Adhesion peptides

Abstract

The present invention relates to a plurality of peptides or polypeptides in or for use in an adhesive, which ideally comprise at least one of the following sequences, including any combination of five: an adhesive sequence, a cross-linking sequence, an elasticity-imparting sequence, and a release or cleavable sequence.

Description

[Technical Field]

[0001] The present invention relates to a plurality of peptides or polypeptides in or for use in an adhesive, which ideally comprise at least one of the following sequences: an adhesive sequence, a cross-linking sequence, an elasticity-imparting sequence, and a release or cleavable sequence, including any combination thereof. [Background technology]

[0002] Mussels produce and secrete a natural protein molecule (Mfp5) that functions as a water-resistant bioadhesive that anchors the mollusk to the seafloor. Adhesive plaques are deposited at the ends of secretory threads, and these plaques contain several proteins with a high proportion of 3,4-dihydroxyphenyl-L-alanine (DOPA), derived from the hydroxylation of tyrosine residues. The DOPA content of mussel adhesive proteins is particularly relevant to their adhesive properties, and DOPA-free analogs of mussel adhesive proteins exhibit significantly reduced adhesive capacity.

[0003] Synthetic peptides are ideal stepping stones for harnessing DOPA-based adhesion. They can be easily synthesized using chemical or recombinant methods, allowing for precise placement of adhesive functional groups. Tyrosine amino acids can be converted to DOPA using the enzyme tyrosinase, conveniently activating adhesives derived from non-adhesive precursor peptides. Furthermore, additional amino acids can be included in the engineered peptides to enhance at least one functionality or provide additional functionality, such as improving the adhesive properties (e.g., to hydrophobic surfaces) or increasing elasticity or resilience of the adhesive material.

[0004] These synthetic peptides are often non-toxic and non-immunogenic to the human body, and therefore can be advantageously used as medical adhesives. Furthermore, their biodegradability makes them environmentally friendly. In fact, peptide degradation can be accelerated by treatment with protease enzymes or chemicals that promote peptide bond hydrolysis. This avoids the use of harsh conditions such as UV radiation or high heat.

[0005] In reconstituted mussel adhesives, DOPA residues are introduced by generating proteins or peptides containing tyrosine (Y) at the required positions and then post-translationally converting the tyrosine to DOPA using the enzyme tyrosinase. DOPA residues enable adhesive and cohesive interactions through surface chelation, hydrogen bonding, the formation of mono-, bidentate, or tridentate metal ion complexes, and through cation-π interactions with positively charged residues, particularly lysine (K) and arginine (R) residues. Furthermore, DOPA residues can enable mussel adhesive protein molecules to cross-link with each other via oxidative conversion to o-quinones. Covalent interactions are important for building high-molecular-weight structures and increase the cohesive strength of the resulting material, but at the expense of adhesive interactions that arise through the DOPA functional groups. However, loss of cross-links results in molecules sliding past each other, producing softer materials with weaker cohesion. Therefore, it is desirable to develop a system that can generate covalent cross-links between peptide chains through chemistries that do not "quench" the adhesive DOPA functional groups.

[0006] The invention described herein relates to a new class of adhesion peptides that possess the above desirable characteristics. Summary of the Invention [Problem to be solved by the invention]

[0007] According to a first aspect of the present invention there is provided a plurality of adhesive hexapeptides in, or for use in, an adhesive, each having a motif comprising three different amino acids, two of which are repeated side by side to form the following sequence pattern: XX-YY- or -XX-YY where X, Y and - are any three different amino acids. Adhesion hexapeptides are provided that provide at least one of the following:

[0008] In a preferred embodiment of the invention, at least two of the adhesion hexapeptides are the same.

[0009] As used herein, a hexapeptide refers to a peptide containing six amino acids.

[0010] As used herein, adhesive hexapeptide motif or adhesive peptide refers to a peptide sequence that has adhesive properties or that has adhesive properties after treatment with an activator such as tyrosinase. Prior to such treatment, the hexapeptide motif is thus a precursor peptide adhesive.

[0011] Thus, the present invention provides an adhesion polypeptide comprising a plurality of hexapeptides, each having a motif comprising three different amino acids, two of which are repeated side by side in the following pattern: XX-YY- or -XX-YY where X, Y and - are any three different amino acids. The adhesive polypeptide includes an adhesion polypeptide that provides at least one of the following:

[0012] In a preferred embodiment, at least two of the adhesion hexapeptides are the same.

[0013] In a preferred embodiment, the amino acid is selected from the group comprising alanine A, arginine R, asparagine N, aspartic acid D, cysteine ​​C, glutamic acid E, glutamine Q, glycine G, histidine H, isoleucine I, leucine L, lysine K, methionine M, phenylalanine F, proline P, serine S, threonine T, tryptophan W, tyrosine Y, and valine V.

[0014] Most preferably, the amino acids are naturally occurring L-amino acids, but may also be D-amino acids, which are amino acids in which the stereogenic carbon alpha to the amino group has the D configuration.

[0015] In a further preferred embodiment, the amino acid is selected from the group comprising glycine G, tyrosine Y, lysine K, alanine A, serine S and arginine R.

[0016] More preferably, the hexapeptide comprises one of the following motifs: GYYGKK, AYYAKK, KKGYYG, KKAYYA, GKKGYY, GYYGRR, GYYGSS, AYYARR AYYASS, RRGYYG, RRAYYA, SSGYYG and SSAYYA.

[0017] The hexapeptide sequences presented in this invention contain a functional (adhesive) domain, which contains a high proportion of DOPA groups that confer adhesive function, or their non-adhesive precursor peptide tyrosine (Y).

