monomer protein subunit present in the protofibril to one or more other monomer protein subunits of the protein fibril, as isopeptide bonds.

[0100] An isopeptide bond is an amide bond formed between a carboxyl group / carboxamide group and an amino group, where at least one of the carboxyl group or amino group is located outside the protein backbone (protein skeleton). In contrast to general peptide (true peptide) bonds, isopeptide bonds can form covalent links between (different) polypeptide backbones, which are very rare covalent interactions, but are well known for post-translational events such as ubiquitination, where ubiquitin at its C-terminal glycine residue interacts with the lysine side chain of the substrate protein to form an intermolecular isopeptide bond, or glutathione, which contains an isopeptide bond between the side chain of a glutamic acid residue and the amino group of a cysteine ​​residue, provides an intramolecular isopeptide bond that is formed autocatalytically. Isopropyl alcohol bonds are chemically irreversible under biological conditions and are resistant to most proteases. While they may be formed through enzymatic catalysis, isopeptide bonds can also be formed spontaneously or autocatalyzed, as used interchangeably herein. Spontaneous isopeptide bond formation was first observed in bacteriophage HK97, and it was revealed that the HK97 capsid structure is assembled by polymerization of covalently crosslinked subunits as a result of autocatalyzed isopeptide bond formation between the Lys and Asn side chains in adjacent subunits, linking between the capsid assemblies to form covalently assembled five-membered and six-membered rings that form a structure called protein chainmail (Wikoff et al., 2000; Science 289, 2129-2133).

[0101] Spontaneous isopeptide bond formation typically occurs after protein folding, via nucleophilic attack of the ε-amino group from lysine on the Cγ group of asparagine, facilitated by the adjacent glutamate.

[0102] As used herein, the term “spontaneous” refers to a bond that can be formed in or between proteins, e.g., an isopeptide bond or covalent bond, in the absence of any other agents or enzymes and / or without any chemical modification of the protein, e.g., without any natural chemical ligation or chemical coupling. Thus, a “spontaneous isopeptide bond” or “autocatalytically formed isopeptide bond” is formed when one or more proteins are isolated by themselves (intramolecularly) or between two proteins when they are alone (without further agents or chemical modifications). Thus, spontaneous isopeptide bonds or covalent bonds can be formed spontaneously in the absence of enzymes or other exogenous substances or without chemical modification. However, spontaneous isopeptide bonds or covalent bonds may, in particular, require the presence of “autocatalytic residues,” such as glutamic acid residues or aspartic acid residues, which are part of the polypeptide chain of the isopeptide bond-forming protein, to enable the formation of the bond in a proximity-inducible manner.

[0103] Therefore, as interchangeably used herein, the autocatalytic formation of isopeptide crosslinks or bindings or interactions involves the conservation of three amino acids: one functioning as a nucleophilic donor, one as an electrophilic acceptor, and one as a catalytically important acidic Glu or Asp residue mediating proton transfer. The latter requirement and the specific arrangement of residues in a hydrophobic environment adjacent to the active site have been observed in numerous immunoglobulin-like pyrine subunits of Gram-positive bacterial pili, where these internal crosslinks provide stabilization against chemical, thermal, and mechanical stresses. The hydrophobic environment is important because it alters the pKa values ​​of the amino acid side chains, resulting in a reverse-protonated state where the lysine or donor residue side chain is deprotonated and the Glu / Asp side chain is protonated (Kang and Baker, 2011-Trends Biochem. Sci. 36, 229-237; Hu et al., 2011-J Am Chem Soc. 133(3):478-85). Furthermore, isopeptide bond formation is proximity-inducible, and therefore the three residues involved in the reaction must have a suitable geometric arrangement. The protein nanofibrils described herein are composed of the monomer protein subunits appearing as coiled coils, and upon self-assembly into protofibrils and further entangled nanofibrils, the positions of the three residues provide the geometric necessity for forming the autocatalytic intermolecular isopeptide bonds described herein. In fact, the amino acid sequences of the A-ENA or A-ENA-based protein monomers defined above include necessary limitations to first assemble into an alpha-helix antiparallel coiled-coil structure within their heptad elements, thereby providing the hydrophobic environment and geometric arrangement necessary to enable proximity-inducible isopeptide linkage when further monomers exist as protofibril subunits.Therefore, in addition to the limitations based on the heptad elements required for coiled-coil assembly, the A-ENA protein sequence is also highly conserved in positions containing donor, acceptor, and catalytic residues for isopeptide bond formation, as observed in protein nanofibrils.

[0104] Therefore, after the monomer coiled-coil structure is assembled, the protofibril polymer fiber structure is assembled, and isopeptide bonds are formed between the monomers of the protofibrils and between the monomers of the second protofibril entangled with the first protofibril, resulting in a tight, strong, and irreversible protein nanofibril. Specifically, the mechanism of autocatalytic isopeptide bond formation is as follows: First, the Cγ carbonyl group of the acceptor or electrophile (preferably Asn / Asp) is polarized via hydrogen bonding to a protonated catalytic Glu / Asp residue; then, the unprotonated Lys ε-amino group of the donor residue nucleophilically attacks the polarized Cγ carbonyl group of the acceptor residue, leading to the formation of a tetrahedral intermediate; from there, a proton is transferred from the Lys ε-amino group to the catalytic Glu / Asp residue, and a proton is transferred from the catalytic Glu / Asp residue to the leaving group of the acceptor residue. Subsequently, depending on the properties of the acceptor residue's side chain, the formation of a Lys-Asn isopeptide bond leads to the release of an NH3 molecule, while Lys-Asp linkage leads to the release of an H2O molecule (Kang and Baker, 2011).

[0105] Accordingly, the protein nanofibrils of the present invention provide a protein structure in which isopeptide bonds are formed intermolecularly, meaning between two protein molecules. Typically, the isopeptide bonds described herein are formed between a lysine residue and a glutamine, glutamic acid, asparagine, or aspartic acid residue, or between the terminal carboxyl group of a protein, or between the alpha-amino terminus of a protein and asparagine, aspartic acid, glutamine, or glutamic acid. Accordingly, the isopeptide bonds provided in the protein nanofibrils described herein may be formed between a lysine residue and an asparagine residue, or between a lysine residue and an aspartic acid residue. In particular, isopeptide bonds may occur between the side-chain amine of lysine and the carboxamide group of asparagine. Each residue in a pair involved in an isopeptide bond is referred to herein as a reactive residue.

[0106] The presence of covalent bonds within or between proteins, particularly isopeptide bond interactions, can be determined by bioinformatics sequence analysis, or preferably by mass spectrometry or structural determination, such as X-ray crystallography, cryo-EM, or NMR.

[0107] Spontaneously formed isopeptide bonds, as a method of covalently linking amino acids, are often observed intramolecularly, but when they appear intermolecularly, as in the case of protein nanofibrils described herein, this is a favorable approach for crosslinking molecules. In contrast to other methods of covalently linking polypeptides, spontaneously formed isopeptide bonds do not require enzyme catalysis or cofactors, are predicted to form specifically due to the need for three reactive residues positioned in a precise conformation surrounded by a hydrophobic environment, are chemically extremely stable as a result of amide-type bonding, are typically resistant to proteolysis because they are outside the protein backbone, and finally, their formation is not redox-dependent and is an irreversible interaction.

[0108] These advantages have likely evolved to play a role, for example, in bacteria lacking a disulfide bond formation "mechanism" to enhance excreted bacterial proteins (Dutton et al., 2008 - Proc. Natl. Acad. Sci. USA 105, 11933~11938). This is because many Gram-positive bacteria have been shown to incorporate intramolecular isopeptide bonds, which are physiologically important for outer membrane proteins in these bacteria, as a means of stabilizing protein structure (Dutton et al., 2008; Hu et al., 2011 - J Am Chem Soc. 133(3):478~85).

[0109] In certain embodiments, the protein nanofibrils described herein are characterized in that monomeric protein subunits, preferably A-ENA monomeric protein subunits, are numbered i, i+n, and in (and subsequently i', i'+n, and in for the monomer units of the protofibril f') with respect to the C or N pole of the fibril f, and each monomer i, i+ / -n, i', and i'+ / -n is covalently linked to at least one further monomer i, i+ / -n, i', and / or i'+ / -n by the presence of an isopeptide bond, and more preferably each monomer is linked to the protein nanofibrils via one or more IPBs. The fibril is covalently linked to one or more further monomers, more preferably each monomer is covalently linked to one further monomer of the protein nanofibril by the presence of one IPB, more preferably each monomer is covalently linked to one or more further monomers of the protein nanofibril via two IPBs, and more preferably each monomer is covalently linked to one or more further monomers of the nanofibril via two IPBs, which may be inter-protofibril or intra-protofibril connections, the intra-protofibril connections may also be called cross-fibril connections.In further embodiments, each monomer is covalently linked to one or more further monomers of the protein nanofibril by the presence of three IPBs; more preferably, each monomer is covalently linked to one or more further monomers of the protein nanofibril via four IPBs; more preferably, each monomer is covalently linked to one or more further monomers of the protein nanofibril via five IPBs; more preferably, each monomer is covalently linked to one or more further monomers of the protein nanofibril via six IPBs; more preferably, each monomer is covalently linked to one or more further monomers of the protein nanofibril via seven IPBs; more preferably, each monomer is covalently linked to one or more further monomers of the protein nanofibril via eight IPBs. The fibril is covalently linked to one or more further monomers, more preferably each monomer is covalently linked to one or more further monomers of the protein nanofibril via nine IPBs, more preferably each monomer is covalently linked to one or more further monomers of the protein nanofibril via ten IPBs, more preferably each monomer is covalently linked to one or more further monomers of the protein nanofibril via eleven IPBs, more preferably each monomer is covalently linked to one or more further monomers of the protein nanofibril via twelve IPBs, and more preferably each monomer is covalently linked to one or more further monomers of the protein nanofibril via thirteen or more IPBs. In this specification, a monomer having one or more IPBs in connection or crosslinking to one or more monomers may mean that one monomer is linked to one further monomer of the fibril via one or more IPBs, or similarly, that one monomer is linked to several other further monomers of the fibril, each of which is linked to one monomer via one or more IPBs.

[0110] In certain embodiments, each monomer of the fibril is therefore linked to one or more further monomers of the fibril via the presence of an IPB, and the crosslinking between two monomers may include one, two, three or more IPBs. In further specific embodiments, the protein nanofibril includes an A-ENA monomer in which each monomer is involved in at least one IPB that covalently links both protofibrils (f) and (f') of the nanofibril. In another embodiment, the protein nanofibril includes an A-ENA monomer in which each monomer is involved in one or more IPBs, at least one of which covalently links both protofibrils (f) and (f') of the nanofibril, and at least one covalently links the monomer subunit together with a further monomer subunit of the same protofibril of the nanofibril. In a further embodiment, the protein nanofibril comprises A-ENA monomers, each monomer of which is involved in 9 or 10 isopeptide bonds together with other monomers of the fibril, and more specifically, the monomer is linked to 3, 4, 5 or 6 other A-ENA monomers of the nanofibril.

[0111] In further specific embodiments, the protein nanofibrils described herein comprise monomeric protein subunits, each monomeric protein subunit, preferably A-ENA or A-ENA-based monomeric protein subunit, having covalent connections to further monomers of the fibril, the IPB having, between the side chains of amino acids that function as nucleophiles and the side chains of amino acids that function as electrophiles, respectively, at the following positions: • The N-terminal residue of the NTL in (i) (as a nucleophilic donor via the free amino-terminal group of the polypeptide backbone) and H1-5b of (i-5), (i-4), (i-3) or (i-2), • H1-4g of (i) and H2-2a of (i-1), • H2-3g of (i) and H2-4a of (i-1), • H2-3f in (i) and H2-4e in (i') • H2-4g of (i) and H1-3a of (i-1), • H2-4g of (i+1) and H1-3a of (i), • NTL of (i+5), (i+4), (i+3) or (i+2) and H1-5b of (i), • H1-4g of (i+1) and H2-2a of (i), • H2-3g of (i+1) and H2-4a of (i), and / or H2-3f of (i'+1) and H2-4e of (i) Formed in, This is read as the position of the nucleophilic donor, which is the position of the first monomer, in connection with the electrophilic acceptor, which is the position of the second monomer's side chain (for example, the donor side chain is at the position of the heptad element H1-4 of monomer (i) in connection with the acceptor side chain at the position of the heptad element H2-2 of monomer (i-1)).

[0112] In another alternative embodiment, the protein nanofibril described herein comprises monomeric protein subunits, each monomeric protein subunit, preferably A-ENA or A-ENA-based monomeric protein subunit, having covalent connections to further monomers of the same protofibril, the IPB having a nucleophilic amino acid side chain and an electrophilic amino acid side chain. These are formed at the heptad element positions of (i) H1-4g and (i-1) H2-2a, and / or (i) H2-3g and (i-1) H2-4a, and / or (i) H2-4g and (i-1) H1-3a, and / or (i+1) H2-4g and (i) H1-3a, and / or (i+1) H1-4g and (i) H2-2a, and / or (i+1) H2-3g and (i) H2-4a.

[0113] In another specific embodiment, the protein nanofibril described herein comprises monomeric protein subunits, each monomeric protein subunit, preferably A-ENA or A-ENA-based monomeric protein subunit, having covalent connections to further monomers of other protofibrils of the nanofibril, and the IPB is formed between the side chain of an amino acid functioning as a nucleophile and the side chain of an amino acid functioning as an electrophile, at the heptad element positions of (i) H2-3f and (i') H2-4e, and / or (i'+1) H2-3f and (i) H2-4e, respectively.

