Alpha-helical protein nanofibrils

Abstract

This invention relates to the field of microbial self-assembled protein fibrils as novel bionanomaterials. More specifically, this invention relates to protein nanofibril materials comprising Bacillus endospore appendage (ENA) proteins, which are spontaneously folded helical hairpin subunits assembled into α-helical multimers (A-ENA) fibrils having a hydrophobic core and covalently linked by one or more isopeptide bonds, providing nanofibrils with high stability and tensile strength. This invention also relates to said protein nanofibrils being engineered or modified to provide functionalized bionanomaterials. In particular, this invention relates to methods for the recombinant preparation of said self-assembled protein nanofibrils, and their use in modifying bacterial endospores and endospore activity or pathogenicity.

Description

Technical Field

[0001] This invention relates to the field of microbial self-assembled protein fibrils as novel bionanomaterials. More specifically, this invention relates to protein nanofibril materials comprising Bacillus endospore appendage (ENA) proteins, which are spontaneously folded helical hairpin subunits assembled into α-helical multimers (A-ENA) fibrils having a hydrophobic core and covalently linked by one or more isopeptide bonds, providing nanofibrils with high stability and tensile strength. This invention also relates to said protein nanofibrils being engineered or modified to provide functionalized bionanomaterials. In particular, this invention relates to methods for recombinantly preparing said self-assembled protein nanofibrils, and their use in modifying bacterial endospores and their endospore activity or pathogenicity. Background Technology

[0002] Nanotechnology facilitates the control of various processes at the nanoscale and is applied to the design of novel functional nanomaterials, including nanofibers, nanowires, nanofibrils, and nanoparticles. Nanofibrils are typically constructed from polysaccharides such as cellulose and chitosan and / or proteins such as silk fibroin, thus possessing not only excellent mechanical properties but also meeting the demand for biosustainable and biodegradable alternative materials. To date, cellulose is the most abundant starting material; as a polysaccharide, cellulose can be isolated and modified for application in the field of biomaterials in a nanoscale form. Secondly, protein nanofibrils, such as silk, spider silk, and collagen, have been characterized in animals and plants and are considered highly promising nanostructures, with potential applications in the development of biomaterials and biomedicine. Furthermore, modifying and developing these biomaterials from a biopreparation perspective further enhances their potential to provide materials with excellent biological properties. Their applications are extensive, and in an economy and society moving towards greater ecological and biosustainability, the value of these materials continues to increase, leading to a tremendous demand for commercially viable solutions.

[0003] Bacterial spores are outstanding examples of the tenacious vitality of organisms, representing a dormant state of survival capable of withstanding harsh environmental conditions for extended periods (decades), including physical and chemical hazards such as high salinity, strong acidity, extreme heat, and drought. Therefore, the protein components constituting the endospore shell and its appendages must possess material properties capable of adapting to such demanding environments. Previously, researchers in *Bacillus cereus* (a generalized type of bacterial spore)... Bacillus cereus sensu latoA novel class of bio-nanofibers was identified through structural analysis of the endospores of *S. sl*. These fibers were named "Endospore Appendages" (ENA) (Pradhan et al., 2021, the EMBO Journal e106887; Remaut et al., WO2022 / 029325). Based on the self-assembled protein fibrous structures present in the endospore samples, the proteins constituting these assemblies were classified as S-ENA and L-ENA proteins. These nanofibrils clearly constitute a novel class of building blocks that can be used in future bio-nanomaterials and further functionalization modifications. Thanks to their extremely high flexibility and elasticity, they exhibit a series of remarkable properties, such as excellent chemical and physical strength, and the ability to withstand low pH environments, protease treatment, pasteurization, drying, and high vacuum conditions. Furthermore, the self-assembly properties of S-ENA and L-ENA protofibrils facilitate the production of high-yield recombinant microorganisms, and the extreme stability of the protofibrils allows for easy separation from cell debris, proteins, nucleic acids, and membranes while preserving native structure and properties. At least for S-ENA-based protofibrils, functionalization has been demonstrated through the insertion of peptides into two distinct loop regions on the protein surface (Remaut et al., WO2022 / 029325). Although the heterologous insertion sequences are inherently limited in length and engineerable fold types to maintain the protein's self-assembly properties and the resulting protofibrils' mechanical properties, N-terminal or C-terminal functionalization is not desirable for such S-Ena or L-Ena-type protofibrils. S-ENA protofibrils have been reported to contain intermolecular disulfide covalent bonds to increase their tensile strength, stability, and stiffness. Such rigid covalent interactions are also known in other types of protein materials, such as the formation of intermolecular isopeptide bonds, a process that also occurs, for example, in the well-known ubiquitination reaction.

[0004] Bacteria, typically Gram-positive, possess pilins, which utilize enzyme-mediated intermolecular isopeptide bonds to covalently link their subunits, thereby constructing slender, elongated fibrous 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 immunoglobulins can be autocatalytically formed, thereby enhancing the protease resistance, thermal stability, and mechanical stability of pili-forming protein domains (e.g., collagen-adhesion domain A (CnaA), such as FimA, and CnaB, such as 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 a proximity-induced reaction; their self-assembly properties depend primarily on the geometry of the amino acids involved in the reaction and the hydrophobic portion in which the isopeptide unit is formed (Kang and Baker, 2011 Trends Biochem Sci. 36(4): 229-37). The autocatalytic isopeptide bond formation configurations identified in the immunoglobulin-like domains of bacterial pili have been successfully applied in the field of biotechnology to achieve covalent coupling of engineered derivatives; for example, the split-domain architecture used in SpyCatcher – SpyTag or SnoopCatcher – SnoopTag technologies (Zakeri et al., 2012, Proc. Nat. Acad. Sci. USA 109: E690-97; Haterm et al., 2019, Int J Mol Sci 20(9): 2129). To date, there is a lack of equivalent isopeptide bond formation geometries that can achieve covalent linkage of α-helical structural units, which limits the stability and application range of natural and synthetic α-helical protein materials.

[0005] Given the need to enhance and further customize protein-based materials and nanofiber innovations, there is an urgent need to develop novel protein fibers; such fibers should exhibit superior performance in terms of chemical stability, mechanical strength, potential for bioengineering applications, as well as sustainable manufacturing and biodegradability. Summary of the Invention

[0006] This invention discloses a novel protein-based nanofiber derived from the endospores of Bacillus, particularly Bacillus thuringiensis. Bacillus thuringiensisThe protofibrils described herein appear on the surface of endospores and are typically associated with parasporal bodies. The protein nanofibrils of this invention are characterized for the first time herein as being composed of α-helical hairpin proteins and consistent with a previously annotated family of endospore accessory proteins (see Pradhan et al., 2021), these newly annotated protofibril-assembling proteins being referred to as α-helical endospore accessory proteins or A-ENA proteins. This novel protein family is unique in that its monomeric or subunit structures are extremely simple and ingenious, consisting of two antiparallel α-helices connected by a short amino acid turn, ending with free N-terminus and C-terminus; wherein the subunits self-assemble to form two helically wound protofilaments or protofibrils (e.g., see...). Figure 3 The resulting nanofibers provide an environment in which the side chains of A-ENA monomers are arranged in a helical wheel pattern, enabling cross-linking through a highly unique isopeptide cross-linking mode (see, for example, [link to other documentation]). Figure 7 The presence of intermolecular covalent interactions between A-ENA subunits within and between precursor fibrils provides irreversibly organized structural components to form A-ENA fibrils with high tensile strength. This invention relates to protein nanofibrils constituting A-ENA or A-ENA-based protein variants defined by rational patterns of residues with nucleophilic, electrophilic, and acid / base properties, for designing covalently stable helical-helical contact isopeptide bond-forming units. Furthermore, these nanofibrils can also be fabricated from chimeric gene constructs expressing recombinant A-ENA or A-ENA-like or A-ENA-based variant proteins through recombinant production in (microbial) hosts. Self-assembly is conserved due to the structural conservation of the protein family. Finally, the simple topology of A-ENA-based variants or engineered A-ENA fibrils allows A-ENA nanofibrils to carry N-termini and C-termini in addition to internal loop or turn-inserted (poly)peptide constructs, resulting in tunable high-density functionalization of the fibrils.

[0007] Therefore, a first aspect of the present invention relates to protein-based protofibrils, referred herein as protein nanofibrils, which consist of or comprise two precursor protofibrils, each of which contains two or more monomeric protein subunits, and thus the protofibrils contain at least one tetramer, wherein each monomeric protein subunit contains a covalently linked amino acid sequence fragment, wherein the covalent link is a peptide bond according to the following formula: N-terminal lock (NTL) – helix 1 – linker (L) – helix 2 – C-terminal tail (CT), wherein the monomeric protein subunits, when expressed or present in aqueous solution, self-assemble into two helices, namely helix 1 and helix 2, forming an α-helix antiparallel coiled-coil protein structure, and wherein helix 1 and helix 2 each contain a consecutive sequence of at least five heptaponic repeat (H) elements, helix 1 following the general formula H1-1 – H1-2 – H1-3 – H1-4 – H1-5, and helix 2 following the general formula H2-1 – H2-2 – H2-3 – H2-4 – H2-5, wherein each of the seven repeating elements comprises seven amino acid residues, labeled “abcdefg”, and has the following common sequence: - Seven-element repeat 1-1: XXX- -XX- , - Seven-element repetition 1-2: -X- - - - - , - Seven-element repetition 1-3: -X- - - -X- , - Seven-element repetition 1-4: -N- - - - - δ, - Seven elements repeat 1-5: - -X- - -XX, - Seven-element repetition 2-1: -XX- - -X- , - Seven-element repetition 2-2: -XX- -XXX, - Seven-element repetition 2-3: -XX- - -δ-δ, - Seven-element repetition 2-4: - - - - -X- δ, - Seven-element repetition 2-5: - -X- - -XX, in: - For at least 70% of the seven repeating elements specified therein Location, It is a hydrophobic amino acid residue, selected from M, V, I, L, A, G, H, W, Y, F; preferably a bulky amino acid selected from L, M, I, V, F or W; - It is a short side chain residue, selected from V, C, G, A, P, S, T, N, D; preferably an amino acid selected from A, G, S, T; - For at least one or more of the seven-element repeating elements specified therein Location, The residues selected for use as acid / base catalysts are Glu (E) or Asp (D). - For one or more of the δ positions specified in the seven-membered repeating element, δ is a residue selected from lysine that can serve as an isopeptide bond "donor" or nucleophilic residue; - For one or more of the seven repeating elements specified therein Location, The residues that can serve as "donors" for isopeptide bonds or as electrophilic residues are selected from Glu (E), or Asp (D), Gln (Q), or Asn (N); - as well as δ and It can be any amino acid at one or more remaining positions that do not participate in IPB formation. - X can be any type of amino acid. Furthermore, the linker (L) segment contains at least 4 amino acids, and the N-terminal lock (NTL) segment and the C-terminal tail (CT) segment contain at least one amino acid, and the monomeric protein subunits are interconnected by at least one or more isopeptide bonds (IPB).

[0008] Specifically, the protein nanofibrils form at least one IPB to covalently interconnect two precursor fibrils (f) and (f'). More specifically, the at least one IPB interconnecting the two precursor fibrils (f) and (f') is respectively located between the monomeric protein subunit (i) of the precursor fibril (f) and the monomeric subunit (i' and / or i' + 1) of the precursor fibril (f'), and between the amino acid side chain as a nucleophilic residue and the amino acid side chain as an electrophilic residue, specifically at the following locations: - Between H2-3f of (i) and H2-4e of (i'); and / or - Between H2-3f of (i'+1) and H2-4e of (i).

[0009] Alternatively, the protein nanofibrils form at least one IPB to covalently interconnect two precursor fibrils (f') and (f), wherein the at least one IPB interconnecting the two precursor fibrils (f') and (f) is respectively located between the monomeric protein subunit (i') of the precursor fibril (f') and the monomeric subunit (i and / or i + 1) of the precursor fibril (f), and between the side chain of an amino acid as a nucleophilic residue and the side chain of an amino acid as an electrophilic residue, specifically at the following locations: - Between H2-3f of (i') and H2-4e of (i); and / or - Between H2-3f of (i+1) and H2-4e of (i').

[0010] In other embodiments, the protein nanofibers have a tertiary structure, possessing, for example... Figure 3 The smallest tetramer shown in c, and / or wherein the monomeric protein subunits (i + / - n) of the precursor fibril (f) and the monomeric subunits (i' + / - n) of the precursor fibril (f') are covalently linked by at least one or more IPBs, preferably 10 IPBs, wherein, for example, Figure 9 As shown, the IPB is formed between the amino acid side chain as a nucleophilic residue and the amino acid side chain as an electrophilic residue, specifically at the following positions: - Between NTL of (i) and H1-5b of (i-5) or (i-4), (i-3) or (i-2); - Between H1-4g of (i) and H2-2a of (i-1); - Between H2-3g of (i) and H2-4a of (i-1); - Between H2-3f of (i) and H2-4e of (i'); - Between H2-4g of (i) and H1-3a of (i-1); - Between H2-4g of (i+1) and H1-3a of (i); - Between the NTL of (i+5) or (i+4), (i+3) or (i+2) and the H1-5b of (i); - Between H1-4g of (i+1) and H2-2a of (i); - Between H2-3g of (i+1) and H2-4a of (i); and / or - Between H2-3f of (i'+1) and H2-4e of (i).

[0011] Alternatively, protein nanofibers possess a tertiary structure, exhibiting properties such as... Figure 3 The minimal tetramer shown in c, and / or wherein the monomeric protein subunits (i + / - n) of the precursor fibril (f) and the monomeric subunits (i' + / - n) of the precursor fibril (f') are covalently linked by at least one or more IPBs, preferably 10 IPBs, wherein the IPBs are formed between amino acid side chains that are nucleophilic residues and amino acid side chains that are electrophilic residues, specifically at the following locations: - Between NTL of (i) and H1-5b of (i-5), (i-4), (i-3) or (i-2); - Between H1-4g of (i) and H2-2a of (i-1); - Between H2-3g of (i) and H2-4a of (i-1); - Between H2-3f of (i) and H2-4e of (i'-1); - Between H2-4g of (i) and H1-3a of (i-1); - Between H2-4g of (i+1) and H1-3a of (i); - Between the NTL of (i+5), (i+4), (i+3) or (i+2) and H1-5b of (i); - Between H1-4g of (i+1) and H2-2a of (i); - Between H2-3g of (i+1) and H2-4a of (i); and / or - Between H2-3f of (i') and H2-4e of (i).

[0012] In other embodiments, the protein nanofibrils comprise hexavalent repeating elements having the following shared sequences α1 and α2: - H1-1: XXX- -XX- , - H1-2: -X-(S / T)- -(A / G)- -(E / Q) - H1-3: EX- X- - X-(H / N)-

[0013] - H1-4: -N-(A / G / S)-E-(G / A)-EK

[0014] - H1-5: -QX- - -XX

[0015] - H2-1: -XX- - -X- , - H2-2:(N / Q / D)-XX- -XXX - H2-3: -XX- - -(K / X)-(K / X) - H2-4: (E / Q)- - -(L)-(Q / X)-XK, - H2-5: - -X- - -XX, Specific residues are represented by single-letter amino acid codes, and " / " is interpreted as "or". - It is a hydrophobic amino acid, selected from M, V, I, L, A, G, H, W, Y, F; preferably a large-volume amino acid selected from L, M, I, V, F or W.

[0016] - The residues used as acid / base catalytic residues are selected from Glu (E), Gln (Q), or Asp (D); - X can be any type of amino acid.

[0017] Other specific embodiments involve the protein nanofibers, wherein the monomeric protein subunits are based on, derived from, or derived from Bacillus proteins, specifically Bacillus endospore appendage (ENA) proteins, or more specifically α-helical (A-)ENA proteins, preferably from Bacillus thuringiensis. Bacillus thuringiensis ).

[0018] Other implementations provide those corresponding to M- - -ZX- The protein nanofibrils of -PP with an N-terminal locked (NTL) amino acid sequence, wherein: - =Short side chain residues, selected from V, C, G, A, P, S, T, N, D; preferably A, G, S or T; - =Hydrophobic amino acids, selected from M, V, I, L, A, G, H, W, Y, F; preferably selected from L, M, I, V, F or W; - X = any amino acid, Z is Ala or proline, and M and P are the single-letter codes for the amino acids methionine and proline, respectively.

[0019] More specifically, the protein nanofibrils may contain components corresponding to M- - -ZX- The NTL of the amino acid sequence of -P, where Selected from S, G, or T, The amino acid is hydrophobic and is selected from M, V, I, L, A, G, H, W, Y, F, preferably from L, M, I, V, F or W; and X is any amino acid, Z is Ala or proline, and M and P are the single-letter amino acid codes for methionine and proline, respectively.

[0020] Other specific embodiments relate to the protein fibrils, wherein the NTL-helix 1-L-helix 2-CT sequence of the monomer is composed of an A-ENA protein sequence, wherein the A-ENA protein sequence is selected from proteins listed in Table 3 by their accession numbers. In a specific embodiment, the A-ENA protein sequence is selected from SEQ ID NO: 1-6, or from modified, engineered, or functionalized fibrils containing an A-ENA protein selected from SEQ ID NO: 1-6, wherein the NTL and / or connector and / or CT are modified.

[0021] Other specific embodiments involve the protein fibrils, wherein the NTL-helix 1-L-helix 2-CT sequence of the monomer is composed of, for example... Figure 11The A-ENA (like) protein composition defined by the Hidden Markov Model (HMM) maps of helices 1 and 2 shown are illustrated in Tables 1 and 2, respectively (obtained from the 593 A-ENA homologs identified in this work), or the modified, engineered, or functionalized protofibril composition of the A-ENA (like) protein composition, wherein the NTL and / or linker and / or CT are modified. Those skilled in the art can use (online) tools and databases to verify whether protein sequences conform to HMM maps representing protein classes or families (e.g., https: / / www.ebi.ac.uk / Tools / hmmer / ), thus allowing the use of Tables 1-3 and... Figure 11 The information disclosed in the literature is used to determine whether the protein belongs to the A-Ena family.

[0022] Other specific implementations involve any of the described protein fibrils, wherein the NTL is truncated, i.e., it is at least 1 but less than 4 amino acids.

[0023] Another embodiment relates to the protein-based fibrils, wherein the monomeric protein subunit comprises an NTL of at least four amino acids, and the monomeric protein is modified on its surface, or is a mutant variant compared to the shared motif of the fragment NTL–helix 1–L–helix 2–CT as defined herein, having at least one or more amino acid substitutions at the following positions of the seven-membered repeat element in 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, the amino acids of which differ from the amino acid list provided in the shared sequence described herein for the positions of the seven-membered repeat element.

[0024] In an alternative embodiment, the protein-based fibril comprises a monomeric protein subunit containing at least one but fewer than four amino acids of NTL, and the monomeric protein is modified on its surface, or is a mutant variant as defined herein compared to the common motif of the fragment NTL–helix 1–L–helix 2–CT, having at least one or more amino acid substitutions at the following positions of the seven-membered repeat element in 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, wherein the amino acid is different from the amino acid list provided in the common sequence for the positions of the seven-membered repeat element described herein.

[0025] Other embodiments involve the protein nanofibrils comprising monomeric proteins corresponding to a common sequence as defined herein as NTL-helix 1-L-helix 2-CT, further linked to a tag or protein domain, or conjugated at the N-terminus, C-terminus, or linker region of the monomer to form a functionalized fibril. The fusion or conjugation can be performed directly or using intermediate (flexible and / or optimized) linker sequences. Thus, the self-assembled protein fibrils comprise one or more monomers with functional fusions to other portions, wherein these other portions can be folded proteins, tags, markers, or functional parts that are fused or conjugated directly or via linkers to the A-ENA-based monomers. In specific embodiments, the monomeric protein of the protein fibril is conjugated to an additional portion to form a functionalized protein nanofibril, the monomeric protein being obtained by (recombinant) expression through gene fusion that effectively connects the monomer to the additional functional portion. More specifically, the genetic fusion of the monomer can be present at its N-terminus or C-terminus, or as an insertion into a linker region. In another specific embodiment, the monomeric protein of the protein nanofibrils is functionalized or modified after fibril formation, and the modified or functionalized protein nanofibrils are obtained from orthogonal conjugation of one or more monomeric protein subunits of the protein nanofibrils after fibril self-assembly. In a preferred embodiment, the further orthogonal conjugation comprises covalent linkage, more preferably isopeptide binding, and even more preferably functionalization comprises modification comprising peptide tag / binding partner pairs derived from isopeptide bond forming chain – fimbriae subunit folding complementary pairs, more specifically (Spy)tag / collagen adherent domain B (CnaB2) catcher type (e.g., SpyCatcher) or (Snoop)tag / D4 domain (RrgA) catcher type (e.g., SnoopCatcher) binding partner type (Hatlem et al., 2019, Int J Mol Sci 20(9): 2129).

[0026] Other embodiments provide protein nanofibrils composed of the monomeric proteins described herein, wherein at least one monomer, preferably A-ENA or an A-ENA-based variant monomer, further comprises a fusion with a heterologous protein domain for display on the surface of the protein nanofibrils. Specific embodiments relate to protein nanofibrils comprising at least one monomer described herein, the monomer further fused to a heterologous protein domain, wherein the fusion occurs at the N-terminus or C-terminus of the monomer, or is inserted into the L- or linker region of the monomer. In further specific embodiments, the heterologous protein domain fused to the at least one protein monomer comprises a VHH for surface display of nanobodies, or comprises a counterpart of a peptide tag / binding partner pair, preferably comprising covalent interactions, such as isopeptide bonds, more specifically, such as tag / catcher binding pairs, such as SpyTag / SpyCatcher or SnoopTag / SnoopCatcher, or other proteins included for surface display when present on the surface of the protein nanofibrils. In further specific embodiments, the heterologous protein comprises a sequence selected from SEQ ID NO: 7-11, 13-15, or 67.

[0027] In one specific embodiment, the protein nanofibril comprises the monomer fused with a heterologous protein domain, wherein each monomer is identical and forms a homopolymeric fibril upon self-assembly. In other alternative embodiments, the protein nanofibril comprises one or more monomeric protein subunits as described herein, wherein each monomer further comprises a heterologous protein domain containing a flexible (optimized) linker between the monomer and the heterologous protein domain as described herein, wherein the hydrodynamic radius of the protein domain is less than 11 nm, and is suitable for surface display when the heterologous protein domain is present on the homopolymeric fibril.

[0028] Another specific embodiment relates to the nanofibrils, wherein the monomer subunits described herein are fused with a heterologous protein domain in at least one monomer to generate protein nanofibrils, wherein after the monomers self-assemble into precursor fibrils and entangle with each other to form nanofibrils, at least two monomeric proteins with different sequences appear as part of heteropolymeric fibrils.

[0029] Other specific embodiments provide the protein nanofibers isolated "as is" from a bacterial or endospore environment, and / or are protein nanofibers composed of the protein material, without additional biological material, such as endospores or companion cell attachment components. In other specific embodiments, the protein nanofibers described herein are isolated from recombinant bacterial cell cultures, preferably from Bacillus spp. ( Bacillus Cell cultures or sporulation cultures of Bacillus thuringiensis, more preferably from Bacillus thuringiensis (Bt). Bacillus thuringiensis The bacterial cell culture contains heterologous A-ENA or A-ENA-based or modified A-ENA monomeric protein subunits for self-assembly of the protein nanofibrils described herein. Alternatively, the protein nanofibrils described herein are recombinant protein nanofibrils, such as protein nanofibrils 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.

[0030] In a second aspect, the present invention relates to bacterial endospores, preferably Bacillus spp. Bacillus Endospores, which are similar to wild-type bacterial endospores and preferably Bacillus spp. Bacillus Endospores are modified because they contain and / or display modified A-ENA monomeric protein subunits or functionalized protein nanofibrils as described herein.

[0031] More specifically, the modified bacterial endospores, preferably Bacillus endospores, may lack an endogenous A-ENA monomeric protein that conforms to the common sequence of the general formula NTL-helix 1-L-helix 2-CT described herein; and the bacterial strain, preferably Bacillus endospores ( Bacillus The strain contains an exogenously introduced A-ENA monomeric protein containing the common sequence described herein in the general formula NTL-helix 1-L-helix 2-CT; or contains the protein nanofibrils displaying the aforementioned heterologous or exogenously introduced modified A-ENA monomeric protein or monomeric mutant or monomeric fusion protein.

[0032] Other embodiments involve the modified bacterial endospores, preferably Bacillus spp. Bacillus The use of endospores in increasing bacterial endospore activity, more preferably in improving or increasing the pathogenicity of Bt.

[0033] Alternative implementation schemes involve the formation of a suspension of the protein nanofibers described herein with bacterial endospores or bacterial spores, preferably Bacillus spp. Bacillus Endospores or Bacillus genus ( Bacillus The suspension is used in combination to enhance the bacteria or endospores, preferably the Bacillus spp. Bacillus The toxicity of Bt spores or endospores can enhance insecticidal activity, with the insecticidal activity of the endospores or suspensions being preferred.

[0034] Other aspects of the invention relate to nucleic acid molecules, vectors, host cells, or compositions for providing A-ENA monomers in modified mutant variants or fusion subunits to spontaneously assemble protein nanofibrils.

[0035] The final aspect of the invention relates to a method for producing A-ENA protein nanofibrils as described herein or A-ENA-based variant protein nanofibrils, the method comprising the steps of: - Introducing a nucleic acid molecule encoding an A-ENA monomer as described herein, or an A-ENA-based monomer or a modified A-ENA monomer, into cells; or recombinantly expressing the monomeric protein as described herein in host cells and incubating the cells to allow the monomeric protein to self-assemble into precursor fibrils and subsequently assemble protein nanofibrils; or alternatively culturing host cells containing the monomeric protein subunits or assembling the protein nanofibrils as described herein. - Obtain self-assembled monomers and / or protein nanofibrils from host cells and / or cell suspensions, preferably through cell lysis, and - Purify self-assembled protein nanofibrils, preferably by resuspending them in insoluble fractions and / or further purifying them from cell lysates.

[0036] Specific implementations also involve methods for generating the recombinant A-ENA protein nanofibrils, wherein one or more protein monomers are modified, mutated variants, or fusion proteins as described herein, also referred to herein as functionalized A-ENA protein nanofibrils. Alternatively, the separation and / or purification of the protein nanofibrils in the final step may be performed in an alternative manner known to those skilled in the art to remove cell debris, DNA, or host cell proteins or compounds, which are undesirable for further application of compositions of protein nanofibrils or functionalized protein nanofibrils obtained by the methods. Attached Figure Description

[0037] The accompanying drawings are illustrative only and not restrictive. For illustrative purposes, some elements may be enlarged and not drawn to scale in the drawings.

[0038] Figure 1 Type A endospore appendages (A-ENA) were identified as Bacillus thuringiensis subsp. Israel. (Bacillus thuringiensis Sv. Israelensis (Bti))A novel family of protein fibrils arising from endospores. Transmission electron microscopy (TEM) characterization of Bti spore biofilms revealed the presence of a nanoscale fibril network. (a) A contamination-free TEM micrograph of a resuspended Bti spore biofilm composed of endospores and parasporal bodies (PSBs), grown on LB agar and deposited on a Formvar / carbon grid; (b) A higher magnification image of the same grid used in (a), showing A-ENA fibrils permeating the biofilm; (c) A negative-stained TEM (nsTEM) micrograph of Bti endospores, showing A-ENA fibrils emanating from the spore surface; (df) A-ENA-mediated spore-PSB, respectively. Examples of spore-spore-PSB and spore-spore coupling; (g) nsTEM micrograph showing a cluster of parasporal bodies decorated with A-ENA filaments extending outward from the surface of the parasporal crystals; (h) nsTEM micrograph showing wild-type A-ENA protofibrils of the corresponding 2D class average (frame size 384 × 384 Å); (i) cryoEM micrograph showing vitrified wild-type A-ENA protofibrils of the corresponding 2D average (frame size 230 × 230 Å).

[0039] Figure 2 Recombinant A-ENA protofibrils were generated. (a) nsTEM micrograph of recombinant A-ENA (UniprotKB Q8KNV8; SEQ ID NO: 1) expressed in the cytoplasm of *E. coli*. The protein nanofibrils were isolated from the culture by resuspending the cell pellet in EDTA-lysozyme, incubating in warm 1% SDS, followed by centrifugation and a series of washing steps in water (resuspend & centrifugation) to remove dissolved lysates (the entire process in Example 2). The resulting A-ENA protofibril suspension was deposited on a copper grid supported by a Formvar / carbon film and stained with 2% (w / v) uranyl acetate solution for observation by nsTEM (scale bar: 100 nm; inset: corresponding 2D classification average plot, frame size 384x384 Å); (b) reconstructed cryoEM volume (resolution: 2.59 Å, according to 0.143 FSC standard) derived from the reconstituted A-ENA protofibrils vitrified in deionized water; (c) the corresponding Fourier shell correlation function of the volume shown in Figure b; (d) a cartoon illustration of the atomic model constructed based on the electrostatic potential plot shown in (b).

[0040] Figure 3Visualization of the architecture and structure of type A endospore appendages (A-ENA). (a, b) Banded and space-filled models of A-ENA protofibrils, viewed (a) perpendicular to the long axis of the protofibrils and (b) along the long axis of the protofibrils. The A-ENA protofibrils consist of two precursor protofibrils (white and dark gray, labeled f and f') entangled together to form a biaxial helical superstructure (i.e., parallel double helix) with torsion angles and ascent distances of approximately 12° and 10.8 Å, respectively. (c) Top view; banded and surface representation of a pair of juxtaposed A-ENA monomers, labeled i and i'. The A-ENA subunits consist of α-helical hairpins with N-termini projecting downward along the protofibril axis. The A-ENA helical hairpins are at an angle of approximately 68° to the long axis of the protofibrils. Within a single vertical plane of the protofibril, the two A-ENA monomers (from the two precursor protofibrils, respectively) interact laterally to form a dimer entity. Middle view; A-ENA is axially stacked, causing each precursor fibril to form a tetrameric complex with a relative twist angle of approximately 12° and a rise distance of 10.8 Å, which can be considered a minimal fibril entity or fibril nucleus. Bottom view; Therefore, fibril elongation is achieved by axially stacking additional dimer units at either pole of the A-ENA tetramer, each with a relative twist angle and rise distance of approximately ±12° and 10.8 Å.

[0041] A-ENA fibrils are stretched in the following manner: the C-end of the subunit faces upward (called the C-end or fibril tip, i.e., the C pole) and the N-end extension of the subunit faces downward (called the N-end or the barbed end of the fibril, i.e., the N pole). A-ENA monomers are referred to as i and i' in the f and f' precursor fibrils, respectively, where each stack of fibrils is numbered in ascending (i.e., i+1; i+2, i+3, etc.) or descending (i.e., i-1, i-2, i-3, etc.) directions toward the C and N poles, respectively.