[0018] Furthermore, increasing the amount of hydrophobic amino acids provides additional adhesion and aggregation potential through hydrophobic interactions, resulting in stronger adhesion, especially when binding low energy hydrophobic surfaces. Thus, in certain embodiments of the invention, the peptides comprise, in addition to tyrosine, amino acids selected from the group comprising the amino acids glycine (G), arginine (R), serine (S), lysine (K) and alanine (A) in the functional adhesive domain.

[0019] Most preferably, a plurality of said hexapeptide motifs are the same, and therefore, ideally, several different hexapeptide motifs are provided.

[0020] Furthermore, each hexapeptide motif is a functional adhesive domain, flanked by N- and / or C-terminal bridging domains.

[0021] Thus, even more preferably, the adhesion peptide comprises an N-terminal and / or C-terminal bridging sequence, whereby the adhesion hexapeptide is cross-linked to other adhesion hexapeptides to form a peptide chain. Preferably, the bridging sequence comprises: Selected from the group comprising SGEGKK, SGEGK, GKK, AKAAK, AKA, SSAKAAK, SSAKA, YFKG, LKG, FKG, YLKG, GQQQLG, YGQQQLG, KKGEGS, AKAAKSS and AKASS and KKGEGS.

[0022] Most ideally, the N-terminal hexapeptide cross-linking sequence is selected from the group comprising SGEGKK, SGEGK, GKK, AKAAK, AKA, SSAKAAK, SSAKA, YFKG, LKG, FKG, YLKG, GQQQLG and YGQQQLG.

[0023] Most ideally, the C-terminal hexapeptide cross-linking sequence is selected from the group comprising GKK, KKGEGS, AKAAK, AKA, AKAAKSS, AKASS, YFKG, LKG, FKG, YLKG, GQQQLG and YGQQQLG.

[0024] Ideally, the N- or C-terminal hexapeptide cross-linking sequences are used in combination with the following cross-linking enzymes: lysyl oxidase, which reacts with lysine (K) to give desmosine cross-links, or transglutaminase, which cross-links glutamine (Q) with lysine (K).

[0025] Referring to Table 1, when the cross-linking enzyme is transglutaminase, the N-terminal cross-linking sequences P12 to P13 are preferably used together with the C-terminal cross-linking sequences S1 to S10, more preferably S7 to S10.

[0026] Furthermore, the inventors have observed that combining a tyrosine / DOPA-rich adhesive motif with an elastic motif in a single polypeptide results in an adhesive material with increased resilience and thermal stability. These observations are believed to be a result of the increased flexibility of the material due to the inclusion of the elastic motif. This adhesive is particularly advantageous for joining joints that must withstand bending, for example, when the joined joint is heated or cooled.

[0027] Thus, in yet a further preferred aspect or embodiment of the present invention, the adhesion hexapeptide is linked to at least one elasticity- or elastomeric property-conferring sequence, the elasticity-conferring sequence being selected from the group comprising GVGVAP, GGRPSDSYGAPGGGN and KKWTWNPATGKWTWQE.

[0028] In particularly preferred embodiments, the adhesion polypeptide comprises a plurality of the elasticity- or elastomeric property-conferring sequences, each of which is located at the N-terminus or C-terminus of at least one of the plurality of adhesive hexapeptide motifs.

[0029] As mentioned above, degradation of adhesive peptides can be accelerated by treatment with protease enzymes or chemicals that promote peptide bond hydrolysis. In this regard, the inventors have observed that combining a tyrosine / DOPA-rich adhesive hexapeptide motif with a release or cleavable motif, ideally in a single polypeptide, results in an adhesive material that can be released from a substrate. This is particularly advantageous when reversible adhesion is required.

[0030] Thus, in yet a further preferred aspect or embodiment of the present invention, the adhesive hexapeptide is linked to at least one release or cleavable sequence, whereby the adhesive containing the plurality of hexapeptides can be released from a substrate, and the release or cleavable sequence is XIEGR / / X or XIDGR / / X XLEVLFQ / / GPX XENLYFQ / / SGX XDDDDK / / X XK / / X or XR / / X XΦK / / ZX or XΦR / / ZX XK / / X or XA / / X or XY / / X where X=any amino acid, Φ=A, V, L, I, F, W or Y, Z=any amino acid except V, and / / is the peptide bond to be cleaved. is selected from the group comprising:

[0031] The above-mentioned release sequences or cleavable sequence motifs contain peptide bonds that are highly susceptible to hydrolysis when treated with specific enzymes. Preferably, the inventors use proteases to cleave the sequence motifs, which are advantageously recognized only by highly specific protease enzymes, thus enabling highly selective degradation of the adhesive polymer. The seven sequence motifs are recognized by factor Xa protease, human rhinovirus 3C protease (HRV-3C), tobacco etch virus (TEV) protease, enterokinase (Enk) protease, trypsin protease, papain protease, and bromelain protease, respectively. The inventors have shown that digestion of adhesive peptides after curing leads to degradation of the material and a decrease in the strength of the adhesive bond or bond, known as release.

[0032] Those skilled in the art will appreciate that the present invention encompasses adhesives comprising any number or combination of sequences listed in Table 1. Preferably, in certain embodiments, at least two of the adhesive hexapeptide motifs [A1-A13] are the same. More preferably, the adhesive further comprises any combination of 2, 4, 6, 8, and 10 sequences of the sequences, with each sequence number and combination of sequences determining the adhesive strength, elastomeric properties, and peel ability of the adhesive.