[0114] In another alternative embodiment, the protein nanofibril described herein comprises a monomeric protein subunit, each monomeric protein subunit, preferably A-ENA or A-ENA-based monomeric protein subunit, having covalent connections to further monomers, wherein the IPB is formed between an amino acid side chain functioning as a nucleophile and an amino acid side chain functioning as an electrophile, at the N-terminal residue of the NTL of (i) (as a nucleophilic donor via a free amino-terminal group of the polypeptide backbone) and H1-5b of (i-5), respectively, via its N-terminal locked amino-terminal group and H1-5b of monomer (i+5) (as a nucleophilic donor via a free amino-terminal group of the polypeptide backbone) and H1-5b of monomer (i+5).

[0115] In certain embodiments, the A-ENA monomer protein is described by the formula NTL-h1-L-h2-CT, and the heptad element is as follows: - Regarding heptad 1-1 (H1-1), XXX-Φ-XX-Φ, - For H1-2, Φ-X-(S or T)-Φ-(A or G)-Φ-(E or Q), - For H1-3, EXX-Φ-X-(H or N)-Φ, - For H1-4, Φ-N-(A or G or S)-E-(G or A)-EK, - Regarding H1-5, Φ-QX-Φ-Φ-XX, - Regarding H2-1, Φ-XX-Φ-Φ-X-Φ, - Regarding H2-2, (N or Q or D)-XX-Φ-XXX, - For H2-3, Φ-XX-Φ-Φ-(K or X)-(K or X), - For H2-4, (E or Q)-Φ-Φ-(L)-(Q or X)-XK, - Regarding H2-5, Φ-γ-X-Φ-Φ-XX Provided by the consensus sequence of residues abcdefg defined as follows: The consensus amino acid sequence residues of the heptad element are defined as follows: - Φ is a hydrophobic amino acid residue selected from the list M, V, I, L, A, G, H, W, Y, F, preferably L, M, I, V, F, or W. - Ψ is a short side-chain amino acid residue selected from the list of V, C, G, A, P, S, T, N, D, preferably A, G, S, or T. - γ is an amino acid residue for isopeptide interactions that function as an acid / base catalyst, selected from E or D, preferably E. - ε is an amino acid residue for isopeptide interactions that functions as an "acceptor" or electrophile, selected from E, Q, D, or N, preferably E, Q, or N. - X may be any type of amino acid residue, the single-letter codes of the amino acid residues are as shown, and the amino acids in parentheses provide alternatives (indicated by "or") to the "abcdegf" residues at their respective positions.

[0116] The "donor" or nucleophile that forms the isopeptide bond is lysine (K) in each heptad, but if this donor K is substituted with any other amino acid (X) in H2-3, and therefore H2-3 heptad residues f and / or g are not lysine (K), then these residues do not form an IPB, and presumably do not form a protein nanofibril either, as this IPB provides interprotofibril connectivity that gives stability to the nanofibril assembly.

[0117] Therefore, in preferred embodiments, the H2-3f and / or H2-3g residues are K, and in alternative embodiments, an A-ENA(like) protein is provided in which each of the donor residues of the heptad element, more specifically H1-4g and / or H2-4g, may be replaced with any other amino acid (X), resulting in fewer IPBs when self-assembled into fibrils. However, it is still essential that at least one K is present in order to form at least one IPB, preferably at the position of the H2-3f or g donor.

[0118] Functionalized A-ENA protein assembly As defined and introduced above, A-ENA protein may be provided as an A-ENA protein conforming to the formula NTL-h1-L-h2-CT as defined and described herein above, wherein the monomer has been modified or manipulated to obtain a “modified A-ENA protein” or “A-ENA-based monomeric protein,” which therefore refers to an A-ENA protein based on a naturally occurring or native A-ENA protein sequence, or at least as defined by the definition of A-ENA provided herein with respect to its sequence and functionality, but modified or manipulated to result in an A-ENA protein that functionalizes A-ENA protein nanofibrils spontaneously assembled at the time of production in the host.

[0119] The aforementioned modification or manipulation strategies are further illustrated and described herein in a non-limiting manner and serve as support for enabling those skilled in the art to design such modified A-ENA proteins.

[0120] Therefore, “modified” or “manipulated” A-ENA protein or “A-ENA-based protein” (as used interchangeably herein) means herein that “modified” A-ENA protein is a protein subunit having a sequence comprising interconnected fragments according to the formula NTL-h1-L-h2-CT, as previously described herein, where at least five heptad elements of the h1 and h2 helices are defined as described herein, thus enabling spontaneous folding of monomers at the helix hairpin, with linkers forming a loop connecting two antiparallel helices, with NTL and CT present on the surface, and further this self-assembles into a fibrous structure as described herein, where each monomer, together with further monomers (which may have identical or different sequences), is involved in at least one intermolecule-intermolecule-branched (IPB), and optimally 10 IPBs. In the comparison, “modified” may then be further specified by the presence of any one of the following manipulation formats: - (1) The NTL is cleaved compared to the native A-ENA protein of the modified A-ENA monomer, preferably the cleavage results in an NTL of fewer than 4 amino acids, preferably 3 amino acids, 2 amino acids, or 1 amino acid. One advantage of providing a modified A-ENA in which the modification is a cleavage of NTL compared to naturally occurring NTL is that the A-ENA monomer is more accessible for post-fibril formation modification of the nanofibril in its folded state. - (2) The A-ENA heptad element sequence conservation is modified to provide an A-ENA mutant variant or surface mutant variant, enabling the production of A-ENA protein nanofibrils having a modified surface structure or properties compared to the native A-ENA sequence of the modified A-ENA, more specifically, (2a) The modified A-ENA sequence comprises at least four amino acid NTLs, and the heptad elements of the modified A-ENA monomer are substituted at at least one of the following positions: H1-1a, H1-1b, H1-1c, H1-3f, H1-4f, H1-5b, H1-5f, H2-1b, H2-1c and / or H2-5b with an amino acid (natural or synthetic) that does not correspond to the consensus sequence of the “conventional” A-ENA heptad as defined herein (i.e., a mutant variant), or (2b) The modified A-ENA sequence contains fewer than four amino acid NTLs, and the heptad elements of the modified A-ENA monomer protein are mutated at at least one of the following positions: H1-1a, H1-1b, H1-1c, H1-1f, H1-2f, H1-3b, H1-3f, H1-4b, H1-4f, H1-5b, H1-5f, H2-1b, H2-1c, H2-2b, H2-2f, H2-3f, H2-4f, H2-5b, H2-5c and / or H2-5f to amino acids that do not correspond to the consensus sequence of the “conventional” A-ENA heptad as defined herein (natural or synthetic), - (3) Modifications comprising the A-ENA protein being further fused to a protein tag or protein domain at its N-terminus or C-terminus, or being inserted into a linker region, wherein the fusion is typically obtained through the expression of a gene fusion, in which a heterologous (poly)peptide sequence capable of itself forming a tag or folded protein domain is fused to or inserted into the native A-ENA sequence (or A-ENA mutant variant). - (4) The A-ENA protein is conjugated to another protein or chemical moiety, such as, in particular, a tag, protein domain, small molecule or label, the conjugation may occur after fibril formation, and / or the conjugation is defined as a covalent linkage (in an orthogonal manner) formed with one or more side chain residues or atoms.

[0121] Modifications involving A-ENA fusion or conjugation with or to heterogeneous or further peptides, proteins, protein domains, tags, labels, or other functional moieties are envisioned herein, and such modifications can be obtained by connecting the heterogeneous moieties directly or via the use of linkers to the A-ENA subunits.

[0122] In certain embodiments disclosed herein, the A-ENA subunit is fused or conjugated to a tag or protein-binding partner for specific tag-protein binding interactions on the fibril surface after fibril formation. In further specific embodiments, more specifically, as known for the tag / protein-binding partner pair SpyTag / SpyCatcher exemplified herein, the tag / protein-binding partner pair comprises a protein and a tag capable of interacting by covalent bonds, for example, through the formation of isopeptide bonds, and the tag or catcher may be fused to one or more A-ENA proteins, resulting in one counterpart of the pair being presented on the surface of a protein nanofibril and the other counterpart forming a covalent interaction, specifically an isopeptide bond (preferably after fibril formation), with a portion of the pair associated with the fibril.

[0123] Accordingly, a further embodiment relates to a protein nanofibril comprising at least one modified A-ENA monomer, wherein the modified format is provided according to (1), (2), (3) or (4) as provided above. A more specific embodiment relates to the protein nanofibrils described herein, wherein at least two different sequences of monomeric proteins form heteropolymeric fibrils upon self-assembly, or alternatively, all monomers are identical to form homopolymeric fibrils.

[0124] In a further specific embodiment, said protein nanofibril comprises or consists of two protofibrils, and each monomeric protein further comprises a flexible linker and / or a protein domain having a hydrodynamic radius of less than 11 nm for surface presentation on the homopolymeric fibril, so as to prevent collision of the protein domains when nanofibril assembly occurs.

[0125] As used herein, a flexible linker is typically provided by an amino acid sequence having a length in the range of 1 to 30 amino acids, which is generally rich in small or polar amino acids such as Ser or Thr, or non-polar amino acids such as Gly, to provide good flexibility and solubility. The small size of these amino acids provides flexibility and enables mobility of the connected functional domains. The incorporation of Ser or Thr can maintain the stability of the linker in aqueous solution by forming hydrogen bonds with water molecules, thus reducing undesirable interactions between the linker and the protein moiety. The most commonly used flexible linker has a sequence consisting primarily of a stretch of Gly and Ser residues ("GS" linker). Examples of the most widely used flexible linkers include (Gly-Gly-Gly-Gly-Ser) nIt has the following sequence. By adjusting the copy number "n", the length of this GS linker can be optimized to achieve proper separation of functional domains or to maintain the necessary interdomain interactions (Chen et al., Adv Drug Deliv Rev. 2013, 65(10):1357~69). In addition to the GS linker, many other flexible linkers have been designed for recombinant fusion proteins, and are also rich in small or polar amino acids, but may also contain amino acids such as Lys and Glu to improve solubility. Alternatively, linker peptides are constructed using computer design by adding a single amino acid or short peptide that extends from the scaffold (see also Example 8).

[0126] In certain embodiments, the protein nanofibril comprises a modified A-ENA monomer containing a heterogeneous protein domain (following the modified format of (3) provided above), wherein the heterogeneous protein domain is fused to the A-ENA monomer via its N-terminus or C-terminus, or is inserted into the linker region of the A-ENA, resulting in a 3D protein structure in which the heterogeneous protein domain is presented on the surface of the nanofibril.

[0127] In further specific embodiments, the protein nanofibril comprises or comprises two protofibrils, each comprising or comprising at least two A-ENA monomers, at least one of which is a modified A-ENA, the modified A-ENA being provided in the format of (3) or (4) above, or specifically a fusion of A-ENA with a heterogeneous protein domain or tag as described herein (and according to (3)). The protein nanofibrils are obtained by combining or mixing an unmodified A-ENA protein (i.e., native A-ENA protein) or a mutant variant A-ENA protein (according to the modified format (2) provided above) with a modified A-ENA protein, such as an A-ENA heterofusion protein (as in the format (3) provided above) or a conjugate (as in the format (4) provided above), which can result in the self-assembly of heteropolymer nanofibrils that can be considered copolymers appearing as alternating blocks of surface-presented and non-presented portions (as visualized, for example, in Figure 16a).

[0128] The mixture of different A-ENA monomers and modified A-ENA monomers of the heteropolymer protein nanofibrils can be obtained in a host through recombinant expression of modified A-ENA protein sequences (e.g., gene fusions of A-ENA to heterologous peptides or protein tags or domains inserted at the N-terminus, C-terminus, or linker region of A-ENA) and unmodified or conventional A-ENA.

[0129] Alternatively, the mixture of different A-ENA monomers and modified A-ENA monomers of the heteropolymer protein nanofibrils can be obtained through recombinant expression of the modified A-ENA protein sequence (e.g., a gene fusion of A-ENA to a heterologous peptide or protein tag or domain inserted into the N-terminus, C-terminus, or linker region of A-ENA) in a host that endogenously expresses native A-ENA, such as a spore-forming bacterial strain, preferably the Bt strain.

[0130] Alternatively, the mixture of different A-ENA monomers and modified A-ENA monomers of the heteropolymer protein nanofibrils can be obtained in a host through recombinant expression of modified A-ENA, which is modified in the sense of the modified format (1) (cleavage-type NTL) or (2) (mutant variant with modified surface) provided above, and modified A-ENA, which provides a heterologous portion (presented) on the surface of the fibril, and which is modified in the sense of the modified format (1) (cleavage-type NTL) or (2) (mutant variant with modified surface).

[0131] Further embodiments relating to A-ENA protein nanofibrils and their production Further aspects of the present invention relate to nucleic acid molecules or nucleic acid sequences encoding A-ENA-based monomers or modified A-ENA protein monomers as described herein for forming recombinant protein nanofibrils as described herein.

[0132] Furthermore, vectors are disclosed comprising nucleic acid molecules encoding naturally occurring A-ENA protein or A-ENA-based monomers or modified A-ENA protein monomers as described herein, which may be for cloning or expression of recombinant A-ENA protein, modified A-ENA protein, as described herein. The vectors may be introduced into a host through techniques for transiently or stably transforming the host, as known to those skilled in the art, to produce exogenously introduced A-ENA or modified A-ENA protein as a secreted protein for obtaining A-ENA protein or modified A-ENA protein in the host, specifically in the cytoplasm of a host cell, or alternatively in a culture medium. During the self-assembly of recombinantly expressed A-ENA or modified A-ENA or A-ENA-based monomers, fibrous structures are formed in the host or host culture, enabling A-ENA polymer assembly, and optimally, extraction of A-ENA or A-ENA-based protein nanofibrils, from the host.