[0042] Figure 4Structural and protomer interactions in A-ENA protofibrils of Bacillus thuringiensis subsp. Israel (Bti). (a) Cartoon schematic of a single Bti A-ENA monomer (UniprotKB Q8KNV8; SEQ ID NO: 1) identified in the cryoEM structure of the recombinant A-ENA protofibril. As shown herein, the A-ENA monomer consists of α-helical hairpins (α1 and α2) with 13 residues of N-terminal extension (NTL; i.e., res 2-14 of SEQ ID NO: 1). Residues shown in a stick model (residue numbers are shown in bold underline) participate in the formation of intermolecular isopeptide bonds (IPB) with adjacent A-ENA monomers (receptor residues of the corresponding A-ENA partner subunits are shown as adjacent to each highlighted residue, with arrows pointing from nucleophilic residues to electrophilic residues); (b) Front view of Bti A-ENA (SEQ ID NO: 1; rotated 90° relative to Figure a), in which residues involved in the formation of intermolecular isopeptide bonds are highlighted and shown in a stick model: the contact between precursor fibrils established by the monomer designated i is formed between monomer i' (i.e., located in the same plane as precursor fibril f') and monomer i'+1 (i.e., translated one unit upward in precursor fibril f'); while the internal contact of the precursor fibril is formed between the A-ENA monomer located directly above (i+1) or directly below (i-1) subunit i, and between monomer i-5 through the second residue in the N-terminal linker region. Therefore, each subunit i participates in up to 10 IPBs, thereby crosslinking 7 different protozoa within and between the precursor fibrils; (c) Illustration view of the top (C pole) and bottom (N pole) surfaces of a single A-ENA monomer: the two surfaces represent the terminal poles of the A-ENA fibrils and form the contact sites of the upper and lower subunits, respectively. These contact surfaces are primarily hydrophobic, except for the residues involved in IPB formation—these residues are highlighted and labeled with arrows; (d) Ultrastructure of A-ENA fibrils, in which precursor fibrils (f' and f) are shown in light gray and gray, with individual A-ENA monomers highlighted in the cartoon illustration; (e) Close-up of the boxed area in Figure d and a cartoon illustration of the subunits in precursor fibrils f (i+1 to i-5), with the residues involved in IPB within the precursor fibrils between i / i+1, i / i-1, and i / i-5 represented by a stick model; (f) Cartoon illustration of IPB between precursor fibrils between i / i' and i / i'+1.

[0043] Figure 5Helical crosslinking via autocatalytic units facilitates isopeptide bond formation. A-ENA interacts with adjacent A-ENA subunits via ten isopeptide bonds. These isopeptide bonds are formed by autocatalytic units composed of acid-base catalytic residues (preferably Glu), nucleophilic residues (preferably Lys or N-terminal amino groups), and electrophilic residues (preferably Glu, Gln, Asn, or Asp). Autocatalytic isopeptide bond formation depends on the close proximity and defined relative positions of the acid-base catalytic residues, which activate the nucleophilic subunits for attack and nucleophilic substitution (i.e., dehydration) of the electrophilic residues. The defined positions of the nucleophilic and electrophilic residues on adjacent helices lead to spontaneous, autocatalytic crosslinking of the A-ENA subunits under physiological conditions. (a) Overview of subunit “i” and its adjacent subunits containing isopeptide bond units mediating intrafiber and interfiber crosslinking of the helical hairpin; (b) Close-up view of the five IPB units (IPB-U1 – IPB-U5) involved in subunit crosslinking.

[0044] Figure 6 Sequence conservation in A-ENA (like) proteins. The shared weblogo for A-ENA originated from multiple sequence alignments of the first 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 cryo-electron microscopy structure of recombinant Q8KNV8 protofilaments) are highlighted with an asterisk. Catalytic acid / base residues (mainly Glu) are highlighted with a plus sign. The residue numbering shown here is inconsistent with the residue numbering system used for Q8KNV8 (with a +4 offset). This difference stems from the multiple sequence alignments used to create the shared identifier.

[0045] Figure 7The helical wheel diagram of the α-ENA interaction network is shown. The helical secondary structure leads to defined side chain positions and defined knock-in-hole interactions formed in the helical-helical contacts. The positions of the side chains can be plotted in the helical wheel when viewed along the long axis of the helix. The uniform helical ascent distance and torsion angle in the α-helix are 1.5 Å and 100°, respectively, resulting in the positioning of residues such that every 8th residue is located at an equivalent position on the helical wheel, with a translation of 10.5 Å along the helical axis. Therefore, the helix can be defined as a continuous heptamer unit or "seven-cell repeat" (H), with side chains named "a" through "g". The protrusions of the side chains on the α-helix form ridge-like and valley-like structures called "knobs" and "holes," which are located in defined positions within the system; defined knock-in-hole interactions occur when two adjacent α-helices interact. Within the A-ENA subunit, α-helices α1 and α2 each consist of five seven-member repeats (H1-1 to H1-5 and H2-1 to H2-5, respectively), and are connected in antiparallel stacking contact through hydrophobic "knobs-in-hole" interactions along the helical length (i.e., position "d" on the helical wheel diagram as defined above) to form a helical hairpin. During the stacking process of the A-ENA subunits (i.e., the i / i+1 and i / i-1 interactions), the α-helices α1 and α2 in the continuous subunits interact along the helical length direction through the main hydrophobic "knobs-in-hole" interactions, achieving paired parallel stacking, including the seven-member repeat positions "a" and "e" on the C-pole side of the A-ENA hairpin and "g" and "c" on the N-pole side. Exceptions to hydrophobic contacts are formed by residues that enter into isopeptide bonds (highlighted by solid arrows pointing from nucleophilic residues to electrophilic residues) and / or form acid-base catalytic residues in IPB units (highlighted and marked with red circles). The stacking of f and f' precursor fibrils in A-ENA is mediated by "knobs-in-hole" interactions between subunits i and i' and i'+1, the "b", "e", and "f" sites in the α2 heptad repeats H2-3 and H2-4, and IPB interactions. Background: Sequence alignment of A-ENA orthologs, showing sequence identity as low as 24% with A-ENA (UniprotKB Q8KNV8; SEQ ID NO:1) (see [link]). Figure 7The A-ENA subunit employs a generic structure consisting of a variable N-terminal lock (referred to as "NTL"), followed by an α-helix "α1" composed of five heptagonal repeating units (H1-1 to H1-5), a variable-length linker sequence (referred to as "L"), a second α-helix "α2", and a variable-length C-terminal tail (referred to as "CT"). Alignment of A-ENA orthologs with the heptagonal repeating units demonstrates a high degree of conservation of the positions of hydrophobic residues, which participate in intra- and inter-helical "button-in-hole" interactions (labeled with f and y, respectively) and form inter-helical isopeptide units (labeled with g, d, and e, respectively, for acid / base catalytic residues, nucleophilic residues, and electrophilic residues, respectively).

[0046] Figure 8 Recombination produces and protofibrils form A-ENA orthologs. (a) Uniprot registry numbers (SEQ ID NO: 3-5) of the presumed A-ENA orthologs and their association with... Bti (a) Sequence identity of A-ENA (Uniprot: Q8KNV8, SEQ ID NO: 1); (b) nsTEM micrographs of self-assembled protofibrils formed by recombinant expression of the corresponding A-ENA ortholog in the cytoplasm of *E. coli*. (The images were taken at 37°C at 1 mg / mL.) -1 After digestion with lysozyme, 5 mM EDTA, 50 mM Tris at pH 6.8, and 50 mM NaCl for 18 h, the fibrils were then purified from the cytoplasm of *E. coli* C43 (DE3) in 1% (w / v) sodium dodecyl sulfate (SDS) at 100 °C for 30 min using detergent (see the full description in Example 2). The diameters of the fibrils obtained from the three tested orthologs were consistent with those observed in recombinant Q8KNV8 and recombinant Q8KNV7. These nsTEM images, taken from samples of the insoluble fraction obtained after SDS / thermal extraction, demonstrate the extremely strong physicochemical stability of the formed fibrils. The latter indicates the existence of one or more autocatalytic formation processes of IPB; (c) AlphaFold2 multimer v1.3 model of the hexamer sequences of each A-ENA ortholog shown in Figure (a); (d) Multiple sequence alignment results of the tested A-ENA ortholog sequences, and Q8KNV8 (A-ENA; SEQ ID NO: 1) and Q8KNV7 (A-ENA-1; SEQ ID NO: 2): residues involved in IPB formation and catalytically active acidic glutamic acid residues are highlighted with black asterisks and red plus signs, respectively.

[0047] Figure 9Conserved architecture and isopeptide bond patterns in A-ENA-like protein nanofibrils. (a, b) The A-ENA-like fibril assembly unit employs a universal structure whose components are, in sequence: an N-terminal lock (NTL) of variable length and sequence; followed by an α-helix (α1), preferably containing five-helix heptavalent repeat units (H1-1 to H1-5); a linker region of variable length (L, typically but not limited to a turn consisting of 4 to 5 residues); an α-helix (α2), preferably containing five-helix heptavalent repeat units (H2-1 to H2-5); and a C-terminal tail (CT) of variable length. In the fibril assembly unit, the α1 and α2 heptavalent repeat units are maintained as follows: Figure 10 The conserved sequence motifs of hydrophobic and short side-chain residues shown facilitate the use of antiparallel helical hairpins in A-ENA-like units and the pairing of protofibril assembly units (i / i+1 and i / i-1) into helical precursor protofibrils (labeled f), with approximate twist angles and ascent distances of 12° and 10.8 Å, respectively. Furthermore, the conserved sequence motifs of hydrophilic residues in the helical seven-membered repeating units H1-2, H1-3, H1-4, and H1-5 in α1 and the helical seven-membered repeating units H2-2, H2-3, H2-4, and H2-5 in α2 (see...) Figure 10The isopeptide bond forming unit is formed, which consists of acid-base catalytic residues (preferably Glu or Asp), nucleophilic residues (preferably Lys or N-terminal amino groups), and electrophilic residues (preferably Glu, Gln, Asp, or Asn). The isopeptide bond pairs are highlighted with boxes, indicating conserved seven-membered repeat sites (e.g., H1-3a in subunit "i") within each subunit of the precursor protofibrils f(i, i+1, i-1, i-5) and f'(i' and i'+1); arrows point from the nucleophilic residues to the electrophilic residues. The schematic diagram shows the positions most favorable for maximizing isopeptide bond pairing. Individual IPB pairings can be considered as a facultative structure and can replace the hydrophobic "button-in-hole" interaction. Complete removal of all IPB pairings results in poor self-assembly of the A-ENA-like structure (see Figure 12) and poor protofibril stability. The N-terminal amino group in the NTL may be involved in the formation of H1-5bIPB with residues of the non-adjacent subunit (i.e., subunit i-5 in the case of Bti A-ENA (SEQ ID NO: 1)). The exact subunit "ix" involved in IPB formation can be altered by lengthening or shortening the NTL; (c, d) side and top views (C-pole) of a cartoon schematic of the hexammeric fragment of the A-ENA-like nanofibrils show the helical stacking of the A-ENA-like units (i, i+1, i-1, etc.) and the binding between two A-ENA-like precursor fibers (f and f'). The protofibrils are extended by adding new A-ENA-like monomer units at the C-pole or N-pole of the protofibrils. The spherical model shows those residues involved in the intrafibril and interfibril isopeptide crosslinking of the A-ENA-like subunits according to the schematic diagrams in Figures a and b.

[0048] Figure 10 Design principles and sequence motifs for self-assembly and self-crosslinking protein nanofibrils using helical hairpins. (a,b) The A-ENA-like fibril assembly unit employs a universal structure, with components in the following order: a variable-length and variable-sequence N-terminal lock (NTL); followed by an α-helix (α1), preferably containing five-helix seven-membered repeat units (H1-1 to H1-5); a variable-length linker region (L); an α-helix (α2), preferably containing five-helix seven-membered repeat units (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 seven-membered repeats maintain a shared sequence motif of hydrophobic and short side chain residues, facilitating the use of antiparallel helical hairpins in the A-ENA-like unit and facilitating the pairing and stacking of fibril assembly units (i / i+1 and i / i-1) into helical precursor fibrils (see...). Figure 7Furthermore, the conserved sequence motifs of hydrophilic residues in the helical seven-membered repeating units H1-2, H1-3, H1-4, and H1-5 in α1 and the helical seven-membered repeating units H2-2, H2-3, H2-4, and H2-5 in α2 form isopeptide bond-forming units, which are composed of acid-base catalytic residues (g; preferably Glu or Asp), nucleophilic residues (d; preferably Lys or N-terminal amino groups), and electrophilic residues (e; preferably Glu, Gln, Asp, or Asn). These isopeptide bond-forming units ( Figure 5 , 7 (as shown in Figure 9) can be individually replaced by hydrophobic residues for hydrophobic convex-concave-recessed interactions. The NTL, connector (L), and C tail element are variable in length and sequence. In a non-limiting manner, the preferred common sequence of the NTL is shown in Figure c.

[0049] Figure 11 HMM maps of helices 1 and 2. Sequence identification of the 35-residue length HMM maps of helices 1 and 2 was established on a set of 593A-ENA (sample) sequences, whose accession numbers are listed in Table 3. Although the paired sequence identity in this protein list is as low as 20-30%, there is strong conservation at key positions in helices 1 and 2, such as the seven-membered repeat elements of each helix as reflected in the general formula provided herein.

[0050] Figure 12 The self-assembly of stable A-ENA-like nanofibrils requires the formation of heteropeptide bonds. (a) SDS-PAGE of purified A-ENA mutant (i.e., A-ENA E29A Q44A N64A E78A Q82A; SEQ ID NO: 15) with residues E29, Q44, N64, E78, and Q82 mutated to alanine residues; (b) and (c) 1 mg / mL in 1xPBS. -1 nsTEM images of A-ENA. Scale bars are shown in 1 μm and 0.1 μm.

[0051] Figure 13Overview of A-ENA engineered sites. (a) Sites for inserting a single amino acid, peptide, or complete domain at the N-terminus, C-terminus, or loop connecting helices α1 and α2 of wild-type A-ENA; (b) Sites for inserting a single amino acid, peptide, or complete domain at the N-terminus or C-terminus of A-ENA ΔNTL (e.g., SEQ ID NO: 12) or loop connecting helices α1 and α2; (c) and (e) Site-directed mutagenesis sites of exposed residues on the upper surface of wild-type A-ENA protofibrils: represented by a stick model in the cartoon illustration of A-ENA (c), and in bold on the five seven-membered repeating units of helices α1 and α2, respectively; (d) and (f) Site-directed mutagenesis sites of exposed residues on the upper surface of ΔNTL A-ENA protofibrils: represented by a stick model in the cartoon illustration of A-ENA (c), and in bold on the five seven-membered repeating units of helices α1 and α2, respectively.

[0052] Figure 14 Post-polymerization functionalization of A-ENA fibrils was performed using the SpyTag / SpyCatcher system. (a) Required genetic building blocks: A-ENA with a C-terminal SpyTag, SpyCatcher with a C-terminal fusion protein (article: superfolded green fluorescent protein (sfGFP); Pédelacq et al., 2006. Nat Biotechnol. 24(1):79-88), and a SpyTagged A-ENA fibril molecular model incorporating a SpyCatcher-sfGFP unit. For illustrative purposes, only a single bound SpyCatcher-sfGFP molecule is shown here; (b) nsTEM micrograph of purified homopolymer A-ENA-SpyTag fibrils; (c) SDS-PAGE analysis of sfGFP (with or without pre-incubation with A-ENA-(1)-SpyTag fibrils), showing that the fluorescence signal of Spycatch-sfGFP bound to A-ENA fibrils appears in the stacked gel (high molecular weight region, not separated) in lanes (2-3); (d) Fluorescence image of A-ENA-SpyTag fibril suspension after incubation with SpyCatcher-sfGFP and centrifugation: the fluorescent insoluble phase consists of A-ENA fibrils.

[0053] Figure 15An optimized connector design strategy for surface display of heterogeneous domains on A-ENA scaffolds. (a) Molecular model of homopolymer A-ENA fibrils displaying fused domains on fibril surfaces: For this strategy, the target domain is fused with A-ENA (located at the N-terminus, C-terminus, or within the α1-α2 ring connector region). When recombinantly expressed, homopolymer A-ENA fibrils with an A-ENA to fused domain ratio of 1:1 are produced. Crucially, flexible connectors are introduced to avoid steric hindrance between the displayed domains. Using this method, fusion domains with hydrodynamic radii less than or equal to 150% (i.e., 10.8 nm) of the A-ENA fibril spacing can be effectively displayed; (b) Examples of surface display by coupling different heterologous domains to the C-terminus of A-ENA: nsTEM micrographs of A-ENA fibrils of p66a (SEQ ID NO: 8), MBD2 (SEQ ID NO: 9), and erythroredoxin (SEQ ID NO: 10), respectively; (c) AlphaFold2 models and molecular weights of the corresponding domains discussed in (b); (d) Eppendorf inversion experiments were performed to investigate hydrogelation; (e) Unwinding curves of wild-type A-ENA and A-ENA-erythroredoxin (RR) fusion proteins.

[0054] Figure 16 A block copolymer design strategy for surface display of heterologous domains on A-ENA scaffolds. (a) Molecular model of hybrid A-ENA protofibrils displaying fusion domains on protofibril surfaces: For this strategy, the target domain is fused with the A-ENA gene (located at the N-terminus, C-terminus, or within the α1-α2 loop linker region). When recombinant expression is performed in a background strain that also expresses wild-type A-ENA, a hybrid A-ENA protofibril is generated in which the ratio of A-ENA to the fusion domain is greater than 1. This method can efficiently display fusion domains with a hydrodynamic radius greater than or equal to 150% (i.e., 10.8 nm) of the A-ENA protofibril spacing; (b) nsTEM micrograph of block copolymer A-ENA protofibrils composed of wild-type A-ENA (block A) and nanobody-TEV-A-ENA (SEQ ID NO: 11) units; (c) Alphafold2 model of the displayed nanobody.

[0055] Figure 17Functional demonstration of camelid antibodies. Schematic diagram of A-ENA-like nanofibrils, where the SpyCatcher (left) or SpyTag (right) sequence is fused with the NTL or CT of the A-ENA monomer (only single fusions are shown for clarity). The SpyCatcher and SpyTag A-ENA fusions form fibrils that can be used to capture SpyTagged and SpyCatcher constructs, respectively. Nb fusions of SpyTagged (left) or SpyCatcher (right) bound to modified A-ENA nanofibrils are shown here.

[0056] Figure 18 The A-ENA spore filament network incorporates Bacillus thuringiensis subsp. Israel (B. thuringiensis). Bacillus thuringienis Sv. israelensis (a) The spores combine with the parasporal body. (a) Developed on LB agar, scraped from the plate and resuspended in water. Bti Unstained TEM image of A-ENA biofilm. The sample was then deposited onto a Formvar / carbon grid. The observed fibrous structures extending from the spores, identified as A-ENA based on their diameter (≈10 nm); (b) grown on LB agar medium, scraped from the plate, resuspended in water, and finally deposited onto a Formvar / carbon grid. BTI Unstained TEM images of the ΔA-ENA strain. This strain shows a complete lack of A-ENA fibrils; (c, d) from Bti WT and Bti Unstained TEM image of spores isolated from strain ΔA-ENA. To isolate spores, 200 μl of spore suspension was added to 1 ml of 50% Histodesnz, and the sample was centrifuged at 16000 × g for 40 min. The supernatant was removed, the precipitate was washed three times with water (16000 x g, 5 min), and then deposited onto a Formvar / carbon grid; (e) using a plasmid carrying the A-ENA gene ORF and its own natural promoter, the spores were... Bti The ΔA-ENA strain was complemented. The A-ENA biofilm was observed to contain spores and PSBs; (f) based on TEM images, spores and PSBs were manually counted, and the PSB / spore ratio is shown in the comparison of spore suspension and spore separation. The experiment was repeated four times, with each count involving more than 100 entities (spores or PSBs) in each group. Figures (a) through (e) show representative images from each group.

[0057] Figure 19 Exogenous A-ENA forms a sporophytic network, which transmits Bacillus thuringiensis subsp. Kustack (… Bacillus thuringiensis Sv. Kurstaki (Btk)(a) WT spores and parasporin crystals bound together. (a) WT cells grown on LB agar, scraped from the plate and resuspended in water. Btk Unstained TEM image of the strain. PSB crystals are free in the culture medium and not directly attached to spores. (b) Expression of PSB derived from its natural promoter under the regulation of its natural promoter. Bti The A-ENA gene Btk Unstained TEM image of the strain. The image clearly shows the spores connected to the PSB via A-ENA filaments, and as... Bti As observed, A-ENA forms a biofilm. (c, d) Isolation of spores from WT Btk and +A-ENA Btk. Spores were isolated using a sucrose pad solution. 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 min. Free PSBs were found at the interface between 70% and 80% sucrose, while spores were found at the precipitate. The supernatant was completely removed, the precipitate was washed three times with water (16000 × g, 5 min), and then deposited onto a Formvar / carbon grid. (e) Detailed image of PSBs from Btk, showing their connection via A-ENA bundles. (f) Manual counting of spores and PSBs based on TEM images, and the PSB / spore ratio shown in the comparison of spore suspension and spore separation. The experiment was repeated four times, with each count involving more than 100 entities (spores or PSBs) in each group. Figures (a) to (d) show representative images of each group.

[0058] Figure 20 A-ENA-mediated spore-parasporin binding enhances the insect pathogenicity of Bacillus thuringiensis. (a) As shown Figure 18 As shown, the April chironomid (April chironomid) was measured in the presence of isolated spores. Chironomus aprilinus (a) Survival curves of larvae; (b) Similarly, survival curves of April chironomid larvae in the presence of spore suspension were evaluated. In experiments a) and b), each group contained at least 8 larvae. The survival rate of all larvae was confirmed before the start of the experiments. Larvae of varying sizes representing different stages of maturity were included in the experiments. The isolated spores and spore suspensions were measured at a final concentration OD. 600 Inoculation was performed at 0.02. Phosphate-buffered saline (PBS) was used as a negative control. Larval survival was monitored daily for one week. The experiment was performed in triplicate at room temperature. In both figures, all three curves were statistically significant according to the Mantel-Cox log-rank test (p<0.0001); (c) shows images of examples of surviving and dead larvae in the midge larval population used in the experimental setup.

[0059] Figure 21 In vivo functionalization of the A-ENA sporophytic network in Bacillus thuringiensis. Electroporation was performed on Bacillus thuringiensis subsp. Israel (Bti) and Bacillus thuringiensis subsp. Kustack (Btk) strains using the pA180 plasmid, which carries an A-ENA fused to the C-terminus of the SpyTag under the control of its natural promoter and terminator (SEQ ID NO:7). After spore isolation as described above, spores were fused to the SpyCatcher at the N-terminus with 100 μM purified sfGFP and incubated at room temperature for 10 min with continuous stirring. Subsequently, the spores were centrifuged at 10000 x g for 5 min and washed with PBS to remove any unbound sfGFP:SpyCatcher. This washing step was repeated three times before spore imaging. Fluorescence microscopy revealed strong fluorescence signals in both the spore clusters and the PSB site in both Bti and Btk strains transformed with A-ENA:SpyTag. To illustrate the potential autofluorescence derived from spores, wild-type (WT) Bti and Btk strains were included as negative controls. The scale bar in the image represents a length of 2.5 μm.

[0060] Figure 22 Stability analysis of recombinant A-ENA. The images in the figure show A-ENA fibrils after the following treatments: (a) incubation at 99°C in 2% (w / v) SDS for 1 h, (b) incubation at room temperature in 8M urea for 1 h, (c) high-temperature drying; i.e., drying A-ENA fibers suspended in miliQ water at 200°C for 15 min and rehydrating in miliQ water for imaging, (d) incubation in 2M NaOH at room temperature for 1 h, (e) autoclaving at 121°C for 20 min, and (f) incubation in 100% (v / v) formic acid at room temperature for 30 min.

[0061] Figure 23 Purified recombinant A-ENA fibrils enhanced the resistance of Bacillus thuringiensis to Spodoptera litura (a type of leafminer moth). Trichoderma ni Insectivorous pathogenicity of (Calophyllum looper) larvae. a. Representative ns-EM images of wild-type Btk spore suspension incubated with purified recombinant A-ENA protofibrils (4.18 mg / mL, produced in E. coli), showing that exogenous addition of protofibrils to the spore formulation resulted in the aggregation of spores and Cryotoxin crystals within the A-ENA protofibril network. b. Dead and surviving *Spodoptera litura* (a type of leafminer moth). Trichoderma ni Representative images of larvae reared on a medium supplemented with Btk spore suspension. c. White-spotted armyworms reared on media supplemented with the following different formulations. Trichoderma niSurvival curves of larvae: spore suspensions of wild-type Bacillus thuringiensis subsp. Kustack (“BTK WT”); recombinant Bacillus thuringiensis subsp. Kustack expressing A-ENA (“BTK + A-ENA”); or wild-type Bacillus thuringiensis subsp. Israel (“BTI”); and phosphate-buffered saline (“PBS”) as a negative control. d. White-spotted armyworms reared on media supplemented with the following formulations ( Trichoderma ni Larval survival curves: spore suspensions of wild-type Bacillus thuringiensis subsp. Kustack (“BTK WT”); spore suspensions of wild-type Bacillus thuringiensis subsp. Kustack (“BTK WT + A-ENA fibrils”) incubated with purified recombinant A-ENA (4.18 mg / mL, produced in Escherichia coli); and purified recombinant A-ENA fibrils (4.18 mg / mL) as a negative control to exclude any toxic effects.

[0062] Figure 24 a.-c. Illustration of the A-ENA adapter insertion mutant (A-ENA_LI). d. Negative stained EM image of an E. coli DH5α cell suspension expressing A-ENA_LI-R4.5-2RFD (SEQ ID NO:67); scale bar = 100 nm. This image shows the presence of assembled A-ENA_LI-R4.5-2RFD fibrils in the cell suspension. e. EM image of purified A-ENA_LI-R4.5-2RFD nanofibrils showing the presence of numerous micron-long A-ENA-like nanofibrils approximately 10 nm thick, confirming that the adapter insertion mutant retains the ability to self-assemble into SDS-stable protein nanofibrils; scale bar = 200 nm (left) and 100 nm (right).

[0063] Figure 25A mutant A_ENA_E28Q_E39Q_E41Q possesses a single IPB for the self-assembly of A-ENA protofibrils. A-ENA is mutated to Gln(Q) at residues E28, E39, and E41, thereby removing the acid-base catalytic residues of IPB1, 2, 4, and 5 that are normally formed in wild-type A-ENA protofibrils. Thus, only IPB3 is retained as the interconnection mechanism between A-ENA monomer subunits, which is sufficient to support fiber assembly. (a, b). A banded diagram (A) and a schematic diagram (b) of A-ENA protofibrils, where the residues forming the IPB3 isopeptide bond (K76 -> Q82; SEQ ID NO: 68) are shown as spheres in Figure (a) and indicated by arrows in Figure (b). IPB3 crosslinks f and f' precursor protofibrils in an i -> i'-1 manner. Therefore, a single isopeptide bond across the f–f' precursor fibril interface generates nanofibrils, in which all the fibrils are cross-linked to form a single covalent unit. To construct an A-ENA variant containing only a single IPB3, the acid-base catalytic residues necessary for the formation of IPB1 (E41), IPB2 (E39), and IPB4 and 5 (E28) in A-ENA (SEQ ID: NO: 1) were removed. Figure 5 (c) Alphafold 3 prediction and associated PAE plot of precursor fibrils of A_ENA_E28Q_E39Q_E41Q, which were mutated to glutamine (Gln) residues with similar properties but no catalytic activity, thus obtaining “A_ENA_E28Q_E39Q_E41Q” (SEQ ID NO 68). (d) Negative staining EM image of purified, SDS-resistant mutant-A-ENA fibrils formed from A_ENA_E28Q_E39Q_E41Q, showing that the IPB (i.e., IPB3) of a single cross-fibril is sufficient to form robust protein nanofibrils. Scale bar = 100 nm.

[0064] Figure 26 Overview of A-ENA's production and processing pipeline. Detailed Implementation

[0065] The invention is described below with reference to specific embodiments and certain accompanying drawings; however, the invention is not limited thereto, but is defined only by the claims. Any reference numerals in the claims should not be construed as limiting the scope. It should be understood, of course, that any specific embodiment of the invention need not achieve all aspects and advantages. Therefore, by way of example, those skilled in the art should recognize that the invention may be practiced or implemented in a manner that achieves or optimizes one or more advantages described in this application without necessarily achieving other aspects or advantages that may be described or implied in this application. The organization and operation of the invention, as well as its features and advantages, can be best understood by referring to the following detailed description when read in conjunction with the accompanying drawings. Aspects and advantages of the invention will become apparent and clear from the embodiments described below. Throughout the specification, the phrase “in one embodiment” or “in an embodiment” means that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of this aspect. Therefore, the phrases “in one embodiment” or “in an embodiment” appearing in various places throughout the specification do not necessarily refer to the same embodiment, but may refer to the same embodiment.

[0066] definition

[0067] Unless otherwise specified, the use of indefinite or definite articles when referring to singular nouns, such as “a”, “an”, or “the,” includes the plural form of the noun. When the term “comprising” is used in this specification and claims, it does not exclude other elements or steps. When the terms “essentially consisting of” or “consisting essentially of” are used herein for chemical substances or compounds such as proteins, it means that specific other components or substances may be present, i.e., those that do not substantially affect the essential characteristics of the chemical substance. When specifically mentioned herein, “essentially consisting of” or “consisting essentially of” refers to the majority or most of a chemical substance such as a polypeptide, where the functional outcome is defined therein, but other substances, such as other amino acids, may be contained. Furthermore, the terms first, second, third, etc., are used in the specification and claims to distinguish similar elements, but are not necessarily used to describe a sequential or chronological order. It should be understood that the terms used herein are interchangeable where appropriate, and embodiments of the invention described herein can be operated in any order other than those described or illustrated herein. The following words or definitions are used only to facilitate understanding of the invention. Unless specifically defined herein, all terms used herein have the same meaning as would be understood by one of ordinary skill in the art. Those skilled in the art should in particular refer 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 (Supplement 114), John Wiley & Sons, New York (2016) for understanding the definitions and terminology in this field. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., molecular biology, biochemistry, structural biology, and / or computational biology).