[0033] In certain embodiments of the invention, the adhesive comprises at least one of the following polypeptide sequences (shown in Table 2): SGEGKK GYYGKK GYYGKK GYYGKK, the sequence is [P1]-[A1]3, known as AP1, which contains an N-terminal cross-linking sequence followed by three adhesion hexapeptide motifs, making this polypeptide suitable for cross-linking and therefore has excellent binding properties; SGEGKK AYYAKK AYYAKK AYYAKK, the sequence is [P1]-[A2]3, known as AP2, which contains an N-terminal cross-linking sequence followed by three adhesion hexapeptide motifs, making this polypeptide suitable for cross-linking and therefore has excellent binding properties; SGEGKK GYYGKK GYYGKK GYYGKK GYYGKK GYYGKK GYYGKK, the sequence is [P1]-[A1]6, known as AP3, which contains an N-terminal cross-linking sequence followed by six adhesion hexapeptide motifs, making this polypeptide suitable for cross-linking and therefore has better binding properties; SGEGKK GYYGKK GYYGKK GYYGKK GYYGKK GYYGKK GYYGKK GYYGKK GYYGKK, the sequence is [P1]-[A1]9, known as AP6, which contains an N-terminal cross-linking sequence followed by nine adhesion hexapeptide motifs, making this polypeptide suitable for cross-linking and therefore has better binding properties; GYYGKK GYYGKK AYYARR GKKGYY GKKGYY AYYARR GKKGYY GKKGYY, the sequence of which contains eight adhesion hexapeptide motifs, [A1]2-[A8]-[A1]2-[A8]-[A1]2, known as AP29, and which polypeptide has excellent binding properties; and SGEGKK AYYARR AYYARR AYYAKK, known as AP30, which contains an N-terminal cross-linking sequence followed by three adhesion hexapeptide motifs, [P1]-[A8]2-[A2], making this polypeptide suitable for cross-linking and therefore has better binding properties.

[0034] In the following alternative embodiment of the invention, the use of cross-linking N- and C-terminal sequences is important because there are no lysines (K) in the adhesive hexapeptide domain for the cross-linking enzyme to act on to form cross-links. The following polypeptide sequences are shown in Table 3: AKA GYYGRR GYYGRR AKA, the sequence is [P5]-[A6]2-S4, known as AP24, which contains N- and C-terminal cross-linking sequences and two adhesion hexapeptide motifs, making this polypeptide highly suitable for cross-linking and therefore has excellent binding properties; and AKA GYYGSS GYYGSS AKA, the sequence is [P5]-[A7]2-S4, known as AP25, which contains N- and C-terminal cross-linking sequences and two adhesion hexapeptide motifs, making this polypeptide highly suitable for cross-linking and therefore has excellent binding properties.

[0035] Thus, one skilled in the art will understand that inclusion of a terminal lysine-containing sequence in the aforementioned peptide sequences allows for the formation of high molecular weight polymers via lysine crosslinking, which can be effected enzymatically using a lysine-mediated crosslinking enzyme or chemically using a suitable chemical crosslinker, such as a compound having two or more Michael acceptors.

[0036] In particular, the absence of lysine (K) or arginine (R) (other than in the N- or C-terminal bridging sequences) means that the peptide is less susceptible to degradation by environmental proteases, making AP25 particularly suitable for use in environments where proteases are present.

[0037] Yet further alternative aspects or embodiments of the present invention are based on the above premise but include one of the following releasable or cleavable polypeptide sequences (shown in Table 4): SGEGKK GYYGKK GYYGKK GYYGKK LEVLFQGP GYYGKK GYYGKK GYYGKK GYYGKK, the sequence of which is [P1]-[A1]3-[D3]-[A1]4, known as ADP1, which contains an N-terminal cross-linking sequence, three adhesion hexapeptide motifs, a releasable region, and four additional adhesion hexapeptide motifs, making this polypeptide highly suitable for cross-linking and therefore has excellent binding properties; or SGEGKK GYYGKK GYYGKK GYYGKK G LEVLFQGP GYYGKK GYYGKK GYYGKK GYYGKK GYY, the sequence of which is [P1]-[A1]3-[D3]-[A1]3GYY, known as ADP1[G], which contains an N-terminal cross-linking sequence, three adhesive hexapeptide motifs, glycine, a releasable region, four additional adhesive hexapeptide motifs, and a glycine-tyrosine-tyrosine tripeptide, making this polypeptide highly suitable for cross-linking and therefore has excellent binding properties; and SGEGKK GYYGKK GYYGKK LEVLFQGP GKKGYY GKKGYY LEVLFQGP GKKGYY GKKGYY, the sequence of which is [P1]-[A1]2-[D3]-[A5]2-[D3]-[A5]2-[S1], known as ADP2, which contains an N-terminal cross-linking sequence, two adhesion hexapeptide motifs, a releasable region, two further adhesion hexapeptide motifs, a further releasable region, and two further adhesion hexapeptide motifs, making this polypeptide highly suitable for cross-linking and therefore has excellent binding properties; or SGEGKK GYYGKK GYYG LEVLFQGP GKKGYY GKKGYY G LEVLFQGP GKKGYY GKKGYY GKK, the sequence of which is [P1]-[A1]-GYYG-[D3]-[A5]2-G-[D3]-[A5]2-[S1], known as ADP2[G], which contains an N-terminal crosslinking sequence, one adhesion hexapeptide motif, a GYYG sequence, a releasable region, two additional adhesion hexapeptide motifs, glycine, an additional releasable region, two additional adhesion hexapeptide motifs, and a C-terminal crosslinking sequence, making this polypeptide highly suitable for crosslinking and therefore has excellent binding properties; and SGEGKK AYYAKK ARA AYYAKK, the sequence of which is [P1]-[A2]-[D9]-[A2], known as ADP5, which contains an N-terminal cross-linking sequence, one adhesion hexapeptide motif, an ARA release sequence, and one adhesion hexapeptide motif, making this polypeptide highly suitable for cross-linking and therefore has excellent binding properties; and SGEGKK GYYGKK ARA GYYGKK, the sequence of which is [P1]-[A1]-[D9]-[A1], known as ADP6, which contains an N-terminal cross-linking sequence, one adhesion hexapeptide motif, an ARA release sequence, and one adhesion hexapeptide motif, making this polypeptide highly suitable for cross-linking and therefore has excellent binding properties; and SGEGKK GYYGKK ENLYFQSG GYYGKK, the sequence of which is [P1]-[A1]-[D4]-[A1], known as ADP7, which contains an N-terminal cross-linking sequence, one adhesion hexapeptide motif, a release sequence, and one adhesion hexapeptide motif, making this polypeptide highly suitable for cross-linking and therefore has excellent binding properties; and FKG KKAYYA ARA AYYAKK GQQQLG, the sequence of which is [P10]-[A4]-[D9]-[A2]-[S11], known as ADP8, also known as ADP-X, which contains an N-terminal cross-linking sequence, one adhesion hexapeptide motif, a release sequence, one adhesion hexapeptide motif, and a C-terminal cross-linking sequence, and this polypeptide is highly suitable for cross-linking and therefore has excellent binding properties; and SGEGKK GYYGRR GYYGRR LEVLFQGP GYYGRR GYYGRR GKK, the sequence of which is [P1]-[A6]2-[D3]-[A6]2-[S1], known as ADP-X, which contains an N-terminal cross-linking sequence, two adhesive hexapeptide motifs, a releasable region, two further adhesive hexapeptide motifs, and a C-terminal cross-linking sequence; this polypeptide is highly suitable for cross-linking and therefore has excellent binding properties.