[0133] Accordingly, in certain embodiments, the Disclosure relates to a host cell comprising a nucleic acid molecule described herein that encodes an A-ENA protein or a modified A-ENA protein or an A-ENA-based protein. Another particular embodiment relates to the host cell comprising a protein nanofibril described herein, wherein the protein nanofibril comprises an A-ENA protein resulting from the recombinant production of an exogenously introduced A-ENA protein or an A-ENA protein-coding nucleic acid molecule or a modified A-ENA protein-coding nucleic acid molecule, an expression cassette or vector (including a chimeric gene construct for encoding the A-ENA protein assembly).

[0134] In a preferred embodiment, the host cell is a bacterial cell, and more preferably, the host cell is E. coli.

[0135] In another preferred embodiment, the host cell is a bacterial cell or a Bacillus cell that enables spore formation, more preferably a Bacillus thuringiensis cell. In another preferred embodiment, the bacterial host cell does not have an endogenous A-ENA protein-coding gene. The host cell may also be provided by other organisms such as plants, fungi, or animals.

[0136] Further embodiments relate to a method for recombinantly producing A-ENA or A-ENA-based protein nanofibrils as described herein, the following steps: a) The step of introducing a nucleic acid molecule encoding the A-ENA protein, an A-ENA-based protein, or a modified A-ENA protein as described herein, for recombinant expression of the A-ENA monomer protein in a host cell, and / or b) Incubating host cells expressing the A-ENA protein under conditions suitable for culturing A-ENA protein or A-ENA-based protein or modified A-ENA protein products that self-assemble and preferentially spontaneously form protein nanofibrils within the host cells or in the host cell culture. c) Preferably the step of releasing the A-ENA protein assembly from the host cells through cell lysis, and / or d) Isolating the self-assembled protein nanofibrils, preferably by resuspension from the insoluble fraction and / or further purification from the cell lysate. Includes.

[0137] In a preferred embodiment, the introduction step in step a) is achieved by transforming the host cell with a chimeric gene containing the A-ENA (or modified A-ENA) protein coding sequence. In another embodiment, the host cell is a bacterial or fungal cell, and in a more preferred embodiment, a microbial-producing host such as E. coli for cytoplasmic expression of A-ENA protein nanofibrils, or a microbial-producing host such as Lactococcus lactis for secretion of A-ENA protein nanofibrils into culture medium.

[0138] In another preferred embodiment, the host is a spore-forming strain, more preferably a Bacillus strain, and more preferably a Bt strain. In another preferred embodiment, the A-ENA protein produced in the bacterial strain, preferably the Bacillus strain, is a heterologous A-ENA protein. Another embodiment provides a host in which the Bacillus strain expresses an endogenous A-ENA protein, which can be used to introduce a heterologous A-ENA protein to produce the (heteropolymer) protein nanofibrils described herein.

[0139] The present invention further provides recombinant protein nanofibrils, preferably A-ENA protein nanofibrils or A-ENA-based protein nanofibrils or modified A-ENA protein nanofibrils, which can be obtained by or from the methods for producing nanofibrils described herein.

[0140] The present invention further provides isolated protein nanofibrils, preferably A-ENA protein nanofibrils or A-ENA-based protein nanofibrils or modified A-ENA protein nanofibrils, which can be obtained by or isolated from the host, provided by the method for producing nanofibrils described herein.

[0141] Bacterial spores containing recombinant A-ENA protein nanofibrils As disclosed in this application, Bti spore cultures have been found to contain protein nanofibrils composed of A-ENA protein or A-ENA-1 protein (as shown in SEQ ID NOs. 1 and 2, respectively), which function to bring spores and parasporial bodies into contact and associate, for example, to ensure their pathogenic activity against lepidopteran insects.

[0142] Since the presence of these protein nanofibrils can be optimized, altered, or modified for specific purposes of providing modified spores, another aspect of the present invention relates to modified spores or modified Bacillus spores or modified Bt spores, where the term “modified” refers to the presence of non-endogenous A-ENA protein or modified A-ENA protein or A-ENA-based protein in the Bacillus strain and / or Bacillus spore. In further specific embodiments, the modified Bacillus spore or spore-forming Bacillus strain or cell comprises an A-ENA protein or encoding nucleic acid molecule that is not naturally occurring or is modified or manipulated.

[0143] As disclosed herein, numerous Bacillus strains lacking endogenous or native genes for producing A-ENA protein or protein nanofibrils have been identified. Accordingly, in certain embodiments, it is provided herein that A-ENA protein or A-ENA-based protein may be introduced or expressed recombinantly (or exogenously) in the bacterial strain or cells lacking the gene or ability to produce native A-ENA for the production of A-ENA protein nanofibrils as described herein.

[0144] Further specific embodiments relate to modified Bacillus spores, which present a portion of the modified A-ENA monomer protein on the surface of the assembled protein nanofibrils described herein. Thus, the modified Bacillus spore nanofibrils presenting a heterologous protein portion of the A-ENA protein (modified as described herein by insertion or fusion of a protein tag or protein domain into the A-ENA protein monomer) are provided herein.

[0145] Finally, as demonstrated herein, the modified bacterial spores, preferably Bacillus spores, may be used to assist, promote, improve, increase, or enhance bacterial spore activity, preferably pathogenic activity, more preferably pesticide activity, and most preferably insecticidal activity. In alternative embodiments, the modified bacterial spores, preferably Bacillus spores, described herein may be used to enhance, promote, increase, or improve the functional effects obtained by recombinantly produced protein nanofibrils in the modified spore culture.

[0146] In certain embodiments, the protein nanofibrils produced in the modified Bacillus spores may exist in a modified form or environment that allows for ex vivo extraction or isolation of the protein nanofibrils themselves from the culture or Bacillus spores, which is also assumed to be from the modified Bacillus spores disclosed herein and described herein.

[0147] Further alternative applications of protein nanofibrils are disclosed herein, wherein the recombinantly produced and purified forms of the protein nanofibrils are applied more closely with, in combination with, or sequentially with, the application of bacterial spore cultures, preferably Bt spore cultures or suspensions, to enhance or improve the pathogenicity of the spores against their targets, compared to applying the spore cultures without the addition of the purified protein nanofibrils.

[0148] Therefore, in certain embodiments, the protein nanofibrils produced recombinantly as described herein, and preferably by the methods described herein, provide additional or synergistic toxicity when the pesticide spore material is applied to their respective pests.

[0149] Therefore, the protein nanofibrils of the present invention may further provide alternative applications in the treatment or improvement of crops and contribute to improved pest management strategies in the future.

[0150] Pharmaceutical compositions and medical uses Further aspects of the present invention relate to pharmaceutical compositions comprising a nucleic acid molecule encoding a protein nanofibril or a monomer of a nanofibril as described herein, and optionally comprising a carrier, diluent, or excipient. In further specific embodiments, the pharmaceutical composition comprises a therapeutically active composition comprising a protein nanofibril as described herein and / or a pharmaceutical composition presented or provided as a drug bound to, inserted into, or attached to the protein fibril of the present invention.

[0151] In further embodiments, the protein nanofibrils or nucleic acid molecules or pharmaceutical compositions described herein are applied as part of a drug or pharmaceutical. Specific embodiments relate to the protein nanofibrils or nucleic acid molecules or pharmaceutical compositions described herein for use in therapeutic, preventive, or protective treatment of a subject. Accordingly, specific embodiments disclose a therapeutic method comprising the step of administering the protein nanofibrils, nucleic acid molecules or pharmaceutical compositions described herein to a subject suffering from or potentially suffering from a disease or disorder in order to cure or prevent the disease or disorder.

[0152] In certain embodiments, protein nanofibrils are modified by covalent or non-covalent addition of bioactive proteins or therapeutic proteins, preferably nanobodies. In these embodiments, the modified protein nanofibrils and their aggregates, assemblies, or hydrogels provide high-density, highly polyvalent (hundreds to thousands) presentation of bioactive proteins. When modified via reversible binding by non-covalent association or proteolytic release, nanofibril aggregates and hydrogels can be used for sustained release of bioactive proteins. A preferred method of reversible non-covalent modification of the protein nanofibrils and bioactive proteins is by gene fusion of a receptor-peptide tag pair to a protein nanofibril monomer and a bioactive protein, where the receptor or peptide tag is attached to the NTL or CT of the nanofibril monomer by a peptide bond, and the second component of the receptor-peptide tag pair is attached to the N-terminus or C-terminus of the bioactive protein. In preferred embodiments, the receptor-peptide tag pair consists of a Strep-tag-(strept)avidin pair, a SUMO-tag-SUMO pair, a coiled-coiled helix pair, or a SpyTag-SpyDock (a mutant SpyTag-SpyCatcher pair that cannot form an isopeptide bond) pair.

[0153] The nature of this disclosure In a first aspect, this disclosure relates to an isolated protein nanofibril comprising two protofibrils, each containing at least two monomeric protein subunits, wherein each monomeric protein subunit comprises an amino acid sequence fragment covalently linked according to the formula:NTL-Helix 1-L-Helix 2-CT, the monomeric protein subunit spontaneously folds in aqueous solution into two helices, Helix 1 and Helix 2, as an alpha-helix antiparallel coiled-coil structure, wherein Helix 1 and Helix 2 each contain a sequence of at least five heptad (H) elements according to the formula H1-1-H1-2-H1-3-H1-4-H1-5 for Helix 1 and H2-1-H2-2-H2-3-H2-4-H2-5 for Helix 2, the heptad elements having the following consensus sequence: - Heptad 1-1:XXX-Φ-XX-Φ, - Heptad 1-2:Φ-X-Ψ-Φ-Ψ-Φ-γ, - Heptad 1-3:ε-X-Ψ-Φ-Ψ-X-Φ, - Heptad 1-4:Φ-N-Ψ-γ-Ψ-γ-δ, - Heptad 1-5:Φ-ε-X-Φ-Φ-XX, - Heptad 2-1:Φ-XX-Φ-Φ-X-Φ, - Heptad 2-2: ε-XX-Φ-XXX, - Heptad 2-3:Φ-XX-Φ-Φ-δ-δ, - Heptad 2-4:ε-Φ-Φ-Φ-ε-X-δ, - Heptad 2-5:Φ-γ-X-Φ-Φ-XX Each of the seven amino acid residues specified as "abcdefg" contains the following: In the formula, Φ is a hydrophobic amino acid selected from the list of M, V, I, L, A, G, H, W, Y, F; Ψ is a short side-chain residue selected from the list of V, C, G, A, P, S, T, N, D; γ is a residue selected from E, D, Q that functions as an acid / base catalyst; δ is a residue selected from K that can function as an isopeptide bond "donor" or nucleophile; ε is a residue selected from E, Q, D, N that can function as an isopeptide bond "acceptor" or electrophile; X may be any amino acid; the linker (L) fragment contains at least four amino acids; the N-terminal lock (NTL) and C-terminal tail (CT) fragments contain at least one amino acid; and the monomeric protein subunits are interconnected via at least one isopeptide bond (IPB).

[0154] In a further embodiment, monomeric protein subunits (i and i+ / -n) of a first protofibril (f) and monomeric subunits (i' and i'+ / -n) of a second protofibril (f') are covalently linked via at least one IPB, wherein the IPB is positioned between a side chain of an amino acid functioning as a nucleophile and a side chain of an amino acid functioning as an electrophile: - (i) NTL and (i-5), (i-4), (i-3) or (i-2) H1-5b, - H1-4g of (i) and H2-2a of (i-1), - H2-3g of (i) and H2-4a of (i-1), - H2-3f in (i) and H2-4e in (i'), - H2-4g of (i) and H1-3a of (i-1), - H2-4g of (i+1) and H1-3a of (i), - NTL of (i+5), (i+4), (i+3) or (i+2) and H1-5b of (i), - H1-4g of (i+1) and H2-2a of (i), - H2-3g of (i+1) and H2-4a of (i), and / or - H2-3f of (i'+1) and H2-4e of (i) The protein nanofibrils formed in the above are disclosed.

[0155] Further embodiments include the heptad elements having the following consensus arrangement: - H1-1:XXX-Φ-XX-Φ, - H1-2:Φ-X-(S / T)-Φ-(A / G)-Φ-(E / Q), - H1-3:EXX-Φ-X-(H / N)-Φ, - H1-4:Φ-N-(A / G / S)-E-(G / A)-EK, - H1-5:Φ-QX-Φ-Φ-XX, - H2-1:Φ-XX-Φ-Φ-X-Φ, - H2-2:(N / Q / D)-XX-Φ-XXX, - H2-3:Φ-XX-Φ-Φ-(K / X)-(K / X), - H2-4:(E / Q)-Φ-Φ-(L)-(Q / X)-XK, - H2-5:Φ-γ-X-Φ-Φ-XX Disclosed are any of the protein nanofibrils having the above characteristics.

[0156] A more specific embodiment is one in which the NTL includes the consensus sequence: M-Ψ-Φ-ZX-Φ-P, where, - Ψ is a short side chain residue selected from the list of V, C, G, A, P, S, T, N, D, - Φ is a hydrophobic amino acid selected from the list of M, V, I, L, A, G, H, W, Y, F, - X is any amino acid, - Z is either Ala or proline, - Further disclosures any of the protein nanofibrils in which M and P are single-letter codes for the amino acids methionine and proline, respectively.