[0068] As used herein, the terms “nucleic acid sequence,” “DNA sequence,” or “nucleic acid molecule” refer to a polymer of nucleotides of any length, which may be ribonucleotides or deoxyribonucleotides. The term refers only to the primary structure of the molecule. Therefore, the term includes double-stranded and single-stranded DNA and RNA. It also includes modifications of known types, such as methylation, “capping” substitution of one or more naturally occurring nucleotides with analogs. A “nucleic acid construct” refers to a nucleic acid molecule that has been constructed to contain one or more functional units not found together in nature. Examples include circular, linear, double-stranded, extrachromosomal DNA molecules (plasmids), plasmids (plasmids containing a COS sequence from λ phage), viral genomes containing non-natural nucleic acid sequences, etc. A “coding sequence” is a nucleotide sequence that, when placed under the control of appropriate regulatory sequences, is transcribed into mRNA and / or translated into polypeptides. The boundaries of a coding sequence are defined by a 5'-terminal translation start codon and a 3'-terminal translation stop codon. Coding sequences may include, but are not limited to, mRNA, cDNA, recombinant nucleotide sequences, or genomic DNA, and in some cases may contain introns. As used herein, "promoter region of a gene" or "regulatory element" refers to a functional DNA sequence unit that, when effectively linked to a coding sequence and possibly placed under suitable inducible conditions, is sufficient to promote transcription of said coding sequence. "Operably linked" refers to a juxtaposition where the components are in a relationship that allows them to function in their intended manner. "Operably linked" to a promoter sequence of a nucleic acid molecule that is a coding sequence is linked in a manner that allows the coding sequence to be expressed under conditions compatible with the promoter sequence. As used herein, "gene" includes both the promoter region and the coding sequence of a gene. It refers both to a genomic sequence (including possible introns) effectively linked to a promoter sequence and to cDNA derived from splicing messengers. The term "terminator" or "transcription termination signal" includes a control sequence, which is a DNA sequence at the end of a transcription unit that signals the 3' processing and polyadenylation of the primary transcript and the termination of transcription. Terminators can be derived from natural genes or from a variety of other (bacterial) genes. A "chimeric gene," "chimeric construct," or "chimeric gene construct" refers to a recombinant nucleic acid sequence molecule in which a promoter or regulatory nucleic acid sequence is effectively linked or bound to a nucleic acid sequence encoding mRNA, enabling the promoter or regulatory nucleic acid sequence to regulate the transcription or expression of the related nucleic acid coding sequence. As found in nature, the regulatory nucleic acid sequence of a chimeric gene is not effectively linked to the related nucleic acid sequence and can be heterologous to the coding nucleic acid sequence molecule, meaning that its sequence does not exist in nature in the same construct as presented in a chimeric construct. More generally, the term "heterologous" is defined herein as a sequence or molecule of a different origin.

[0069] The terms “protein,” “polypeptide,” and “protein domain” are further used interchangeably herein to refer to polymers of amino acid residues and their variants and synthetic analogs. A monomer or protomer is defined as a single polypeptide chain extending from the amino terminus (also referred to herein as the N-terminus or N-terminus) to the carboxyl terminus (also referred to herein as the C-terminus or C-terminus). As used herein, a “protein subunit” refers to a monomer or protomer that may form part of a multi-protein complex or assembly. As used herein, a “protein domain” refers to a folded protein, or a folded portion of a protein that serves as a unique functional and / or structural unit within a protein. Typically, protein domains are responsible for specific functions or interactions that contribute to the overall function of the protein. Domains can be found in a wide variety of biological environments, with similar domains being found in proteins with different functions. Secondary structural elements of proteins typically form intermediates spontaneously before the protein folds into its three-dimensional tertiary structure. The two most common secondary structural elements of proteins are the α-helix and β-sheet, but β-turns and Ω-loops also exist. A β-sheet consists of at least two or three β-chains (also called β-chains) linked laterally by hydrogen bonds in the main chain, forming a typically twisted, wrinkled sheet. A β-chain is a polypeptide chain, usually 3 to 10 amino acids long, with the main chain in an extended conformation. A β-turn is a non-regular secondary structure in proteins that causes a change in the orientation of the polypeptide chain. β-turns (β-bends, β-turns, sharp turns, reverse turns) are very common motifs in proteins and polypeptides, primarily responsible for linking β-chains.

[0070] A coiled helix is ​​a tertiary protein structure in which α-helices intertwine to form a supercoil bundle, typically consisting of two or three helices oriented in parallel or antiparallel configurations. Coiled helices are usually formed from multimeric proteins of the same (homologous) or different (heterologous) chains. Continuous helices from the same polypeptide chain can also fold into coiled helical structures, typically with helices of the same monomers then in an antiparallel orientation. The helices constituting the coiled helix interact through a "button-in-hole" geometry of the amino acid side chains at their interfaces (Crick FHC (1952) Nature 170:882–883), where residues from one helix (button) are inserted into the space surrounded by the four side chains of the facing helix (hole). This regular stacking also satisfies the necessary condition for a heptad repeat; a heptad repeat is specifically defined as a side chain position that precisely and periodically repeats every seven residues along the direction of the helical interface. Therefore, this seven-repetition regularity of the side chain positions of coiled helices is reflected in the repeating units of their seven-residue sequences, with each position within the unit labeled a through g; where hydrophobic residues typically occupy the core-forming positions (a and d), while the remaining positions exposed to the solvent environment (b, c, e, f, and g) are primarily occupied by hydrophilic residues. Through this strict pattern, coiled helices are predictable based on their amino acid sequences, with a level of detail allowing for the assignment of individual residues to the positions of the seven-repetition units. 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).

[0071] In the context of this invention, "self-assembly" refers to the spontaneous organization of molecules within an ordered supramolecular structure, thanks to non-covalent interactions between them in the absence of external control or a template. The chemical and conformational structures of individual molecules carry instructions on how to assemble these molecules. Identical or dissimilar molecules can constitute the building blocks of a molecular self-assembly system. Typically, interactions are established in less ordered states, such as solutions, random coils, or disordered aggregates leading to an ordered final state, which can be crystalline or folded macromolecules, or further assembly of macromolecules. The binding of small molecules or proteins into ordered structures is driven by thermodynamic principles, and therefore based on energy minimization. The interactions involved in molecular assembly are electrostatic, hydrophobic, hydrogen bonding, van der Waals interactions, aromatic stacking, and / or metal coordination. Although these forces are non-covalent and weak individually, they can produce highly stable assemblies and control the shape and function of the final assembly (Lombardi et al., 2019; Pharmaceutics, 11, 166). The self-assembling protein subunits described herein, and the A-ENA monomeric protein referred to herein, are capable of self-assembling into monomers, further polymerizing and folding, and binding to the protein fibrils described herein. Fibril assemblies can be obtained from pre-existing components referred to as building units or subunits, and more specifically, from the isolated self-assembling A-ENA monomeric protein described herein.

[0072] The terms “chimeric polypeptide,” “chimeric protein,” “chimera,” “fusion polypeptide,” “fusion protein,” or “heterologous fusion” are used interchangeably herein to refer to a protein comprising at least two independent and distinct polypeptide components, which may be derived from the same protein or preferably not from the same protein. The term also refers to a non-naturally occurring molecule, meaning it is artificial. When referring to chimeric or fusion polypeptides (as defined herein), the terms “fused to” and other grammatical equivalents such as “covalently linked,” “connected,” “attached,” “ligated,” “conjugated,” and “insertion” as specifically used herein refer to any chemical or recombination mechanism used to link two or more polypeptide components. The fusion of two or more polypeptide components can be a direct fusion of sequences, or it can be an indirect fusion, such as by intercalating amino acid sequences or linker sequences, or chemical linkers. The fusion of amino acid residues or (poly)peptides with the Ena protein, their insertion into the Ena protein sequence, or their fusion with another target protein described herein can be a covalent peptide bond connection or a fusion achieved through chemical linkage. The term “fusion to” as used herein is interchangeable with “linked to,” “conjugated to,” or “linked to,” specifically referring to a “genetic fusion” that results in a stable covalent link, such as through recombinant DNA technology, and “chemical and / or enzymatic conjugation.”

[0073] As used herein, the terms “protein complex” or “protein assembly” or “polymer” refer to a group of two or more associated macromolecules, at least one of which is a protein. As used herein, a protein complex or assembly generally refers to the binding or association of macromolecules that can form under physiological conditions. The individual members of a protein complex, such as protein subunits or protomolecules, are linked by non-covalent or covalent interactions. “Binding” refers to any interaction, whether direct or indirect. A direct interaction means contact between the binding partners. An indirect interaction is any interaction through which the binding partners interact in a complex of two or more molecules. With the help of one or more bridging molecules, interactions can be completely indirect; interactions can also be partially indirect, where direct contact remains between the partners, which is stabilized by additional interactions of one or more molecules. Binding or association can be non-covalent, where the juxtaposition is supported energetically by, for example, hydrogen bonds or van der Waals forces or electrostatic interactions, or it can be covalent, such as peptide bonds, disulfide bonds, or isopeptide bonds.

[0074] It should be understood that protein complexes can be multimers. Assembly of protein complexes can lead to the formation of homopolymers or heteropolymers. Furthermore, interactions can be stable or transient. The terms "multimer," "multimer complex," or "multimer protein or assembly" encompass multiple identical or heterologous polypeptide monomers. Polypeptides are capable of self-assembling into multimer assemblies (i.e., dimers, trimers, pentamers, hexamers, heptamers, octamers, etc.) formed by the self-assembly of multiple single polypeptide monomers (i.e., "homogeneous assemblies") or by the self-assembly of multiple different polypeptide monomers (i.e., "heterogeneous assemblies"). As used herein, "multiple" means two or more. Multimer assemblies contain 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or preferably more polypeptide monomers. Multimer assemblies can be used for any purpose and provide a method for developing a variety of protein "nanomaterials." In addition to a limited number of mesh-like or shell-like protein assemblies, they can also be designed by selecting suitable target symmetry structures. The monomeric or proto- and / or polymeric assemblies of this invention can be used to design higher-order assemblies, such as precursor fibrils, fibril assemblies, or fibrils and other fibers, with the added advantage of hierarchical assembly. The resulting polymeric or fibrous assemblies are highly ordered materials with excellent rigidity and monodispersity, and can function as polymeric or fibrous structures themselves, or form the basis for advanced functional materials, such as modified surfaces containing polymeric assemblies or fibrous structures, and custom-designed molecular machines with a wide range of applications. More specifically, the polymers used herein refer to homopolymers or hybrids, or homopolymeric or hybrid protein complexes, which are bound together by non-covalent and / or covalent interactions to form organized structures; and / or further modified to grow or evolve into self-assembled nanofibrils, or to trigger the formation of nanofibrils. The multimer assembly may contain a monomeric ENA fusion protein as defined herein, or ENA protein variants, mutants and / or engineered ENA proteins, such as those exemplified and described herein, as well as other proteins that can bind to the ENA-based or particularly A-ENA-based multimer (referred to as the engineered multimer), thereby enabling the multimer to be further modified to meet the needs of a specific application.

[0075] "Recombinant polypeptide" refers to a polypeptide prepared using recombinant technology, i.e., a polypeptide prepared by expressing recombinant or synthetic polynucleotides, which can be obtained in vitro and / or in a cellular environment. When recombination produces chimeric or fusion polypeptides or their biologically active (i.e., functional) portions, it is also preferable to enrich, purify, or substantially remove them from the culture medium, i.e., impurities constitute less than about 20% of the volume of the protein preparation, more preferably less than about 10%, and most preferably less than about 5%. "Isolated" or "purified" means a substance that is substantially or substantially free of the components that normally accompany it in its native state.

[0076] The term "homology" of proteins includes peptides, oligopeptides, polypeptides, proteins, and enzymes that have amino acid substitutions, deletions, and / or insertions relative to the unmodified or wild-type protein in question, and that have similar biological and functional activities to the unmodified protein from which they are derived. As used in this application, "amino acid identity" refers to the degree of sequence similarity when compared amino acid-by-amino acid within a comparison window. Therefore, the "sequence identity percentage" is calculated as follows: Two sequences that are maximally aligned within the comparison window are compared: the number of positions in the two sequences containing the same amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, Met, as indicated by the single-letter codes commonly used herein) is determined to obtain the number of matching positions; the number of matching positions is divided by the total number of positions within the comparison window (i.e., the window size); and the result is multiplied by 100 to obtain the sequence identity percentage. As used herein, a “substitution” or “mutation” results from the substitution of one or more amino acids or nucleotides, respectively, by a different amino acid or nucleotide sequence compared to the amino acid or nucleotide sequence of the parent protein or a fragment thereof. It should be understood that proteins or fragments thereof may have conserved amino acid substitutions that have substantially no effect on protein activity. The percentage of amino acid identity provided herein preferably takes into account a comparison window corresponding to the total length of the native or native wild-type protein or the specific amino acid sequence referred to.

[0077] The term "wild-type" refers to a gene or gene product isolated from a natural source, or a gene or gene product contained in a cell, cell line, or organism. Wild-type genes or gene products are the most commonly observed in populations and are therefore arbitrarily engineered to be the "normal" or "wild-type" form of genes or gene products observed in nature. Conversely, the terms "modified," "engineered," "mutant," or "variant" refer to a gene or gene product that exhibits alterations (i.e., characteristics) in sequence, post-translational modifications, and / or functional properties compared to a wild-type or naturally occurring gene or gene product. Knockout refers to a modified, mutated, or deleted gene to provide a non-functional gene product and / or function. It is important to note that naturally occurring mutants or variants can be isolated; these are identified by the fact that they possess altered characteristics compared to a wild-type gene or gene product and have a different sequence compared to a reference gene or protein. The terms “functional homolog” or “functional variant” herein refer to a modified, engineered, or mutated gene product that retains its functionality compared to the wild-type or original A-ENA sequence on which its modification or mutation is based. “Functionality” herein refers to the self-assembly properties of monomeric protein subunits, and the further spontaneous assembly of said monomers into nanofibril structures, as described herein.

[0078] As used herein, the terms "vector," "vector construct," or "recombinant vector" can be double-stranded or single-stranded and can be DNA, RNA, or DNA / RNA hybrid molecules of any conformation, including but not limited to linear, circular, coiled, supercoiled, twisted, nicked, etc. These vectors of the present invention include, but are not limited to, plasmid vectors, granular vectors, phage vectors such as λ 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 commercially available. Any vector can be used to construct and express the fusion molecules used in the present invention. General categories of vectors of particular interest include: prokaryotic and / or eukaryotic cloning vectors, expression vectors, fusion vectors, phage and yeast display vectors, shuttle vectors for different hosts, mutagenesis vectors, transcription vectors, vectors for accommodating large insert sequences, etc. Most of the required methods can be found in the article by Ausubel et al., 2007. The preparation of vector constructs for introduction into prokaryotic or eukaryotic hosts typically includes a host-recognized replication system, comprising the intended DNA fragment encoding the nucleic acid molecule of the present invention, and preferably including transcription and translation initiation regulatory sequences effectively linked to the molecular coding fragment. The expression system may include, for example, an origin of replication or autonomous replication sequence (ARS) and expression control sequences, promoters, enhancers, and essential processing information sites, such as ribosome binding sites, RNA splicing sites, polyadenylation sites, transcription terminator sequences, and mRNA stabilizing sequences. Where appropriate, signal peptides derived from secreted polypeptides of the same or related species may also be included; these signal peptides enable proteins to cross and / or lodge within the cell membrane, or be secreted from the cell. Appropriate promoters and other essential vector sequences are selected to function in the host. Other vectors may integrate into the host cell's genome upon introduction into the host cell, thereby replicating along with the host genome. Examples of feasible combinations of cell lines and expression vectors are described, for example, in Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 4th edition, Cold SpringHarbor Press, Plainsview, New York (2012); and Ausubel et al., *Current Protocols in Molecular Biology* (Supplement 114), John Wiley & Sons, New York (2016). Many useful vectors for expression in bacteria, yeast, fungi, mammals, insects, plants, or other cells are well known in the art.

[0079] "Host cell" can be a prokaryotic or eukaryotic cell. Transfection can be transient or stable. The expression vector can be transfected into prokaryotic and eukaryotic cells using any technique known in the art, including but not limited to standard bacterial transformation, calcium phosphate coprecipitation, electroporation or liposome-mediated transfection, DEAE-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 (Supplement 114), John Wiley & Sons, New York (2016). In this document, recombinant host cells are those cells that have been genetically modified to contain the isolated DNA molecule, nucleic acid molecule, or expression construct or vector of the present invention. DNA can be introduced using any method known in the art suitable for a particular cell type, including but not limited to transformation, lipid transfection, electroporation, or virus-mediated transduction. DNA constructs capable of expressing the chimeric proteins of the present invention can be readily prepared using techniques known in the art, such as cloning, hybridization screening, and polymerase chain reaction (PCR). Standard techniques for cloning, DNA isolation, amplification, and purification, standard techniques for enzymatic reactions including DNA ligases, DNA polymerases, restriction endonucleases, etc., and various isolation techniques are known and commonly used by those skilled in the art. Many standard techniques are described in Sambrook et al. (2012), Wu (ed.) (1993), and Ausubel et al. (2016). Representative host cells suitable for use 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 in the present invention include: Escherichia coli (… Escherichia spp .) cells, Bacillus spp. ( Bacillus spp. ) cells, Pasteurella spp. ( Pasteuria spp .) cells, Streptomyces ( Streptomyces spp .) cells, Erwinia spp. ( Erwinia spp .) cells, Klebsiella spp. ( Klebsiella spp .) cells, Serratia spp. ( Serratia spp .) cells, Pseudomonas spp. ( Pseudomonas spp .) cells, Lactococcus spp. ( Lactococcus spp .) cells, Lactobacillus spp. ( Lactobacillusspp.) cells and Salmonella spp. ( Salmonella spp Cells. Animal host cells suitable for this invention include insect cells and mammalian cells (more specifically derived from Chinese hamster cell lines such as CHO and human cell lines such as HeLa). Yeast host cells suitable for this invention include yeast genus (…). Saccharomyces ), genus *Fissionyomyces* Schizosaccharomyces ), Kluyveromyces ( Kluyveromyces ), Pichia pastoris ( Pichia (For example, Pichia pastoris) Pichia pastoris )), Hansenula genus ( Hansenula (For example, Hansenula polymorpha) Hansenula polymorpha )) Yarowia genus *Schefflera* ( Schwaniomyces ), genus *Fissionyomyces* Schizosaccharomyces ), Zygosaccharomyces ( Zygosaccharomyces Species within the range of ), including *Saccharomyces cerevisiae*, *Saccharomyces carlsbergensis*, and others. K. lactis It is the most commonly used yeast host and also a convenient fungal host. Host cells can be provided in suspension or flask cultures, tissue cultures, organ cultures, etc. Alternatively, host cells can also be transgenic animals, plants, or parts or derived materials thereof.

[0080] As used herein, the terms “medicine” or “drug” refer to a substance / composition intended for the treatment, i.e., prevention or treatment of a subject potentially suffering from a disease or condition. The terms “disease” or “condition” refer to any pathological state, particularly a disease or condition as defined herein.

[0081] The terms “subject,” “individual,” or “patient” are used interchangeably herein to refer to any living organism, such as a vertebrate, particularly any mammal, including humans and another mammal, for whom a diagnosis, treatment, or prevention is required, such as animals, such as rodents, rabbits, cattle, sheep, horses, dogs, cats, lamas, pigs, or non-human primates (e.g., monkeys). 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 condition, or is expected to be at high risk of developing a disease or condition, particularly those disclosed herein, also referred to herein as a “patient.”

[0082] The term "treatment" and its grammatical variants, or "a treatment method," are used interchangeably. It is defined as a therapeutic intervention designed to slow, interrupt, prevent, control, stop, alleviate, or reverse the progression or severity of a sign, symptom, condition, illness, or disease, but does not necessarily involve the complete elimination of all signs, symptoms, conditions, or illnesses associated with the disease. Therefore, therapeutic treatment is designed to treat disease or improve the health of a person or animal, rather than to prevent disease. Treatment can also be preventative, referring to medications or treatments designed to prevent the occurrence of disease; in this text, these are collectively referred to as "prevention."

[0083] The term "composition" refers to a combination of one or more active molecules, preferably comprising one or more protein nanofibers of the present invention, and may also include buffer solutions and / or solutes, such as pH buffers, water, saline, physiological saline solutions, glycerol, preservatives, etc., which are known to those skilled in the art to be suitable for obtaining optimal performance. Suitable conditions as used herein may also refer to conditions under which the protein nanofibers as described herein are used, applied, or administered, and may include bacterial cultures or endospore material, or residues from bacterial cultures when the protein nanofibers are produced in spore-forming bacteria and / or isolated from sporogenic bacteria.

[0084] A “pharmaceutical composition” is a therapeutically active composition comprising the therapeutically active agent or composition provided by the present invention, and optionally comprising a carrier, diluent, or excipient. A “carrier” or “adjuvant,” particularly a “pharmaceutically acceptable carrier” or “pharmaceuticalally acceptable adjuvant,” is any suitable excipient, diluent, carrier, and / or adjuvant that itself neither induces antibodies harmful to the individual receiving the composition nor induces a protective effect. “Pharmaceutically acceptable” means a substance that has no adverse effects in biological or other respects; that is, the substance can be administered to an individual with the compound without causing any adverse biological effects or harmful interactions with any other component in the pharmaceutical composition. A pharmaceutically acceptable carrier is preferably a carrier 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 caused by the carrier do not detract from the beneficial effects of the active ingredient. Preferably, a pharmaceutically acceptable carrier or adjuvant can enhance the immune response induced by the antigen. Suitable carriers or adjuvants typically contain one or more compounds from a non-exhaustive list of the following: slowly metabolizing macromolecules, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, and inactive viral particles. The term "excipient" as used herein is intended to include all substances that may be present in a pharmaceutical composition and are not active ingredients, such as salts, binders (e.g., lactose, dextrose, sucrose, trehalose, sorbitol, mannitol), lubricants, thickeners, surfactants, preservatives, emulsifiers, buffers, stabilizers, flavoring agents, or coloring agents. "Diluents" include solvents such as water, saline, physiological saline solutions, glycerol, ethanol, etc. These solvents may include auxiliary substances such as wetting agents or emulsifiers, pH buffers, or preservatives. The effective amount of the polypeptide or conjugate and pharmaceutically acceptable carrier of the present invention is preferably an amount that produces a result or exerts an effect on the specific condition being treated. For treatment, the pharmaceutical compositions of the present invention can be administered to any patient according to standard techniques. Administration can be performed by any suitable means, including oral, parenteral, topical, nasal, ocular, intrathecal, intraventricular, sublingual, rectal, and vaginal administration. Other formulation techniques such as nanotechnology, as well as aerosols and inhalers, are also within the scope of this invention. The dosage and frequency of administration will depend on the patient's age, sex, condition, whether other medications are being used concurrently, contraindications, and other relevant factors that the clinician must consider. The pharmaceutical compositions of this invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. If the pharmaceutical compositions of this invention are prepared as lyophilized or liquid dosage forms, physiologically acceptable carriers, excipients, and stabilizers must be added (Remington's Pharmaceutical Sciences, 22nd edition, Allen, ed., Loyd V, Jr. (2012)).The dosage and concentration of carriers, excipients, and stabilizers should be safe for subjects (humans, mice, and other mammals), including buffers such as phosphates, citrates, and other organic acids; antioxidants such as vitamin C, small peptides, proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as PVP, amino acids such as glycine, glutamate, asparagine, arginine, and lysine; glucose, disaccharides, other carbohydrates such as glucose, mannose, or dextrin, 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.

[0085] Detailed description

[0086] This invention is based on a novel endospore appendage (ENA) protein, particularly the α-helical endospore appendage protein, or A-ENA, which has been found to possess self-assembly capabilities, forming multifibrillary structures that bind to Bacillus thuringiensis (Bt) endospores and parasporal bodies, contributing to the pathogenicity of these bacterial spores, for example, acting as insecticides. This paper provides the first resolution of these fibrous structures appearing in Bt endospore cultures, revealing that they contain proteinaceous material composed of A-ENA proteins that spontaneously fold into highly ordered, striking protofibrillary structures, providing extremely high stability and rigidity for tight binding of spores to parasporal bodies. Investigations into the biological function of these fibrous assemblies using Bt A-ENA knockout strains and complementation experiments with sporulating bacteria lacking endogenous A-ENA proteins indeed demonstrate that bipyramidal crystals containing toxic proteins used to kill lepidopteran insects, i.e., parasporal bodies, bind to Bt spores precisely through A-ENA-based protofibrils.

[0087] The rigidity, thermal stability, mechanical stability, and tensile strength of A-ENA-based protein protofibrils appear to originate from several heteropeptide covalent interactions within the protofibrils, leading to the immense potential of biostructured materials in various applications. However, isolating A-ENA protofibrils from parasporal bodies for in vitro separation is theoretically impossible, as these naturally occurring A-ENA-based protofibrils, such as those found in Bt spore cultures, are always tightly bound to parasporal bodies in other potential components of the sporogenic culture, playing a crucial role in the toxicity of Bt spores to insects, but also complicating their isolation and structural analysis. Finally, thanks to more advanced technological improvements in the Cryo-EM field, structural analysis of Bacillus thuringiensis subsp. Israel (Bti) spore cultures confirmed that these Bti protofibrils are indeed A-ENA protein-based protofibrils, and two A-ENA genes in the genome of this Bacillus strain encode these two proteins (described herein as A-ENA and A-ENA-1, SEQ ID NO: 1 and 2, respectively). Based on new structural insights, further comparative analysis of homologous bacterial protein sequences enabled the determination of the structural features of this newly annotated “A-ENA” protein family.

[0088] α-helix endospore appendage (A-ENA) protein

[0089] This paper defines A-ENA proteins as protein monomers that exhibit self-assembling α-helical antiparallel coil-and-coil monomers. Their structure presents a helical pattern determined by their amino acid sequence (as is well-known for coil-and-coil structural proteins). Furthermore, as further defined herein, the monomer consists of a shared sequence defined by NTL-helix 1, L, helix 2, and CT segments. Each helix segment is defined by a sequence of at least five heptagonal repeating elements (H1-5). Each heptagonal repeating element contains seven amino acids, with their side chain positions defined as “abcdefg”, as further defined herein. In addition to their sequence-based α-helical coil-and-coil structural features, A-ENA proteins are characterized by the presence of multiple amino acid residues geometrically positioned within the hydrophobic environment of the self-assembling antiparallel coil-and-coil helix. This facilitates the autocatalytic formation of one or more isopeptide covalent bonds within or with other A-ENA monomers, resulting in “functional” A-ENA proteins capable of spontaneously forming fibrous assemblies, as described herein.

[0090] Therefore, this invention provides a newly annotated family of bacterial proteins, defined as "A-ENA proteins"; this family of proteins is capable of spontaneously forming protein fibrils, with intermolecular cross-linking between fibrils via isopeptide bonds, which have been demonstrated herein to impart irreversible properties to these highly assembled fibrous structures. Further analysis of the fibrils recombined by exogenous introduction of Bti A-ENA into *E. coli*, based on the heptatomic repeating unit elements and coiled-helical monomer structures defined herein, yielded a matrix-based structural design for obtaining the protein nanofibrils described herein, thereby providing the required rigidity through autocatalytic association of at least one or more isopeptide bonds (IPBs) during their self-assembly. Although the wild-type A-ENA (SEQ ID NO:1) identified in this paper contains multiple IPBs in assembling these Bti protein filament structures connecting spores and parasporins, interestingly, mutations of all but one conserved acid / base autocatalytic residues revealed that nanofibrils could still spontaneously assemble as long as at least one IPB was formed between the A-ENA protein monomer subunits of the two precursor fibrils. This requirement for at least one (cross-fibril) IPB further supports the finding that even with low sequence homology among A-ENA structural homologs, as long as they conform to the sequence motifs provided for the heptadic repeat elements and contain at least one acid / base catalytic residue of an IPB in the heptadic repeat (…),… ), donor (δ) and acceptor ( If the positions, which respectively conform to E / D (as γ), K (as donor) and E / Q / D / N (as acceptor amino acid identity), generate at least one covalent isopeptide bond linking that provides the stability required for the assembly of protein nanofibrils, then they can still self-assemble into nanofibrils.

[0091] Therefore, a first aspect of the invention relates to a protein fibril constructed or composed of two precursor fibrils, wherein each precursor fibril contains two or more monomeric protein subunits based on or derived from A-ENA protein subunits or composed of A-ENA protein subunits, the monomeric protein subunits spontaneously folding into an α-helix antiparallel coil-and-coil structure monomer and having an amino acid sequence composed of protein fragments of the following formula: NTL-helix 1 (h1) - L-helix 2 (h2) - CT, wherein each fragment NTL, L and CT contains at least 1, 1 and 4 amino acids, respectively, and wherein fragments h1 and h2 (also referred to herein as helix α1 and helix α2) contain at least 5 heptaponic repeating elements to allow the formation of helical hairpins, and wherein the amino acids are defined as further detailed below, and wherein the monomeric protein subunits are interconnected by at least one or more isopeptide bonds (IPB). The self-assembling monomeric protein subunits are A-ENA proteins as defined herein, or A-ENA-based proteins or proteins derived from A-ENA protein subunits as further defined herein, wherein the protein fibrils thus form the basic A-ENA protein structural scaffold as defined herein, and when A-ENA or A-ENA-derived protein monomer sequences are used to form fibrils, they also have similar functions to wild-type A-ENA proteins in assembling into fibrils, but may contain modifications, such as insertions, mutations, fusions, or conjugations to A-ENA monomers (therefore referred to as "A-ENA-based" monomers or "A-ENA-derived" monomer sequences, i.e., functional homologs or functional variants, or engineered A-ENA proteins), said modifications being provided by engineered A-ENA monomer sequences or proteins, as further described herein.

[0092] Specific implementations may also involve protein nanofibrils constructed from two precursor fibrils, each precursor fibril comprising two or more monomeric protein subunits based on, derived from, or composed of A-ENA protein subunits. The protein nanofibrils are "A-ENA-based protein nanofibrils," meaning that, in addition to A-ENA protein subunits and / or their derivatives or variants, the fibrils may also contain other components from the production host, from the attached sporogenic culture, or other components intentionally attached to or in contact with the protein nanofibrils. These components may be synthetic materials, such as chemicals, plastics, surfaces, etc., or natural materials, such as other proteins, carbohydrates, lipids, LPS, metabolites, bacterial components, etc.

[0093] To clearly define the basic structural features of the A-ENA protein monomer subunits, based on the structural information of BtiA-ENA nanofibrils disclosed in this paper, features considered essential for folded proteins to achieve spontaneous fibril assembly were derived. These features were then validated through structural modeling of potential A-ENA orthologs, followed by recombinant production and self-assembly into fibrils. In fact, comparisons of A-ENA orthologs based on heptagonal repeating unit elements indicate that they participate in intrahelical and interhelical "convex-button-in-concave" interactions (respectively using...). and The positions of hydrophobic residues (labeled with hydrophobic residues and short chain residues) are highly conserved, and these interactions are essential for obtaining coiled-helical structures; simultaneously, the residues involved in the interhelical isopeptide bond units (labeled with...) , and The positions of labeled acid / base catalytic residues, nucleophilic residues, and electrophilic residues also show high conservation.