[0038] In still further preferred aspects or embodiments of the invention, the adhesive comprises at least one elasticity- or elastomeric property-imparting sequence, such as at least one of the following polypeptide sequences (shown in Table 5): KKGYYG KKGYYG GVGVAP GVGVAP GVGVAP GYYGKK GYYGKK, the sequence is [A3]2-[E1]3-[A1]2, known as AEP1, which contains two adhesive hexapeptide motifs, three elastomeric sequences and two further adhesive hexapeptide sequences, this polypeptide being suitable for use where the adhesive needs to be flexible; or G KKGYYG KKGYYG GVGVAP GVGVAP GVGVAP G GYYGKK GYYGKK, the sequence is [A3]2-[E1]3-[G]-[A1]2, known as AEP1[G], which contains a glycine, two adhesive hexapeptide motifs, three elastomeric sequences, a glycine, and two additional adhesive hexapeptide sequences, this polypeptide being suitable for use when the adhesive needs to be flexible; and SGEKK GYYGKK GYYGKK GVGVAP GVGVAP GVGVAP GYYGKK GYYGKK, the sequence of which is [1P]-[A1]2-[E1]3-[A1]2, known as AEP1b, which contains an N-terminal bridging sequence, two adhesive hexapeptide motifs, three elastomeric sequences, and two additional adhesive hexapeptide sequences, this polypeptide being suitable for use in situations where the adhesive needs to be flexible; and KKGYYG GGRPSDSYGAPGGGN GYYGKK, the sequence is [A3]-[E2]-[A1], known as AEP3, which contains an adhesive hexapeptide motif, an elastomeric sequence, and a further adhesive hexapeptide sequence, making this polypeptide suitable for use in situations where the adhesive needs to be flexible; and KKGYYG KKWTWNPATGKWTWQE GYYGKK, the sequence of which is [A3]-[E3]-[A1], known as AEP4, which contains an adhesive hexapeptide motif, an elastomeric sequence, and a further adhesive hexapeptide sequence, making this polypeptide suitable for use in situations where the adhesive needs to be flexible; and GKK GYYGKK GYYGKK GGRPSDSYGAPGGGN GGRPSDSYGAPGGGN GKKGYY GKKGYY GKK, the sequence is [1P]-[A1]2-[E3]2-[A5]2-[S1], known as AEP5, which contains an N-terminal bridging sequence, two adhesive hexapeptide motifs, two elastomer sequences, two further adhesive hexapeptide sequences, and GKK, this polypeptide is suitable for use when the adhesive needs to be flexible; and GKK GYYGKK GYYGKK KKWTWNPATGKWTWQE GKKGYY GKKGYY GKK, the sequence is [P3]-[A3]2-[E3]-[A5]2-[S1], known as AEP7, which contains an N-terminal bridging sequence, two adhesive hexapeptide motifs, an elastomeric sequence, two further adhesive hexapeptide sequences and a C-terminal bridging sequence; this polypeptide is suitable for use in situations where the adhesive needs to be flexible.

[0039] In still further preferred aspects or embodiments of the present invention, the adhesive comprises at least one elasticity-imparting or elastomeric property-imparting sequence and at least one release or cleavable region, such as at least one of the following polypeptide sequences (shown in Table 6): GKKGYY GKKGYY G GVGVAP LEVLFQGP GVGVAP G GYYGKK GYYGKK, the sequence is [A5]2-G-[E1]-[D3]-[E1]-G-[A1]2, known as AEDP1[G], which comprises two adhesive hexapeptide motifs, glycine, an elastomeric sequence, a release sequence, an elastomeric sequence, glycine, and two additional adhesive hexapeptide sequences, this polypeptide being suitable for use when the adhesive is flexible and needs to be selectively released from a substrate; or GKKGYY GKKGYY GVGVAP LEVLFQGP GVGVAP GYYGKK GYYGKK, the sequence is [A5]2-[E1]-[D3]-[E1]-[A1]2, known as AEDP1, which contains two adhesive hexapeptide motifs, an elastomeric sequence, a release sequence, an elastomeric sequence, and two additional adhesive hexapeptide motifs, making this polypeptide suitable for use when the adhesive is flexible and needs to be selectively released from a substrate; and AKA GYYGSS GYYGSS GVGVAP LEVLFQGP GVGVAP GYYGSS GYYGSS AKA, the sequence is [P5]-[A7]2-[E1]-[D3]-[E1]-[A7]2, known as AEDP4, which contains an N-terminal bridging sequence, two adhesive hexapeptide motifs, an elastomeric sequence, a release sequence, an elastomeric sequence, two further adhesive hexapeptide sequences and a C-terminal bridging sequence; this polypeptide is suitable for use when the adhesive is flexible and needs to be selectively released from a substrate.