[0157] Another embodiment discloses any of the protein nanofibrils wherein the monomer is an A-ENA protein selected from the list of proteins shown in Table 3, and / or consists of an amino acid sequence selected from the list of SEQ ID NOs: 1 to 6.

[0158] In certain embodiments, the protein nanofibril has an NTL consisting of fewer than four amino acids. Alternatively, a monomeric protein subunit may be used. The NTL and monomeric protein of at least four amino acids is a mutant variant at at least one of the following positions: H1-1a, H1-1b, H1-1c, H1-3f, H1-4f, H1-5b, H1-5f, H2-1b, H2-1c and / or H2-5b, or The NTL has fewer than 4 amino acids, and the monomeric protein is a mutant variant at at least one of the following positions: H1-1a, H1-1b, H1-1c, H1-1f, H1-2f, H1-3b, H1-3f, H1-4b, H1-4f, H1-5b, H1-5f, H2-1b, H2-1c, H2-2b, H2-2f, H2-3f, H2-4f, H2-5b, H2-5c and / or H2-5f. Includes, A protein nanofibril having a modified surface is disclosed.

[0159] Further embodiments disclose any of the protein nanofibrils, wherein the monomer protein further comprises a protein tag or domain fused or conjugated within the N-terminus, C-terminus, or linker region to form a functional fibril.

[0160] An alternative embodiment discloses a protein nanofibril comprising the same monomeric protein that forms a homopolymer fibril by self-assembly, or comprising at least two different monomeric proteins that form a heteropolymer fibril by self-assembly.

[0161] In a further embodiment, any of the protein nanofibrils is disclosed, wherein the fibril is a recombinant protein nanofibril.

[0162] Another embodiment discloses a modified Bacillus spore comprising and / or presenting either the monomeric protein subunit or (modified) protein nanofibrils.

[0163] An alternative embodiment discloses a modified Bacillus spore in which the Bacillus strain lacks the endogenous monomer protein of the protein nanofibril and contains an exogenously introduced monomer protein subunit or contains the protein nanofibril disclosed herein.

[0164] Further embodiments disclose the use of the modified Bacillus spores to increase bacterial spore activity, preferably pathogenic activity.

[0165] Further embodiments disclose nucleic acid molecules encoding monomeric protein subunits for forming the (modified) protein nanofibrils.

[0166] The final aspect relates to a method for producing any of the disclosed protein nanofibrils, comprising the following steps: recombinant expression of the disclosed nucleic acid molecule encoding the protein, or expression of the monomer protein for forming the nanofibrils, in a host cell; release of the self-assembled protein nanofibrils from the host cell, preferably by cell lysis; and isolation of the self-assembled protein nanofibrils, preferably by resuspension from an insoluble fraction and / or further purification from the cell lysate.

[0167] While specific embodiments, specific configurations, and materials and / or molecules of methods and products relating to this disclosure have been described herein, it should be understood that various changes or modifications may be made in form and detail without departing from the scope of the invention. The following examples are provided to better illustrate specific embodiments and should not be considered as limiting this application. This application is limited only by the claims. [Examples]

[0168] [Example 1] Characterization of A-ENA nanofibrils in Bacillus thuringiensis serovar israelensis spore biofilms Here, the inventors summarize the characterization of nanofibrils found in the spore biofilm of Bacillus thuringiensis cerovale islaerensis (Bti). Bacterial lawns of Bti spores were prepared on LB agar as follows: Starting with a single Bti colony pre-grown on LB agar, it was inoculated into 10 mL of liquid LB and grown overnight at 37°C until stationary. Next, 2 mL of the liquid culture was spread onto a 250 mL square LB agar plate and incubated at 30°C for 1 week. The resulting spore biofilm was collected using a cell scraper and resuspended in 10 mL of deionized water (miliQ). The spore suspension was used with bright-field microscopy and negative-stained transmission electron microscopy (nsTEM). Phase-contrast microscopy imaging using a 100x oil immersion objective revealed the presence of two types of refractive objects (quasi-spherical and elliptical) with typical diameters of 0.5 μm and 1.5 μm, respectively. The latter species are enclosed in a sac-like structure and correspond to Bti spores. The spores associate with the refractive bodies of the former class, which the inventors identified as parasporial bodies (PSBs; see below). Bti-derived PSBs are condensed, aggregated structures consisting of different crystalline subdomains composed of multiple proteins that are presumed to have larval killing activity against insect larvae such as mosquitoes and black flies. To further investigate the spore-PSB association, the inventors performed nsTEM analysis of resuspended spore biofilm samples. For this purpose, the inventors collected nsTEM micrographs of Bti spore samples placed on a Formvar / Carbon grid (400 mesh, Cu; Electron Microscopy Sciences) and stained with 2% (w / v) uranyl acetate. Low-magnification TEM images (2500x) revealed the presence of a fibrous network permeating the entire biofilm enclosing both spores and PSBs (Figure 1). Based on high-magnification cryoEM imaging (60k), the inventors calculated a 3D reconstruction map that enables partial de novo residue assignment of fibril monomers. This allowed for subsequent sequence-based searching of the Bti genome and clear identification of the constituent subunit as UniProt:Q8KNV8 (SEQ ID NO: 1), which was referred to here as "A-ENA".Genomic analysis revealed the presence of a second A-ENA-like sequence adjacent to A-ENA, consisting of Q8KNV7 (SEQ ID NO: 2), which was referred to here as "A-ENA1". Based on nsTEM analysis, the inventors concluded that the rich fibril network is composed of individual A-ENA protein filaments consisting of A-ENA and / or A-ENA-1. The A-ENA nanofibrils are characterized by an apparent diameter of ±8 nm and a clear projection map in nsTEM or cryoEM. The A-ENA fibrils shown in the model in Figure 3 consist of two protofibrils (f and f') that intertwine into a 2-start helix superstructure with twists and rises of approximately 12° and 10.8 Å, respectively. The A-ENA monomer subunits (labeled i and i') self-assemble into an alpha-helix hairpin with an N-terminal extension projecting downward along the fibril axis. A-ENA monomers interact laterally between protofibrils to form dimeric entities, which then stack axially, thereby extending each protofibril and resulting in a tetrameric complex as shown in Figure 3c, providing the minimum essential fibril units formed by self-assembling A-ENA.

[0169] The inventors have clearly confirmed the binding of A-ENA filaments to the surface of PSBs, not only to the outermost layer of spores, and for example, clusters of A-ENA fibrils originate from both the PSB and / or the spore outer membrane. Furthermore, it has been found that PSBs are often bound to mature spores via A-ENA filaments. Such fibril-mediated PSB-spore or spore-spore connections can extend for several micrometers and effectively function as reinforced anchorages between various structures.

[0170] [Example 2] Recombinant production and purification of A-ENA and A-ENA-1 fibrils assembled in incelo Native wild-type sequences of A-ENA (gene: ATN07_33990; protein: Q8KNV8; SEQ ID NO: 1) and A-ENA-1 (gene: ATN07_33980; protein: Q8KNV7; SEQ ID NO: 2) were PCR amplified from pBtoxis plasmid (CP013279; pAM65-52-4-128K) using primer set 1 (SEQ ID NOs: 16-17) and primer set 2 (SEQ ID NOs: 18-19), respectively. The amplified fragments were cloned into pASK vectors using Gibson Assembly (https: / / doi.org / 10.1038 / nmeth.1318) and introduced into E. coli Top10. The resulting transformants were screened by colony PCR and Sanger sequencing (pASK sequencing primers; SEQ ID NOs: 20-21). Competent cells of E. coli C43 (DE3) were transformed using the obtained plasmid (pASK_A-ENA; pASK_A-ENA-1). Single colonies were used to initiate overnight (ON) LB culture. 10 mL of ON culture was inoculated with 1 L of LB and 50 μg / ml ampicillin at 37°C. 0.8 OD was reduced by adding 50 μM anhydrotetracycline. 600 Recombinant expression was induced, and the culture was incubated at 18°C. Cells were divided into 1 mg / mL. -1The lysozyme was resuspended in 5 mM ethylenediaminetetraacetic acid (EDTA), 50 mM Tris pH 6.8, and 50 mM NaCl, and incubated at room temperature under stirring conditions for 18 hours. Next, sodium dodecyl sulfate (SDS) was added to the solution to a final concentration of 1% (w / v), and the solution / SDS mixture was heated to 100°C. The mixture was then allowed to cool at room temperature for 15 minutes and centrifuged at 20,000 rcf for 30 minutes in a Beckman JA 14.50 50 mL falcon rotor. The supernatant was discarded, and the pellet (i.e., the insoluble fraction consisting of A-ENA(-1) fibrils) was returned to the suspension (5 mL of miliQ per gram of wet fibril pellet) using an IKA overhead stirring system. The resulting suspensions were subjected to three additional rounds of centrifugation (20,000 rcf, 30 min) and resuspension of each pellet in miliQ to remove residual SDS. After the final washing step, the resulting suspensions were diluted 10-fold in miliQ, placed on a Formvar / Carbon grid (400 mesh, Cu; Electron Microscopy Sciences), stained with 2% (w / v) uranyl acetate, and absorbed and dried. Negative-stained transmission electron microscopy (nsTEM) analysis revealed the presence of micrometer-long fibrils with a diameter of approximately 8 nm (Figure 2). The 2D classification of the framed fibril segments (Figure 2a) was consistent with 2D class-mean images previously obtained in fibril spore suspensions in exovivo extracted from Bacillus thuringiensis cerovar islaerensis spore biofilms (Figure 1h-i).

[0171] CryoEM structural analysis of these recombinant A-ENA fibrils confirmed the A-ENA monomer subunit alpha-helix antiparallel coiled-coil structure, as observed in wild-type A-ENA fibril monomers, as well as their protofibril indirect points and isopeptide bonds (Figures 4 and 5). Furthermore, protofibril intracontact points and isopeptide bonds were also visible from A-ENA(i) with monomers i-5 via residue 2 in the N-terminal connector, and from the A-ENA monomer directly above (i+1) or directly below (i-1) subunit i. These observations demonstrate that A-ENA nanofibril formation occurs spontaneously and efficiently in unrelated recombinant host cells, independent of native Bti cell cofactors. Therefore, A-ENA subunits possess intrinsic self-assembly properties and the ability for autocatalytic formation of intermolecular and interfibril isopeptide bonds. The contact surfaces of A-ENA monomers are primarily hydrophobic, excluding some residues involved in covalent interactions. Furthermore, the observation of the highly stable properties of wild-type A-ENA fibrils can be explained by the fact that these covalent interactions form irreversible isopeptide bonds (IPBs), with each subunit i participating in up to 10 IPBs, resulting in a stable fibril structure that bridges seven different subunits within and across the protofibril (Figures 4 and 9).

[0172] [Example 3] Isopeptide bond formation is necessary for the self-assembly of stable A-ENA-like nanofibrils. The A-ENA monomer subunit, which self-assembles into nanofibrils, contains 10 isopeptide bonds together with adjacent A-ENA subunits. These isopeptide bonds are formed by an autocatalytic unit consisting of an acid-base catalyst (preferably Glu), a nucleophilic residue (preferably Lys or an N-terminal amine), and an electrophilic residue (preferably Glu, Gln, Asn, or Asp), and are proximity-inducible, defined by the relative positions of each acid-base catalyst that activate the nucleophilic residue for electrophilic attack. In addition to lysine in the helix, the N-terminal amine in the NTL may also be involved as a nucleophilic donor in IPB formation, and the exact subunit "ix" involved in IPB can be altered by elongating or shortening the NTL (Figures 4a, 9). The exact residue positions of each type of residue are annotated for A-ENA (SEQ ID NO: 1) as shown in Figures 4a, 5, and 7.

[0173] To measure the role of isopeptide bonds in the self-assembly of A-ENA monomers into stable A-ENA nanofibrils, the inventors mutated electrophilic residues of the isopeptide bond pairs. For this purpose, E29, Q44, N64, E78, and Q82 were mutated to alanine residues (SEQ ID NO: 15). A 7×His tag was also added to the C-terminus to facilitate purification from E. coli lysate. The obtained DNA sequences were ordered as double-stranded synthetic DNA fragments (gblock, IDT), cloned into pASK vectors, and expressed by recombination as outlined in Example 2. The harvested cells were resuspended in 100 mM K-phosphate (pH 7.2) buffer supplemented with 8 M urea and 1 M NaCl at a buffer concentration of 10 ml per gram of wet cell pellet. The resulting cell suspensions were sonicated on ice for 5 minutes using a 500 W Qsonica probe sonicator with 30 sec / 15 sec on / off pulses set to 50% amplitude. Next, the cell lysates were centrifuged at 20,000 rcf for 45 minutes to pellet insoluble cell debris. The cleared lysates were loaded onto a pre-equalized 5 mL Histrap column and washed with 20 column volumes of 100 mM K-phosphate (pH 7.2), 8 M urea, and 1 M NaCl. The column was eluted with 100 mM K-phosphate (pH 7.2), 8 M urea, and 400 mM imidazole, and the resulting fractions were analyzed using SDS-PAGE. The fractions containing A-ENA molecules were pooled and dialyzed against 100 mM Hepes (pH 8.0) and 8 M urea. The dialyzed proteins were loaded onto a pre-equalized 1 mL Q anion exchange column with 100 mM Hepes (pH 8.0) and 8 M urea, and the flow-through was collected. Next, the column was eluted with 100 mM Hepes (pH 8.0), 8 M urea, and 1 M NaCl. SDS-PAGE analysis of the flow-through and eluted fractions showed that A-ENA was found as a pure monomer species during the flow-through (Figure 12a). Next, using a 40 K MWCO Zeba spin-desalting column, the A-ENA monomer was buffered from 100 mM Hepes (pH 8.0) and 8 M urea to 1 × PBS and incubated at room temperature for 24 hours.Desalted A-ENA samples were applied to a Formvar / Carbon grid, stained with 2% uranyl acetate, and imaged using a 120kV JEOL electron microscope (Figures 12b and c). The resulting micrographs showed no fibril structures. Rather, the A-ENA E29A Q44A N64A E78A Q82A mutants formed abundant fractal-like aggregates. From this, the inventors concluded that isopeptide bonds are extremely important for the formation of stable A-ENA fibrils.