[0094] The protein nanofibrils of the present invention comprise or are composed of two precursor fibrils, each precursor fibril comprising at least two monomeric A-ENA or A-ENA-based protein subunits, said protein subunits comprising covalently linked amino acid sequence fragments according to the general formula NTL-helix 1(h1)-L-helix 2(h2)-CT, wherein said monomeric protein subunits spontaneously fold in aqueous solution into two α-helices, referred to as Helix 1(h1) and Helix 2(h2), which are antiparallel coiled-up α-helical structures as defined herein. Helices H1 and H2, or alternatively referred to as α-helices. 1 and 2. This document defines a continuous heptameric unit or “seven-membered repeat” or “seven-membered repeating unit” or “seven-membered repeating element” (H) containing amino acid residues with side chains designated as a to g, hence “abcdefg”. Each of the two helices contains a continuous sequence of at least five seven-membered repeating (H) elements, defining Helix 1 according to the general formula H1-1 - H1-2 - H1-3 - H1-4 - H1-5, and Helix 2 according to the general formula H2-1 – H2-2 – H2-3 – H2-4 – H2-5; wherein each seven-membered repeating element contains seven amino acid residues named “abcdefg”, each having a common sequence as defined below, thereby connecting in an antiparallel stacked contact manner through hydrophobic “button-in-hole” interactions to form a helical hairpin structure. This is the necessary mode to provide an antiparallel coiled helical structure and to generate a hydrophobic environment by providing said residues to autocatalytically form intermolecular isopeptide bonds in the presence of more monomers.

[0095] Therefore, in the A-ENA subunit, the primary amino acid sequences of the h1 and h2 helices of the NTL-h1-L-h2-CT formula determine the 3D structure to obtain the helical wheel diagram along the helical length, thereby assembling into a coiled helical structure, wherein IPB formation is triggered in the presence of other A-ENA or A-ENA-based monomers.

[0096] According to conventional understanding, coiled helices are typically formed by the folding of seven-membered repeating elements in an amino acid sequence, resulting in a tightly packed helical structure in a "knobs-in-hole" pattern. This structural feature is attributed to the systematic arrangement of residues in the "a to g" seven-membered repeating elements in the side chains, thus forming corresponding ridge and valley structures. Therefore, for those skilled in the art who possess the amino acid sequence, the spontaneous folding process of this monomeric coiled helical structure is predictable, for example, by applying predictive tools such as those described by Lupas et al. (2017; The Structure and Topology of α-Helical Coiled Coils. Subcell Biochem; 82: 95-129). Thus, determined by the conserved motifs in each subsequently defined seven-membered repeating element of the monomeric amino acid sequence, and using tools available in the art, those skilled in the art can infer the coiled helical structural features of the polypeptide. In this invention, due to the presence of intramolecular antiparallel coiled helical structures induced by the sequences h1 and h2 in the NTL-h1-L-h2-CT common sequence of the A-ENA protein, the monomer forms a helical hairpin, as defined herein.

[0097] As disclosed herein, the A-ENA monomeric protein subunits contained in the precursor fibrils and nanofibrils of the present invention were determined based on preliminary characterization after structural analysis of Bti fiber assemblies derived from spore cultures; thereby revealing that their main components are subunits provided by A-ENA protein sequences (SEQ ID NO: 1 and 2), which form fibers at the endospore junctions, thereby enabling the spores to be tightly bound to the parasporal bodies. Based on structural analysis results and protein sequences, to provide the coiled-helical basis for the self-assembled protein nanofibrils structure of this invention, this invention provides a monomeric protein sequence common protein, which is composed of an NTL-h1-L-h2-CT fragment, wherein the NTL, h1, L, h2, and CT fragments are interconnected by peptide bonds to form a polypeptide chain, and wherein helices 1 and 2 consist of at least five heptagonal repeating elements (H). The common sequence of each of these heptagonal repeating elements is provided herein to generate monomeric protein subunits of the A-ENA protein family as defined herein. These monomeric protein subunits are capable of forming antiparallel coiled-helices, which further assemble into precursor fibrils, which then entangle with each other to form the protein nanofibrils of this invention. Furthermore, the heptagonal repeating elements as defined herein contain the necessary structural features to facilitate spontaneous intermolecular heteropeptide interactions, thereby irreversibly linking the monomers within the aforementioned protein nanofibrils together.

[0098] The monomeric protein subunits of the protein nanofibrils presented in this article are referred to as α-helical-endospore appendages (A-ENA) polypeptides. These are a family of proteins derived from bacteria, particularly sporogenic bacteria, and most specifically from the genus *Bacillus*. In this article, they are defined as spontaneously folding monomeric protein subunits forming two α-helices assembled in an antiparallel coiled-coil structure, stacked in a "button-in-hole" manner. The amino acid sequence comprises fragments according to the general formula NTL-h1-L-h2-CT, which are covalently linked by peptide bonds to form a polypeptide chain. - NTL refers to an N-terminal lock or N-terminal amino acid sequence that contains at least one amino acid preceding the sequence that constitutes helix 1 and that provides a variable fragment; - L refers to a linker fragment that forms a loop or turn in the monomer's 3D structure and contains at least four amino acid residues located between helix 1 and helix 2, providing (through peptide bonds) a covalent link to provide an α-helix intramolecular coiled helix, and the linker is usually variable in its sequence; - CT refers to the C-terminal tail or C-terminus of the polypeptide of the A-ENA monomer, containing at least one amino acid residue following the sequence constituting helix 2, and providing a variable fragment; and - h1 and h2 refer to helix 1 and helix 2, respectively, also known as the α1 helix or α2 helix of the coiled helix, which is further defined by (at least) 5 seven-membered repeating elements (described as H1-5) present in each of helix 1 and helix 2, and is further defined for the A-ENA protein family as follows: Helix 1 comprises H1-1 -H1-2 -H1-3 -H1-4 -H1-5 or thereof, wherein the seven-membered repeating elements are defined by residues “abcdefg” and are linked in this specific order from the N-terminus to the C-terminus of the helix, and wherein the common amino acid sequence “abcdefg” of each seven-membered repeating element (H1-1 to H1-5) is or respectively corresponds to: - Seven-element repeating 1-1 of XXX- -XX- , - Seven elements repeating 1-2 -X- - - - -, - Seven elements repeating 1-3 -X- - - -X- , - Seven elements repeating 1-4 -N- - - - - δ, - Seven elements repeating 1-5 - -X- - -XX, Helix 2 comprises H2-1 -H2-2 -H2-3 -H2-4 -H2-5 or thereof, wherein the seven-membered repeating element is defined by residues “abcdefg” and linked in this specific order from the N-terminus to the C-terminus of the helix, and wherein the common amino acid sequence “abcdefg” of each seven-membered repeating element is or respectively corresponds to: - Seven-element repeating 2-1 -XX- - -X- , - Seven elements repeating 2-2 -XX- -XXX, - Seven elements repeating 2-3 times -XX- - -δ-δ, - Seven elements repeating 2-4 - - - - -X-δ, - Seven elements repeating 2-5 - -X- - -XX, Furthermore, the shared amino acid sequence residues are defined as follows: For at least 70% of the seven repeating elements specified therein Location," "Represents a hydrophobic amino acid residue selected from M, V, I, L, A, G, H, W, Y, F; preferably selected from L, M, I, V, F, W; “ "" indicates a short side-chain amino acid residue selected from V, C, G, A, P, S, T, N, D; preferably selected from amino acids A, G, S, and T; “γ” is an amino acid residue, selected from E or D, that corresponds to at least one or more of the γ positions shown in the seven-membered repeating element and serves as an isopeptide interaction of an acid / base catalytic residue; preferably, the amino acid representing γ is E. “δ” is an amino acid residue corresponding to at least one or more of the δ positions shown in the seven-membered repeating element, which serves as a “donor” or nucleophilic residue for heteropeptide interaction, preferably a lysine residue (K). “ "is corresponding to at least one or more of the seven-element repeating elements shown in the original text. The position of the amino acid residue used as a "receptor" or electrophilic residue for heteropeptide interaction is selected from the E, Q, D and N tables; preferably selected from E, Q and N. Furthermore, X represents an amino acid, which can be any type of amino acid residue known in the art, and its identity does not limit the function of the A-ENA protein.

[0099] Therefore, the amino acid “X” or “any type of amino acid residue” as used herein can be defined as any of the 20 naturally occurring amino acid residues commonly known and listed herein (hereinafter), along with its different isomers or enantiomers and its synthetic analogs or variants. Amino acids are represented herein by their 3- or 1-letter codes, as defined and provided by the IUPAC-IUB Joint Committee on Biochemical Nomenclature (Nomenclature and Symbolism for Amino Acids and Peptides). All amino acids mentioned herein are represented by their three-letter or single-letter abbreviations, which are derived from the IUPAC-IUB Joint Committee on Biochemical Nomenclature (Nomenclature and Symbolism for Amino Acids and Peptides. Eur. J. Biochem. 138: 9-37). (1984) defines and provides the following: 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), histidine (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).

[0100] In another specific implementation plan, " indicates that it corresponds to the seven-element repeating element shown in the figure. At least 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the position consists of hydrophobic amino acid residues selected from M, V, I, L, A, G, H, W, Y, and F.

[0101] In a specific implementation, the monomeric protein subunits are interconnected in the protein nanofibers described herein via a single IPB in each monomer, defined as " “δ” and “ The positions of “” will respectively provide the corresponding acid / base catalytic residues, donor residues, and acceptor residues, at least in H2-5 “ The above is true, where the "γ" site is E or D; at least H2-3 "δ" provides lysine, and at least H2-4 " "Provides E, Q, D, or N receptors, thereby allowing for the covalent formation of cross-linked precursor fibrils (see...") Figure 25 In this case, the motifs in the seven-member repeating motif are defined as "γ", "δ", and "". The residue position of “X” can be any amino acid (defined herein as “X” above), preferably an amino acid that does not spatially impede folding or fibril formation, for example, for acid / base catalytic residues, Q replaces E or D.

[0102] In a more specific embodiment, the monomeric protein subunits of the protein nanofibrils described herein are covalently interconnected with other monomers via multiple IPBs of each monomer, wherein the monomer sequence comprises the "γ", "δ", and "" subunits in its heptagonal repeating elements. "The residues are defined to provide acid / base catalytic residues, donor residues, and acceptor residues, respectively, which are linked between one or more of the following specific side chain residues to form the IPB (also as Figure 7 (As shown) Required: - Between NTL of (i) and H1-5b of (i-5), (i-4), (i-3) or (i-2); - Between H1-4g of (i) and H2-2a of (i-1); - Between H2-3g of (i) and H2-4a of (i-1); - Between H2-3f of (i) and H2-4e of (i'); - Between H2-4g of (i) and H1-3a of (i-1); - Between H2-4g of (i+1) and H1-3a of (i); - Between the NTL of (i+5), (i+4), (i+3) or (i+2) and H1-5b of (i); - Between H1-4g of (i+1) and H2-2a of (i); - Between H2-3g of (i+1) and H2-4a of (i); and / or - Between H2-3f of (i'+1) and H2-4e of (i).

[0103] like Figure 7 As shown, the "γ", "δ", and "" positions required to form the IPB between some or all of these IPB connections. "The residues are therefore present in these monomers as catalytic residues E or D, as donor lysine, and as acceptors E, Q, D, or N. For monomers, all 'γ', 'δ', and 'N' that are not seven-membered repeating elements are present." "The residues all participate in IPB linkage, therefore, the position may also contain alternative amino acids that do not have catalytic residue (e.g., Gln), donor or acceptor functions; that is, it can be any type of amino acid, preferably an amino acid whose side chain structure is similar to the side chain structure of an amino acid that has catalytic residue, donor or acceptor functions."

[0104] In other embodiments, the protein nanofibrils comprise or consist of two precursor fibrils, each precursor fibril comprising at least two A-ENA monomers as defined above, and wherein the helix 1 and helix 2 of the monomeric protein subunits comprise or consist of a common sequence of seven-membered repeating elements according to the following general formula, or are defined by a common sequence of seven-membered repeating elements according to the following general formula: - Spiral 1, containing XXX - -XX- - -X- - - - - -ε-X- - - -X- - -N- - - - -δ- - ε-X- - -XX as well as - Spiral 2, containing -XX- - -X- - ε-XX- -XXX- -XX- - -δ-δ- ε- - - - ε-X- δ- - -X- - -XX, The shared amino acid sequence residues are defined as follows: - It is a hydrophobic amino acid residue, selected from M, V, I, L, A, G, H, W, Y, F; preferably selected from L, M, I, V, F or W; - It consists of short side-chain amino acid residues, selected from V, C, G, A, P, S, T, N, and D; preferably A, G, S, or T; - the At least one of the positions is an amino acid residue selected from E or D that serves as an acid / base catalytic residue for heteropeptide interaction; preferably E; the remaining... Each residue can be any type of amino acid residue; - At least one of the δ sites is an amino acid residue selected from K that acts as a "donor" or nucleophilic residue in an isopeptide interaction; the remaining δ residues can be any type of amino acid residue. - the At least one or more of the positions are amino acid residues selected from E, Q, D, or N that act as "receptors" or electrophilic residues in heteropeptide interactions; preferably E, Q, or N; the remaining... The residue can be any type of amino acid residue; - X can be any type of amino acid residue.

[0105] In a specific implementation, the A-ENA protein monomer initially characterized herein is used to form the isolated protein nanofibrils of the present invention. Thus, the isolated protein nanofibrils are obtainable from a host, wherein the A-ENA protein is recombinantly generated by expressing a nucleic acid molecule encoding the A-ENA protein, and / or wherein the A-ENA protein monomer is provided as a monomer subunit selected from the A-ENA protein whose protein sequence is represented by any of the accession numbers listed in Table 3.

[0106] This list, containing 593 accession numbers, represents 593 different proteins, which are categorized herein as A-ENA(like) proteins or A-ENA protein orthologs. This list was compiled through analysis of structurally identified Bti A-ENA proteins, combined with the detection results of potential orthologs in Example 7 (SEQ ID NO: 3-5 is also used as an example herein). Based on the shared sequence motifs of the NTL-h1-L-h2-CT fragment and the seven-membered repeat element described herein, this list provides a self-assembling protein unit capable of spontaneously forming fibrils and achieving irreversible crosslinking through the presence of one or more autocatalytically formed isopeptide bonds, preferably forming cross-linked precursor fibrils.

[0107] Furthermore, alignments of the A-ENA protein family (including members with accession numbers provided in Table 3) resulted in pairwise sequence identity of only 20-30% for the entire sequence (NTL-h1-L-h2-CT). Despite the low sequence conservation, as discussed herein, the shared motifs structurally necessary for self-assembly and IPB formation are conserved; in particular, the heptate repetition element residues located in helix 1 and helix 2 provide the foundation for establishing a methodology to determine whether a protein sequence can be classified as A-ENA or A-ENA-like protein. This paper defines “A-ENA-like protein” as an A-ENA protein conforming to the general formula NTL-h1-L-h2-CT and containing a scaffold motif; wherein the heptate repetition element is defined as described herein, and although the lengths of its NTL, L, and CT regions may vary naturally, and the protein itself may contain certain modifications or additions, these variations, modifications, or additions do not affect the scaffold structure or self-assembly structural features of A-ENA. In fact, the data in Tables 1 and 2 contain Hidden Markov Model (HMM) spectra, which are constructed for helices 1 and 2 of the A-ENA monomer composed of the general formula NTL-h1-L-h2-CT as defined herein. The HMM calculations are based on the 593A-Ena(sample) sequence and sequence identifiers of the 35-residue-length HMM spectra of helices 1 and 2, respectively, and should be interpreted, as Wheeler et al., (2014), as: "The Hidden Markov Model is shown by drawing a stack of letters for each position, where the height of the stack corresponds to the conservatism of that position, and the height of each letter in the stack depends on the frequency of that letter at that position."

[0108] In a specific implementation, the A-ENA protein monomer subunit is defined by an amino acid sequence according to the general formula NTL-h1-L-h2-CT, wherein NTL, CT, and L each contain at least 1, 1, or 4 amino acids, and h1 and h2 provide helix 1 and helix 2, respectively, each containing or substantially composed of five heptagonal repeating elements H1, H2, H3, H4, and H5 according to the common motif provided below. The heptagonal repeating elements composed of residues ABCDEFG can be defined more specifically as follows: - Seven-element repeating 1-1(H1-1) of XXX- -XX- , - H1-2 -X-(S or T)- -(A or G)- -(E or Q), - EXX of H1-3- -X-(H or N)- , - H1-4 -N-(A or G or S)-E-(G or A)-EK, - H1-5 -QX- - -XX, - H2-1 -XX- - -X- , - H2-2 of (N or Q or D)-XX- -XXX, - H2-3 -XX- - -(K or X)-(K or X), - H2-4 (E or Q) - - -(L)-(Q or X)-XK, - H2-5 - -X- - -XX, The common amino acid sequence residues of the seven-membered repeating element are defined as follows: - It is a hydrophobic amino acid residue, selected from M, V, I, L, A, G, H, W, Y, F; preferably selected from L, M, I, V, F or W; - It consists of short side-chain amino acid residues, selected from V, C, G, A, P, S, T, N, and D; preferably A, G, S, or T; - The amino acid residues that serve as acid / base catalytic residues for heteropeptide interactions are selected from E or D; preferably E. - The amino acid residues that act as "receptors" or electrophilic residues in heteropeptide interactions are selected from E, Q, D, or N; preferably E, Q, or N. - X can be any type of amino acid residue. - The single-letter code for amino acid residues is shown in the figure. - and the amino acid in parentheses provides alternative options for the “abcdegf” residue at the corresponding position (indicated by “or”).

[0109] In other specific embodiments, the A-ENA protein monomer subunit is defined by the NTL-h1-L-h2-CT common formula as defined herein, and is further specifically defined by the following h1 and h2 polypeptide sequences, namely comprising or substantially consisting of the seven-membered repeat 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, and H2-5 is LEEAKSL (corresponding to SEQ ID NO: 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 SEQ ID NO: 34-43 respectively). It is a seven-membered repeat element sequence “abcdefg”, which corresponds to those seven-membered repeat elements present in the A-ENA protein of Bacillus thuringiensis subspecies Israel ATCC35646 (defined by UniprotKB Q8KNV8-SEQ ID NO: 1 and UniprotKB Q8KNV7-SEQ ID NO: 2, respectively); Furthermore, the monomer spontaneously folds into an antiparallel coiled-coil α-helix structure, and allows for autocatalytically induced isopeptide bond formation during further assembly into a fibrous structure.

[0110] In this paper, the fragments NTL, CT, and L are used to define the N-terminal NTL preceding helix 1, the C-terminal CT following helix 2, and the polypeptide sequence L, the internal linker sequence connecting helix 1 and helix 2, respectively. The minimum number of amino acid residues constituting these fragments is defined as follows: NTL and CT fragments each contain one amino acid, while the linker contains at least four amino acids. The maximum number of amino acids is not limited; the only requirement is that the A-ENA monomeric protein should spontaneously fold into an antiparallel α-helix coil-and-coil structure, allowing it to further self-assemble into precursor fibrils and ultimately nanofibrils.

[0111] Observations have shown that NTLs play a role in the covalent linkage of protein nanofibrils, thus acting as a "lock" for the fibrous structure; therefore, based on its sequence, this fragment can be used to regulate the structure of the fibrils. In specific embodiments, the length of the NTL sequence is at least 1 and at most (or maximum) 3 amino acids; or at least 1 and at most 4 amino acids; or at least 1 and at most 5 amino acids; or at least 1 and at most 6 amino acids; or at least 1 and at most 7 amino acids; or at least 1 and at most 8 amino acids; or at least 1 and at most 9 amino acids; or at least 1 and at most 10 amino acids; or at least 1 and at most 11 amino acids; or at least 1 and at most 12 amino acids; or at least 1 and at most 13 amino acids; or at least 1 and at most 14 amino acids. The NTL may contain at least one and at most 15 amino acids; or at least one and at most 16 amino acids; or at least one and at most 17 amino acids; or at least one and at most 18 amino acids; or at least one and at most 19 amino acids; or at least one and at most 20 amino acids; or at least one and at most 21 amino acids; or at least one and at most 22 amino acids; or at least one and at most 23 amino acids; or at least one and at most 24 or more amino acids; or at least 2, 3, 4, 5, 6, or 7 amino acids, and at most 3, 4, 5, 6, 7, or 8 or more amino acids. The total length or number of amino acids in the NTL may include or exclude the amino acid sequence of an additional tag, protein domain, or peptide attached to the A-ENA NTL fragment to provide a modified A-ENA or an A-ENA-based monomeric protein.

[0112] The C-terminal tail (CT) provides a segment structurally exposed on the surface of a monomer or protofibril, and is therefore suitable for attachment, linkage, or fusion to other parts, such as protein domains, tags, or other molecules. Thus, in another specific embodiment, the length of the C-terminal tail sequence is at least 1 and at most (or at most) 2 amino acids; or at least 1 and at most 3 or 4 amino acids; or at least 1 and at most 5 amino acids; or at least 1 and at most 6 amino acids; or at least 1 and at most 7 amino acids; or at least 1 and at most 8 amino acids; or at least 1 and at most 9 amino acids; or at least 1 and at most 10 amino acids; or at least 1 and at most 11 amino acids; or at least 1 and at most 12 amino acids; or at least 1 and at most 13 amino acids; or at least 1 and at most 14 amino acids. The total length or number of amino acids in the CT may include or exclude amino acid sequences of additional tags, protein domains, or peptides attached to the A-ENA CT fragment to provide a modified A-ENA or an A-ENA-based monomeric protein.

[0113] The linker (L) sequence appears in the structure as a loop or turn, providing a covalent connection between the two helices of the hairpin and allowing, in parallel, the insertion or attachment of other proteins, molecules, or tags to the A-ENA protein. Therefore, in a specific embodiment, the linker sequence located between the helix 1 and helix 2 sequences is at least 4 amino acids long and at most (or at most) the number of amino acids that produce a 3D structure that prevents helix 1 and helix 2 from forming a coiled helix. 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 in the linker may or may not include an amino acid sequence of an additional tag, protein domain, or peptide inserted into the A-ENA linker fragment to provide a modified A-ENA or an A-ENA-based monomeric protein.

[0114] As described herein, the A-ENA-based monomeric protein subunits constituting the protein nanofibrils of the present invention provide A-ENA proteins with sequences based on or derived from primitive bacterial A-ENA proteins; however, certain portions of the common sequence of these segments can be adjusted, modified, or altered by mutation, substitution, addition, or deletion of other amino acids, peptides, or proteins to obtain functionalized A-ENA proteins or fibrous structures composed thereof. In some embodiments, heterologous amino acid sequences or proteins or tags can be fused, added, or inserted into any of the NTL, CT, or L fragments, resulting in A-ENA-based protein monomers or modified or engineered A-ENA, ultimately providing functionalized proteins that self-assemble into functionalized protein fibrils and / or fibrils with modified surfaces.

[0115] Specifically, the N-terminus of A-ENA or an A-ENA-based monomeric protein subunit may be further defined as an amino acid sequence of at least one amino acid, preferably at least seven amino acids, wherein the NTL fragment contains an amino acid sequence defined as M- - -ZX- -P is a common sequence or is composed of it, where: - The amino acid residues are short side chains, selected from V, C, G, A, P, S, T, N, and D; preferably A, G, S, or T. - It is a hydrophobic amino acid residue, selected from M, V, I, L, A, G, H, W, Y, F; preferably selected from L, M, I, V, F or W; - X = any amino acid residue; - Z stands for Ala or proline, and M and P are the single-letter codes for the amino acids methionine and proline, respectively.

[0116] In other specific embodiments, the NTL segment includes segments defined as M- - -ZX- -P is a common sequence or is composed of it, where

[0117] - Selected from S, G, or T; - These are hydrophobic amino acid residues selected from M, V, I, L, A, G, H, W, Y, and F; - X = any amino acid residue; - Z stands for Ala or proline, and M and P are the single-letter codes for the amino acids methionine and proline, respectively.

[0118] A-ENA proteins with an NTL defined by the shared motif provide the option to form an IPB, wherein the donor or nucleophilic residue is provided by an N-terminal free amino group (NH2-) present on the peptide backbone. The shared NTL motif forms a structural unit that facilitates the NTL stacking on the protofibril surface, thereby helping the N-terminal amine to be oriented and spaced optimally for the formation of an isopeptide bond.

[0119] In a specific implementation, the NTL comprises or consists of MG-(M or I)-P-(T or N)-IP, wherein a single-letter code is applied to each amino acid, the amino acid in parentheses provides an alternative (indicated by "or") at the position, and wherein the NTL may contain other amino acids, such as those NTL fragments of SEQ ID NO:1 and SEQ ID NO:2, which also contain EGLDIT (SEQ ID NO:44) as part of the NTL.

[0120] In some embodiments, an A-ENA monomeric protein subunit comprising an amino acid sequence corresponding to or composed of the general formula NTL-h1-L-h2-CT described herein, comprising other amino acid residues located at the N-terminus and / or C-terminus of the h1 and / or h2 helical sequences at the seven-membered repeat elements H1 to H5, but not part of the NTL, CT, or linker region, wherein helix 1 and helix 2 therefore correspond to the general formula of helix 1: [X] n1 -H1-1 -H1-2 -H1-3 -H1-4 -H1-5 –[X] n2 , and / or the general formula corresponding to helix 2: [X] n1 -H2-1 -H2-2 -H2-3 -H2-4 -H2-5 –[X] n2 The seven-membered repeating element is defined by residues “abcdefg”, covalently linked in the aforementioned order as described herein, and wherein [X] n The additional amino acid present as part of the helical structure h1 and / or h2, wherein X can be any amino acid, and n1 and n2 refer to the number of different amino acids of the helix before or after the seven-membered repeating element, wherein X in the n amino acids can be the same or different to result in helix 1 or helix 2 polypeptide chains forming the α-helix, respectively.

[0121] In the specific implementation scheme, the [X] of h1 n1 It is a single arginine (R), leucine (L), asparagine (N), or lysine (K) residue, and / or helix 1 [X]. n2 It contains or is composed of 3 amino acids, such as FEK or FKK, and / or a 2-helix [X]. n2 It contains or is composed of 3 amino acids, such as IQS or LKL.

[0122] A-ENA protein fiber

[0123] This invention relates to protein fibrils, preferably nanofibrils, wherein “nanofibrillar” is defined herein as a fibrous assembly with a diameter on the nanometer scale and a length significantly up to several micrometers. The terms “fibril,” “filament,” “fibrous assembly,” or “fibrous structure,” as used interchangeably herein, refer to structured biological compounds, such as protein assemblies or protein-based assemblies, preferably composed of protein materials, forming elongated, ordered structures with diameters up to 100 nanometers, and possibly as part of a larger hierarchical structure.

[0124] Given the terminology used herein to define (nanofibrillar) fibers, we refer to "fiber" herein as a potential plurality of nanofibers, where a fiber is generally considered to represent a structure with a larger diameter (micrometer to millimeter scale) compared to a fibril, and where the "fiber" therefore preferably constitutes a higher-order hierarchical structure formed by the aforementioned plurality of fibrils. In a specific embodiment, such a fiber comprises a plurality of A-ENA nanofibers, wherein each fibril is further laterally bonded to another fibril to provide a structured fiber. Another example of a fiber is a woven assembly of protein nanofibers.

[0125] Nanofibrils possess unique and intriguing properties, making them an important subject of research and application in various fields such as microbiology, biomechanics, and materials science. This invention provides protein nanofibrils obtained through the spontaneous entanglement of two precursor fibril structures. The term "protofibril" refers to a fibrous self-assembly of a multimer or polymer, essentially composed of monomeric protein subunits described herein, referred to as A-ENA proteins or A-ENA-based proteins, containing protein fragments of the general formula NTL-helix 1(h1)-L-helix 2(h2)-CT as further detailed herein, interconnected by at least one or more isopeptide bonds. Specifically, the precursor fibrils are composed of functional A-ENA proteins or their orthologs; functionality refers to their ability to self-assemble as coiled helical structures into entangled precursor fibril structures as described herein to form nanofibrils.

[0126] Previously, it has been reported that based on Bacillus cereus ( Bacillus cereusThe self-assembled S-ENA and L-ENA proteins of endospore appendages (Pradhan et al., 2021; Remaut et al., WO2022 / 029325) can also form self-assembled proteins that polymerize and assemble into protein fibrils. These fibril structures based on S-ENA and L-ENA proteins actually provide nanofibril structures in the sense of this invention, wherein the S-ENA and L-ENA fibrils are composed of multiple polypeptide chains that behave as helical polymers or disks, rather than stacked into helical (S-ENA) or ladder-like (L-ENA) polymer assemblies or fibrils, wherein the polymers of the fibrils are interconnected by disulfide bonds as covalent intermolecular rigid connections. Although Pradhan et al. (2021) and Remaut et al. (WO2022 / 029325) mention S-ENA or L-ENA “fibers”, we have chosen to redefine these structures as nanofibers or “fibers” in order to better align with the conventional usage of the term fiber, which in many fields usually refers to an aggregate of multiple fibers.

[0127] In this invention, the α-helical endospore appendage protein, referred to as A-ENA protein and first mentioned in Bacillus thuringiensis, differs significantly in its protein structure from the previously discovered S-ENA and L-ENA proteins. Similarly, for the A-ENA protein, the structural appearance of the resulting assembled fibrils is determined or driven by the amino acid sequence of monomeric proteins that not only self-assemble into monomers forming precursor fibrils, but also that these monomers are intermolecularly linked by one or more isopeptide bonds. The resulting A-ENA multimeric assemblies have a (precursor) fibrous structure, while the S-ENA and L-ENA multimeric assemblies form helical arcs or disks, respectively, which can be visually distinguished from the fibrous structure (under a microscope). Due to the structural differences, nanofibrils composed of S-ENA or L-ENA proteins are respectively considered as multiple stacked helical arcs or disks, which further extend into fibrous assemblies covalently interconnected by disulfide bonds between different polymeric assemblies; while nanofibrils composed of A-ENA proteins contain at least two entangled α-helical precursor fibrils covalently linked by isopeptide bonds.

[0128] One of the advantages of using the fiber materials for at least some biotechnological applications is that the formation of isopeptide bonds is irreversible, thus providing extremely high tensile strength to the assembled protofibrils and excellent mechanical and thermal stability to protein nanofibrils. Even protofibrils based on S-ENA and L-ENA are considered to be rigid and stable bionanomaterials, and the properties of isopeptide covalent linkages are stronger than those of disulfide bonds.

[0129] From a phenotypic perspective, A-ENA protofibrils can be easily distinguished from any other protofibrils in bacterial or, more specifically, genus *Sporobacter* endospore samples through simple microscopic analysis. In fact, when observing 2D images of the microscopic analysis as shown in this paper, for example in… Figure 2 In contrast to the fibrous structures described for, for example, S-ENA and L-ENA proteins, A-ENA nanofibrils reveal a highly ordered and unique fibril structure (see Pradhan et al., 2021 or Remaut et al., WO2022 / 029325).