[0040] In yet a further preferred aspect or embodiment of the invention, the adhesive comprises at least one sequence (shown in Table 7) that can be crosslinked with transglutaminase: FKG GYYGRR GYYGRR GQQQLG, the sequence of which is [P10]-[A6]2-[S11], known as AP26, which contains an N-terminal bridging sequence, two adhesion hexapeptide motifs, and a C-terminal bridging sequence; or FKG GYYGSS GYYGSS GQQQLG, the sequence is [P10]-[A7]2-[S11], known as AP27, containing an N-terminal bridging sequence, two adhesion hexapeptide motifs and a C-terminal bridging sequence.

[0041] FKG KKGYYG KKGYYG GQQQLG, the sequence is [P10]-[A3]2-[S11], known as AP28, which contains an N-terminal bridging sequence, two adhesion hexapeptide motifs and a C-terminal bridging sequence.

[0042] According to yet a further aspect of the present invention, Each has a motif containing three different amino acids, two of which are repeated side by side in the following pattern: XX-YY- or -XX-YY where X, Y and - are any three different amino acids. a plurality of hexapeptides resulting in at least one of: 0.5-2% gelatin in aqueous solution; and and optionally tyrosinase.

[0043] In a preferred embodiment, at least two of the hexapeptides are the same.

[0044] According to a further aspect of the present invention, Each has a motif containing three different amino acids, two of which are repeated side by side in the following pattern: XX-YY- or -XX-YY where X, Y and - are any three different amino acids. an adhesive comprising a plurality of hexapeptides resulting in at least one of: The aforementioned formulations are provided.

[0045] In a preferred embodiment, at least two of the hexapeptides are the same.

[0046] In a preferred embodiment of the present invention, the adhesive or formulation comprises a plurality of adhesive hexapeptides, including any number or combination of adhesive hexapeptides described herein, and preferably at least two of the hexapeptides are the same.

[0047] More preferably, the adhesive or formulation comprises a plurality of adhesive hexapeptides, at least one of which comprises an N-terminal and / or C-terminal bridging sequence.

[0048] Even more preferably, the adhesive or formulation comprises a plurality of adhesive hexapeptides, a plurality of which comprise a plurality of N-terminal and / or C-terminal cross-linking sequences.

[0049] Even more preferably, the adhesive or formulation comprises a plurality of adhesive hexapeptides and at least one, and ideally a plurality of release or cleavage sequences, which allow the adhesive to be released from a substrate.

[0050] Even more preferably, the adhesive or formulation comprises a plurality of adhesive hexapeptides and at least one, and ideally a plurality of elasticity- or elastomeric property-imparting sequences.

[0051] Most ideally, the adhesive or formulation will include a plurality of adhesive hexapeptides and at least one, ideally a plurality of release or cleavage sequences and / or at least one, ideally a plurality of elasticity or elastomeric property-imparting sequences.

[0052] Most ideally, the adhesive or formulation will comprise a plurality of adhesive hexapeptides and at least one, ideally a plurality of release or cleavage sequences and / or at least one, ideally a plurality of elasticity or elastomeric property-imparting sequences and / or at least one N-terminal or C-terminal crosslinking sequence.

[0053] As stated, one skilled in the art will understand that the present invention encompasses adhesives or formulations that include multiple sequences, including any number or combination, listed in Table 1. Preferably, at least two of the same adhesive hexapeptides are provided. More preferably, the adhesive or formulation includes any combination of 2, 4, 6, 8, and 10 sequences of the sequence, with each sequence number and combination of sequences determining the adhesive strength, peel ability, and elastomeric properties of the adhesive.

[0054] The above sequences can be used to create polypeptide sequences that incorporate two preferred features of the present invention. One feature is reducing the lysine (K) content in the adhesive hexapeptide sequence, and the other is allowing cross-linking to occur solely through sequence motifs at the beginning (N-terminus) and / or end (C-terminus) of the hexapeptide. This is exemplified by AP24 and AP25 in Table 3. Another notable feature of the present invention is that by excluding all lysine (K) (except when used in the N- and / or C-terminal cross-linking sequences) or arginine (R) from the hexapeptide motif, the hexapeptide, and therefore the adhesive made therefrom, is less susceptible to degradation by environmental proteases. This is exemplified by AP25 in Table 3.

[0055] In the following claims and the foregoing description of the invention, unless otherwise required by context, express language, or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" are used in their inclusive sense, i.e., to specify the presence of stated features but do not exclude the presence or addition of further features in various embodiments of the invention.

[0056] All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. Further, no admission is made that any of the prior art constitutes part of the common general knowledge in the art.

[0057] Preferred features of each aspect of the invention may be as described in relation to any of the other aspects.

[0058] Other features of the present invention will become apparent from the following examples. Generally speaking, the present invention extends to every novel, or every novel combination of, features disclosed in this specification (including the accompanying claims and drawings). Accordingly, any feature, integer, property, compound, or chemical moiety described in connection with a particular aspect, embodiment, or example of the present invention should be understood to be applicable to any other aspect, embodiment, or example described herein, except to the extent that it is incompatible therewith.