[0174] [Example 4] The A-ENA spore silk network induces Bacillus thuringiensis spore biofilms and associates spores with parasporial bodies. To produce A-ENA KO (ΔA-ENA) in Bacillus thuringiensis cerovale islaerensis (Bti), the inventors followed the procedure described by Wang et al. (2019 Front Microbiol. 10:1932) based on the pJOE8999 plasmid possessing a CRISPR / Cas9 system (Altenbuchner, 2016. Appl Environ Microbiol. 82(17):5421~7) purchased from the Bacillus Genetic Stock Center. First, plasmid pJOE8999 was linearized with primer pairs p551 and p552. Using the Bti spore stock as a gDNA donor template, approximately 1kb upstream and 1kb downstream of the A-ENA gene were amplified using primer pairs p588 and p633 and p634 and p635, respectively. Both PCR fragments were cloned into a linearized pJOE8999 vector by Gibson assembly according to the manufacturer's instructions. Plasmid sequencing was confirmed using oligonucleotides p525 and p526. Next, the inventors cloned an sgRNA designed using the web server: http: / / www.rgenome.net / cas-designer / . The selected sgRNA (SEQ ID NO: 22) was cloned into the plasmid designed above by PCR using primer pair p594 and p595. Plasmid sequencing was confirmed using oligonucleotide p531. The plasmid was transformed into E. coli strain dam- / dcm- and grown in LB supplemented with 50 μg / ml kanamycin to produce an unmethylated plasmid. The plasmid was then electroporated in Bti WT, and transformants were selected at 30°C on BHI plates supplemented with 0.5% glycerol and 25 μg / ml kanamycin. The following day, a single colony was inoculated into 10 ml of liquid BHI supplemented with 25 μg / ml kanamycin and incubated at 37°C for 3 hours. To induce Cas9 expression, mannose was added at a final concentration of 0.4% w / v and incubated for a further 3 hours at 37°C. Serial dilutions of the culture were plated at 37°C onto LB agar containing 25 μg / ml kanamycin and 0.4% w / v mannose.The following day, mutants were selected by colony PCR using oligonucleotides p602 and p650. A single colony was selected, and the sequence of the A-ENA locus was confirmed using oligonucleotides p602 and p650. Finally, the KO A-ENA strain was imaged by TEM to confirm the absence of A-ENA fibrils (Figure 18b).

[0175] Complementation of A-ENA KO was performed by replacing the cas9 gene in the pJOE8999 plasmid with the promoter, CDS and terminator of the A-ENA gene derived from Bti. First, plasmid pJOE8999 was linearized using the oligonucleotide pair p622 and p623. The A-ENA locus containing the promoter, CDS and terminator was amplified using the oligonucleotide pair p683 and p684, and cloned into the above linearized plasmid by Gibson assembly according to the manufacturer's instructions. The mixture was transformed into E. coli dam / dcm- strain, selected on LB plates supplemented with 50 μg / ml kanamycin, and incubated at 37°C. The next day, positive colonies were selected by colony PCR using the primer pair p617 and p631, grown in 10 ml of LB containing 50 μg / ml kanamycin, and the plasmid was extracted using the NucleoSpin Plasmid EasyPure kit to obtain plasmid A176. The sequence of the plasmid was verified (Eurofins), and transformed into electrocompetent Bti cells. A single positive colony was inoculated into BHI supplemented with 25 μg / ml kanamycin and grown overnight at 30°C. The next day, 2 mL of the liquid culture was spread onto a 250 mL square LB agar plate supplemented with 25 μg / ml kanamycin, and incubated at 30°C for 1 week. Spores were resuspended in water, and the presence of A-ENA fibrils was evaluated by TEM. The spore sample was mounted on a Formvar / Carbon grid (400 mesh, Cu; Electron Microscopy Sciences) and stained using 2% (w / v) uranyl acetate. TEM images revealed the presence of a fibril network retaining the entire biofilm encompassing both spores and PSB, as observed for WT (Figure 18e), confirming the correct complementation of A-ENA KO.

[0176] [Example 5] Exogenous A-ENA forms a spore-silk network that associates spores and Cry toxin crystals in Bacillus thuringiensis Sv. kurstaki. The A176 plasmid used to complement the A-ENA knockout (KO) described in Example 4 was electroporated into Bacillus thuringiensis Sv. Krustaki cells, which naturally lack A-ENA. After electroporation, the transformed cells were selected on a Brain Heart Infusion (BHI) plate supplemented with 25 μg / ml kanamycin and incubated overnight at 30°C. The following day, a single colony was inoculated into 10 ml of BHI medium containing 25 μg / ml kanamycin and grown overnight at 30°C. Subsequently, 200 μl of the overnight culture was plated onto a 245 × 245 × 25 mm square plate containing Luria-Bertani (LB) medium supplemented with 25 μg / ml kanamycin and spore formation was allowed to occur for 1 week at 30°C. The obtained spore biofilms were collected using a cell scraper, resuspended in 10 mL of deionized water (miliQ), and used for TEM imaging and optical microscopy. A-ENA Bti The Btk strain expressing this gene exhibited a biofilm similar to that of Bti, in addition to PSB crystals of both cones connected to the A-ENA bundle, as shown in Figure 19.

[0177] [Example 6] A-ENA-mediated association of spores and parasporal body toxins enhances entomopathogenicity in Bacillus thuringiensis. To investigate the role of A-ENA in the association between PSB and spores, the inventors evaluated the toxicity of Bacillus thuringiensis sv. islaerensis strains expressing or lacking A-ENA (wild-type vs. A-ENA knockout) using the insect model Chironomus aprilinus. Fresh Chironomus aprilinus larvae were obtained from Zooschatz (Berlin, Germany) in batch number 1223153. Bti wild-type (WT) and A-ENA knockout (KO) strains were cultured on LB plates for 1 week. The resulting spore biofilm was scraped off and resuspended in 10 ml of deionized water to prepare a spore suspension. In parallel, spores were isolated from 1 ml of the spore suspension by centrifugation at 16,000 × g for 40 minutes using a 50% histodens cushion. Next, the isolated spores were washed three times with deionized water by centrifugation at 5000 × g for 10 minutes. To determine mortality, each group contained at least eight Thyronomus aprilinus larvae, and the final volume was 3 ml containing the sea salt and vitamin premix provided with the larvae. The spore suspension and isolated spore samples were then measured to a final optical density (OD) of 0.02. 600) was added to achieve the desired concentration. Phosphate-buffered saline (PBS) was used as a negative control. Larval survival was monitored daily for 7 days. The study was performed in biological triplicates under room temperature conditions. As shown in Figure 20, the loss of A-ENA fibrils results in a significant reduction in the killing activity of Bti spores. The killing time point of larvae exposed to Bti ΔA-ENA is extended by at least 1 day compared to larvae exposed to WT Bti, and insect larvae have a higher probability of surviving at least 1 week of exposure to Bti spores. This effect is most pronounced in the isolated spore preparation of Bti ΔA-ENA, where precipitation and isolation of spores from the growth medium results in a dramatic reduction of PSB due to the lack of A-ENA fibrils (Figure 18f). A slight delay in killing activity is still observed in the spore suspension of Bti ΔA-ENA, where PSB remains present in the suspension but is no longer associated with the spores (Figures 18d, f). Therefore, these experiments demonstrate that the inability of Bt spores to retain secreted toxins in proximity to the spores leads to a significant reduction in virulence and insecticidal activity.

[0178] [Example 7] A-ENA Protein Sequence Conservation. As shown in Figure 6, a multiple sequence alignment was performed based on the top 593 BLAST hits using Q8KNV8 (SEQ ID NO: 1) as the query sequence to generate an A-ENA consensus web logo. Looking at the alignment of A-ENA orthologs with only 24% sequence identity to A-ENA (SEQ ID NO: 1) (see the lower panel of Figure 7), it was found that the A-ENA subunits adopt a general structure consisting of a variable N-terminal lock ("NTL"), followed by an α-helix "α1" composed of 5 heptad repeats (H1-1 to H1-5), followed by a variable-length linker sequence (designated "L"), followed by a second α-helix "α2", and terminating in a variable-length C-terminal tail ("CT"). Alignment of A-ENA orthologs according to the heptad repeats demonstrates strong conservation of the positions of hydrophobic residues that participate in knobs-into-holes interactions within and between helices and form the isopeptide bonding unit.

[0179] These residues involved in IPB formation (derived from the cryoEM structure of recombinant Q8KNV8 fibril) were found to be largely conserved in the conserved sequence logo, and the conservation of hydrophobic residues within the helix suggests that these A-ENA orthologues also likely function in the formation of self-assembled nanofibrils. To test this hypothesis, A-ENA orthologues (SEQ ID NOs. 3-5 shown in Figures 7 and 8), as well as Bti A-ENA (Uniprot:Q8KNV8, SEQ ID NO. 1) and A-ENA-1 (Uniprot:Q8KNV7; SEQ ID NO. 2) were produced by recombination. As shown in the image in Figure 8, self-assembled fibrils were indeed formed in the cytoplasm of E., which may contain IPBs. This is because sequence conservation makes this possible, and the shown fibrils represent the insoluble fraction after SDS / thermal extraction, thus indicating the autocatalytic formation of one or more IPBs.

[0180] Furthermore, based on the alpha-helix coiled-coil structure of the A-ENA monomer, the inventors were able to outline the relationship between the amino acid sequence and structure of the A-ENA protein by representing the monomer as a helix wheel in which intermolecular interactions are depicted (Figure 7). In fact, the knob-in-hole interactions at the helix-helix junctions can be precisely defined by the side chains at each position in the sequence. Based on insights from the cryo-EM structure, which shows fibrils with uniform helical ascent and twist of 1.5 Å and 100°, respectively, in the alpha-helix, the monomer residues were arranged so that every eighth residue is located at an equivalent position on the helix wheel and adopts a translation of 10.5 Å along the helix. Accordingly, in this specification, the inventors define helices h1 and h2 or α1 and α2 by a series of five heptameric units or "heptads" (H) having side chains named "a" through "g", respectively, and provide a conserved array motif that enables self-assembly to helix hairpins or alpha-helix coiled coils used interchangeably herein (see Figures 7 and 10). For further fibril formation, the A-ENA subunits are further involved in stacking interactions (i.e., i / i+1 and i / i-1 interactions) by alpha-helices α1 and α2 in successive subunits involved in pairwise parallel packing by primarily hydrophobic knob-in-hole interactions along the length of the helix, and are involved in the heptad positions "a" and "e" on the C-pole side heptads, and "g" and "c" on the N-pole side heptads of the A-ENA hairpin. The presence of the aforementioned consensus sequence motifs of hydrophobic and short side-chain residues in the α1 and α2 heptads shown in Figure 10 facilitates the adoption of antiparallel helix hairpins within the A-ENA-like unit, as well as pairwise stacking of fibril assembly units (i / i+1 and i / i-1) into helix protofibrils (labeled f) with twists and rises of approximately 12° and 10.8 Å.

[0181] Exceptions at hydrophobic junctions are formed by residues involved in isopeptide bond formation and / or residues that form acid-base catalysts in the IPB unit (Figure 9). Furthermore, the packing of f and f' protofibrils in A-ENA is mediated by knob-in-hole and IPB interactions at positions "b", "e", and "f" of the α2 heptad H2-3 and H2-4 in subunit i with i' and i'+1.

[0182] [Example 8] Operation of A-ENA fibril. Based on structural insights gained from the analysis of A-ENA nanofibrils, the inventors tested different locations within the A-ENA protein sequence for the addition / insertion of a single amino acid, peptide, or folded domain, and also tested different sites for site-directed mutagenesis, from the perspective of functionalizing A-ENA nanofibrils. Considering that both the N-terminus and C-terminus are exposed on the surface in the final double protofibril assembly, both ends are labeled as potential sites for the addition of a single amino acid, peptide, or folded domain. In addition to the ends, the inventors also identified the loop (L, linker) connecting helices α1 and α2 as a potential site for the insertion of a single amino acid, peptide, and folded domain (Figure 13a). In addition to these sites, the inventors also propose solvent-exposed residues found on the surface of the A-ENA fibril as potential mutational possibilities for altering the surface properties (hydrophobicity, electrostatics, etc.) of the resulting A-ENA fibril, forming sites for targeted modification by alteration to natural or non-natural amino acids (i.e., residues including primary amines, thiols, azides, alkyne groups, etc., as examples not exhaustively) suitable for chemical conjugations known to those skilled in the art. As shown in Figure 13c, these possible mutational sites are presented herein by stick representations annotated with residue numbers according to the A-ENA sequence (SEQ ID NO: 1). In Figure 13e, the inventors map these residues onto five heptads of helices α1 and α2, which were previously defined and shown in Figures 9 and 10. In wild-type A-ENA fibrils, heptads H1-2 (and to a lesser extent H1-1 and H1-3) are hidden from the solvent by an N-terminal lock docking on the fibril surface. However, deletion of the N-terminal lock by cleaving residues 1-12 exposes these residues to the solvent, making them more resistant to modification. In Figure 13b, we have defined A-ENA-delta NTL (ΔNTL; SEQ ID NO: 12) and its corresponding site for the addition of a single amino acid, peptide, or folded domain.This allowed the inventors to then determine additional residues for site-directed mutagenesis (Figure 13d) and their corresponding positions in each heptad (Figure 13f).