[0130] In one embodiment described herein, the protein nanofibrils comprise or consist of two precursor fibrils, each precursor fibril comprising or consisting of at least two monomeric protein subunits, wherein each monomeric protein subunit comprises two helices h1 and h2, covalently interconnected fragments according to the general formula NTL-h1-L-h2-CT, spontaneously folding in aqueous solution into an α-helical antiparallel coil-and-coil structure, and each helical comprises five or more heptavalent repeating elements conforming to the general formulas H1 to H5 described above herein, according to a shared sequence provided for its “abcdefg” sidechain format; and wherein L, NTL, and CT are defined as described herein; and wherein the monomeric protein subunits interact with each other through at least one or more isopeptide bonds. In a specific implementation, the nanofibrils are therefore composed of A-ENA monomers, wherein the A-ENA monomers are as defined above, and / or wherein the A-ENA monomers may also be A-ENA-based monomers, which are defined as containing one or more modified A-ENA proteins required for functionalizing (nanofibrillar)fibrils, although the functionality of self-assembling into protein nanofibrils is thus preserved.

[0131] Therefore, the fibrils of the present invention are preferably formed from two precursor fibrils, each of which has at least two or more A-ENA 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, or 400 or more A-ENA or A-ENA-based monomers. Thus, the basic structure of the protein nanofibrils described herein comprises two precursor fibrils, each with at least two monomers, and is therefore a tetrameric structure. In embodiments where the protein nanofibrils comprise two precursor fibrils or each with at least three monomers, a hexameric structure is provided, and so on. The A-ENA monomers or A-ENA-based monomers of the precursor fibrils may be the same or different, as further described herein.

[0132] To visualize the 3D structure of the protein nanofibrils, the A-ENA monomers in the fibrils are referred to as i and i' in the precursor fibrils f and f', respectively, forming intertwined helical fibrils. For fibrils further stacked towards the C and N poles of the fibrils, incremental (i.e., i+1; i+2, i+3, etc., or i'+1; i'+2; i'+3, etc.) or decremental (i.e., i-1, i-2, i-3, etc., or i'-1; i'-2; i'-3, or) numbering is used. Herein and throughout this application, the A-ENA fibrils are oriented in the figures with the C-terminus of their subunits facing upwards (referred to as the C-terminus or fibril tip, i.e., the C pole), and the N-terminal extensions or ends of the subunits facing downwards (referred to as the N-terminus or the barbed end of the fibril, i.e., the N pole).

[0133] In other specific embodiments, the protein nanofibrils are isolated protein nanofibrils, where "isolated" means that the nanofibrils do not contain other compounds, such as those derived from bacterial spore cultures or from bacterial components such as parasporins, spores, or other bacterial-derived materials. In fact, it is impossible to extract or isolate A-ENA fibrils from naturally occurring bacterial endospores in vitro. This is believed in retrospect to be due to their unique structure and the presence of isopeptide bonds. Since A-ENA nanofibrils cannot be obtained "as is" from bacterial spores, i.e., fibrils without any other bacterial components (e.g., without attached parasporins or spores) cannot be isolated, the protein nanofibrils of this invention are isolated or obtained by other means, such as through recombinant fibrils, thereby achieving isolation without the need for a spore formation process or the presence of related bacterial components. Therefore, in specific embodiments, the protein nanofibrils of this invention, also referred to as A-ENA protein nanofibrils or A-ENA-based protein nanofibrils, are recombinant A-ENA nanofibrils or A-ENA-based nanofibrils.

[0134] Isopeptide bonds (IPB)

[0135] This invention relates to protein nanofibrils as described herein, which have extremely high strength and stability, attributed to the spontaneous formation of covalent bonds, i.e., isopeptide bonds, between the monomeric protein subunits in the precursor fibrils and one or more other monomeric protein subunits in the protein fibrils.

[0136] An isopeptide bond is an amide bond formed between a carboxyl / carboxamide group and an amino group, wherein at least one of the carboxyl or amino groups is located outside the protein backbone. Unlike common peptide bonds (true peptides), isopeptide bonds can form covalent links between (different) polypeptide backbones. Although such covalent interactions are rare, they are well-known in certain specific contexts, such as in post-translational modifications like ubiquitination, where the C-terminal glycine residue of the ubiquitin molecule interacts with the side chain of a lysine residue in the substrate protein, forming an intermolecular isopeptide bond; or, as in the glutathione molecule, an isopeptide bond exists between the side chain of the glutamate residue and the amino group of the cysteine ​​residue, formed internally by autocatalysis. Isopeptide bonds are chemically irreversible under biological conditions and are resistant to most proteases. Isopeptide bonds can be formed by enzymatic catalysis; however, they can also form spontaneously or autocatalytically, and these terms are used interchangeably in this paper. The spontaneous formation of isopeptide bonds was first observed in bacteriophage HK97. The HK97 capsid structure was found to be assembled through subunit polymerization. These subunits undergo covalent cross-linking. The mechanism is that autocatalytic isopeptide bond formation occurs between the Lys and Asn side chains in adjacent subunits. These covalent bonds form covalently assembled 5-membered and 6-membered rings, which catenate with each other during capsid assembly, ultimately forming a structure called protein chainmail (Wikoff et al., 2000; Science 289, 2129-2133).

[0137] Spontaneous isopeptide bond formation typically occurs after protein folding, via the nucleophilic attack of the ε-amino group of lysine on the Cγ group of asparagine, promoted by nearby glutamate.

[0138] As used herein, the term "spontaneous" refers to a bond, such as an isopeptide bond or covalent bond, that can form within or between proteins without the presence of any additional reagents or enzymes, and / or without chemical modification of the proteins, such as the absence of natural chemical linkages or chemical couplings. Thus, "spontaneous isopeptide bonds" or "autocatalytically formed isopeptide bonds" are formed when one or more proteins exist alone in their isolated state (intramolecularly), or when two proteins coexist in their isolated state (without additional reagents or chemical modification). Therefore, spontaneous isopeptides or covalent bonds can form on their own without the presence of enzymes or other exogenous substances or without chemical modification. However, in particular, spontaneous isopeptide bonds or covalent bonds may require the presence of "autocatalytic residues," such as glutamate or aspartic acid residues, which are part of the polypeptide chain of the isopeptide bond-forming protein to allow bond formation in a proximity-induced manner.

[0139] The key to the autocatalytic formation of the interchangeable isopeptide crosslinks, isopeptide bonds, or isopeptide interactions described in this paper lies in the conservation of three amino acids: one acts as a nucleophilic donor, one as an electrophilic acceptor, and the other as an important acidic Glu or Asp residue in the catalytic process, mediating proton transfer. The necessity of the latter and the specific positions of the residues in hydrophobic environments near the active sites were observed in various immunoglobulin-like fimbriae of Gram-positive bacteria, where these internal crosslinks provide stability against chemical, thermal, and mechanical stresses. The hydrophobic environment is crucial because it alters the pKa value of the amino acid side chains, leading to an antiprotonated state where the lysine or donor residue side chains are unprotonated, and the Glu / Asp side chains are protonated (Kang and Baker, 2011- Trends Biochem.Sci.36, 229-237; Hu et al., 2011- J Am Chem Soc.133(3):478-85). Furthermore, the formation of isopeptide bonds is proximity-induced, therefore the three residues involved in the reaction must have a suitable geometry. Protein nanofibrils, as described herein, consist of coiled-helical monomeric protein subunits, wherein the positions of the three residues satisfy the geometry required for the formation of autocatalytic intermolecular isopeptide bonds during self-assembly into precursor fibrils and further entanglement to form nanofibrils, as described herein. Indeed, A-ENA or A-ENA-based protein monomers, as defined above, contain the necessary structural constraints within their seven-membered repeating elements, enabling them to first assemble into an α-helical antiparallel coiled-helical structure, thus providing the required hydrophobic environment and geometry to allow proximity-induced isopeptide linkage when other monomers are present as precursor fibril subunits. Therefore, in addition to the seven-membered repeating element constraints required for coiled-helical assembly, the A-ENA protein sequence is also highly conserved in the positions containing donor, acceptor, and catalytic residues to form isopeptide bonds, as observed in protein nanofibrils.

[0140] Therefore, after assembling the monomer coiled helical structure, the precursor protofibrils assemble into a polymeric fibrous structure, and heteropeptide bonds are formed between the monomers of the precursor protofibrils and between the monomers of the second precursor protofibrils that are already entangled with the first precursor protofibrils, resulting in tightly packed, highly irreversible protein nanofibrils. Specifically, the mechanism of autocatalytic heteropeptide bond formation includes: first, the Cγ carbonyl group of the acceptor or electrophilic residue (preferably Asn / Asp) is polarized by a protonated catalytic Glu / Asp residue through hydrogen bonding; subsequently, the unprotonated Lys ε-amino group in the donor residue nucleophilically attacks the polarized Cγ carbonyl group in the acceptor residue, thereby forming a tetrahedral intermediate; immediately afterward, a proton is transferred from the Lys ε-amino group to the catalytic Glu / Asp residue, while another proton is transferred from the catalytic Glu / Asp residue to the leaving group of the acceptor residue. Subsequently, depending on the nature of the receptor residue side chains, the formation of Lys-Asn isopeptide bonds leads to the release of NH3 molecules, while Lys-Asp bonds lead to the release of H2O molecules (Kang and Baker, 2011).

[0141] Therefore, the protein nanofibers of the present invention provide a protein structure in which isopeptide bonds are formed intermolecularly, i.e., between two protein molecules. Typically, isopeptide bonds are formed between a lysine residue and a glutamine, glutamic acid, asparagine, or aspartic acid residue, or the terminal carboxyl group of a protein; or between the α-amino terminus of a protein and asparagine, aspartic acid, glutamine, or glutamic acid. Therefore, the isopeptide bonds provided in the protein nanofibers described herein can be formed between a lysine residue and an asparagine residue, or between a lysine residue and an aspartic acid residue. In particular, isopeptide bonds can occur between the side-chain amine of lysine and the carboxamide group of asparagine. Each residue in this residue pair involved in the formation of the isopeptide bond is referred to herein as a reactive residue.

[0142] The presence of covalent (especially heteropeptide) interactions within or between proteins can be determined by bioinformatics sequence analysis, or preferably by mass spectrometry or structural determination, such as by X-ray crystallography, Cryo-EM or NMR.

[0143] As a method for covalently linking amino acids, spontaneously formed isopeptide bonds are typically observed intramolecularly, but when they occur intermolecularly, as in the case of protein nanofibrils described herein, this is an advantageous method for cross-linking molecules. Compared to other methods of covalently linking peptides, spontaneously formed isopeptide bonds have the following characteristics: they do not require enzyme catalysis or cofactors; they are expected to form specifically because three reactive residues need to be correctly conformated within a hydrophobic environment; they are chemically extremely stable because they are amide bonds; they are generally resistant to proteolytic degradation because these bonds are located outside the protein backbone; and finally, their formation is redox-independent and is an irreversible interaction.

[0144] These advantages may have played an important role in evolution for bacteria that lack disulfide bond formation "mechanisms" to enhance, for example, export proteins (Dutton et al., 2008 - Proc. Natl. Acad. Sci. US A 105, 11933-11938), since many Gram-positive bacteria have been shown to use intramolecular isopeptide bonds as a means of stabilizing protein structures, which is also physiologically important for outer membrane proteins in these bacteria (Dutton et al., 2008; Hu et al., 2011 - J Am Chem Soc. 133(3):478-85).

[0145] In a specific implementation, the protein nanofibrils described herein, wherein the monomeric protein subunits, preferably A-ENA monomeric protein subunits, are numbered i, i+n, and in respectively in the direction toward the C or N pole of the fibril f (for the monomeric units of the precursor fibril f', they are numbered i', i'+n, and i'-n sequentially), each monomer i, i+ / -n, i', and i'+ / -n is covalently linked to at least one other monomer (i, i+ / -n, i', and / or i'+ / -n) through the presence of isopeptide bonds. More preferably, each monomer is covalently linked to one or more IPBs. One or more other monomers of the protein nanofibrils are covalently linked; more preferably, each monomer is covalently linked to one other monomer of the protein nanofibrils through the presence of one IPB; more preferably, each monomer is covalently linked to one or more other monomers of the protein nanofibrils through two IPBs; more preferably, each monomer is covalently linked to one or more other monomers of the nanofibrils through two IPBs, the linking can be between precursor fibrils or within precursor fibrils, wherein the linking within precursor fibrils can also be referred to as cross-fibril linking. In other embodiments, each monomer is covalently linked to one or more other monomers of the protein nanofibrils via three IPBs; more preferably, each monomer is covalently linked to one or more other monomers of the protein nanofibrils via four IPBs; more preferably, each monomer is covalently linked to one or more other monomers of the protein nanofibrils via five IPBs; more preferably, each monomer is covalently linked to one or more other monomers of the protein nanofibrils via six IPBs; more preferably, each monomer is covalently linked to one or more other monomers of the protein nanofibrils via seven IPBs; more preferably, each monomer is covalently linked to one or more other monomers of the protein nanofibrils via eight IPBs. One or more other monomers of the protein nanofiber are covalently linked; more preferably, each monomer is covalently linked to one or more other monomers of the protein nanofiber via nine IPBs; more preferably, each monomer is covalently linked to one or more other monomers of the protein nanofiber via ten IPBs; more preferably, each monomer is covalently linked to one or more other monomers of the protein nanofiber via eleven IPBs; more preferably, each monomer is covalently linked to one or more other monomers of the protein nanofiber via twelve IPBs; more preferably, each monomer is covalently linked to one or more other monomers of the protein nanofiber via thirteen or more IPBs. In this document, a monomer having more than one IPB linking or crosslinking with one or more monomers may mean that the monomer is linked to another monomer of the fibril via more than one IPB, or it may mean that the monomer is linked to several other monomers of the fibril, wherein each other monomer is linked to the monomer via one or more IPBs.

[0146] In specific embodiments, each monomer of the protofibril is thus linked to more than one other monomer of the protofibril via the presence of an IPB, wherein crosslinking between two monomers may involve 1, 2, 3 or more IPBs. In other specific embodiments, the protein nanofibrils comprise A-ENA monomers, wherein each monomer participates in the formation of at least one IPB, which covalently links the precursor protofibrils (f) and (f') of the nanofibril. In another embodiment, the protein nanofibrils comprise A-ENA monomers, wherein each monomer participates in the formation of more than one IPB, wherein at least one IPB covalently links both the precursor protofibrils (f) and (f') of the nanofibril, and at least one IPB covalently links a monomer subunit to other monomer subunits of the same precursor protofibril of the nanofibril. In other embodiments, the protein nanofibrils comprise A-ENA monomers, wherein each monomer participates in the formation of 9 or 10 isopeptide bonds with other monomers of the protofibril, more specifically, linking the monomer to 3, 4, 5 or 6 other A-ENA monomers in the nanofibril.

[0147] In other specific embodiments, the protein nanofibrils described herein comprise monomeric protein subunits, wherein each monomeric protein subunit, preferably A-ENA or A-ENA-based monomeric protein subunit, is covalently linked to other monomers in the fibril, wherein isopeptide bonds are formed between amino acid side chains that are nucleophilic residues and amino acid side chains that are electrophilic residues, specifically at the following locations: (i) Between the N-terminal residue of the NTL (via the free amino terminal group of the polypeptide backbone as a nucleophilic donor) and H1-5b of (i-5), (i-4), (i-3) or (i-2); Between H1-4g of (i) and H2-2a of (i-1); Between H2-3g of (i) and H2-4a of (i-1); Between H2-3f of (i) and H2-4e of (i'); Between H2-4g of (i) and H1-3a of (i-1); Between H2-4g of (i+1) and H1-3a of (i); Between the NTL of (i+5), (i+4); (i+3) or (i+2) and the H1-5b of (i); Between H1-4g of (i+1) and H2-2a of (i); Between H2-3g of (i+1) and H2-4a of (i); and / or Between H2-3f of (i'+1) and H2-4e of (i), It is read as the binding of the nucleophilic donor side chain position of the first monomer to the electrophilic acceptor side chain position of the second monomer (e.g., the donor side chain is located at position g of the heptad repeating element H1-4 of monomer (i) and binds to the acceptor side chain at position a of the heptad repeating element H2-2 of monomer (i-1)).

[0148] In another alternative embodiment, the protein nanofibrils described herein comprise monomeric protein subunits, wherein each monomeric protein subunit, preferably A-ENA or A-ENA-based monomeric protein subunit, is covalently linked to other monomers of the same precursor fibril, wherein isopeptide bonds are formed between amino acid side chains as nucleophilic residues and amino acid side chains as electrophilic residues, specifically at the following positions: between the heptaponic repeating element position H1-4g of (i) and H2-2a of (i-1), and / or between H2-3g of (i) and H2-4a of (i-1), and / or between H2-4g of (i) and H1-3a of (i-1), and / or between H2-4g of (i+1) and H1-3a of (i), and / or between H1-4g of (i+1) and H2-2a of (i), and / or between H2-3g of (i+1) and H2-4a of (i).

[0149] In another specific embodiment, the protein nanofibrils described herein comprise monomeric protein subunits, wherein each monomeric protein subunit, preferably A-ENA or A-ENA-based monomeric protein subunit, is covalently linked to other monomers in another precursor fibril of the nanofibril, wherein IPBs are formed between amino acid side chains as nucleophilic residues and amino acid side chains as electrophilic residues, specifically at the following positions: between the hepta-repetitive element position H2-3f of (i) and H2-4e of (i'), and / or between H2-3f of (i'+1) and H2-4e of (i).

[0150] In another alternative embodiment, the protein nanofibrils described herein comprise monomeric protein subunits, wherein each monomeric protein subunit (preferably A-ENA or A-ENA-based monomeric protein subunits) is covalently linked to other monomers; wherein IPBs are formed between amino acid side chains as nucleophilic residues and amino acid side chains as electrophilic residues, specifically between: (i) the N-terminal residue of the NTL (using its free amino-terminal group of the polypeptide backbone as a nucleophilic donor) and (i-5) H1-5b, formed by using the N-terminal amino-terminal group of monomer (i) as a nucleophilic donor and (i-5) H1-5b; and / or formed between the N-terminal residue of the NTL of monomer (i+5) (using its free amino-terminal group of the polypeptide backbone as a nucleophilic donor) and (i) H1-5b.

[0151] In a specific implementation, the A-ENA monomeric protein is described by the formula NTL-h1-L-h2-CT, and the seven-membered repeat element is provided by a concordant sequence of residues Abcdefg as defined below: - Seven-element repeating 1-1(H1-1) of XXX- -XX- , - H1-2 -X-(S or T)- -(A or G)- -(E or Q), - EXX of H1-3- -X-(H or N)- , - H1-4 -N-(A or G or S)-E-(G or A)-EK, - H1-5 -QX- - -XX, - H2-1 -XX- - -X- , - H2-2 of (N or Q or D)-XX- -XXX, - H2-3 -XX- - -(K or X)-(K or X), - H2-4 (E or Q) - - -(L)-(Q or X)-XK, - H2-5 - -X- - -XX, The common amino acid sequence residues of the seven-membered repeating element are defined as follows: - It is a hydrophobic amino acid residue, selected from M, V, I, L, A, G, H, W, Y, F; preferably selected from L, M, I, V, F or W; - It consists of short side-chain amino acid residues, selected from V, C, G, A, P, S, T, N, and D; preferably A, G, S, or T; - The amino acid residues that serve as acid / base catalytic residues for heteropeptide interactions are selected from E or D; preferably E. - The amino acid residues that act as "receptors" or electrophilic residues in heteropeptide interactions are selected from E, Q, D, or N; preferably E, Q, or N. - X can be any type of amino acid residue, the single-letter codes for amino acid residues are as shown, and the amino acid in parentheses provides alternative options for the "abcdegf" residue at the corresponding position (indicated by "or").

[0152] In each heptapeptide repeat unit, the "donor" or nucleophilic residue that forms the isopeptide bond IPB is lysine (K); however, if the donor K is located in the H2-3 region, it will be replaced by any other type of amino acid (X). Therefore, if the H2-3 heptapeptide repeat residues f and / or g are not lysine (K), these residues will not be able to form IPB, and thus are unlikely to form protein nanofibrils; this is because IPB is responsible for establishing the connections between precursor fibrils, thereby providing stability for the assembly of nanofibrils.

[0153] Therefore, in the preferred embodiment, H2-3f and / or H2-3g residues are K. In an alternative embodiment, an A-ENA(like) protein is provided, wherein each donor residue of the seven-membered repeat element, more specifically H1-4g and / or H2-4g, can be replaced by any other amino acid (X), resulting in fewer IPBs when self-assembling into protofibrils. However, it is important that at least one K is still present to form at least one IPB, preferably with the H2-3f or g donor site being K.

[0154] Functionalized A-ENA protein assemblies

[0155] As defined and described above, A-ENA proteins can be provided as A-ENA proteins according to the general formula NTL-h1-L-h2-CT as defined and described above, wherein the monomer is modified or engineered to obtain a “modified A-ENA protein” or “A-ENA-based monomeric protein”, and thus these terms refer to A-ENA proteins based on naturally occurring or native A-ENA protein sequences, or at least as defined herein for their sequence and function, but modified or engineered to produce an A-ENA protein capable of functionalizing spontaneously assembled A-ENA protein nanofibrils when produced in a host.

[0156] The modification or engineering strategies described herein are further illustrated and discussed in a non-limiting manner and serve as support for enabling those skilled in the art to design such modified A-ENA proteins.

[0157] Therefore, the terms “modified” or “engineered” A-ENA protein, or “A-ENA-based protein” (which are used interchangeably herein), as used herein, mean that the reference A-ENA protein compared to the “modified” A-ENA protein is a protein subunit whose sequence comprises segments interconnected according to the general formula NTL-h1-L-h2-CT as described above, wherein at least five heptagonal repeating elements of the h1 and h2 helices are as defined herein, thus allowing the monomers to spontaneously fold into helical hairpins, wherein the connector forms loops connecting two antiparallel helices, and NTL and CT are present on the surface, and which further self-assemble into a fibrous structure as described herein, wherein each monomer participates in the formation of at least one intermolecularly formed IPB, preferably 10 IPBs, with other monomers (whose sequences may be identical or different from each other). In the comparison, the “modified” can then be further specified by the presence of any of the following engineered forms: - (1) The NTL is truncated compared to the native A-ENA protein of the modified A-ENA monomer; preferably, the truncation results in the NTL being less than 4 amino acids in length, preferably 3 amino acids, 2 amino acids or 1 amino acid.

[0158] One advantage of providing modified A-ENA (where the modification is a truncation of the NTL compared to the naturally occurring NTL) is that the A-ENA monomer, in its folded state, has greater accessibility for modification after the formation of nanofibrils.

[0159] - (2) The sequence conservation of the A-ENA heptavalent repeat element is altered—and an A-ENA mutant variant or surface mutant variant is provided—so that A-ENA protein nanofibrils with modified surface structures or properties can be generated compared to the native A-ENA sequence of the modified A-ENA; more specifically, wherein: (2a) The modified A-ENA sequence contains an NTL of at least 4 amino acids, and the seven-membered repeat element of the modified A-ENA monomer is replaced (natural or synthetic) at one or more of the following positions with an amino acid (i.e., a mutant) that is not part of the common sequence of the "conventional" A-ENA seven-membered repeat at that position (i.e., a mutant): H1-1a, H1-1b, H1-1c, H1-3f, H1-4f, H1-5b, H1-5f, H2-1b, H2-1c and / or H2-5b; or (2b) The modified A-ENA sequence contains an NTL of fewer than 4 amino acids, and the seven-membered repeat element of the modified A-ENA monomeric protein is mutated (natural or synthetic) at one or more of the following positions to amino acids that are not part of the common sequence of the “regular” A-ENA seven-membered repeat at that position: 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; - (3) The A-ENA protein contains a modification in which a protein tag or protein domain is further fused into its N-terminus, C-terminus or linker region, wherein the fusion is typically obtained by expressing a gene fusion in which a heterologous (poly)peptide sequence (which itself can form a tag or folded protein domain) is fused to the end of or inserted into the native A-ENA sequence (or an A-ENA mutant). - (4) The A-ENA protein is conjugated to another protein or chemical part (e.g., a tag, protein domain, small molecule or marker, etc.), wherein the conjugation may occur after fibril formation, and / or wherein the conjugation is defined as a covalent connection (in an orthogonal manner) formed with one or more side chain residues or atoms.

[0160] The present invention considers modifications involving the fusion or conjugation of A-ENA with heterologous or additional peptides, proteins, protein domains, tags, labels or other functional parts, which can be achieved by directly linking the heterologous part to the A-ENA subunit or by using a connector.

[0161] In the specific embodiments disclosed herein, the A-ENA subunit is fused or conjugated to a tag or protein-binding partner to enable post-fibril-specific tag-protein binding interactions on the fibril surface. In other specific embodiments, the tag / protein-binding partner pair comprises a protein and a tag capable of covalently interacting, for example, by forming isopeptide bonds. More specifically, as exemplified herein by the tag / protein-binding partner pair SpyTag / SpyCatcher, the tag or catcher may be fused to one or more A-ENA proteins such that one corresponding portion of the binding partner pair is exposed on the surface of the protein nanofibril, while another corresponding portion covalently interacts with the fibril-binding portion of the binding partner pair, specifically by isopeptide bonds (preferably after fibril formation).

[0162] Therefore, other embodiments relate to protein nanofibrils comprising at least one modified A-ENA monomer, wherein the modification is performed according to (1), (2), (3), or (4) provided above. More specific embodiments relate to the protein nanofibrils described herein, wherein at least two monomeric proteins with different sequences form heteropolymeric fibrils upon self-assembly; or, wherein all monomers are identical to form homopolymeric fibrils.

[0163] In other specific embodiments, the protein nanofibrils comprise or consist of two precursor fibrils, wherein each monomeric protein further comprises a flexible linker and / or a protein domain with a hydrodynamic radius of less than 11 nm, for display on the surface of the homopolymer fibrils to prevent collisions of the protein domains when the nanofibrils assemble.

[0164] The flexible linkers used in this paper are typically provided by amino acid sequences of 1-30 amino acids in length, and are usually enriched with small or polar amino acids such as Ser or Thr, or nonpolar amino acids such as Gly, to provide good flexibility and solubility. The small size of these amino acids provides flexibility and allows for the mobility of the linked functional domains. Incorporation of Ser or Thr can maintain the stability of the linker in aqueous solution by forming hydrogen bonds with water molecules, thereby reducing unfavorable interactions between the linker and protein moieties. The most commonly used flexible linkers have sequences consisting primarily of segments of Gly and Ser residues (“GS” linkers). The most widely used example of a flexible linker has the sequence (Gly-Gly-Gly-Gly-Ser). nBy adjusting the copy number "n", the length of the GS linker can be optimized to achieve proper separation of functional domains or maintain necessary interdomain interactions (Chen et al., Adv Drug Deliv Rev. 2013, 65(10):1357-69). Besides the GS linker, many other flexible linkers have been designed for recombinant fusion proteins. These 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 can be constructed using computational design by adding single amino acids or short peptides extending from the scaffold (see also Example 8).

[0165] In a specific implementation, the protein nanofiber comprises an A-ENA monomer modified with a heterologous protein domain (in accordance with the modification form of (3) provided above), wherein the heterologous protein domain is fused to or inserted into the junction region of the A-ENA via its N-terminus or C-terminus to produce a 3D protein structure, wherein the heterologous protein domain is displayed on the surface of the nanofiber.

[0166] In other specific embodiments, the protein nanofibrils comprise or consist of two precursor fibrils, each precursor fibril comprising or consisting of at least two A-ENA monomers, wherein at least one A-ENA monomer is a modified A-ENA, and wherein said modified A-ENA is provided in the form provided above (3) or (4), or specifically as a fusion of an A-ENA with a heterologous protein domain or tag as described herein (and according to (3)). The protein nanofibrils can be obtained by combining or mixing unmodified A-ENA protein (i.e., native A-ENA protein) or mutant variant A-ENA protein (according to the modified form (2) provided above) and modified A-ENA protein (which is an A-ENA heterologous fusion protein (as provided above (3))) or conjugate (as provided above (4)) to produce the self-assembly of hybrid nanofibrils, which can be considered as copolymers appearing as alternating blocks of surface-displayed and non-displayed portions (e.g., as Figure 16 (as seen in a).

[0167] The mixture of different A-ENA monomers and modified A-ENA monomers in the heteropolymer protein nanofibrils can be obtained by recombinant expression of modified A-ENA protein sequences (e.g., gene fusion of A-ENA with heteropeptides, protein tags or domains at the N-terminus and C-terminus of A-ENA, or insertion into the adapter region) and unmodified or conventional A-ENA in the host.

[0168] Alternatively, the mixture of different A-ENA monomers and modified A-ENA monomers of the heteropolymeric protein nanofibrils can be obtained by recombinantly expressing a modified A-ENA protein sequence in a host that endogenously expresses natural A-ENA (e.g., a bacterial strain that forms endospores, preferably a Bt strain) (e.g., by gene fusion of A-ENA with a heteropeptide or protein tag or domain at the A-ENA, C-terminus, or insertion into a linker region).

[0169] Alternatively, the mixture of different A-ENA and modified A-ENA monomers of the heteropolymeric protein nanofibrils can be obtained by recombinant expression of the following two proteins in a host: a modified A-ENA protein sequence (e.g., gene fusion of A-ENA with a heteropeptide, protein tag or domain at the N-terminus or C-terminus of A-ENA, or insertion into the adapter region), which provides a heterologous portion on the fiber surface (shown); and a modified A-ENA modified according to the modification forms (1) (truncated NTL) or (2) (mutant variant with modified surface) provided above.

[0170] Other aspects related to A-ENA protein nanofibers and their production

[0171] Another aspect of the invention relates to nucleic acid molecules or nucleic acid sequences that encode the A-ENA-based monomers or modified A-ENA protein monomers described herein for use in forming the recombinant protein nanofibrils described herein.

[0172] Furthermore, a vector comprising a nucleic acid molecule encoding a naturally occurring A-ENA protein as described herein, or an A-ENA-based monomer or modified A-ENA protein monomer, is disclosed. This vector can be used to clone or express recombinant A-ENA proteins or modified A-ENA proteins as described herein. As known to those skilled in the art, the vector can be introduced into a host via transient or stable transformation techniques to generate exogenously introduced A-ENA or modified A-ENA proteins in the host, particularly in the cytoplasm of host cells, or to obtain A-ENA or modified A-ENA proteins as secreted proteins in a culture medium. Following self-assembly of the recombinantly expressed A-ENA or modified A-ENA or A-ENA-based monomer, fibrous structures are formed in the host or host culture, allowing for the extraction of A-ENA multimeric assemblies from the host and optimal extraction of A-ENA or A-ENA-based protein nanofibrils.

[0173] Therefore, in specific embodiments, this disclosure relates to host cells comprising nucleic acid molecules as described herein, said nucleic acid molecules encoding A-ENA protein or modified A-ENA protein or A-ENA-based protein. Another specific embodiment relates to said host cells comprising protein nanofibrils as described herein, said protein nanofibrils comprising A-ENA protein, said A-ENA protein being recombinantly produced from exogenously introduced A-ENA protein, A-ENA protein-encoding nucleic acid molecules, modified A-ENA protein-encoding nucleic acid molecules, expression cassettes or vectors (containing chimeric gene constructs for encoding said A-ENA protein assemblies).