[0059] Moreover, unless stated otherwise, any feature disclosed in this specification may be replaced by an alternative feature serving the same or a similar purpose.

[0060] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating the plural as well as the singular unless the context otherwise requires.

[0061] Referring now to the following, one embodiment of the present invention will now be described, by way of example only. [Brief explanation of the drawings]

[0062] [Figure 1A] This shows data showing the increase in adhesive strength of gelatin due to the addition of the adhesive peptide AEP1. P = adhesive peptide AEP1, P + T = adhesive peptide AEP1 + tyrosinase, G = gelatin, G + T = gelatin + tyrosinase, P + G = adhesive peptide AEP1 + gelatin, P + G + T = adhesive peptide AEP1 + gelatin + tyrosinase. Error bars indicate standard deviation from n = 5 samples. [Figure 1B]Figure 1 shows the time-dependent oxidation of exemplary adhesive hexapeptides ADP2, AP2, AEP3, AEDP1, AP25 and AP28 by tyrosinase, resulting in the formation of high molecular weight structures by oxidative cross-linking. [Figure 2] Data to determine the optimal cross-linking time of the adhesive peptide (AP) mixture in the crucible before applying it to the surface. Errors are calculated from the standard deviation (n=5). [Figure 3] A comparison of AEP1 and naturally occurring Mfp5 is shown. Dataset 1 shows the force at break after curing at ambient conditions (as described in Methods). Dataset 2 shows the force at break after heating at 150 °C for 60 minutes. Error bars indicate standard deviation (n = 8-10). The thin line graph is the test adhesive peptide AEP1, and the dotted graph is the mussel protein adhesive. [Figure 4A] Figure 1 shows the peeling of AEP1. Dataset 1 shows the force at break after curing at ambient conditions (described in Methods). Dataset 2 shows the force at break after treatment with the peeling solution (described in Methods). Error bars indicate standard deviation (n=6-7). [Figure 4B] This shows a comparison of the detachment performance of trypsin and PBS for AEP1. The detachment time after adding the detaching agent was significantly shorter with trypsin. Error bars indicate the standard deviation for n=5. [Figure 4C] This figure shows a comparison of the stripping performance of papain and PBS for ADP5. The stripping time after adding the stripping agent was significantly shorter with papain. Error bars indicate the standard deviation for n=5. [Figure 5A] Figure 1 shows the lap shear strength of various adhesives according to the present invention compared to gelatin. This shows that the addition of various adhesives made according to the present invention to gelatin all improves the adhesive properties of gelatin. Error bars indicate standard deviation for n=5. Tensile strength is also reported for all peptides. [Figure 5B] The lap shear data in Figure 5A is shown with adhesives categorized by function, thus showing adhesives with various numbers of adhesive hexapeptides and N- or C-terminal cross-linking sequences to enable the formation of high molecular weight structures. [Figure 5C] Lap shear data in Figure 5A shows the strength of selected elastomeric adhesives compared to gelatin. This shows that the addition of various elastic motifs all improves the adhesive properties of gelatin. Error bars indicate standard deviation for n=5. Tensile strength is also reported for all peptides. [Figure 5D] Figure 5A shows the strength of selected peel adhesives compared to gelatin for lap shear data. This shows that the addition of various release motifs all improves the release properties of the adhesive gelatin. Error bars indicate standard deviation for n=5. Tensile strength is also reported for all peptides. [Figure 6A] Detachment of AEDP1 by 3C protease over time. [Figure 6B] The shedding or digestion of AEDP1 and the formation of digestion products have been shown to occur simultaneously. [Figure 6C] The peeling time after the addition of the stripping agent was shorter for 3C. Error bars indicate the standard deviation of n=5. DETAILED DESCRIPTION OF THE INVENTION

[0063] Table 1: All peptide examples are made from the "building block" motifs presented in the table below. From the motifs presented below, it is possible to generate peptides with any number and / or combination of hexapeptide adhesive / detachment / elasticity motifs. They may also contain prefix and / or suffix motifs that allow adhesive peptides to be crosslinked to form higher molecular weight structures, either by chemical or enzymatically induced reactions. In the detachment motif, X = any amino acid, Φ = A, V, L, I, F, W, or Y, and Z = any amino acid except V.

[0064] Table 2: Examples of adhesive peptides containing any number (or combination) of adhesive hexapeptide motifs.

[0065] Table 3: Examples of adhesion peptides containing prefix or suffix motifs that may be required to ensure cross-linking of peptide chains.

[0066] Table 4: Examples of releasable adhesives. Releasable sequence motifs are underlined.

[0067] Table 5: Examples of elastic adhesives. Elastic sequence motifs are in italics.

[0068] Table 6: Examples of releasable elastic adhesives. Elastic sequence motifs are italicized, and releasable sequence motifs are underlined.

[0069] Table 7: Examples of adhesive peptides that could be cross-linked with transglutaminase.

[0070] Methods and Materials Peptide Production Peptides can be obtained from commercial suppliers such as Sigma-Aldrich, Genscript, etc. Alternatively, they can be produced in-house using standard synthetic methods such as solid-phase synthesis as described by Mollica et al., Current Bioactive Compounds, 2013, 9, 184, or by recombinant methods as described by Mateja Zorko and Roman Jerala, Methods in Molecular Biology, 2009, 618, pp. 61-76.

[0071] tyrosinase Mushroom tyrosinase obtained from suppliers such as Sigma Aldrich can be used.