[0183] -Post-polymerization functionalization of A-ENA fibrils: As an example of A-ENA manipulation, the inventors cloned the SpyTag sequence (SEQ ID NO: 23) to the C-terminus of A-ENA and A-ENA-1 and investigated the ability of the resulting constructs to self-polymerize and to form a covalent complex with a two-domain construct consisting of SpyCatcher003 and superfolder green fluorescent protein (sfGFP) separated by an SG linker (Figure 14a). Here, A-ENA-SpyTag and A-ENA-1-SpyTag were recombinately expressed in the cytoplasm of E. coli, and the resulting fibrils were purified using the protocol outlined in Example 2 (Figure 14b). Next, the inventors tested whether the fibrils could selectively capture SpyCatcher-sfGFP. To this end, suspensions of A-ENA-SpyTag and A-ENA-1-SpyTag fibrils were co-incubated with purified SpyCatcher-sfGFP at room temperature for 30 minutes. A 10 μl aliquot of the reaction mixture was analyzed by SDS-PAGE, and the resulting gel was analyzed using the Li-COR M Odyssey imaging system (green fluorescence channel at 488 nm). The SpyCatcher-sfGFP sample (lane 1 in Figure 14c) flows with an apparent molecular weight of 40 kDa, consistent with its theoretical molecular mass of 40.19 kDa. The inventors also note that the SpyCatcher-sfGFP band still fluoresces at the expected wavelength, demonstrating that the sfGFP moiety remains folded even when bound to the fibril. Co-incubation with A-ENA-SpyTag (lane 2) and A-ENA-1-SpyTag (lane 3) revealed a second fluorescence band, particularly in the gel region corresponding to the stacked gel. This section of the SDS-PAGE far exceeds the high molecular weight range that can be calibrated with molecular markers, but it is clear that these are high molecular weight species.The fact that these species (i) remained trapped within the pore network of the stacked gel after boiling in NuPage LDS sample buffer, and (ii) emitted fluorescence, led us to conclude that they correspond to A-ENA and A-ENA-1 fibrils covalently bound to SpyCatcher-sfGFP. This is further illustrated by larger-scale co-incubation experiments with A-ENA-SpyTag fibrils and soluble SpyCatcher-sfGFP. After incubation at room temperature for 30 minutes, the fibril suspension was centrifuged at 20,000 rcf for 30 minutes. The resulting pellet (insoluble) and supernatant (soluble) were irradiated using a Clare chemical DR46B blue LED transilluminator, which excited the sample with pure visible blue light and filtered the signal through an amber screen, allowing for visualization of fluorescence (Figure 14d). The inventors recorded a clear fluorescence signal in the fibril pellet after centrifugation, demonstrating that the SpyCatcher-sfGFP molecule was efficiently sequestered by the A-ENA fibril.

[0184] - Pre-polymerization functionalization of A-ENA fibrils - Optimized linker strategy: The SpyTag approach outlined above can be used for post-polymerization functionalization of A-ENA fibrils. Using SpyCatcher fusion constructs, virtually any passenger domain can be efficiently bound to the A-ENA scaffold without any limitations on size or molecular weight. Since the presentation interval or "rise" of the SpyTag peptide is approximately 10.8 Å per protofibril, we predict that this approach will not result in complete stoichiometric labeling of all available binding sites for presentation domains with a hydrodynamic radius significantly exceeding 10.8 Å. In cases where a one-to-one ratio between A-ENA and the desired presentation domain is required (e.g., to minimize the distance between domains along the length of the fibril), or where pre-polymerization functionalization is preferred, we have developed a second method for A-ENA-based functional presentation (Figure 15a). For this purpose, we constructed gene fusions between A-ENA separated by a flexible linker and various presentation domains. This approach is 0.108 Å -1To obtain a homopolymer with a presentation density, an optimized linker is required to avoid steric collisions between the presentation domains of adjacent units. By including a flexible linker, the inserted presentation domain gains conformational freedom to pack in zigzag or alternating stacking rather than pure translational stacking along the length of the fibril. In this way, domains with heights exceeding the 10.8 Å rise of the A-ENA subunit in the protofibril can be accommodated by alternating stacking. Preferred linkers are between 1 and 20 amino acid residues in length, but can be longer if higher degrees of freedom and / or longer distances from the A-ENA fibril backbone are desired. Preferred linkers mainly have small hydrophilic amino acids (G, A, S, T, N, D) rather than in a specific preferred sequence, but may also contain larger hydrophilic residues (K, E, R, Q, H, Y) and tend to have few or no proline. Linker design may be performed randomly or, in particular, guided through state-of-the-art protein modeling and design tools such as protein MPNN (Dauparas et al., Science 378, 49-56 (2022)) and Rosetta (https: / / github.com / RosettaCommons), as are known to those skilled in the art. One approach is to position the insertion domain in silico along the A-ENA backbone in a preferred orientation and position, and to construct in silico the spacing between the N-terminal, C-terminal, or linker residues connected to the C-terminal or N-terminal residues of the insertion domain using a protein design tool. The linker design may be validated in the first step by using de novo structural prediction tools such as Alphafold (Jumper, J., Evans, R., Pritzel, A. et al., Nature 596, 583-589 (2021)) or RosettaFold (Baek et al., Science 373, 871-876 (2021)), as well as by those skilled in the art, and / or by experimental validation through negative staining EM testing of the culture supernatant.Here, we report the successful recombinant production and polymerization of three different A-ENA fusion constructs (p66α, MBD2, and rubredoxin; SEQ ID NOs: 8, 9, and 10). Figure 15b shows nsTEM micrographs of self-assembled A-ENA-TEV-p66α, A-ENA-TEV-pMBD2, and A-ENA-TEV-rubredoxin fibrils purified from the cytoplasm of E. coli.

[0185] Next, the inventors performed a functional assay to demonstrate the folded nature of the presented domain. p66α and MBD2 are two Homo sapiens-derived alpha-helix proteins that form antiparallel coiled-coil complexes with nanomolar affinity. Here, the inventors predicted that a comix of A-ENA-TEV-p66α and A-ENA-TEV-pMBD2 would result in a non-covalently crosslinked fibril network driven by the formation of interfibril complexes of p66α and MBD2. To demonstrate such interfibril binding, both A-ENA coiled-coil fibrils were purified as described in Example 2, and then subjected to additional freeze-thaw cycles using 12% (w / v) urea and 8% (w / v) NaOH to achieve higher purity. After three washing steps involving continuous centrifugation (20,000 rcf for 30 minutes) and resuspension in 1×PBS buffer, the fibril-insoluble pellet was collected, and 100 mg of A-ENA-p66α and A-ENA-MBD2 protein pellets were separately resuspended in 180 μl of 50 mM Tris buffer (pH 7.0). 60 μl from each solution were combined and thoroughly mixed. After 30 minutes, the Eppendorf inversion test was performed to examine hydrogel formation (Figure 15d). The suspensions of A-ENA-p66α and A-ENA-MBD2 did not remain fixed to the base of the Eppendorf during the inversion test, but the mixed suspension formed a turbid, stable hydrogel and remained stably at the base of the Eppendorf. This indicates that each presented domain is (i) folded and (ii) accessible for complex formation, resulting in interfibril bonding, kinetic termination of the polymer network, and establishment of a mixed A-ENA hydrogel.

[0186] To establish the folded nature of the rubredoxin passenger domain (derived from Desulfovibrio vulgaris), a thermal denaturation assay based on SYPRO orange fluorescence was performed. For this purpose, 25 μl of a sample containing the fibrils of interest, combined with 1×SYPRO® protein gel staining (Thermo Fisher Scientific; Waltham, Massachusetts, United States), was packed into a Hard-Shell 96-well microplate (Bio-rad; Hercules, California, United States). Using a CFX opus 96 qPCR plate reader system (Bio-rad; Hercules, California, United States), the temperature was increased from 25°C to 100°C in 0.5°C increments every 10 seconds, and the fluorescence emission spectrum was measured at each temperature interval. The melting curve was calculated as the first derivative of the RFU in order of temperature (Figure 15e). The reported melting temperature (Tm) corresponds to a local maximum value of the melting curve when the value exceeds 1000 RFU / dT. In A-ENA wild-type fibrils, no clear maximum value greater than 1000 RFU / dT was measured, indicating no significant unfolding events within the measured temperature range (Figure 15e). However, in the A-ENA-TEV rubredoxin sample, a clear maximum value (1435 dRFU / dT) was detected at 94.5°C, indicating unfolding of the fused rubredoxin domain. From this, we concluded that the rubredoxin domain grafted onto the A-ENA scaffold is naturally folded and exhibits unfolding at a Tm of 94.5°C. This Tm is similar to the thermal unfolding temperature previously reported for bacterial rubredoxin. Although precise unfolding data is not available for the rubredoxin protein of Desulfovibrio vulgaris used here, it is known to unfold rapidly at 100°C (Lazaridis, I. Lee and Karplus 1997, Protein Sci. 6(12):2589~605).By comparison, homologous rubredoxin from Clostridium pasteurianum (with 70% amino acid sequence identity) has unfolding temperatures of 69°C in the reduced state and 83°C in the oxidized state (Bonomi et al., 2000, Protein Sci. 9(12):2413~26).

[0187] - Pre-polymerization functionalization of A-ENA fibrils with loop insertion To evaluate whether A-ENA fibers can be modified by inserting a recombinant sequence into the linker loop connecting helix 1 and helix 2 of the A-ENA-like fibril subunit (Figure 24), a gene fusion construct was created in which an exogenous sequence was inserted between the valine 55 and threonine 56 residues of Bacillus thuringiensis Sv. islaerensis A-ENA (UniprotKB Q8KNV8; SEQ ID NO: 1). As a non-limiting example, the inventors inserted a sequence corresponding to a beta-solenoid domain containing two curlin-like repeats, having the amino acid sequence shown in SEQ ID NO: 66 (referred to as "R4.5-2RFD"). The inserted curl-like repeat domain and A-ENA monomer are linked by a two-amino acid linker (GG) connecting the C-terminus of helix 1 to the N-terminus of the insertion sequence, and by a two-amino acid linker (SG) connecting the C-terminus of the insertion sequence to the N-terminus of helix 2, resulting in a fusion protein referred to herein as "A-ENA_LI-R4.5-2RFD" (SEQ ID NO: 67). In these A-ENA loop insertion (LI) designs, the length and sequence of linking residues connecting helices 1 and 2 to the insertion sequence are variable and can be shortened or lengthened depending on the desired distance between the A-ENA fibril scaffold and the insertion sequence presented on the A-ENA fibril ridge (Figure 24).

[0188] Constructs containing the coding sequence A-ENA_LI-R4.5-2RFD (SEQ ID NO: 67) were prepared by PCR linearization of a plasmid vector (pASK-A-ENA) containing the coding sequence of A-ENA, using outward-facing primers adjacent to residues 55 and 56 (primer LI_1 and primer LI_2 - SEQ ID NOs: 69-70, respectively). Subsequently, Gibson assembly was performed on the vector and DNA fragments adjacent to complementary sequences equivalent to LI_1 and LI_2, corresponding to the coding sequences of the desired insert and linker.

[0189] To evaluate whether A-ENA_LI insertion mutants (hereinafter also referred to as A-ENA sequences in which a heterologous or exogenous sequence is inserted into the linker connecting helix 1 and helix 2) retain their self-assembly properties, E. coli DH5α cells were transformed with a plasmid encoding the sequence A-ENA_LI-R4.5-2RFD (SEQ ID NO: 67). Expression of A-ENA_LI-R4.5-2RFD was increased to 0.8 OD by adding 50 μM anhydrotetracycline. 600Cells were induced and incubated overnight at 18°C. The cells were resuspended in 1 mg / mL-1 lysozyme, 5 mM ethylenediaminetetraacetic acid (EDTA), 50 mM Tris (pH 6.8), and 50 mM NaCl, and incubated at room temperature under stirring conditions for 18 hours. Next, sodium dodecyl sulfate (SDS) was added to the lysate to a final concentration of 1% (w / v), and the lysate / SDS mixture was heated to 100°C. The mixture was then cooled at room temperature for 15 minutes and centrifuged at 20,000 rcf for 30 minutes in a Beckman JA 14.50 50 mL falcon rotor. The supernatant was discarded, and the pellet consisting of A-ENA fibrils was returned to suspension at a concentration of 5 mL of miliQ water per gram of wet fibril pellet. The resulting suspension was subjected to three additional rounds of centrifugation (20,000 rcf, 30 min) and resuspension of each pellet in miliQ water to remove residual SDS. After the final washing step, the resulting suspension was diluted 10-fold with miliQ water, placed on a Formvar / Carbon grid (400 mesh, Cu; Electron Microscopy Sciences), stained with 2% (w / v) uranyl acetate, and absorbed and dried. Negative-stained transmission electron microscopy (nsTEM) analysis revealed the presence of micrometer-length fibrils with a diameter of approximately 10 nm (Figure 24d, e), demonstrating that the A-ENA linker insertion variant retains the ability to self-assemble into SDS-stable A-ENA-like nanofibrils.