[0174] In a preferred embodiment, the host cell is a bacterial cell, and more preferably, the host cell is an Escherichia coli cell.

[0175] In another preferred embodiment, the host cell is a spore-forming bacterial cell, or a Bacillus cell, more preferably a Bacillus thuringiensis cell. In another preferred embodiment, the bacterial host cell does not contain the endogenous A-ENA protein-coding gene. The host cell may also be provided by other organisms such as plants, fungi, or animals.

[0176] Other aspects involve methods for producing recombinant protein nanofibers such as A-ENA or A-ENA-based fibers as described herein, including the following steps: a) Introducing a nucleic acid molecule encoding an A-ENA protein, or an A-ENA-based or modified A-ENA protein, as described herein, to recombinantly express the A-ENA monomeric protein in a host cell, and / or b) Culture host cells expressing the A-ENA protein under suitable conditions to promote the production of A-ENA protein, A-ENA-based protein, or modified A-ENA protein within the host cells or in the host cell culture, so that it self-assembles and preferably spontaneously forms protein nanofibrils.

[0177] c) Release the A-ENA protein assembly from the host cell, preferably via cell lysis, and / or

[0178] d) Isolate self-assembled protein nanofibrils, preferably by resuspending in an insoluble fraction and / or further purifying from cell lysates.

[0179] In a preferred embodiment, the substance introduced in step a) is obtained by transforming the host cell using a chimeric gene containing the protein-coding sequence of the A-ENA (or a modified A-ENA). In another embodiment, the host cell is a bacterial or fungal cell. In a more preferred embodiment, the microorganism that produces the host is, for example, *Escherichia coli* for cytoplasmic expression of A-ENA protein nanofibrils, or *Lactococcus lactis* for secreting A-ENA protein nanofibrils in a culture medium. Lactococcus lactis ).

[0180] 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 (hybrid) protein nanofibrils described herein.

[0181] The present invention also provides recombinant protein nanofibrils, preferably A-ENA or A-ENA-based or modified A-ENA protein nanofibrils, which can be obtained by or from the methods described herein for producing the nanofibrils.

[0182] The present invention also provides isolated protein nanofibrils, preferably A-ENA or A-ENA-based or modified A-ENA protein nanofibrils, which can be obtained or isolated from the host, as provided by the methods for generating nanofibrils described herein.

[0183] Bacterial endospores with recombinant A-ENA protein nanofibrils

[0184] As disclosed in this application, Bti spore cultures have been found to contain protein nanofibrils composed of A-ENA or A-ENA-1 proteins (as shown in SEQ ID NO:1 and 2, respectively), which function to enable spores and parasporal bodies to bind and contact each other to ensure their pathogenicity against insects such as Lepidoptera.

[0185] Because the presence of these protein nanofibrils can be optimized, altered, or modified for specific efficacy purposes to provide modified endospores, another aspect of the invention relates to modified endospores or modified Bacillus endospores or modified Bt endospores, wherein the term "modified" means the presence of a non-endogenous A-ENA protein or a modified A-ENA protein or an A-ENA-based protein in the said Bacillus strains and / or Bacillus endospores. In other specific embodiments, the modified Bacillus endospores or the Bacillus strains or cells that form endospores contain a non-naturally occurring or modified or engineered A-ENA protein or a nucleic acid molecule encoding it.

[0186] As disclosed herein, numerous Bacillus strains lacking endogenous or natural genes for producing A-ENA proteins or protein nanofibrils have been identified. Therefore, in specific embodiments, this document provides for the recombinant (or exogenous) introduction or expression of A-ENA proteins or A-ENA-based proteins in said bacterial strains or cells lacking the genes or ability to produce natural A-ENA for the production of the A-ENA protein nanofibrils described herein.

[0187] Other specific embodiments involve modified Bacillus endospores that display a portion of their modified A-ENA monomeric protein on the surface of protein nanofibrils assembled as described herein. Therefore, this document provides the aforementioned modified Bacillus endospore nanofibrils that display a heterologous protein as part of the A-ENA protein (i.e., a modified product obtained by inserting or fusing a protein tag or protein domain into the A-ENA protein monomer, as described herein).

[0188] Finally, as also demonstrated herein, the modified bacterial endospores, preferably Bacillus endospores, can be used to assist, promote, improve, enhance, or improve bacterial spore activity, preferably pathogenic activity, more preferably insecticidal activity, and most preferably insecticidal activity. In alternative embodiments, the modified bacterial spores, preferably Bacillus spores, as described herein, can be used to obtain, promote, enhance, or improve the functional effects obtained by recombination of protein nanofibrils generated in the modified spore culture.

[0189] In some embodiments, protein nanofibrils generated in the modified Bacillus endospores may be present in a modified form or in a modified environment, thereby allowing “as is” extraction or separation of protein nanofibrils from the culture or Bacillus spores, which is also within the scope of the concept of modified Bacillus endospores disclosed and described in this application.

[0190] This article also discloses another alternative use of the protein nanofibrils, wherein the protein nanofibrils, in their recombinant-prepared purified form, when applied together with, in combination with or after a bacterial endospore culture (preferably a Bt endospore culture or suspension), can enhance or improve the pathogenicity of the endospores to their targets compared to spore cultures without the addition of purified protein nanofibrils.

[0191] Therefore, in specific implementations, the protein nanofibers described herein (preferably recombined by the methods described herein) can provide additional or synergistic toxicity when applied to their respective pests as insecticidal spores.

[0192] Therefore, the protein nanofibers of the present invention also provide alternative uses in agricultural crop treatment or crop improvement, and may contribute to future improved pest control strategies.

[0193] Pharmaceutical Compositions and Medical Uses

[0194] Another aspect of the invention relates to pharmaceutical compositions comprising the protein nanofibrils described herein or nucleic acid molecules encoding monomers of the nanofibrils described herein, and optionally comprising a carrier, diluent, or excipient. In other specific embodiments, the pharmaceutical composition comprises a therapeutically active composition comprising the protein nanofibrils described herein, and / or a pharmaceutical composition wherein the therapeutically active composition is presented or provided as an active agent coupled, inserted, or attached to the protein nanofibrils of the invention.

[0195] In other respects, protein nanofibers or nucleic acid molecules or pharmaceutical compositions as described herein are used as part of a drug or pharmaceutical agent. Specific embodiments relate to protein nanofibers or nucleic acid molecules or pharmaceutical compositions as described herein for therapeutic, prophylactic, or preventative treatment of a subject. Therefore, in specific embodiments, a treatment method is disclosed comprising the steps of administering a protein nanofiber, nucleic acid molecule, or pharmaceutical composition as described herein to a subject who has or may have a disease or condition for the treatment or prevention of said disease or condition.

[0196] In specific implementations, protein nanofibrils are modified by covalently or nonvalently incorporating bioactive or therapeutic proteins, preferably nanobodies. In these implementations, the modified protein nanofibrils and their conjugates, aggregates, or hydrogels can achieve high-density, high-multivalent (hundreds to thousands) display of bioactive proteins. When modified via reversible bonds, the nanofibril aggregates and hydrogels can be used for the slow release of bioactive proteins through nonvalent association or protein hydrolysis. A preferred method for reversibly, nonvalently modifying the protein nanofibrils and bioactive proteins involves gene-fusing a receptor-peptide tag pair to the protein nanofibril monomer and the bioactive protein, wherein the receptor or peptide tag is linked to the NTL or CT of the nanofibril monomer via a peptide bond, and wherein a second component of the receptor-peptide tag pair is linked to the N-terminus or C-terminus of the bioactive protein. In a preferred embodiment, the receptor-peptide tag pair consists of: a Strep tag-(Strept) avidin pair, a SUMO tag-SUMO pair, a coil-coil pair, or a SpyTag-SpyDock pair (a mutated SpyTag-SpyCatcher pair that cannot form heteropeptide bonds).

[0197] All aspects of this disclosure

[0198] This disclosure relates, in a first aspect, to an isolated protein nanofibril comprising two precursor fibrils, each precursor fibril comprising at least two monomeric protein subunits, wherein each monomeric protein subunit comprises a covalently linked amino acid sequence fragment according to the following general formula: NTL – Helix 1 – L – Helix 2 – CT, wherein the monomeric protein subunit spontaneously folds in aqueous solution into two helices, namely Helix 1 and Helix 2, forming an α-helix antiparallel coil-up helical structure, and wherein each of Helix 1 and Helix 2 comprises a continuous sequence of at least five heptad repeat (H) elements, Helix 1 following the general formula H1-1 – H1-2 – H1-3 – H1-4 – H1-5, and Helix 2 following the general formula H2-1 – H2-2 – H2-3 – H2-4 – H2-5, wherein each heptad repeat element comprises seven amino acid residues, labeled “abcdefg”, and having the following common sequence: - Seven-element repeat 1-1: XXX- -XX- , - Seven-element repetition 1-2: -X- - - - - , - Seven-element repetition 1-3: -X- - - -X- , - Seven-element repetition 1-4: -N- - - - - δ, - Seven elements repeat 1-5: - -X- - -XX, - Seven-element repetition 2-1: -XX- - -X- , - Seven-element repetition 2-2: -XX- -XXX, - Seven-element repetition 2-3: -XX- - -δ-δ, - Seven-element repetition 2-4: - - - - -X- δ, - Seven-element repetition 2-5: - -X- - -XX, in: These are hydrophobic amino acids, selected from M, V, I, L, A, G, H, W, Y, and F; These are short side chain residues selected from V, C, G, A, P, S, T, N, and D; Residues selected from E, D, and Q that can be used as acid / base catalytic residues; δ residues selected from K that can be used as isopeptide bond "donors" or nucleophilic residues. It is a residue that can be used as an isopeptide bond "receptor" or an electrophilic residue, selected from E, Q, D, N; X can be any amino acid, and the linker (L) fragment contains at least 4 amino acids, and the N-terminal lock (NTL) fragment and the C-terminal tail (CT) fragment contain at least one amino acid, and the monomeric protein subunit is interconnected by at least one or more isopeptide bonds (IPB).

[0199] In other embodiments, the protein nanofibrils are disclosed, wherein the monomeric protein groups (i and i+ / -n) of the first precursor fibril (f) and the monomeric subunits (i' and i'+ / -n) of the second precursor fibril (f') are covalently linked by at least one or more IPBs, wherein the IPBs are formed between amino acid side chains as nucleophilic residues and amino acid side chains as electrophilic residues, specifically at the following locations: - Between NTL of (i) and H1-5b of (i-5), (i-4), (i-3) or (i-2); - Between H1-4g of (i) and H2-2a of (i-1); - Between H2-3g of (i) and H2-4a of (i-1); - Between H2-3f of (i) and H2-4e of (i'); - Between H2-4g of (i) and H1-3a of (i-1); - Between H2-4g of (i+1) and H1-3a of (i); - Between the NTL of (i+5), (i+4), (i+3) or (i+2) and H1-5b of (i); - Between H1-4g of (i+1) and H2-2a of (i); - Between H2-3g of (i+1) and H2-4a of (i); and / or - Between H2-3f of (i'+1) and H2-4e of (i).

[0200] Other embodiments disclose any of the said protein nanofibrils, wherein the seven-membered repeating elements have the following common sequence: - H1-1: XXX- -XX- , - H1-2: -X-(S / T)- -(A / G)- -(E / Q) - H1-3: EXX- -X-(H / N)-

[0201] - H1-4: -N-(A / G / S)-E-(G / A)-EK

[0202] - H1-5: -QX- - -XX

[0203] - H2-1: -XX- - -X- , - H2-2:(N / Q / D)-XX- -XXX - H2-3: -XX- - -(K / X)-(K / X) - H2-4: (E / Q)- - -(L)-(Q / X)-XK, - H2-5: - -X- - -XX.

[0204] More specific embodiments also disclose any of the said protein nanofibrils, wherein the NTL contains a consensual sequence: M- - -ZX- -P, where: - These are short side chain residues, selected from V, C, G, A, P, S, T, N, and D; - These are hydrophobic amino acids, selected from M, V, I, L, A, G, H, W, Y, and F; - X can be any amino acid. - Z represents Ala or proline, and - M and P are the single-letter codes for the amino acids methionine and proline, respectively.

[0205] Another embodiment discloses any of the said protein nanofibrils, wherein the monomer is an A-ENA protein selected from the protein list described in Table 3, and / or consists of an amino acid sequence selected from SEQ ID NO: 1-6.

[0206] In a specific embodiment, the protein nanofibrils have an NTL of less than 4 amino acids. Alternatively, the protein nanofibrils are disclosed, wherein the monomeric protein subunits comprise: The NTL has at least 4 amino acids, and the monomeric protein is a mutant variant at at least one or more 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 is less 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. The protein nanofibers described therein have modified surfaces.

[0207] Another embodiment discloses any of the said protein nanofibrils, wherein the monomeric protein further includes a protein tag or domain fused or conjugated to the N-terminus, C-terminus, or linker region for forming a functionalized fibril.

[0208] An alternative embodiment discloses the protein nanofibrils comprising the same monomeric protein that forms homopolymeric fibrils upon self-assembly, or comprising at least two different monomeric proteins that form heteropolymeric fibrils upon self-assembly.

[0209] In other embodiments, any of the said protein nanofibers are disclosed, wherein the fibers are recombinant protein nanofibers.

[0210] On the other hand, a modified Bacillus endospore is disclosed, which includes and / or displays any of the said monomeric protein subunits or (modified) protein nanofibrils.

[0211] An alternative embodiment discloses a modified Bacillus endospore, wherein the Bacillus strain lacks the endogenous monomeric protein of the said protein nanofibrils and contains exogenously introduced monomeric protein subunits, or contains the protein nanofibrils disclosed herein.

[0212] Other embodiments disclose the use of the modified Bacillus endospores in increasing bacterial spore activity, preferably pathogenic activity.

[0213] Other aspects disclose nucleic acid molecules that encode monomeric protein subunits for forming the (modified) protein nanofibrils.

[0214] The final aspect relates to a method for generating any of the disclosed protein nanofibrils, comprising the steps of: recombinantly expressing the disclosed nucleic acid molecule encoding the protein, or expressing the monomeric protein for forming the nanofibrils in a host cell; releasing the self-assembled protein nanofibrils from the host cell, preferably by cell lysis; and isolating the self-assembled protein nanofibrils, preferably by resuspending in an insoluble component and / or further purifying from cell lysates.

[0215] It should be understood that although specific embodiments, configurations, and materials and / or molecules have been discussed herein with respect to methods and products according to this disclosure, various changes or modifications in form and detail may be made 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.

[0216] Example

[0217] Example 1. Characterization of A-ENA nanofibrils in the biofilm of Bacillus thuringiensis var. Israel spores

[0218] Here, we summarize the characteristics of the nanofibrils found in the spore biofilm of *Bacillus thuringiensis* var. *Israel* (Bti). The Bti spore biofilm was prepared on LB agar plates as follows: a single pre-cultured Bti colony was inoculated into 10 mL of LB liquid medium and incubated overnight at 37°C to the stationary phase. Subsequently, 2 mL of this 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 observed using bright-field microscopy and negative-stain transmission electron microscopy (nsTEM). Phase-contrast microscopy imaging using a 100× oil immersion lens revealed the presence of two types of refractive bodies (quasi-spherical and elliptical), with typical diameters of 0.5 μm and 1.5 μm, respectively. The latter (elliptical) was enclosed in a sac-like structure and corresponded to the Bti spore. The spores bind to the aforementioned refractive bodies, which we identified as parasporal bodies (PSBs; see below). Bti-derived PSBs are dense, aggregated structures composed of distinct crystalline subdomains made up of various types of proteins that confer larval-killing activity against insect larvae such as mosquitoes and midges. To further investigate the spore-PSB binding in detail, we performed nsTEM analysis on resuspended spore biofilm samples. For this purpose, we acquired nsTEM micrographs of Bti spore samples fixed on a Formvar / carbon grid (400 mesh, copper; Electron Microscopy Sciences) and stained with 2% (w / v) uranium acetate. Low-magnification TEM images (2500×) show a pervasive network of protofibrils throughout the biofilm, encapsulating the spores and PSBs. Figure 1Based on high-magnification CryoEM imaging (60k), we calculated 3D reconstructed maps, enabling partial de novo residue allocation of the fibrillary monomers. This subsequently allowed us to perform a sequence-based search of the Bti genome and definitively identify the constituent subunit as UniProt: Q8KNV8 (SEQ ID NO:1), referred to herein as “A-ENA”. Genome analysis revealed the presence of a second A-ENA-like sequence adjacent to A-ENA, composed of Q8KNV7 (SEQ ID NO:2), referred to herein as “A-ENA1”. Based on nsTEM analysis, we concluded that this rich protofibrillary network consists of individual A-ENA protein filaments composed of A-ENA and / or A-ENA-1. The A-ENA nanofibrils are characterized by an apparent diameter of ±8 nm and distinct projection maps in nsTEM or CryoEM. A-ENA as Figure 3 As shown in the model, the A-ENA fibril consists of two precursor fibrils (f and f') that are intertwined to form a bi-helical superstructure with a twist angle and a rise distance of 12° and 10.8 Å, respectively. A-ENA monomer subunits (labeled i and i') self-assemble into an α-helical hairpin with an N-end extending downward along the fibril axis. Figure 3 As shown in c, A-ENA monomers interact laterally between precursor fibrils to form dimer entities, which then stack axially, thereby extending each fibril and producing a tetramer complex that constitutes the minimum necessary fibril unit formed by self-assembled A-ENA.

[0219] We found a clear connection between A-ENA fibrils and the outermost layer of the endospore, as well as with the PSB surface; for example, A-ENA protofibril clusters extend outward from the PSB and / or spore membrane. Furthermore, PSBs are often connected to mature spores via A-ENA fibrils. This protofibril-mediated PSB-spore or spore-spore connection can span several micrometers and effectively acts as a reinforcing link, connecting various structures.

[0220] Example 2. Recombinant production and purification of intracellularly assembled A-ENA and A-ENA-1 fibrils

[0221] The natural 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 amplified by PCR from the pBtoxis plasmid (CP013279; pAM65-52-4-128K) using primer set 1 (SEQ ID NO:16-17) and primer set 2 (SEQ ID NO:18-19), respectively. The amplified fragments were cloned into the pASK vector using Gibson Assembly (https: / / doi.org / 10.1038 / nmeth.1318) and introduced into *E. coli* Top10. Transformants were screened by colony PCR and Sanger sequencing (pASK sequencing primers; SEQ ID NO:20-21). The obtained plasmids (pASK_A-ENA; pASK_A-ENA-1) were used to transform competent *E. coli* C43(DE3) cells. Single colonies were used to begin overnight (ON) LB culture. 10 mL of the ON culture was inoculated into 1 L of LB medium containing 50 μg / mL ampicillin and cultured at 37°C. When OD600 reached 0.8, 50 µM tetracycline was added to induce recombinant expression, followed by ON incubation at 18°C. The cells were then resuspended in medium containing 1 mg / mL ampicillin. -1 Lysozyme, 5 mM EDTA, 50 mM Tris pH 6.8, and 50 mM NaCl buffer were added to the lysate and incubated with stirring at room temperature for 18 h. Then, 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 to room temperature for 15 min and centrifuged at 20,000 rcf for 30 min using a Beckman JA 14.50 50 mL Falcon rotor. The supernatant was discarded, and the precipitate (i.e., the insoluble fraction consisting of A-ENA(-1) fibrils) was resuspended in a suspension (5 mL miliQ / g wet fibril precipitate) using an IKA overhead stirring system. The resulting suspension was centrifuged three more times (20,000 rcf, 30 min), and each precipitate was resuspended in miliQ to remove residual SDS. The suspension obtained after the final washing step was diluted tenfold in miliQ, deposited on a Formvar / carbon grid (400 mesh, Cu; Electron Microscopy Sciences), stained with 2% (w / v) uranyl acetate, and blotted dry. Negative staining transmission electron microscopy (nsTEM) analysis revealed the presence of fibrils with a diameter of approximately 8 nm and a length in the micrometer range. Figure 2 Two-dimensional classification of framed fibril fragments ( Figure 2 a) is consistent with the average 2D classification image previously obtained from the isolated fibrils-spore suspension extracted from the biofilm of Bacillus thuringiensis subsp. Israel spores. Figure 1 hi).

[0222] CryoEM structural analysis of these recombinant A-ENA fibrils confirmed the antiparallel coiled-helical structure of the A-ENA monomer subunits, as observed in wild-type A-ENA fibril monomers, as well as the contacts and isopeptide bonds between their precursor fibrils. Figure 4 5). Furthermore, contacts and isopeptide bonds within the precursor fibrils were observed in A-ENA: (i) these included contacts between A-ENA monomers and monomers located directly above (i+1) or below (i-1) subunit i, as well as contacts between monomer i-5 and the second residue in the N-terminal linker. These observations suggest that the formation of A-ENA nanofibrils does not depend on cofactors in native Bti cells, but occurs spontaneously and efficiently in unrelated recombinant host cells. Therefore, A-ENA subunits possess inherent self-assembly properties and the ability to autocatalytically form intermolecular and interfibrillary isopeptide bonds. The contact surfaces of A-ENA monomers are predominantly hydrophobic, except for some residues involved in covalent interactions. Furthermore, the high stability exhibited by wild-type A-ENA fibrils can be attributed to the nature of these covalent interactions, namely, the formation of irreversible isopeptide bonds (IPBs), resulting in a robust fibril structure in which each subunit participates in the formation of up to 10 IPBs, thereby achieving cross-linking with 7 different subunits both within and between precursor fibrils. Figure 4 ,9).

[0223] Example 3. Self-assembly of stable A-ENA-like nanofibers requires the formation of heteropeptide bonds.

[0224] The A-ENA monomer subunits that self-assemble into nanofibers contain ten isopeptide bonds with adjacent A-ENA subunits. These isopeptide bonds are formed by autocatalytic units, which consist of acid-base catalytic residues (preferably Glu), nucleophilic residues (preferably Lys or N-terminal amino groups), and electrophilic residues (preferably Glu, Gln, Asn, or Asp). Their formation follows a proximity-induced mechanism, meaning that the relative positions of the acid-base catalytic residues activate the nucleophilic residues to attack the electrophilic residues. Besides lysine residues in the helix, the N-terminal amino group in the NTL can also act as a nucleophilic donor in the formation of IPB. The specific subunit "ix" involved in IPB can be varied by lengthening or shortening the NTL. Figure 4a, 9). For A-ENA (SEQ ID NO:1), the exact locations of various residues were marked, such as... Figure 4 As shown in a, 5, and 7.

[0225] To evaluate the role of isopeptide bonds in the self-assembly of A-ENA monomers into stable A-ENA nanofibrils, we mutated the electrophilic residues in the isopeptide bonds. For this purpose, E29, Q44, N64, E78, and Q82 were mutated to alanine residues (SEQ ID NO: 15). A 7×His tag was added to the C-terminus to facilitate purification from *E. coli* lysates. The resulting DNA sequence was ordered as a double-stranded synthetic DNA fragment (gblock, IDT) and cloned into the pASK vector for recombinant expression as described in Example 2. Harvested cells were resuspended in 100 mM K-phosphate pH 7.2 buffer supplemented with 8 M urea and 1 M NaCl at a concentration of 10 ml buffer per gram of wet cell pellet. The resulting cell suspension was sonicated on ice for 5 min using a 500W Qsonica probe sonicator at 50% amplitude with a 30 s on / 15 s off pulse mode. Cell lysates were then centrifuged at 20,000 rcf for 45 min to precipitate insoluble cell debris. The clarified lysates were loaded onto a pre-equilibrated 5 mL Hispace 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 fractions were analyzed by SDS-PAGE. Fractions containing A-ENA molecules were combined and dialyzed against a buffer containing 100 mM Hepes (pH 8.0) and 8 M urea. The dialyzed proteins were loaded onto a pre-equilibrated 1 mL Q anion exchange column, pre-equilibrated with 100 mM Hepes (pH 8.0) and 8 M urea, and the flowthrough was collected. The column was then eluted with 100 mM Hepes (pH 8.0), 8 M urea, and 1 M NaCl. SDS-PAGE analysis of the flow-through and elution fractions showed that A-ENA existed in the flow-through in pure monomer form. Figure 12 a). Next, the A-ENA monomer was exchanged from 100 mM Hepes pH 8.0, 8M urea buffer to 1×PBS using a 40K MWCO Zeba rotary desalting column and incubated at room temperature for 24 h. The desalted A-ENA sample was applied to a Formvar / carbon grid, stained with 2% uranium acetate, and imaged using a 120kV JEO electron microscope. Figure 12(b) and (c). No fibrous structures were observed in the resulting micrographs. In contrast, the A-ENA E29AQ44A N64A E78A Q82A mutants formed numerous fractal-like aggregates. Therefore, we conclude that isopeptide bonds are crucial for the formation of stable A-ENA protofibrils.

[0226] Example 4. A-ENA spore filament network induces the formation of Bacillus thuringiensis spore biofilm, and combines spores with companions. spore body binding

[0227] In order to study Bacillus thuringiensis subspecies Israel ( Bti) To generate A-ENA KO (ΔA-ENA), we followed the experimental procedure described by Wang et al. (2019 Front Microbiol. 10: 1932), which was based on the pJOE8999 plasmid purchased from the Bacillus Genetic Stock Center and carrying the CRISPR / Cas9 system (Altenbuchner, 2016. Appl Environ Microbiol. 82(17): 5421-7). First, the pJOE8999 plasmid was linearized using primer pairs p551 and p552. Using Bti spore stock solution as a gDNA donor template, the regions approximately 1 kb upstream and 1 kb downstream of the A-ENA gene were amplified using primer pairs p588 & p633 and p634 & p635, respectively. The two PCR fragments were cloned into the linearized pJOE8999 vector using Gibson Assembly, following the manufacturer's instructions. The plasmid was sequenced and validated using oligonucleotides p525 and p526. Next, the designed sgRNA was cloned using the following web server: http: / / www.rgenome.net / cas-designer / . Using primer pairs p594 & p595, the selected sgRNA (SEQ ID NO: 22) was cloned into the designed plasmid via PCR. The sgRNA was sequenced and validated using oligonucleotide p531. The plasmid was transformed into *E. coli* strain dam- / dcm- and cultured in LB broth supplemented with 50 μg / ml kanamycin to produce an unmethylated plasmid. Then... BtiElectroporation was performed on plasmids containing Wt, and transformants were selected on BHI plates supplemented with 0.5% glycerol and 25 μg / mL kanamycin at 30°C. The next day, single colonies were inoculated into 10 mL of liquid BHI medium supplemented with 25 μg / mL kanamycin and incubated at 37°C for 3 h. To induce Cas9 expression, mannose was added to a final concentration of 0.4% (w / v), and incubation was continued at 37°C for another 3 h. The culture was serially diluted and inoculated onto LB agar plates containing 25 μg / mL kanamycin and 0.4% (w / v) mannose and incubated at 37°C. The next day, mutants were selected by colony PCR using oligonucleotide pairs p602 and p650. Single colonies were selected, and oligonucleotide pairs p602 and p650 were used to select mutants. A-ENA The locus was sequence verified. Finally, KO was analyzed using TEM. A-ENA Strain imaging to ensure the absence of A-ENA fibrils ( Figure 18 b).

[0228] A-ENA The KO complementation experiment was performed by adding plasmid pJOE8999 to the KO plasmid. cas9 Gene replacement from Bti The promoter, CDS, and terminator of the A-ENA gene were determined. First, plasmid pJOE8999 was linearized using oligonucleotide pairs p622 & p623. Then, the promoter, CDS, and terminator were amplified using oligonucleotide pairs p683 & p684. A-ENA The gene locus was determined and cloned into the linearized plasmid using Gibson Assembly according to the manufacturer's instructions. The mixture was transformed into *E. coli dam / dcm-* strain, and selected on LB agar plates supplemented with 50 μg / ml kanamycin and incubated at 37°C. The next day, positive colonies were selected by colony PCR using primer pairs p617 & p631, and cultured in 10 ml LB agar plates containing 50 μg / ml kanamycin. The plasmid A176 was extracted using the NucleoSpin Plasmid EasyPure kit. The plasmid was then sequenced (Eurofins) and transformed into electrocompetent cells. BtiIn cells. Single positive colonies were inoculated into BHI medium supplemented with 25 μg / mL kanamycin and incubated overnight at 30°C. The next day, 2 mL of 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 protofibrils was assessed by TEM. Spore samples were deposited on Formvar / carbon grid (400 mesh, Cu; Electron Microscopy Sciences) and stained with 2% (w / v) uranyl acetate. TEM images showed the presence of a protofibril network that held the entire biofilm containing spores and PSB together, as observed in WT ( Figure 18 e), and confirmed that A-ENA KO has been correctly complemented.

[0229] Example 5. Exogenous A-ENA formation of spores and Cryotoxin crystals from Bacillus thuringiensis subsp. Israel. Combined sporophyll network

[0230] The A176 plasmid described in Example 4, used for complementary A-ENA knockout (KO), was introduced into *Bacillus thuringiensis* subsp. *kustag*, which naturally lacks A-ENA, via electroporation. Bacillus thuringiensis Sv. kurstaki Transformed cells were selected after electroporation and screened on brain heart infusion (BHI) plates supplemented with 25 μg / mL kanamycin, and incubated overnight at 30°C. The next day, single colonies were 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 245×245×25 mm square plates of Luria-Bertani (LB) medium supplemented with 25 μg / mL kanamycin and allowed to spore for one week at 30°C. The resulting spore biofilm was harvested using a cell scraper, resuspended in 10 mL of deionized water (miliQ), and used for TEM imaging and optical microscopy examination. Figure 19 As shown, A-ENA is expressed. Bti In addition to having a biofilm similar to that of Bti, the Btk strain also exhibits biconical PSB crystals connected to the A-ENA bundle.

[0231] Example 6. A-ENA-mediated spore-parasporin binding enhances the insect pathogenicity of Bacillus thuringiensis. sex

[0232] To investigate the role of A-ENA in the binding of PSB to spores, we used the April midge ( Chironomus aprilinusThe virulence of *Bacillus thuringiensis* subspecies *Israeloidea* (wild-type vs. A-ENA knockout) strains expressing or lacking A-ENA was evaluated using an insect model. Fresh *Centella asiatica* larvae were purchased from Zooschatz (Berlin, Germany), lot number 1223153. Wild-type (WT) and A-ENA knockout (KO) strains were cultured on LB agar plates for one week. The resulting spore biofilm was scraped off and resuspended in 10 ml of deionized water to prepare a spore suspension. Simultaneously, 1 ml of the spore suspension was centrifuged at 16,000 × g for 40 min using 50% Histodenz buffer to isolate spores. The isolated spores were then washed three times with deionized water by centrifugation (5000 × g, 10 min). To determine mortality, each group contained at least 8 *Centella asiatica* larvae. Chironomus aprilinus Larvae, with a final volume of 3 ml, containing sea salt and vitamin premix provided with the larvae, were added to the spore suspension and spore separation sample until the final optical density (OD) was reached. 600 The value was 0.02. Phosphate-buffered saline (PBS) was used as a negative control. Larval survival was monitored daily for 7 consecutive days. This study was conducted at room temperature, with three biological replicates for each experiment. Figure 20 As shown, the absence of A-ENA protofibrils significantly reduced the bactericidal activity of Bti spores compared to larvae exposed to WT Bti. The mortality time of A-ENA larvae was delayed by at least one day, and the larvae had a higher survival probability after at least one weak exposure to Bti endospores. This effect was observed in Bti. The most significant reduction was observed in A-ENA spore isolation preparations, where the lack of A-ENA protocellulose led to a substantial decrease in PSB during the precipitation and isolation of endospores from the growth medium. Figure 18 f). Bti A slight delay in insecticidal activity was still observed in the spore suspension of A-ENA, in which PSB remained in the suspension, although it was no longer bound to endospores. Figure 18 (d, f). Therefore, these experiments show that if Bt spores cannot retain the toxins they secrete in a position adjacent to the endospores, it will lead to a significant reduction in their toxicity and insecticidal activity.