[0072] Tyrosinase cross-linking Tyrosinase-induced peptide cross-linking was observed by adding tyrosinase (0.125 units / µL) to peptide (4 mg / mL) in 25 mM ammonium acetate, pH 6. At time intervals, the progress of the reaction was monitored by SDS-PAGE analysis. Five µL aliquots were quenched by adding 5 µL of 50 mM NH4OAc (pH 6) and 10 µL of 2X Laemmli sample buffer. They were then heated to 103°C for 8 minutes, followed by centrifugation at 14,500 rpm for 5 minutes in a tabletop centrifuge. Five µL of each sample was analyzed by SDS-PAGE.

[0073] Preparation of Adhesion Peptide Test Formulations Test adhesives were prepared by mixing adhesive peptides (2–10 g / L) with a suspension of gelatin (0.5–2%) in aqueous solution buffered to pH 6. Tyrosinase (1–10 μg) was then added, and the samples were mixed and incubated at room temperature before preparation of test specimens.

[0074] Preparation of lap shear specimens Surfaces for adhesion experiments were prepared by washing in two successive acetone baths for 30 minutes, followed by a single 30-minute wash in a 70% ethanol bath, followed by drying in a desiccator for 16 hours. 7.5 μL of adhesive formulation (as described above) was then added to the edge of a glass slide, which was then sandwiched with a second slide in a 3D-printed fixture. A square polythene sheet was placed between each pair of glass slides. Ten pairs of glass slides were placed in a single fixture and compressed with a single 1 kg mass in a Memmert UT 30 plus oven at 30°C for 18 hours.

[0075] heat curing The specimens were prepared as described in the previous paragraph and further incubated in a Memmert UT 30 plus oven at 150° C. for 1 hour.

[0076] Lap shear test Lap shear tests were performed using a Shimadzu Autograph AGS-X testing machine. Samples prepared as described above were loaded into the testing machine and subjected to a maximum load of 500 N at a loading rate of 1 mm / min. Ten technical replicates were performed for each experiment, and the force at break was observed. Glass slides were prepared according to the ASTM D1002 standard with an overlap area of ​​12 x 26 mm.

[0077] Peeling by immersion Specimens prepared as described in the method above were immersed in 2.5% trypsin for 30 seconds. After storing the specimens under ambient conditions for 16-24 hours, the force at break was measured using a lap shear test.

[0078] AEP1 peeling time The lap shear specimen was loaded into the testing machine and loaded at a loading rate of 1 N / s to a fixed load of 30 N, which was maintained. 50 μL of release agent was then added to the leading edge of the joint of the lap shear specimen. The time to failure was then recorded. The release agent used included PBS and 2.5% trypsin.

[0079] AEDP1 shedding time The lap shear specimen was loaded into the testing machine and loaded at a loading rate of 1 N / s to a fixed load of 10 N AEDP1, which was maintained. 50 μL of release agent was then added to the leading edge of the joint of the lap shear specimen. The time to failure was then recorded. The release agent used included PBS and 2.5% papain.

[0080] ADP5 peeling time The lap shear specimen was loaded into the testing machine and loaded at a loading rate of 1 N / s to a fixed load of 30 N, which was maintained. 50 μL of release agent was then added to the leading edge of the bond of the lap shear specimen. The time to failure was then recorded. The release agent used included PBS and 2.5% papain.

[0081] result Demonstration of adhesion promotion in gelatin formulations Here, our adhesive peptides are evaluated for their adhesion-promoting ability. The preparation of our test formulations is described in "Preparation of Adhesion Peptide Test Formulations." The data shown in Figure 1 demonstrate that peptide mixtures with and without tyrosinase do not effectively adhere under our current test conditions (Samples P and P+T). Gelatin exhibits moderate adhesion (Sample G), and this adhesion strength is unaltered by the addition of tyrosinase, making it an important control. The advantage of our peptides as adhesion promoters is observed, as mixing adhesive peptides, gelatin, and tyrosinase (Sample P+G+T) resulted in an increase in adhesion strength greater than the sum of the individual components.

[0082] The addition of tyrosinase acts to post-translationally convert tyrosine to DOPA, resulting in DOPA residues. DOPA residues enable adhesive and coagulation interactions through surface chelation, hydrogen bonding, the formation of mono-, bidentate, or tridentate metal ion complexes, and through cation-π interactions with positively charged residues, particularly lysine (K) and arginine (R) residues. This results in cross-linking and the adhesive properties of the peptides. Figure 1B shows how the addition of tyrosinase to the hexapeptides ADP2, AP2, AEP3, AEDP1, AP25, and AP28 leads to the formation of high molecular weight complexes over time.

[0083] Optimizing curing time before application Typically, 30 minutes of reaction time after adding tyrosinase before applying the adhesive mixture to the specimen surface is optimal (Figure 2). Longer reaction times result in decreased adhesive strength. This optimal 30 minutes allows DOPA to form and initiate some cross-linking. Longer reaction times may allow the cross-linking reaction to proceed beyond the optimal amount.

[0084] Heat cure improves adhesive performance A comparison of the exemplary adhesive peptide AEP1 with the naturally occurring mollusk Mfp5 (Figure 3) shows the force at break after curing at ambient conditions. A second data set shows the force at break after heating at 150 °C for 60 minutes. Error bars indicate standard deviation (n = 8-10). For both types of adhesive, it can be seen that the curing process improves adhesive performance by approximately 50%.