[0190] - Pre-polymerization functionalization of A-ENA fibrils - Block copolymer strategy: The optimized linker design strategy outlined above is ideally suited for surface presentation of heterologous domains having molecular weights equivalent to or smaller than A-ENA. For the presentation of proteins considerably larger than A-ENA, we developed a block copolymer design strategy (Figure 16a). As the core of this strategy, we performed co-expression of wild-type A-ENA and a gene fusion construct of A-ENA in E. coli. As proof of principle, we performed this using a camelid VHH antibody (nanobody) (hereinafter designated as nanobody-TEV-A-ENA; SEQ ID NO: 11) bound to the N-terminus of A-ENA with a TEV protease cleavage site as a linker. Nanobody-TEV-A-ENA was cloned into a pBAD vector and transformed into Top10 E. coli strains holding the pASK-A-ENA wild-type construct. The resulting co-expression leads to the formation of a heteropolymer A-ENA fibril composed of A-ENA subunits and nanobody-TEV-A-ENA subunits (Figure 16b). The inventors predicted that the distribution of both subunit types within the A-ENA fibril would be random and similar to the topology of a block copolymer. The average distance between the two presentation domains depends on the block size within the copolymer. Careful control of the expression levels of both proteins (by selecting promoter strength, plasmid copy number, inducer concentration, and induction time) controls the ratio of A-ENA to A-ENA fusion constructs. This strategy has the advantage of not imposing an upper limit on the size of the passenger domain due to the random distribution of A-ENA and A-ENA fusion constructs within the fibril assembly.

[0191] [Example 9] In vivo functionalization of A-ENA spore silk in Bacillus thuringiensis. In experiments shown in Figures 14, 15, 16, and 17, the inventors demonstrated how A-ENA subunits can be functionalized by direct fusion of polypeptides, including, but not limited to, affinity reagents such as nanobodies, metal-binding proteins such as rubredoxin, affinity tags such as SpyTag or SpyCatcher, and coiled-coil pairs. The inventors demonstrated that the modified A-ENA retains the ability to self-assemble into homopolymer and / or heteropolymer (i.e., block copolymer) nanofibrils, and that the fused polypeptides retain their activity. Thus, A-ENA nanofibrils can be formed by gene fusion, or, in not limited to, by covalent or non-covalent addition of functional polypeptides through means such as the SpyTag-SpyCatcher technique, the StrepTag-Streptavidin technique, and coiled-coil pairing. These principles are illustrated in Figures 14-17 for in vitro assembly of A-ENA nanofibrils.

[0192] In the experiment shown in Figure 21, the inventors aimed to determine whether the A-ENA fusion protein could result in Bt spores formed in vivo during the spore formation process and modified with A-ENA fibrils as described above. To evaluate this, Bti and Btk were genetically modified using plasmids containing expression constructs for A-ENA:SpyTag under the control of native A-ENA promoter and terminator (SEQ ID NOs. 45 and 46, respectively). Transformed cells exhibited expression of A-ENA fibrils with morphological features indistinguishable from wild-type (WT) A-ENA fibrils, similar to those observed for A-ENA-SpyTag nanofibrils grown in vitro (Figure 14). Subsequently, isolated spores from these transformed cells were exposed to purified SpyCatcher-sfGFP (approximately 240 μM, approximately 5 mg / mL) for 30 minutes, followed by thorough washing with PBS (four times). In parallel, the inventors included wild-type Bti and Btk spores as negative controls. Upon completion of the washing step, the inventors observed fluorescence labeling only on the surface of spores expressing A-ENA:SpyTag, while spores derived from wild-type Btk or Bti did not exhibit fluorescence.

[0193] These experiments demonstrate that, similar to what was observed with A-ENA:SpyTag nanofibrils in vitro, the A-ENA:SpyTag subunit is incorporated into WT A-ENA nanofibrils in vivo and is accessible for modification by SpyCatcher and SpyCatcher fusions. Thus, and through extensions using the alternative labeling methods described above, A-ENA nanofibrils on Bt spores can be functionalized in vivo. Using such an approach, Bt spores can be functionalized with affinity reagents having specific binding properties to biological and non-biological surfaces, such as, in non-limiting examples, the gastrointestinal epithelium of certain insects and insect larvae, and the surfaces of certain plants and crops. In such a method, functionalized Bt spores with increased tropism to specific insect groups and / or increased affinity and retention time to desired crops or surfaces can be derived.

[0194] [Example 10] Physicochemical stability of A-ENA fibrils. To test the chemical stability of recombinant A-ENA fibrils, fibrils were recovered from the concentrated stock solution by centrifugation (20,000 rcf, 1 hour, supernatant discarded). The pellets obtained after centrifugation of 100 μl aliquots were resuspended and incubated in various conditions: 100 μl of 2% (w / v) SDS, 8M urea, 2M NaOH, or 100% (v / v) formic acid, as shown in Figure 22, before negative staining TEM sample preparation. To test the physical robustness of recombinant A-ENA fibrils, 100 μl aliquots of the fibril stock solution were dried in an open glass vial at 200°C for 15 minutes in an oven. The resulting dried material was rehydrated with 100 μl of miliQ water before nsTEM imaging. Furthermore, 100 μl aliquots were autoclaved at 121°C for 20 minutes. Next, 3 μl aliquots of the treated sample were placed on a Cu mesh Formvar grid, washed twice with 20 μl of miliQ water, incubated with 2% (w / v) uranyl acetate for 1 minute, and absorbed and dried on Whatmann2 filter paper. Micrographs were collected using a 120kV JEOL 1400 microscope equipped with LaB6 filament and a TVIPS F416 CCD camera, operating at 60,000x magnification. As shown in Figure 22, in each of these treatments, where the protein typically denatures or undergoes hydrolysis, the A-ENA fibrils were intact and abundantly retained.

[0195] [Example 11] A-ENA increases the insecticidal activity of Bacillus thuringiensis Sv. crustaki. As shown in Example 6, A-ENA forms a virulence factor for the insecticidal activity of Bacillus thuringiensis Sv. israelensis, and as shown in Examples 5 and 9 (Figures 19, 21), recombinant expression of A-ENA in Bt Sv. kurstaki resulted in a spore-silk network similar to Bti, in which the bipyramidal PSB crystals are connected to A-ENA bundles. Next, to test whether the presence of native A-ENA in these Bt strains could result in a gain of function and potentially enhance their toxicity and thus their effectiveness in pest control, recombinant expression of A-ENA was applied.

[0196] We used Trichoplusia ni, commonly known as the nettle moth, as an insect model. Trichoplusia ni is a highly destructive foliivorous organism that has a catastrophic impact on cruciferous crops such as cabbage and broccoli, as it is a natural target of Btk. We tested Btk strains expressing A-ENA on the plasmid described above (gain-of-function approach) and compared the survival of larvae feeding on media supplemented with spores of either WT Btk or Btk Wt strains expressing A-ENA recombinantly (Btk+A-ENA). Bti naturally contains A-ENA, but its PSB is ineffective against T. ni, so it served as a negative control (Figure 23c). We used one-week-old larvae and tracked their survival for one week after adding a spore suspension to solid feed medium. The results clearly showed that approximately 50% (15 / 28) of larvae in the Btk WT strain were still alive on day 4, compared to approximately 10% (23 / 26) in the Btk strain expressing A-ENA (Figure 23c). Based on the observation that the clustering activity of A-ENA enhances toxicity, the inventors investigated the effect of adding purified A-ENA fibrils (recombinantly produced in E. coli) to WT Btk spores. ns-EM showed that the addition of purified A-ENA mimicked the aggregation of spores and PSBs, similar to that observed in Btk expressing A-ENA by recombinant (Figures 19 and 23a). The results demonstrate that the addition of recombinant A-ENA fibrils effectively clusters Btk spores and PSBs (Figure 23a). Next, the inventors repeated the larval death assay using WT Btk spores and PSB suspensions with and without the addition of recombinant A-ENA fibrils. Consistent with the inventors' previous findings, approximately 50% (8 / 16) of larvae survived on day 4 in the WT Btk strain, but the survival rate decreased to approximately 18% (3 / 16) in the presence of recombinant A-ENA fibrils (Figure 23d). As a negative control, the inventors confirmed that A-ENA fibrils, including recombinant A-ENA, do not exert a direct toxic effect on their own.

[0197] [Example 12] A single isopeptide bond is sufficient for the self-assembly of SDS-resistant A-ENA-like nanofibrils. To determine the minimum number of isopeptide bonds required for the formation of A-ENA-type fibrils with high physicochemical stability (see Example 10), we selectively mutated the acid / base catalysts in the three IPB residues required for the formation of five different IPBs present in A-ENA (UniprotKB Q8KNV8; SEQ ID NO: 1). Based on the structures of the A-ENA nanofibrils disclosed herein, it becomes clear that IPB3 may be sufficient to form an IPB network that crosslinks all protomers and the two A-ENA protofibrils f and f' into an A-ENA nanofibril molecule linked by a single covalent bond (Figure 25a, b). None of the other IPBs observed in wild-type A-ENA nanofibrils, either as a single IPB or in combination with one or more IPBs other than IPB3, result in crosslinking of A-ENA subunits within the A-ENA protofibril, but not crosslinking across the A-ENA protofibril. Therefore, the inventors modified A-ENA (UniprotKB Q8KNV8; SEQ ID NO: 1) by mutating residues E28, E39, and E41 to Gln, which are similar but catalytically inactive residues, essentially resulting in the inactivation of IPB4 and 5, and IPB2 and IPB1, respectively (see Figure 5b). To do this, the coding sequence of A-ENA in the pASK_A-ENA plasmid was mutated by directed PCR-based mutagenesis to obtain the plasmid pASK_E-ENA E28Q_E39Q_E41Q. Alphafold 3 predicted that the mutant protein would form a protein fibril nearly identical to that of wild-type A-ENA (Figure 25c). To evaluate this prediction, the A_ENA_E28Q_E39Q_E41Q mutant (SEQ ID NO: 68) was produced by recombinant expression in E. coli C43 transformed with pASK_E-ENA_E28Q_E39Q_E41Q, and the OD was 0.8. 600The cells were then induced for at least 1 hour by adding 50 μM anhydrotetracycline. The cell pellets were treated as described above for A-ENA, and negative staining EM testing of the resulting pellets demonstrated the presence of SDS-resistant nanofibrils with diameter and morphology comparable to those of wild-type A-ENA fibrils (Figure 25d).

[0198] [Example 13] A general flow and process used for the preparation, processing, and storage of ENA nanofibrils. A-ENA nanofibrils are typically produced by biological fermentation via recombinant expression in bacterial (preferred), yeast, plant, or animal hosts. To do this, A-ENA protein subunits are expressed from one or more plasmid-containing or genomically inserted copies of the A-ENA coding sequence, and the expression is induced in the host cytoplasm or efflux system (i.e., the general secretion (SEC) or twin arginine (TAT) pathway), where the subunits self-assemble into robust nanofibrils ranging in length from nanometers to micrometers (Figure 26I). A-ENA nanofibrils are recovered from the culture by gentle lysis of cells. Cells can be lysed in situ in culture medium or in a concentrated suspension after sedimentation or centrifugation and resuspension in a desired buffer. Methods for gentle cell lysis include, in non-limiting examples, spontaneous or induced autolysis (e.g., by co-expression of endolysin, lysozyme, or other cell wall-degrading proteins known in the art), suspension in hypotonic buffer, freeze-thaw cycle, gentle sonication, surfactant treatment, and EDTA-lysozyme treatment (Figure 26II). Cell debris and other insoluble contaminants are then removed from the cell lysates by methods known in the art, including sedimentation, slow centrifugation (i.e., 2,000–10,000 g), cross-flow filtration, and enzymatic treatment (i.e., using proteases, glycosyl hydrolases, nucleases, cellulases, esterases, etc.) in non-inclusive subjects (Figure 26III). The suspended fibrils are further purified from soluble and smaller suspended impurities by surfactants (i.e., in particular SDS, LDS, DDM, terditol, Tween®, Triton®, Elugent) and / or chemical washing (i.e., NaOH, NH4OH, urea, formic acid, alcohol and / or organic solvents) and / or enzymatic decomposition (i.e., using in particular proteases, glycosyl hydrolases, nucleases, cellulases, and esterases) (Figure 26IV(a)).The fibrils are separated by sedimentation of soluble or small suspended impurities (including chemically induced sedimentation) or by faster centrifugation (typically higher than 10,000 g), cross-flow, or dead-end centrifugation (Figure 26IV(b)). The desired purity of the A-ENA fibrils is obtained by the sequential washing and separation steps described above. Finally, the purified A-ENA fibrils can be recovered and / or stored via drying and / or lyophilization (i.e., resulting in dry pellets, flakes, films, or powders) or as a suspension in pure water, aqueous buffers, and / or acid, alkali, or salt solutions, and in organic solvents or combinations thereof, at room temperature, frozen, cooled, or ambient temperature (Figure 26V).