[0233] Example 7. Conservation of A-ENA protein sequence

[0234] Using Q8KNV8 (SEQ ID NO: 1) as the query sequence, the first 593 BLAST hit sequences were selected for multiple sequence alignment to construct an A-ENA conserved sequence identifier (web logo). The results are as follows: Figure 6As shown. The sequence alignment results of A-ENA homologs with only 24% sequence identity to A-ENA (SEQ ID NO: 1) are observed (see...). Figure 7 As shown in the figure below, the A-ENA subunit was found to adopt a general structure consisting of the following: a variable N-terminal lock (“NTL”), followed by an α-helix “α1” composed of five heptagonal repeating units (H1-1 to H1-5), followed by a variable-length linker sequence (called “L”), followed by a second α-helix “α2”, and terminating at a variable-length C-terminal tail (CT’). Alignment of the heptagonal repeating units with A-ENA orthologs demonstrated the strong conservation of the positions of the hydrophobic residues involved in intrahelical and interhelical “button-and-hole” interactions and the formation of isopeptide bond units.

[0235] The residues involved in IPB formation (derived from the cryoEM structure of recombinant Q8KNV8 protofibrils) were found to be mostly conserved in their conserved sequence identifiers, and the conservation of hydrophobic residues in the helix suggests that these A-ENA orthologs may also be functional in the formation of self-assembled nanofibrils. Recombination produces A-ENA orthologs (SEQ ID NO: 3-5, such as...). Figure 7 and Figure 8 (as shown) and Bti A-ENA (Uniprot: Q8KNV8, SEQ ID NO: 1) and A-ENA-1 (Uniprot: Q8KNV7; SEQ ID NO: 2) were used to verify the hypothesis. For example... Figure 8 As shown in the image, self-assembled protofibrils do indeed form in the cytoplasm of E. coli. These protofibrils may contain IPB because sequence conservation allows for their formation, and the protofibrils shown in the figure represent insoluble fractions after SDS / thermal extraction, thus indicating the autocatalytic formation of one or more isopeptide bonds.

[0236] Furthermore, based on the α-helical coil-coil structure of the A-ENA monomer, we can outline the connections between the amino acid sequence and structure of the A-ENA protein by representing the monomer as a helical wheel diagram, in which intermolecular interactions are depicted. Figure 7In fact, the “convex-concave” interaction in the helical-helical contact can be precisely defined by the side chains at each position in the sequence based on the Cryo-EM structure, which shows that the fibrils have uniform helical ascent distances of 1.5 Å and twist angles of 100° in the α-helix, respectively, with monomer residues positioned such that every 8th residue is at an equivalent position on the helical wheel diagram and translated 10.5 Å along the helical direction. Therefore, we define helices H1 and H2, or α1 and α2, each formed by a series of five heptamer units or “seven-cell repeats” (H), with side chains named “a” through “g”, providing a conserved sequence motif that allows self-assembly into helical hairpins or α-helical coiled helices, which are used interchangeably as described herein (see [link]). Figure 7 and 10 For further fibril formation, the A-ENA subunits further participate in stacking interactions (i.e., i / i+1 and i / i-1 interactions) through α-helices α1 and α2 in the continuous subunits, achieving paired parallel stacking through the predominantly hydrophobic "knob-in-hole" interaction along the helical length direction. This includes the seven-membered repeating positions "a" and "e" on the C-pole facing side of the A-ENA hairpin and the seven-membered repeating positions "g" and "c" on the N-pole facing side. Figure 10 As shown, the presence of the conserved sequence motif consisting of hydrophobic residues and short side chain residues in the α1 and α2 heptal repeats promotes the formation of antiparallel helical hairpin structures within the A-ENA-like units, and also promotes the pairwise stacking of protofibrillary assembly units (i / i+1 and i / i-1) into helical precursor protofibrils (labeled f), with an approximate twist angle of 12° and an ascent distance of approximately 10.8 Å.

[0237] Exceptions in hydrophobic contacts are caused by residues that participate in isopeptide bond formation and / or form acid-base catalytic residues in the IPB unit. Figure 9 Furthermore, the accumulation of f and f' precursor fibrils in A-ENA is mediated by the "knob-in-hole" interaction between subunits i and i' and i'+1, the "b", "e" and "f" sites in the seven-membered repeats H2-3 and H2-4 in α2, and IPB interactions.

[0238] Example 8. Engineering of A-ENA fibrils

[0239] Based on the structural resolution obtained from the analysis of A-ENA nanofibrils, we tested different positions in the A-ENA protein sequence to explore the addition / insertion of single amino acids, peptides, or folded domains, and tested different sites for site-directed mutagenesis to functionalize the A-ENA nanofibrils. Since both the N-terminus and C-terminus of the A-ENA protein are exposed on the surface in the final precursor fibril assembly, both ends were labeled as potential sites for the addition of single amino acids, peptides, or folded domains. In addition to the ends, we also identified the loop (L, linker) connecting helices α1 and α2 as a potential site for the insertion of single amino acids, peptides, and folded domains. Figure 13 a). In addition to these sites, we propose that solvent-accessible residues on the surface of A-ENA fibrils can serve as potential mutation sites to alter the surface properties (hydrophobicity, electrostatic properties, etc.) of the A-ENA fibrils, and to form targeted modification sites by changing them to readily chemically conjugated natural or non-natural amino acids known to those skilled in the art (i.e., residues containing primary amines, thiols, azides, alkynyl groups, etc., in a non-exhaustive manner). Figure 13 As shown in Figure c, this paper presents these potential mutation sites in the form of a "stick model" and labels the corresponding residues according to the A-ENA sequence (SEQ ID NO: 1). Figure 13 In e, we map these residues to as previously defined and Figure 9 and 10 The five heptagonal repeats of helices α1 and α2 are shown. For wild-type A-ENA protofibrils, the heptagonal repeats H1-2 (and to a lesser extent H1-1 and H1-3) are anchored to the N-terminal locks on the protofibril surface, buried outside the solvent. However, by truncating residues 1-12, the N-terminal locks are lost, exposing these residues to the solvent, thus making them more easily altered. Figure 13 In section b, we defined the 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, in turn, allows us to identify additional residues for site-directed mutagenesis. Figure 13 d) and the corresponding position in their respective seven-element repeats ( Figure 13 f).

[0240] Post-polymerization functionalization of A-ENA fibrils: As an example of A-ENA engineering, we cloned the SpyTag sequence (SEQ ID NO:23) into the C-terminus of A-ENA and A-ENA-1 and examined the resulting constructs to investigate their self-polymerization ability and their ability to form a covalent complex with a dual-domain construct consisting of SpyCatcher003 separated by an SG linker and a superfolded green fluorescent protein (sfGFP). Figure 14 a). Here, A-ENA-SpyTag and A-ENA-1-SpyTag were recombinantly expressed in the cytoplasm of E. coli, and the resulting protofibrils were purified using the method outlined in Example 2. Figure 14 b). Next, we tested whether the fibrils could selectively capture SpyCatcher-sfGFP. For this purpose, we co-incubated a suspension of A-ENA-SpyTag and A-ENA-1-SpyTag fibrils with purified SpyCatcher-sfGFP for 30 min at room temperature. Aliquots of the reaction mixture (10 µl each) were analyzed by SDS-PAGE, followed by detection of the resulting gels using a Li-COR M Odyssey imaging system (488 nm green fluorescence channel). SpyCatcher-sfGFP samples ( Figure 14Lane 1) of the sample ran at an apparent molecular weight of 40 kDa, consistent with the theoretical molecular weight of 40.19 kDa. We also note that the SpyCatcher-sfGFP band still fluoresced at the expected wavelength, indicating that the sfGFP portion maintained correct folding when the fusion protein was coupled to the protofibrils. A second fluorescent band became visible upon co-incubation with A-ENA-SpyTag (lane 2) and A-ENA-1-SpyTag (lane 3), particularly in the gel region corresponding to the stacked gel. This portion of the SDS-PAGE is well beyond the high molecular weight range that can be calibrated with molecular markers, but these are clearly high molecular weight substances. Given that these species (i) were trapped in the pore network of the stacked gel after boiling with NuPage LDS sample buffer and (ii) exhibited fluorescence, we conclude that they are A-ENA and A-ENA-1 protofibrils covalently coupled to SpyCatcher-sfGFP. A large-scale co-incubation experiment with A-ENA-SpyTag fibrils and soluble SpyCatcher-sfGFP further confirmed this conclusion. After incubation at room temperature for 30 min, the fibril suspension was centrifuged at 20,000 rcf for 30 min. The resulting precipitate (insoluble) and supernatant (soluble) were irradiated under a Clare Chemicals DR46B blue LED transilluminator; this instrument uses pure visible blue light to excite the sample and filters the signal through an amber filter, thus enabling fluorescence visualization. Figure 14 d). We recorded a clear fluorescence signal in the protofibril precipitate after centrifugation, indicating that the SpyCatcher-sfGFP molecule was effectively chelated by the A-ENA protofibrils.

[0241] Prepolymerization functionalization of A-ENA fibrils – Optimized splicing strategy: The SpyTag method described above can be used for post-polymerization functionalization of A-ENA fibrils. Using the SpyCatcher fusion construct, virtually any "passenger domain"—regardless of size or molecular weight—can be efficiently coupled to the A-ENA scaffold. Since the display spacing, or "rise," of the SpyTag peptide on each precursor fibril is approximately 10.8 Å, we anticipate that this method will not be able to achieve complete stoichiometric labeling of all available binding sites for display domains with hydrodynamic radii far exceeding 10.8 Å. For cases requiring a 1:1 stoichiometric ratio between the A-ENA and the target display domain (e.g., to minimize the distance between domains distributed along the fiber axis), or for cases where a "pre-polymerization functionalization" strategy is preferred, we have developed a second A-ENA-based functionalization display method. Figure 15 a). To this end, we constructed genetic fusions between A-ENA and multiple display domains, separated by flexible joints. This method yielded homologous polymers with a display density of 0.108 Å. -1 Therefore, it is necessary to optimize the linker to avoid steric hindrance between adjacent display domains. By introducing a flexible linker, the inserted display domains gain conformational freedom, allowing them to be arranged in a zigzag or staggered stacking manner, rather than simply translated stacking along the fiber axis. In this way, domains with heights exceeding the 10.8 Å rise distance of the A-ENA subunits in the precursor fibrils can be accommodated through staggered stacking. The preferred linker length is 1 to 20 amino acid residues, but its length can be appropriately increased if higher degrees of freedom are required and / or if a greater distance from the A-ENA fibril backbone is needed. The preferred linker mainly consists of small and hydrophilic amino acids (G, A, S, T, N, D), with no specific preference in their arrangement; however, it may also contain larger hydrophilic residues (K, E, R, Q, H, Y), and is typically low in proline or absent. The design of the linker can be randomized or guided by cutting-edge protein modeling and design tools such as Protein MPNN (Dauparas et al., Science 378, 49-56 (2022)) and Rosetta (https: / / github.com / RosettaCommons), as is well known to those skilled in the art. In one approach, the insertion domain is positioned along the A-ENA backbone in a predetermined orientation and location using computer simulation (in silico); and the N-terminus, C-terminus, or spacer residues connecting to the C-terminal or N-terminal residues of the insertion domain can be constructed in a computer using protein design tools. Validation of the connector design can be performed firstly 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 is well known to those skilled in the art; alternatively, it can be validated experimentally by observing the culture supernatant using negative-stain electron microscopy (EM). Here, we report the successful recombinant expression and polymerization of three different A-ENA fusion constructs (p66a, MBD2, and erythroredoxin; SEQ ID NO: 8, 9, 10). Figure 15Figure b shows nsTEM images of self-assembled A-ENA-TEV-p66a, A-ENA-TEV-pMBD2, and A-ENA-TEV-erythroredoxin fibrils purified from E. coli cytoplasm.

[0242] Next, we conducted functional experiments to demonstrate the folding properties of the domains. p66a and MBD2 are two α-helical proteins derived from Homo sapiens, forming an antiparallel coiled-coil complex with nanomolar affinity. Here, we expected that a blend of A-ENA-TEV-p66a and A-ENA-TEV-pMBD2 would result in a non-covalently cross-linked fibrillary network driven by the formation of an interfibrillary complex between p66a and MBD2. To demonstrate this interfibrillary coupling, A-ENA coiled-coil fibrils were purified as described in Example 2, followed by additional freeze-thaw cycles of the sample with 12% (w / v) urea and 8% (w / v) NaOH to obtain higher purity. After three washing steps, the insoluble precipitates of fibrils were harvested by sequential centrifugation (20,000 rcf, 30 min) and resuspending in 1×PBS buffer. The protein precipitates of 100 mg A-ENA-p66α and A-ENA-MBD2 were resuspended separately in 180 μl of 50 mM Tris buffer, pH 7.0. 60 μL of each solution was combined and thoroughly mixed. After 30 min, an Eppendorf inversion assay was performed to detect hydrogelation. Figure 15 d). In the inverted test, the suspensions of A-ENA-p66α and A-ENA-MBD2 did not remain fixed at the bottom of the Eppendorf tube, while the mixed suspension formed a turbid yet stable hydrogel that remained stably at the bottom of the Eppendorf tube. This indicates that the corresponding display domains (i) are folded and (ii) readily composite, leading to interfibrillary coupling, kinetic trapping of the polymer network, and the formation of the mixed A-ENA hydrogel.

[0243] To determine the folding properties of the erythroredoxin transit domain (from *Vibrio sulphureus*), a thermogravimetric assay based on SYPRO orange fluorescence was performed. For this purpose, 25 μl of a sample containing target fibrils combined with 1X SYPRO™ Protein Gel Stains (Thermo Fisher Scientific; Waltham, MA, USA) was loaded into a Hard-Shell 96-well microplate (Bio-rad; Hercules, CA, USA). Using a CFX opus 96 qPCR reader system (Bio-rad; Hercules, CA, USA), the temperature was increased from 25°C to 100°C at a rate of 0.5°C every 10 seconds, and fluorescence emission spectra were measured at each temperature interval. Melting curves were obtained by calculating the first derivative of the relative fluorescence units (RFU) in order of temperature. Figure 15 e). When the value is >1000 RFU / dT, the reported melting temperature corresponds to a local maximum in the melting curve. For A-ENA wild-type fibrils, no significant maximum value greater than 1000 RFU / dT was detected, indicating that no significant unfolding events occurred within the measured temperature range. Figure 15 e). However, for the A-ENA-TEV erythrocyclic protein sample, a significant maximum value (1435 dRFU / dT) was detected at 94.5 °C, indicating that the fused erythrocyclic protein domain is unfolded. Therefore, we infer that the erythreotropic protein domain grafted onto the A-ENA scaffold is natively folded and shows an unfolding Tm of 94.5 °C. This Tm is similar to the previously reported thermal folding temperature of bacterial erythreotropic proteins. Although there is currently no information regarding the common desulfurization Vibrio (Cervus vulgaris) used in this paper... Desulfovibrio vulgaris The exact unfolding data for erythrocyclooxygenase are unknown, but it is known that this protein rapidly unfolds at 100°C (Lazaridis, I. Lee and Karplus 1997, Protein Sci. 6(12):2589-605). By comparison, from Clostridium pasteurellum ( Clostridium pasteurianum The reduced state of the homologous erythroredoxin (70% amino acid sequence identity) has an unfolding temperature of 69 °C, while the oxidized state has an unfolding temperature of 83 °C (Bonomi et al., 2000, Protein Sci. 9(12): 2413-26).

[0244] Prepolymerization functionalization of A-ENA fibrils – with loop insertion

[0245] To evaluate whether A-ENA fibrils can be modified by inserting recombinant sequences into the joint loops of helices 1 and 2 connecting A-ENA-like fiber subunits ( Figure 24A gene fusion construct was prepared in which a foreign sequence was inserted between valine 55 and threonine 56 residues of Bacillus thuringiensis subsp. Israel A-ENA (UniprotKB Q8KNV8; SEQ ID No:1). As a non-limiting example, a sequence corresponding to a solenoid domain was inserted, which contains two curlin-like repeat sequences with the amino acid sequence shown in SEQ ID NO: 66 (hereinafter referred to as "R4.5-2RFD"). The inserted curlin-like repeat domain was linked to the A-ENA monomer via two amino acid linkers: a GG linker connecting the C-terminus of helix 1 to the N-terminus of the inserted sequence, and an SG linker connecting the C-terminus of the inserted sequence to the N-terminus of helix 2, thereby producing a fusion protein referred to herein as "A-ENA_LI-R4.5-2RFD" (SEQ ID NO:67). In these A-ENA cyclic insertion designs, the length and sequence of the linking residues connecting helices 1 and 2 to the insertion sequence are variable and can be shortened or increased depending on the required distance between the A-ENA fibril scaffold and the insertion sequence displayed on the A-ENA fibril ridge. Figure 24 ).

[0246] The construct containing the coding sequence A-ENA_LI-R4.5-2RFD (SEQ ID NO: 67) was prepared by PCR linearization of a plasmid vector carrying the coding sequence A-ENA (pASK-A-ENA) using outward primers (primers LI_1 and LI_2, SEQ ID NO: 69-70) located flanking residues 55 and 56. Subsequently, the vector was assembled with a DNA fragment using Gibson Assembly; this DNA fragment corresponded to the coding sequence and adapter of the desired insert sequence, and was flanked by complementary sequences corresponding to LI_1 and LI_2, respectively.

[0247] To assess whether the A-ENA_LI insertion mutant (also referred to herein as the A-ENA sequence, in which a heterologous or exogenous sequence is inserted into the linker connecting helix 1 and helix 2) retains its self-assembly properties, a plasmid encoding the A-ENA_LI-R4.5-2RFD (SEQ ID NO: 67) sequence was transformed into *E. coli* DH5α cells. When OD 600When the concentration reached 0.8, 50 µM tetracycline was added to induce A-ENA_LI-R4.5-2RFD expression, and the culture was then incubated overnight at 18°C. Cells were resuspended in a buffer containing 1 mg / mL lysozyme, 5 mM EDTA, 50 mM Tris pH 6.8, and 50 mM NaCl, and incubated with stirring at room temperature for 18 h. 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 min and centrifuged at 20,000 rcf for 30 min using a Beckman JA 14.50 50 mL Falcon rotor. The supernatant was discarded, and the precipitate consisting of A-ENA fibrils was resuspended at a concentration of 5 mL miliQ water per gram of wet fibril precipitate. The resulting suspension was centrifuged three more times (20,000 rcf, 30 min), and each precipitate was resuspended in miliQ water to remove residual SDS. The suspension obtained after the final washing step was diluted tenfold in miliQ water, deposited on a Formvar / carbon grid (400 mesh, Cu; Electron Microscopy Sciences), stained with 2% (w / v) uranyl acetate, and blotted dry. Negative staining transmission electron microscopy (nsTEM) analysis revealed the presence of micron-long fibrils with a diameter of approximately 10 nm and a length on the micrometer scale. Figure 24 (d, e) demonstrate that the A-ENA connector insertion variant retains its ability to self-assemble into SDS-stable A-ENA-like nanofibrils.

[0248] Prepolymerization functionalization of A-ENA fibrils – Block copolymer strategy: The optimized linker design strategies outlined above are ideally suited for displaying heterologous domains with molecular weights comparable to or smaller than A-ENA. To display proteins much larger than A-ENA, we developed a block copolymer design strategy (…). Figure 16a). At the heart of this strategy is the co-expression of a wild-type A-ENA gene fusion construct in *E. coli*. As evidence of this principle, we worked with a camel VHH antibody (nanobody) coupled to the A-ENA terminus using a TEV-protease cleavage site as a linker (hereinafter referred to as nanobody-TEV-A-ENA; SEQ ID NO: 11). Nanobody-TEV-A-ENA was cloned into the pBAD vector and transformed into Top10 *E. coli* strains carrying the wild-type pASK-A-ENA construct. The resulting co-expression led to the formation of hybrid A-ENA fibrils composed of the A-ENA and nanobody-TEV-A-ENA subunits. Figure 16 (b) We expect the distribution of the two subunit types in the A-ENA protofibrils to be random and similar to the topology of block copolymers. The average distance between the two display domains depends on the block size within the copolymer. The ratio between the A-ENA and A-ENA fusion constructs is modulated by carefully controlling the expression levels of the two proteins (by selecting their respective promoter strength, plasmid copy number, inducer concentration, and induction time). The advantage of this strategy is that it does not impose an upper limit on the size of the guest domains due to the random distribution of the A-ENA and A-ENA fusion constructs within the protofibril assembly.

[0249] Example 9. In vivo functionalization of A-ENA sporophytic network in Bacillus thuringiensis

[0250] exist Figure 14 , 15 In the experiments shown in 16 and 17, we have demonstrated how to functionalize A-ENA subunits through direct fusion of peptides, including but not limited to affinity reagents such as nanobodies, metal-binding proteins such as erythroredoxin, affinity tags such as SpyTag or SpyCatcher, and coil-coil pairs. We demonstrate that the modified A-ENA retains its property of self-assembling into homopolymeric and / or heteropolymeric (i.e., block copolymer) nanofibrils, and the fused peptide retains its activity. In this way, A-ENA nanofibrils can be formed, which are functionalized by gene fusion or by covalent or non-covalent addition of functional peptides, with functionalization methods not limited to SpyTag–SpyCatcher technology, StrepTag–Streptavidin technology, and coil-coil pairing. These principles are based on… Figure 14-17 The in vitro assembly of A-ENA nanofibers is shown in the figure.

[0251] exist Figure 21In the experiments shown, we aimed to determine whether the A-ENA fusion protein could form in vivo during sporulation, producing Bt endospores modified with engineered A-ENA protofibrils as described above. To evaluate this, Bti and Btk were genetically modified to introduce a plasmid carrying an A-ENA:SpyTag expression construct regulated by endogenous A-ENA promoters and terminators (SEQ ID NO: 45 and 46, respectively). Transformed cells showed expression of A-ENA protofibrils with morphological characteristics indistinguishable from wild-type (WT) A-ENA protofibrils and similar to those observed in in vitro-grown A-ENA-SpyTag nanofibrils. Figure 14 Subsequently, isolated endospores derived from these transformed cells were placed in purified SpyCatcher-sfGFP (approximately 240 μM, ~5 mg / mL) for 30 minutes, followed by thorough washing with PBS (four times). Meanwhile, WT Bti and Btk spores were introduced as negative controls. After the washing steps, fluorescent labeling was observed only on the surface of endospores expressing A-ENA:SpyTag, while endospores from WT Btk or Bti showed no fluorescence.

[0252] These experiments demonstrate that, similar to what has been observed in vitro with A-ENA:SpyTag nanofibers, WT A-ENA nanofibers can be incorporated into the A-ENA:SpyTag subunit and modified by SpyCatcher and SpyCatcher fusions. In this way, and further by employing the aforementioned alternative labeling methods, A-ENA nanofibers attached to the surface of Bt endospores can be functionalized in vivo. Using these methods, Bt endospores can be functionalized with affinity reagents containing specific binding properties to biotic and abiotic surfaces (e.g., in a non-limiting manner, the gastrointestinal epithelium of specific insects and insect larvae, and the surfaces of specific plants and crops). In this way, functionalized Bt spores with increased tropism for specific insect populations and / or increased affinity and retention time on desired crops or surfaces can be obtained.

[0253] Example 10. Physicochemical stability of A-ENA fibrils

[0254] To test the chemical stability of recombinant A-ENA fibrils, fibrils were harvested from the concentrated stock solution by centrifugation (20,000 rcf, 1 h, supernatant discarded). 100 µl aliquots of the sample were resuspended after centrifugation and incubated under different conditions: 100 µl of 2% (w / v) SDS, 8 M urea, 2 M NaOH, or 100% (v / v) formic acid were added, followed by negative staining TEM sample preparation. Specific steps are as follows: Figure 22 As shown. To test the physical stability of recombinant A-ENA fibrils, 100 μl aliquots of the fibril storage solution were dried in an open glass vial at 200 °C for 15 min in an oven. The resulting dried material was rehydrated in 100 μl of MiliQ water and then subjected to nsTEM imaging. Additionally, 100 μl aliquots were autoclaved at 121 °C for 20 min. Then, 3 μl aliquots of the treated sample were deposited on a Cu-mesh Formvar grid, washed twice with 20 μl of MiliQ water, incubated with 2% (w / v) uranyl acetate for 1 min, and blotted dry with Whatmann 2 filter paper. Micrographs were collected at 60,000 × magnification using a 120 kV JEOL 1400 microscope equipped with a LaB6 filament and a TVIPS F416 CCD camera. Figure 22 As shown, under each of these treatment conditions, which would typically lead to protein denaturation or hydrolysis, A-ENA fibrils remained intact and were largely preserved.

[0255] Example 11. A-ENA increases the insecticidal activity of Bacillus thuringiensis subsp. Kustack.

[0256] As shown in Example 6, A-ENA constitutes a virulence factor contributing to the insecticidal activity of Bacillus thuringiensis subsp. Israel; furthermore, as shown in Examples 5 and 9 ( Figure 19 , 21 As shown in the figure, recombinant expression of A-ENA in *Bacillus thuringiensis* subsp. *kustaq* produced a sporophytic network similar to that of the *Israeli* subsp. *japonica*, in which biconical PSB crystals were connected to bundles of A-ENA. Next, the recombinant expression of A-ENA was used to test whether the naturally occurring A-ENA in Bt strains could confer "functional gain" properties, potentially enhancing their virulence and thus improving their efficacy in pest control.

[0257] Powdered Noctuid moth ( Trichoplusia ni The cabbage looper (Btk), commonly known as the cabbage inchworm, is a highly destructive leaf-eating pest that wreaks havoc on cruciferous crops such as cabbage and broccoli. Because it is a natural target for Btk, it is used as an insect model.

[0258] The Btk strain expressing A-ENA on the plasmid (gain-of-function strategy) was tested, and its larval survival was compared with that of larvae fed on spores supplemented with Btk WT or recombinant Btk WT strain expressing A-ENA (Btk + A-ENA). Although Bti naturally contains A-ENA, its PSB is ineffective against the white armyworm and was therefore used as a negative control. Figure 23 c). We used 1-week-old larvae and followed up on their survival over the following week after adding a spore suspension to their solid feed medium. The results clearly showed that on day 4, approximately 50% (15 / 28) of the larvae in the Btk WT strain remained alive, while in the Btk strain expressing A-ENA, this number decreased to approximately 10% (23 / 26). Figure 23 c). Based on observations of enhanced virulence from A-ENA aggregation activity, we investigated whether purified A-ENA protofibrils (recombinantly generated in *E. coli*) were added to WT Btk spores. ns-EM showed that the aggregation of PSB-mimicking spores with added purified A-ENA was similar to that observed in Btk spores recombinantly expressing A-ENA. Figure 19 and 23 a). The results showed that the addition of recombinant A-ENA protofibrils effectively induced the clustering of Btk spores and PSB. Figure 23 a). We then repeated the killing test with WT Btk spores and PSB suspension, with and without the addition of recombinant A-ENA fibrils. Consistent with our earlier findings, approximately 50% (8 / 16) of the larvae in the WT Btk strain remained alive on day four, while the survival rate decreased to approximately 18% (3 / 16) in the presence of recombinant A-ENA fibrils. Figure 23 d). As a negative control, we included recombinant A-ENA to confirm that the A-ENA fibrils themselves do not exert a direct toxic effect.

[0259] Example 12. A single isopeptide bond is sufficient to self-assemble SDS-resistant A-ENA-like nanofibrils

[0260] To determine the minimum number of isopeptide bonds required to form A-ENA-type protofibrils with high physicochemical stability (see Example 10), we selectively mutated the acid-base catalytic residues in the IPB triplet, which are essential for the formation of the five different isopeptide bonds present in A-ENA (UniprotKB Q8KNV8; SEQ ID NO: 1). Based on the A-ENA nanofibril structure disclosed herein, it can be seen that IPB3 may be sufficient to form an IPB network that crosslinks all protofibrils and two A-ENA precursor protofibrils f and f' into a single covalently linked A-ENA nanofibril molecule. Figure 25(a, b) Any other IPB observed in wild-type A-ENA nanofibrils, either as a single IPB or in combination with one or more IPBs other than IPB3, results in the A-ENA subunits being cross-linked within the A-ENA precursor fibrils, rather than cross-linked across the A-ENA precursor fibrils. Therefore, we modified A-ENA (UniprotKB Q8KNV8; SEQ ID NO 1) to mutate residues E28, E39, and E41 to similar but non-catalytically inactive residues Gln, essentially leading to the inactivation of IPB4 and 5, IPB2, and IPB1, respectively (see [link to relevant documentation]). Figure 5 b). Therefore, using directional PCR-based mutagenesis, the coding sequence for A-ENA in plasmid pASK_A-ENA was mutated, resulting in plasmid pASK_E-ENA E28Q_E39Q_E41Q. Alphafold 3 predicted that the protofibrils formed by this mutant protein are nearly isomorphic to the protofibrils of the wild-type A-ENA protein. Figure 25 c). To verify this prediction, we prepared the A_ENA_E28Q_E39Q_E41Q mutant (SEQ ID NO:68) using recombinant expression technology. Specifically, the pASK_E-ENA_E28Q_E39Q_E41Q plasmid was transformed into E. coli C43, and the bacterial culture was allowed to oxidize to 0.05% OD200. 600 When the concentration reached 0.8, 50 μM dehydrotetracycline was added for at least 1 hour to induce expression. The cell pellet was then treated with A-ENA as described above, and the resulting pellet was examined by negative staining EM, confirming the presence of anti-SDS nanofibrils, whose diameter and morphology were consistent with wild-type A-ENA fibrils. Figure 25 d).

[0261] Example 13. General procedures and processes for the preparation, processing, and storage of ENA nanofibers.