[0199] [Table 1] JPEG2026536460000002.jpg184120 JPEG2026536460000003.jpg184121 JPEG2026536460000004.jpg184120 JPEG2026536460000005.jpg184121 JPEG2026536460000006.jpg184120 JPEG2026536460000007.jpg70120

[0200] [Table 2] JPEG2026536460000009.jpg184121 JPEG2026536460000010.jpg184120 JPEG2026536460000011.jpg184120 JPEG2026536460000012.jpg184120 JPEG2026536460000013.jpg184120 JPEG2026536460000014.jpg70120

[0201] [Table 3] JPEG2026536460000016.jpg217150 JPEG2026536460000017.jpg204141 JPEG2026536460000018.jpg203146 JPEG2026536460000019.jpg38150

[0202] Sequence List >Sequence ID 1: Bacillus thuringiensis Sv. islaerensis strain ATCC35646 A-ENA amino acid sequence (UniProt: Q8KNV8) >Sequence ID 2: Bacillus thuringiensis Sv. israerensis strain ATCC35646 A-ENA-1 amino acid sequence (UniProt: Q8KNV7) >Sequence ID 3: A-ENA homolog UniprotKB A0A0R3K429 >Sequence ID 4: A-ENA homolog UniprotKB A0A172TKT0 >Sequence ID 5: A-ENA homolog UniprotKB A0A2X4WSQ5 >Sequence ID 6: A-ENA homolog UniprotKB A0A0A3J768 >Sequence ID 7: Chimeric protein of Bacillus thuringiensis Sv islaerensis strain ATCC35646 A-ENA (Sequence ID 1) fused to SpyTag >Sequence ID 8: Chimeric protein of Bti A-ENA (Sequence ID 1) fused to p66α >Sequence ID 9: A chimeric protein of Bti A-ENA (Sequence ID 1) fused at the C-terminus of MBD2. >SEQ ID NO: 10: Chimeric protein of Bti A-ENA (SEQ ID NO: 1) fused to rubredoxine desulfovibrio vulgaris >Sequence ID 11: Chimeric protein of anti-GFP nanobody 207 fused to cleaved Bti A-ENA-ΔNTL >Sequence ID 12: A-ENA-ΔNTL >Sequence ID 13: Chimeric protein of Bacillus thuringiensis Sv. islaerensis strain ATCC35646 A-ENA-1 (Sequence ID 2) fused to SpyTag >Sequence ID 14: Chimeric protein of superfolder green fluorescent protein fused to SpyCatcher003 >Sequence ID 15: A-ENA mutant variant with a C-terminal 7×His tag (E29A Q44A N64A E78A Q82A) >Sequence ID 16: FW Primer Set 1 >Sequence ID 17: REV Primer Set 1 >Sequence ID 18: FW Primer Set 2 >Sequence ID 19: REV Primer Set 2 >Sequence ID 20: pASK forward sequencing primer >SEQ ID NO: 21: pASK Reverse Sequence Determination Primer >Sequence code 22: sgRNA >Sequence ID 23: SpyTag amino acid sequence >Sequence ID 24: Heptad H1-1 Bti A-ENA >Sequence ID 25: Heptad H1-2 Bti A-ENA >Sequence ID 26: Heptad H1-3 Bti A-ENA >Sequence ID 27: Heptad H1-4 Bti A-ENA >Sequence ID 28: Heptad H1-5 Bti A-ENA >Sequence ID 29: Heptad H2-1 Bti A-ENA >Sequence ID 30: Heptad H2-2 Bti A-ENA >Sequence ID 31: Heptad H2-3 Bti A-ENA >Sequence ID 32: Heptad H2-4 Bti A-ENA >Sequence ID 33: Heptad H2-5 Bti A-ENA >Sequence ID 34: Heptad H1-1 Bti A-ENA-1 >Sequence ID 35: Heptad H1-2 Bti A-ENA-1 >Sequence ID 36: Heptad H1-3 Bti A-ENA-1 >Sequence ID 37: Heptad H1-4 Bti A-ENA-1 >Sequence ID 38: Heptad H1-5 Bti A-ENA-1 >Sequence ID 39: Heptad H2-1 Bti A-ENA-1 >Sequence ID 40: Heptad H2-2 Bti A-ENA-1 >Sequence ID 41: Heptad H2-3 Bti A-ENA-1 >Sequence ID 42: Heptad H2-4 Bti A-ENA-1 >Sequence ID 43: Heptad H2-5 Bti A-ENA-1 >Sequence ID 44: NTL of Bti A-ENA >Sequence ID 45: Bacillus thuringiensis Sv. israerensis strain ATCC35646 A-ENA promoter >Sequence ID 46: Bacillus thuringiensis Sv. israerensis strain ATCC35646 A-ENA Terminator >Sequences 47-65: Oligoprimer sequences used in Example 4. >Sequence ID 66: Two Carlin-like repeats (with calcium and calcium carbonate binding properties) >Sequence ID 67: A-ENA_LI-R4.5-2RFD (=Sequence ID 66 inserted between residues 55 and 56 of A-ENA (Sequence ID 1), with GG and SG linkers adjacent to each other at the N-terminus and C-terminus of the inserted sequence) >Sequence ID 68: A_ENA_E28Q_E39Q_E41Q (= As shown in bold, A-ENA Sequence ID 1 with Glu28, Glu39 and Glu41 replaced by Gln; K76 and Q82 are underlined to form further subunits and IPB3) MGMPTIPEGLDITRDQAINIILASIGLQELGLAHVINAQGQKVQAVVAGFEKETVTFDQLLATNESVTQTLKTVI K KEMLL Q FKLEEAKSLIQSSSPPSIS >Sequence IDs 69-70: Primers

Claims

1. An isolated protein nanofibril comprising two protofibrils, each containing at least two monomeric protein subunits, wherein each monomeric protein subunit contains a covalently linked amino acid sequence fragment according to the formula: NTL-helix1-L-helix2-CT, The monomeric protein subunit spontaneously folds in aqueous solution into two helices, helix 1 and helix 2, as an alpha-helix antiparallel coiled-coil structure, with helix 1 and helix 2 each containing a sequence of at least five heptad (H) elements according to formula H1-1-H1-2-H1-3-H1-4-H1-5 for helix 1 and H2-1-H2-2-H2-3-H2-4-H2-5 for helix 2, wherein the heptad elements have the following consensus sequence: - Heptad 1-1: X-X-X-Φ-X-X-Φ, - Heptad 1-2: Φ-X-Ψ-Φ-Ψ-Φ-γ, - Heptad 1-3: ε-X-Ψ-Φ-Ψ-X-Φ, - Heptad 1-4: Φ-N-Ψ-γ-Ψ-γ-δ, - Heptad 1-5: Φ-ε-X-Φ-Φ-X-X, - Heptad 2-1: Φ-X-X-Φ-Φ-X-Φ, - Heptad 2-2: ε-X-X-Φ-X-X-X, - Heptad 2-3: Φ-X-X-Φ-Φ-δ-δ, - Heptad 2-4: ε-Φ-Φ-Φ-ε-X-δ, - Heptad 2-5: Φ-γ-X-Φ-Φ-X-X Each of the seven amino acid residues specified as "abcdefg" has the following characteristics: During the ceremony, - Φ is a hydrophobic amino acid selected from the list of M, V, I, L, A, G, H, W, Y, F for at least 70% of the Φ positions shown in the heptad element. - Ψ is a short side chain residue selected from the list of V, C, G, A, P, S, T, N, D, - γ is a residue that functions as an acid / base catalyst, selected from E or D, at least one of the γ positions shown in the heptad element. - δ is a lysine (K) residue capable of functioning as an isopeptide bond "donor" or nucleophile at at least one of the δ positions shown in the heptad element, - ε is a residue capable of functioning as an "acceptor" or electrophile for an isopeptide bond selected from E, Q, D, and N at least one of the ε positions shown in the heptad element, - X may be any amino acid. The linker (L) fragment contains at least four amino acids, and the N-terminal lock (NTL) and C-terminal tail (CT) fragments contain at least one amino acid. A protein nanofibril in which the monomeric protein subunits are interconnected via at least one isopeptide bond (IPB).

2. The protein nanofibril according to claim 1, wherein at least one IPB interconnects two protofibrils (f) and (f') by covalent bonds.

3. The above-mentioned at least one IPB is positioned between the side chain of an amino acid acting as a nucleophile and the side chain of an amino acid acting as an electrophile: - H2-3f of (i') and H2-4e of (i), and / or - H2-3f of (i+1) and H2-4e of (i') The protein nanofibril according to claim 2, wherein the (f) and (f') protofibrils are covalently interconnected via the monomeric protein subunit (i') of the protofibril (f) and the monomeric subunit (i and / or i+1) of the protofibril (f).

4. The monomeric protein subunits (i and i+ / -n) of protofibril (f) and the monomeric subunits (i' and i'+ / -n) of protofibril (f') are covalently linked via at least one IPB, where the IPB is located between the side chain of an amino acid acting as a nucleophile and the side chain of an amino acid acting as an electrophile, respectively: - NTL of (i) and H1-5b of (i-5), (i-4), (i-3) or (i-2), - H1-4g of (i) and H2-2a of (i-1), - H2-3g of (i) and H2-4a of (i-1), - H2-3f of (i) and H2-4e of (i'-1), - H2-4g of (i) and H1-3a of (i-1), - H2-4g of (i+1) and H1-3a of (i), - NTL of (i+5), (i+4), (i+3) or (i+2) and H1-5b of (i), - H1-4g of (i+1) and H2-2a of (i), - H2-3g of (i+1) and H2-4a of (i), and / or - H2-3f of (i') and H2-4e of (i) Protein nanofibrils according to claims 1 to 3, formed in the above.

5. The aforementioned heptad elements are in the following consensus sequence: - H1-1: X-X-X-Φ-X-X-Φ, - H1-2: Φ-X-(S / T)-Φ-(A / G)-Φ-(E / Q), - H1-3: E-X-X-Φ-X-(H / N)-Φ, - H1-4: Φ-N-(A / G / S)-E-(G / A)-E-K, - H1-5: Φ-Q-X-Φ-Φ-XX, - H2-1: Φ-X-X-Φ-Φ-X-Φ, - H2-2: (N / Q / D)-X-X-Φ-X-X-X, - H2-3: Φ-X-X-Φ-Φ-(K / X)-(K / X), - H2-4: (E / Q)-Φ-Φ-(L)-(Q / X)-X-K, - H2-5: Φ-γ-X-Φ-Φ-X-X A protein nanofibril according to claims 1 to 4, having the properties described above.

6. NTL includes the consensus sequence: M-Ψ-Φ-Z-X-Φ-P, where, - Ψ is a short side chain residue selected from the list of V, C, G, A, P, S, T, N, D, - Φ is a hydrophobic amino acid selected from the list of M, V, I, L, A, G, H, W, Y, F, - X is any amino acid, - Z is either Ala or proline, - A protein nanofibril according to any one of claims 1 to 5, wherein M and P are one-letter codes for the amino acids methionine and proline, respectively.

7. The protein nanofibril according to any one of claims 1 to 6, wherein the monomer is an A-ENA protein selected from the list of proteins shown in Table 3, and / or consists of an amino acid sequence selected from the list of SEQ ID NOs: 1 to 6, or any one functional homolog or variant thereof.

8. A protein nanofibril according to any one of claims 1 to 7, wherein the NTL of the monomer protein subunit is less than four amino acids.

9. Monomeric protein subunits a. The NTL and monomer protein of at least four amino acids are mutant variants at at least one of the following positions: H1-1a, H1-1b, H1-1c, H1-3f, H1-4f, H1-5b, H1-5f, H2-1b, H2-1c and / or H2-5b, or b. The NTL has fewer than four amino acids, and the monomeric protein is a mutant variant at at least one of the following positions: H1-1a, H1-1b, H1-1c, H1-1f, H1-2f, H1-3b, H1-3f, H1-4b, H1-4f, H1-5b, H1-5f, H2-1b, H2-1c, H2-2b, H2-2f, H2-3f, H2-4f, H2-5b, H2-5c and / or H2-5f. A protein nanofibril according to any one of claims 1 to 7, comprising:

10. A protein nanofibril according to any one of claims 1 to 9, further comprising a protein tag or domain in which a monomer protein is fused or conjugated within the N-terminus, C-terminus, or linker region in order to form a functional fibril.

11. A protein nanofibril according to any one of claims 1 to 10, comprising an identical monomer protein that forms a homopolymer fibril by self-assembly, or comprising at least two different monomer proteins that form a heteropolymer fibril by self-assembly.

12. The protein nanofibril according to any one of claims 1 to 11, wherein the fibril is a protein nanofibril produced by recombination.

13. A modified bacterial spore, preferably a Bacillus spore, comprising and / or presenting the protein nanofibrils described in any one of claims 8 to 12.

14. A modified bacterial spore, preferably a Bacillus spore, wherein the bacterial strain lacks the endogenous monomer protein of the protein nanofibril described in claims 1 to 7, and has been exogenously introduced a self-assembled monomer protein that forms the protein nanofibril described in any one of claims 1 to 12.

15. Use of a modified bacterial spore, preferably a Bacillus spore, according to claim 13 or 14, to increase bacterial spore activity, preferably pathogenic activity.

16. Use of protein nanofibrils according to any one of claims 1 to 12 to enhance the pesticide activity of bacterial spores, preferably the insecticidal activity of B. thuringiensis spores.

17. A host cell that recombinatively expresses a self-assembled monomer protein that forms protein nanofibrils according to any one of claims 1 to 12.

18. A method for producing a protein nanofibril according to any one of claims 1 to 12, comprising the following steps: a. The step of culturing the host cells according to claim 17, b. The step of releasing the self-assembled protein nanofibrils from host cells, preferably through cell lysis, and c. The self-assembled protein nanofibrils are isolated, preferably by resuspension from an insoluble fraction and / or further purification from a cell lysate. Methods that include...

19. A method for producing protein nanofibrils according to any one of claims 1 to 12, according to claim 18, wherein, after cell lysis in step b) and before isolation in step c), cell debris and other impurities are removed from the lysate, preferably by sedimentation, centrifugation, cross-flow filtration and / or enzymatic treatment.

20. A method for producing a protein nanofibril according to any one of claims 1 to 12, according to claim 18 or 19, wherein the release of the self-assembled protein nanofibril in step b) is carried out by cell lysis using a surfactant, preferably SDS, DDM, Triton, Tergitol or Tween.

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