[0262] A-ENA nanofibrils are typically prepared through biofermentation involving recombinant expression in bacteria (preferred), yeast, plant, or animal hosts. For this purpose, A-ENA protein subunits are expressed via plasmids carrying the A-ENA coding sequence or through genomic insertion copies, directing expression to the host cytoplasm or export apparatus (i.e., general secretion (SEC) or diarginine (TAT) pathways). The subunits self-assemble into robust nanofibrils of nanometer to micrometer scale lengths. Figure 26I.). A-ENA nanofibrils are collected from cultures by mild cell lysis. Cells can be lysed in situ in culture medium or in a concentrated suspension after precipitation or centrifugation and resuspending in the desired buffer. Methods of mild lysis include (not exhaustive): spontaneous or induced autolysis (e.g., by co-expression of endosomalin, lysozyme, or other cell wall degradation proteins known in the art), suspension in hypotonic buffer, freeze-thaw cycles, mild sonication, detergent treatment, and EDTA-lysozyme treatment (…). Figure 26 II.) Cell debris and other insoluble contaminants are then removed from the cell lysate by methods known in the art, including (in a non-exhaustive manner) sedimentation, low-speed centrifugation (e.g., 2,000 – 10,000 g), cross-flow filtration, and enzymatic treatment (i.e., using proteases, glycosylhydrolases, nucleases, cellulases, esterases, etc.). Figure 26 III.) Further purification of suspended fibrils from soluble and smaller suspended contaminants is achieved through detergents (i.e., SDS, LDS, DDM, tergitol, Tween™, Triton™, Elugent, etc.) and / or chemical detergents (i.e., NaOH, NH4OH, urea, formic acid, alcohols, and / or organic solvents) and / or enzymatic degradation (i.e., using proteases, glycosylhydrolases, nucleases, cellulases, esterases, etc.). Figure 26 IV(a)). The fibrils are separated from soluble or small suspended contaminant precipitates (including chemically induced precipitates) or from high-speed centrifugation (typically above 10,000 g), cross-flow, or dead-end centrifugation. Figure 26 IV(b)). The desired A-ENA fibrils purity is obtained through a series of washing and separation steps as described above. Finally, the purified A-ENA fibrils are collected and / or stored by drying and / or freeze-drying (i.e., obtaining dried pellets, flakes, films, or powder), or they can be stored in suspension in pure water, aqueous buffer solutions, and / or acid, alkali, salt solutions, and organic solvents or combinations thereof, under frozen, refrigerated, or ambient temperature conditions. Figure 26 V).

[0263] Table 1: HMM pattern of Helix 1 (H1)

[0264] Table 2: HMM pattern of Helix 2 (H2)

[0265] Table 3: NCBI Reference Sequence Accession Numbers for A-ENA-like Proteins (n ​​= 593)

[0266] >WP_087876290.1; >WP_000525743.1; >OUB6455ssaid A-ENA proteinsequence is selected from the list of SEQ ID NOs: 1-6, or a modified orengineered or functionalized fibril consisting of A-ENA proteins selectedfrom SEQ ID NO: 1-6, wherein the NTL, and / or Linker, and / or CT aremodified.said A-ENA protein sequence is selected from the list of SEQ ID NOs:1-6, or a modified or engineered or functionalized fibril consisting of A-ENAproteins selected from SEQ ID NO: 1-6, wherein the NTL, and / or Linker, and / orCT are modified.said A-ENA protein sequence is selected from the list of SEQID NOs: 1-6, or a modified or engineered or functionalized fibril consistingof A-ENA proteins selected from SEQ ID NO: 1-6, wherein the NTL, and / orLinker, and / or CT are modified.aid A-ENA protein sequence is selected fromthe list of SEQ ID NOs: 1-6, or a modified or engineered or functionalizedfibril consisting of A-ENA proteins selected from SEQ ID NO: 1-6, wherein theNTL, and / or Linker, and / or CT are modified.9.1; >OUB63489.1; >RGP45207.1; >PFK04390.1; >OUB60984.1; >OUB63491.1; >WP_098733738.1; >MRB61591.1; >WP_088055169.1; >WP_002187581.1; >WP_078205751.1; >WP_088081642.1; >WP_000525745.1; >WP_000512651.1; >ANN35806.1; >WP_076514003.1; >WP_242175597.1;>WP_214484605.1; >WP_214484129.1; >WP_069717931.1; >WP_069717930.1; >WP_069716202.1; >WP_069716955.1; >WP_069715754.1; >WP_204594723.1; >WP_162037491.1; >WP_160646769.1; >OUM94251.1; >MBO8142698.1; >HIV86996.1; >WP_186888340.1; >URN93840.1; >WP_258200105.1; >WP_100402143.1; >MCL1803234.1; >MCL1989263.1; >MCL2620379.1; >MCL2202208.1; >MCL2188398.1; >MCL2253796.1; >MCL2637362.1; >MCL1914620.1; >MBG9510702.1; >EOO23151.1; >MCL2839321.1; >WP_051685315.1; >MCL2839204.1; >MCL2397714.1; >ETT57678.1; >MCL2194671.1; >MCL1865810.1; >MCL1803896.1; >MCL2611298.1; >MCL2611297.1; >MCL1804267.1; >MCL2637361.1; >MCL1903715.1; >MCL1865811.1; >MCL2188397.1; >MCL2253797.1; >MCL1881257.1; >MCL1918021.1; >WP_193734953.1; >WP_202621541.1; >WP_207707864.1; >WP_059049831.1; >WP_018749816.1; >WP_259617704.1; >WP_190920736.1; >WP_137185573.1; >WP_128657586.1; >WP_020616847.1; >WP_018886156.1; >WP_028590715.1; >WP_049741784.1; >WP_106838507.1; >WP_197240296.1; >WP_088910015.1; >WP_173620449.1; >WP_144618486.1; >WP_144991172.1; >WP_239334048.1; >WP_127585514.1; >WP_138186406.1; >WP_127456484.1; >WP_171681843.1; >WP_161411874.1; >WP_079412741.1; >WP_091176140.1; >OAB30305.1; >WP_106838506.1; >WP_106835505.1; >WP_106838505.1;>WP_007720346.1; >OBZ18015.1; >OBZ19495.1; >WP_270170783.1; >WP_028562892.1;>WP_149453541.1; >WP_028778804.1; >WP_240877222.1; >WP_149453540.1; >WP_246861293.1; >WP_223069556.1; >WP_251637931.1; >WP_251477650.1; >WP_003350018.1; >WP_274854078.1; >WP_090915043.1; >WP_090915041.1; >WP_213144224.1; >WP_213117746.1; >WP_217036363.1; >WP_251637948.1; >WP_126145139.1; >WP_213656143.1; >WP_028987879.1; >CCQ93995.1; >WP_138224389.1;>WP_072581397.1; >WP_213656140.1; >WP_272560816.1; >WP_213117745.1; >WP_213144223.1; >MBK5491850.1; >PEW69385.1; >WP_098650236.1; >MBK5491845.1; >WP_144509082.1; >MBY0037307.1; >WP_206656005.1; >WP_155701568.1; >WP_113029033.1; >WP_238188342.1; >WP_197936188.1; >WP_128896304.1; >WP_128896303.1; >WP_227874317.1; >SFX69187.1; >WP_236818349.1; >WP_256761492.1;>WP_119150865.1; >WP_256761488.1; >WP_068775895.1; >RUT46111.1; >WP_215175550.1; >RXZ80750.1; >WP_068661315.1; >WP_093293777.1; >WP_131848861.1;>WP_093292633.1; >WP_197935515.1; >WP_119150866.1; >WP_005549089.1; >WP_244715230.1; >SFX69347.1; >WP_131849659.1; >WP_249075847.1; >WP_091176133.1;>WP_018885230.1; >WP_238189196.1; >WP_238189194.1; >WP_041058496.1; >WP_133378365.1; >WP_147208463.1; >NMA89768.1; >WP_036201651.1; >WP_121214240.1;>AIM15264.1; >NEX80099.1; >WP_126294795.1; >WP_071392824.1; >WP_151701241.1;>WP_210471316.1; >WP_040376149.1; >WP_212924438.1; >WP_204668115.1; >WP_212976039.1; >WP_066186838.1; >WP_152657822.1; >WP_040982409.1; >WP_188733585.1; >WP_066142189.1; >WP_115749335.1; >WP_020616168.1; >WP_128658904.1; >WP_238650419.1; >WP_173140208.1; >WP_010268044.1; >WP_047980762.1; >WP_263706280.1; >MBO2505129.1; >WP_016839178.1; >WP_141601888.1; >WP_208650703.1; >WP_090774452.1; >WP_090776151.1; >PFB49380.1;>WP_257209516.1; >OQR53105.1; >WP_068775901.1; >WP_142506947.1; >TQR39522.1;>WP_119150851.1; >WP_215175558.1; >WP_215175557.1; >WP_211350104.1; >WP_175371047.1; >PGQ44283.1; >WP_181538938.1; >WP_044896080.1; >MCL6586930.1; >WP_181538937.1; >WP_012957170.1; >KQU17358.1; >KRF52451.1; >KRF52448.1; >KQU17360.1; >WP_066154797.1; >WP_155477750.1; >NLX02164.1; >NLK52489.1; >NLV22477.1; >WP_134230475.1; >WP_134230476.1; >WP_224876227.1; >WP_064094024.1; >WP_224876226.1; >WP_197315488.1; >WP_224809048.1; >WP_066265084.1; >WP_270407384.1; >WP_134230544.1; >WP_069644379.1; >WP_069703205.1; >WP_069644377.1; >WP_153725722.1; >NLF45454.1; >WP_221860360.1;>WP_019155143.1; >NLT40941.1; >MBE3101226.1; >MBE3101227.1; >WP_235222005.1;>OQB14150.1; >MCL2703241.1; >WP_242057465.1 / 6-7; >WP_019155144.1; >WP_080630428.1 / 4-4; >WP_183242236.1; >WP_042536196.1 / 4-4; >KIP20212.1 / 11-11; >HBR31256.1; >OON92172.1; >ONI43887.1; >HIS65799.1; >HIU33750.1; >NLK86606.1;>WP_090446350.1; >QGU95879.1; >WP_072907480.1; >MCL6617755.1 / 104-1; >WP_181520874.1; >WP_055441798.1; >AST05588.1; >WP_035048978.1; >WP_184664076.1;>WP_184664075.1; >WP_110609538.1; >WP_093051849.1; >WP_106589654.1; >WP_010239793.1; >MCK9536559.1; >WP_078665439.1; >NMB44602.1; >NLJ90181.1; >OQA15291.1; >NLO10347.1; >WP_236914082.1; >WP_252225997.1; >WP_204489239.1; >HHU63352.1; >WP_254495860.1; >HBI56858.1; >WP_008908033.1; >SEF39824.1; >WP_242971176.1; >WP_122963797.1; >WP_122903756.1; >WP_007783061.1; >WP_122961155.1; >WP_122925796.1; >WP_163859565.1; >WP_134757257.1; >WP_173140217.1; >WP_216857486.1; >WP_049741785.1; >WP_088910016.1; >WP_106657270.1; >WP_172139574.1; >WP_137031666.1; >WP_016739500.1; >WP_106784580.1; >WP_219661378.1; >WP_047070179.1; >TQR34900.1; >WP_087348525.1;>WP_199929547.1; >WP_017246715.1; >WP_174226400.1; >WP_144618485.1; >WP_007720341.1; >WP_056488977.1; >WP_197936189.1; >WP_010268048.1; >OAB30303.1;>WP_138224403.1; >WP_091176138.1; >WP_130607412.1; >WP_087443534.1; >WP_127585513.1; >WP_161411873.1; >WP_173215395.1; >WP_079412742.1; >WP_171414263.1; >WP_021254599.1; >WP_270170784.1; >OBZ18017.1; >OBZ18016.1; >WP_028562891.1; >WP_209971550.1; >WP_028546586.1; >WP_232275159.1; >WP_028595674.1; >WP_258279168.1; >WP_175373344.1; >WP_058303866.1; >WP_048744837.1; >CDN41938.1 / 4-4; >WP_036651907.1; >WP_113029034.1; >MBD2860923.1; >WP_223836348.1; >WP_144509072.1; >WP_098688323.1; >MBK5491851.1; >WP_252211459.1; >PFI81502.1; >PGK33495.1; >WP_098650108.1; >WP_197722729.1; >WP_216636299.1 / 22-; >WP_197478852.1 / 21-; >KXG08769.1; >WP_115749336.1; >WP_246020340.1; >WP_104059545.1; >WP_151701235.1; >WP_212508844.1; >WP_174521724.1; >WP_191814960.1; >WP_244811852.1; >WP_173660201.1; >WP_036198767.1; >WP_208650728.1; >WP_019155127.1; >WP_212924437.1; >WP_040982410.1; >WP_229720117.1; >WP_235817440.1; >WP_244704997.1; >WP_244853042.1; >WP_239587967.1; >SKB05896.1; >WP_244715222.1;>WP_185959608.1; >NMA90008.1; >MBP1969341.1 / 5-5; >WP_209462530.1; >WP_257230192.1; >MBG0967840.1; >WP_236686903.1; >WP_019244172.1; >ALP35341.1; >WP_238354028.1; >WP_044648569.1; >WP_212734477.1; >WP_211556547.1; >SIS52057.1; >WP_234969477.1; >WP_245629781.1; >AEJ44700.1; >WP_237700123.1; >ACV59513.1; >WP_245530800.1; >MCD8500378.1; >WP_071313875.1; >WP_089023434.1;>WP_197315495.1; >WP_134230541.1; >WP_064094028.1; >WP_224876222.1; >WP_217225283.1; >WP_246421570.1; >WP_242175596.1; >WP_274069240.1; >TQR39524.1;>WP_255504946.1; >MCD8503411.1; >WP_166246166.1; >WP_131015779.1; >WP_270407379.1; >WP_232317625.1; >NLC94992.1; >REK56576.1; >WP_113805716.1; >WP_078665440.1; >WP_188038901.1; >WP_075387276.1; >RBW69966.1 / 7-7; >WP_245947291.1; >WP_171719036.1; >WP_209879878.1; >ETT74612.1; >WP_256761495.1;>RXZ80755.1; >WP_119150854.1; >WP_127738572.1; >WP_127738463.1; >WP_131015782.1; >WP_166246067.1; >MBG9580252.1; >WP_173658896.1; >NLJ96303.1; >MCL1913461.1; >MCL2840958.1 / 19-19; >PKM89043.1; >WP_013278103.1; >WP_248625689.1; >WP_248662742.1; >WP_015326943.1; >WP_015326949.1; >WP_015326948.1; >WP_248625692.1; >WP_248662330.1; >WP_248625694.1; >WP_248662329.1; >WP_013278105.1; >WP_197079058.1; >WP_248625696.1; >WP_248662326.1; >WP_248662328.1; >MTI81694.1; >WP_153725708.1; >MTI81688.1; >BCS80379.1; >WP_011915850.1; >WP_127351039.1; >WP_240666752.1; >WP_240877221.1; >WP_149452665.1; >WP_213656139.1; >WP_155701569.1; >WP_259617707.1; >WP_018886158.1; >WP_128657583.1; >WP_063824367.1; >WP_190920739.1; >WP_028987877.1; >CCQ93392.1; >WP_272560814.1; >WP_066325799.1;>MCL6586927.1; >WP_205536689.1; >WP_235820437.1; >WP_238941729.1 / 17-; >WP_198508029.1; >WP_220129278.1; >WP_246861292.1; >WP_223661909.1; >WP_082028094.1; >WP_039237096.1; >WP_119112635.1; >WP_273127134.1; >WP_069642565.1; >WP_069703207.1; >BDH61653.1; >WP_248573988.1; >WP_251637951.1;>MCM3693956.1; >WP_270407375.1; >WP_166246065.1; >WP_131015781.1; >ONI38470.1; >HIS64903.1; >WP_071219938.1 / 6-6; >WP_127596680.1 / 6-6; >WP_213614477.1; >WP_076358068.1; >WP_007726804.1; >MBU3806290.1; >WP_058963077.1; >HJA24947.1; >HJB67667.1; >GKI15227.1; >MBP8856243.1; >WP_009325957.1; >HIY11658.1; >WP_087292624.1; >MCI2047631.1; >NCC08039.1; >WP_072831610.1; >WP_072832354.1; >WP_242860591.1; >WP_008790747.1; >WP_270640527.1; >MCL2703097.1; >MBP1736326.1; >MCL2859314.1; >MCL2619294.1; >MCL2853074.1; >WP_016147410.1; >WP_242825622.1; >WP_118643908.1; >WP_071430535.1; >WP_087336448.1; >HIZ93229.1; >WP_204706061.1; >WP_087257125.1;>MCI6990316.1; >RHO54856.1; >WP_205506418.1; >MBM6917444.1; >WP_207750792.1;>WP_256316062.1; >WP_249028699.1; >MBS5634142.1; >WP_102049151.1; >MCL2053073.1; >WP_220431088.1; >WP_227060586.1; >WP_216239665.1; >WP_033143250.1; >WP_025489548.1; >MBE5966785.1; >HIX91572.1; >HIU76297.1; >CVI73436.1; >MCL2086502.1; >MBP6887289.1; >WP_052233149.1; >MBS5050812.1; >HIW80245.1; >MBP3487684.1; >HIR70200.1; >MBQ4522206.1; >HIZ82779.1; >WP_230105157.1; >WP_004608502.1; >MBP3469868.1; >HIR75596.1; >MBQ8189914.1; >WP_132281607.1; >NLZ81297.1; >MBS6684747.1; >WP_204215261.1; >MCL2105870.1; >MCL2495712.1; >MCL2300332.1; >WP_080023359.1; >WP_002599444.1; >SCJ97176.1; >MCI7444005.1; >MCI5726701.1; >MBQ8731896.1; >GHU69093.1; >WP_195926118.1; >WP_196371248.1; >WP_216304752.1;>WP_236914053.1; >WP_273962471.1; >WP_103895560.1; >GHU73310.1; >GHU72531.1;>MBP3399735.1; >MBR5290415.1; >MBE6107699.1; >WP_020225511.1; >GHU80979.1;

[0267] The unique accession number representing the A-Ena-like protein sequence is provided between ">" and ";".

[0268] sequence list

[0269] >SEQ ID NO:1: A-ENA amino acid sequence of Bacillus thuringiensis subsp. Israel ATCC35646 (UniProt: Q8KNV8)

[0270] >SEQ ID NO:2: A-ENA-1 amino acid sequence of Bacillus thuringiensis subspecies ATCC35646 (UniProt: Q8KNV7)

[0271] >SEQ ID NO: 3: A-ENA homolog UniprotKB A0A0R3K429

[0272] >SEQ ID NO: 4: A-ENA homolog UniprotKB A0A172TKT0

[0273] >SEQ ID NO: 5: A-ENA homolog UniprotKB A0A2X4WSQ5

[0274] > SEQ ID NO: 6: A-ENA homolog UniprotKB A0A0A3J768

[0275] > SEQ ID NO: 7: Chimeric protein of Bacillus thuringiensis subspecies Israel strain ATCC35646A-ENA (SEQ ID NO:1) fused to SpyTag

[0276] >SEQ ID NO: 8: Fusion to p66a Bti A-ENA (SEQ ID NO:1) chimeric protein

[0277] >SEQ ID NO: 9: C-end integration with MBD2 Bti A-ENA (SEQ ID NO:1) chimeric protein

[0278] >SEQ ID NO: 10: Fusion-to-erythroid desulfovibrio (V. fusible) Desulfovibrio vulgaris )of Bti A-ENA (SEQ ID NO:1) chimeric protein

[0279] >SEQ ID NO: 11: Fused to truncated Bti Chimeric protein of A-ENA-ΔNTL anti-GFP nanobody 207

[0280] >SEQ ID NO: 12: A-ENA-ΔNTL

[0281] >SEQ ID NO:13: Chimeric protein of Bacillus thuringiensis subspecies Israel strain ATCC35646 A-ENA-1 (SEQ ID NO:2) fused to SpyTag

[0282] >SEQ ID NO: 14: Chimeric protein of a superfolded green fluorescent protein fused to SpyCatcher003

[0283] >SEQ ID NO: 15: A-ENA mutant variant with a C-terminal 7×His-tag (E29A Q44A N64AE78A Q82A)

[0284] >SEQ ID NO:16: FW primer set 1

[0285] >SEQ ID NO:17: REV Primer Set 1

[0286] >SEQ ID NO:18: FW primer set 2

[0287] >SEQ ID NO:19: REV Primer Set 2

[0288] >SEQ ID NO:20: pASK forward sequencing primers

[0289] >SEQ ID NO:21: pASK reverse sequencing primers

[0290] >SEQ ID NO:22: sgRNA

[0291] >SEQ ID NO:23: SpyTag amino acid sequence

[0292] >SEQ ID NO:24: Seven-character repeat H1-1 Bti A-ENA

[0293] >SEQ ID NO:25: Seven-character repeat H1-2 Bti A-ENA

[0294] >SEQ ID NO:26: Heptavieric repeat H1-3 Bti A-ENA

[0295] >SEQ ID NO:27: Seven-character repeat H1-4 Bti A-ENA

[0296] >SEQ ID NO:28: Heptavieric repeat H1-5 Bti A-ENA

[0297] >SEQ ID NO:29: Seven-character repeat H2-1 Bti A-ENA

[0298] >SEQ ID NO:30: Seven-character repeat H2-2 Bti A-ENA

[0299] >SEQ ID NO:31: Heptavieric repeat H2-3 Bti A-ENA

[0300] >SEQ ID NO:32: Heptaminorepetition H2-4 Bti A-ENA

[0301] >SEQ ID NO:33: Heptaminode repeat H2-5 Bti A-ENA

[0302] >SEQ ID NO:34: Heptagonal repeat H1-1 Bti A-ENA-1

[0303] >SEQ ID NO:35: Heptavieric repeat H1-2 Bti A-ENA-1

[0304] >SEQ ID NO:36: Heptavieric repeat H1-3 Bti A-ENA-1

[0305] >SEQ ID NO:37: Heptavieric repeat H1-4 Bti A-ENA-1

[0306] >SEQ ID NO:38: Heptavieric repeat H1-5 Bti A-ENA-1

[0307] >SEQ ID NO:39: Heptavieric repeat H2-1 Bti A-ENA-1

[0308] >SEQ ID NO:40: Heptavalent repeat H2-2 Bti A-ENA-1

[0309] >SEQ ID NO:41: Heptad repeat H2-3 Bti A-ENA-1

[0310] >SEQ ID NO:42: Heptavalent repeat H2-4 Bti A-ENA-1

[0311] >SEQ ID NO:43: Heptavalent repeat H2-5 Bti A-ENA-1

[0312] >SEQ ID NO:44: NTL of Bti A-ENA

[0313] >SEQ ID NO:45: Bacillus thuringiensis Israel subspecies strain ATCC35646 A-ENA promoter

[0314] >SEQ ID NO:46: Bacillus thuringiensis subspecies Israel strain ATCC35646 A-ENA terminator

[0315] >SEQ ID NO:47-65: Oligonucleotide primer sequences used in Example 4.

[0316] SEQ ID NO: 66: Two coiled repeating units (with calcium and calcium carbonate binding properties)

[0317] >SEQ ID NO: 67: A-ENA_LI-R4.5-2RFD (= SEQ ID NO: 66 is inserted between residues 55 and 56 of A-ENA (SEQ ID NO 1), and the N-terminus and C-terminus of the inserted sequence are respectively flanked by GG and SG linkers)

[0318] >SEQ ID NO: 68: A_ENA_E28Q_E39Q_E41Q (=A-ENA SEQ ID NO:1, where Glu28, Glu39 and Glu41 are replaced by Gln, as shown in bold; K76 and Q82 are used to form IPB3 with further subunits, as shown in underline)

[0319] MGMPTIPEGLDITRDQAINIILASIGLQELGLAHVINAQGQKVQAVVAGFEKETVTFDQLLATNESVTQTLKTVI K KEMLL Q FKLEEAKSLIQSSSPPSIS

[0320] >SEQ ID NO:69-70: Primers

Claims

1. An isolated protein nanofibril comprising two precursor fibrils, each precursor fibril comprising 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 into two helices, helix 1 and helix 2, in aqueous solution, forming an α-helix antiparallel coiled-coil structure. Helix 1 and helix 2 each contain a continuous sequence of at least five seven-membered repeat (H) elements. The at least five seven-membered repeat (H) elements of helix 1 follow the formula H1-1 - H1-2 - H1-3 - H1-4 - H1-5, and the at least five seven-membered repeat (H) elements of helix 2 follow the formula H2-1 – H2-2 – H2-3 – H2-4 – H2-5. Each of the seven-membered repeat elements contains seven amino acid residues, respectively named "abcdefg," which have the following common sequence: -Seven-element repetition 1-1: XXX- -XX- , -Seven-element repetition 1-2: -X- - - - - , -Seven-element repetition 1-3: -X- - - -X- , -Seven-element repetition 1-4: -N- - - - - δ, -Seven elements repeat 1-5: - -X- - -XX, -Seven-element repetition 2-1: -XX- - -X- , -Seven-element repetition 2-2: -XX- -XXX, -Seven-element repetition 2-3: -XX- - -δ-δ, -Seven-element repetition 2-4: - - - - -X- δ, -Seven-element repetition 2-5: - -X- - -XX, in: -For at least 70% of the seven repeating elements specified therein Location, These are hydrophobic amino acids, selected from M, V, I, L, A, G, H, W, Y, and F; - These are short side chain residues, selected from V, C, G, A, P, S, T, N, and D; -For at least one or more of the seven repeating elements specified therein Location, For use as an acid / base catalytic residue selected from E or D; - For at least one or more of the δ positions specified in the seven-membered repeating element, δ is a lysine (K) residue that can serve as an isopeptide bond "donor" or a nucleophilic residue. -For at least one or more of the seven repeating elements specified therein Location, These are residues that can act as "receptors" or electrophilic residues of isopeptide bonds, selected from E, Q, D, and N; -X can be any amino acid. Furthermore, the linker (L) fragment contains at least 4 amino acids, and the N-terminal lock (NTL) fragment and the C-terminal tail (CT) fragment each contain at least 1 amino acid. Furthermore, the monomeric protein subunits are interconnected by at least one or more isopeptide bonds (IPB).

2. The protein nanofibrils according to claim 1, wherein at least one IPB covalently interconnects the two precursor fibrils (f) and (f').

3. The protein nanofibrils according to claim 2, wherein the at least one IPB is covalently interconnected between the precursor fibrils (f) and (f') via monomeric protein subunits (i') of the precursor fibrils (f) and monomeric subunits (i and / or i + 1) of the precursor fibrils (f) at the following positions: Between H2-3f of (i') and H2-4e of (i); and / or Between H2-3f of -(i+1) and H2-4e of (i').

4. The protein nanofibrils according to claims 1 to 3, wherein the monomeric protein subunits (i and i+ / -n) of the precursor fibril (f) and the monomeric subunits (i' and i'+ / -n) of the precursor fibril (f') are covalently linked by at least one or more IPBs, wherein the IPBs are formed at positions between amino acid side chains that are nucleophilic residues and amino acid side chains that are electrophilic residues, respectively: Between -(i)'s NTL and (i-5), (i-4), (i-3) or (i-2)'s H1-5b; Between H1-4g of (i) and H2-2a of (i-1); Between H2-3g of (i) and H2-4a of (i-1); Between H2-3f of -(i) and H2-4e of (i'-1); Between H2-4g of (i) and H1-3a of (i-1); Between H2-4g of -(i+1) and H1-3a of (i); -(i+5), (i+4), (i+3) or (i+2) NTL and (i) H1-5b; Between H1-4g of -(i+1) and H2-2a of (i); Between H2-3g of (i+1) and H2-4a of (i); and / or Between H2-3f of (i') and H2-4e of (i).

5. The protein nanofibers according to claims 1-4, wherein the seven-membered repeating elements have the following common sequence: -H1-1: XXX- -XX- , -H1-2: -X-(S / T)- -(A / G)- -(E / Q) -H1-3: EXX- -X-(H / N)- -H1-4: -N-(A / G / S)-E-(G / A)-EK -H1-5: -QX- - -XX -H2-1: -X-X- - -X- , -H2-2:(N / Q / D)-X-X- -X-X-X -H2-3: -X-X- - -(K / X)-(K / X) -H2-4:(E / Q)- - -(L)-(Q / X)-X-K, -H2-5: - -X- - -X-X。 6. The protein nanofiber according to any one of claims 1 to 5, wherein the NTL comprises a consensual sequence: M- - -ZX- -P, where: - These are short side chain residues, selected from V, C, G, A, P, S, T, N, and D; - These are hydrophobic amino acids, selected from M, V, I, L, A, G, H, W, Y, and F; -X can be any amino acid. -Z represents Ala or proline, and -M and P are the single-letter codes for the amino acids methionine and proline, respectively.

7. The protein nanofibers according to any one of claims 1 to 6, wherein the monomer is a protein selected from A-ENA proteins in the protein list shown in Table 3, and / or consists of an amino acid sequence selected from SEQ ID NO:1-6 or a functional homolog or variant of any of the amino acid sequences.

8. The protein nanofibers according to any one of claims 1 to 7, wherein the NTL of the monomeric protein subunit is less than 4 amino acids.

9. The protein nanofiber according to any one of claims 1 to 7, wherein the monomeric protein subunit comprises: a. An NTL of at least 4 amino acids, wherein the monomeric protein 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 b. The NTL is less 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.

10. The protein nanofibrils according to any one of claims 1 to 9, wherein the monomeric protein further comprises a protein tag or domain fused or conjugated to the N-terminus, C-terminus, or linker region to form a functionalized fibril.

11. The protein nanofibrils according to any one of claims 1 to 10, comprising the same monomeric protein that forms homopolymeric fibrils upon self-assembly, or comprising at least two different monomeric proteins that form heteropolymeric fibrils upon self-assembly.

12. The protein nanofibers according to any one of claims 1 to 11, wherein the fibers are recombinant protein nanofibers.

13. A modified bacterial endospore, preferably from the genus Bacillus (Bacillus). Bacillus Endospores comprising and / or displaying any one of claims 8 to 12.

14. A modified bacterial endospore, preferably a Bacillus endospore, wherein the bacterial strain lacks the endogenous monomeric protein of the protein nanofibrils of any one of claims 1 to 7, wherein the self-assembling monomeric protein forming the protein nanofibrils of any one of claims 1 to 12 is exogenously introduced.

15. Use of the modified bacterial endospores, preferably Bacillus endospores, according to claim 13 or 14, for increasing bacterial spore activity, preferably pathogenic activity.

16. Use of protein nanofibers according to any one of claims 1 to 12, for enhancing the insecticidal activity of bacterial endospores, preferably Bacillus thuringiensis (Bt). B. thuringiensis Insecticidal activity of endospores.

17. A host cell that recombinantly expresses a self-assembling monomeric protein that forms the protein nanofibers of any one of claims 1 to 12.

18. A method for preparing protein nanofibers according to any one of claims 1 to 12, comprising the following steps: a. Culturing the host cells of claim 17, b. Release of self-assembled protein nanofibrils from host cells, preferably via cell lysis, and c. Isolate self-assembled protein nanofibrils, preferably by resuspending in an insoluble fraction and / or further purifying from cell lysates.

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

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

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