[0085] Demonstration of adhesion to different substrates We have performed lap shear testing of AEP1 on a number of different substrates, including glass, stainless steel, aluminum, and painted steel. A summary of the bond strengths observed using the lap shear method detailed above is included in the table below. [Table 1]

[0086] Demonstration of peeling effectiveness Peeling of AEP1 from a substrate. In Figure 4A, Dataset 1 on the left shows the force at break of the AEP1 adhesive after curing (described in Methods) at ambient conditions. Dataset 2 on the right shows the force at break after curing and then treatment with the stripping solution (described in Methods); error bars indicate standard deviation (n=6-7). It can be seen that once the ability to peel the cured adhesive is incorporated, the force required to break the bond is approximately halved.

[0087] Figure 4B compares the detachment performance of trypsin and PBS for AEP1. The detachment time after addition of the detaching agent was significantly shorter with trypsin, indicating that trypsin is a better detacher for these adhesive peptides. Error bars indicate the standard deviation for a 5-point analysis.

[0088] Figure 4C compares the stripping performance of papain and PBS for ADP5. Papain significantly shortens the stripping time after the addition of the stripping agent. Error bars indicate the standard deviation (n=5).

[0089] Figure 5 shows examples of 13 different adhesive peptides that exhibit improved adhesive properties compared to gelatin. The data demonstrate that any combination of prefix (P), suffix (S), adhesive (A), elastic (E), and releasable (D) motifs can be combined to provide adhesives based on a variety of peptide combinations. Figure 5B shows the same data, but compares the improved performance of only the adhesive hexapeptides {A1}, {A2}, {A3}, {A4}, {A6}, {A7}, {A8}, {A10}, and {A12}}. Figure 5C shows the same data, but compares the improved performance of only the adhesive hexapeptides [E2] and [E3], which contain elasticity-imparting sequences. Figure 5D shows the same data, but compares the improved performance of only the adhesive hexapeptides [D4] and [D9], which contain releasable sequences.

[0090] Figure 6 shows the digestion of AEDP1 by 3C protease over time. A. Digestion of AEDP1 and formation of digested products are shown to occur simultaneously. C. The detachment time after addition of the detaching agent is shorter with 3C compared to trypsin. Error bars indicate standard deviation for n=5. [Table 2] [Table 3] [Table 4] [Table 5] [Table 6] [Table 7] [Table 8]

Claims

1. A peptide adhesive comprising a plurality of adhesive hexapeptides, each having a motif of three different amino acids, two of which are repeated side-by-side resulting in at least one of the following sequence patterns: XX-YY- or -XX-YY wherein the motif of the hexapeptide comprises at least one of the following motifs: GYYGKK, AYYAKK, KKGYYG, KKAYYA, GKKGYY, GYYGRR, GYYGSS, AYYARR AYYASS, RRGYYG, RRAYYA, SSGYYG, and SSAYYA; A non-adhesive precursor peptide tyrosine (Y) is converted to 3,4-dihydroxyphenyl-L-alanine (DOPA) to have adhesive function. Peptide adhesive.

2. The peptide adhesive of claim 1 , wherein at least two of the adhesive hexapeptides are the same.

3. The peptide adhesive of claim 1 , wherein the amino acids are naturally occurring L-amino acids or D-amino acids.

4. 4. The peptide adhesive of claim 1, wherein at least one of the hexapeptides comprises an N-terminal and / or C-terminal bridging sequence, whereby the adhesive hexapeptide is crosslinked to other adhesive hexapeptides to result in a peptide chain.

5. The bridging sequence is 5. The peptide adhesive of claim 4, selected from the group comprising SGEGKK, SGEGK, GKK, AKAAK, AKA, SSAKAAK, SSAKA, YFKG, LKG, FKG, YLKG, GQQQLG, YGQQQLG, KKGEGS, AKAAKSS, AKASS and KKGEGS.

6. 5. The peptide adhesive of claim 4, wherein the N-terminal sequence is selected from the group comprising SGEGKK, SGEGK, GKK, AKAAK, AKA, SSAKAAK, SSAKA, YFKG, LKG, FKG, YLKG, GQQQLG and YGQQQLG.

7. 5. The peptide adhesive of claim 4, wherein the C-terminal sequence is selected from the group comprising GKK, KKGEGS, AKAAK, AKA, AKAAKSS, AKASS, YFKG, LKG, FKG, YLKG, GQQQLG and YGQQQLG.

8. 8. The peptide adhesive of any one of claims 4 to 7, wherein the N-terminal bridging sequence GQQQLG or YGQQQLG is used together with one of the C-terminal bridging sequences GKK, KKGEGS, AKAAK, AKA, AKAAKSS, AKASS, YFKG, LKG, FKG or YLKG.

9. The peptide adhesive of any one of claims 1 to 8, wherein at least one of the adhesive hexapeptides is linked to at least one elasticity-conferring or elastomeric property-conferring sequence.

10. 10. The peptide adhesive of claim 9, wherein the elasticity or elastomeric property imparting sequence is selected from the group comprising GVGVAP, GGRPSDSYGAPGGGGN, and KKWTWNPATGKWTWQE.

11. The peptide adhesive of any one of claims 1 to 10, wherein at least one of the adhesive hexapeptides is linked to a plurality of elasticity-conferring or elastomeric property-conferring sequences.

12. 12. The peptide adhesive of claim 11, wherein each of the elasticity-imparting or elastomeric property-imparting sequences is provided at the N-terminus and / or C-terminus of at least one of the adhesive hexapeptides.

13. 13. The peptide adhesive of any one of claims 1 to 12, wherein at least one of the adhesive hexapeptides is linked to at least one release or cleavable sequence, thereby enabling the adhesive to be released from a substrate.

14. The release or cleavable sequence is XIEGR / / X or XIDGR / / X; XLEVLFQ / / GPX; XENLYFQ / / SGX; XDDDDK / / X; XK / / X or XR / / X; XΦK / / ZX or XΦR / / ZX; and XK / / X or XA / / X or XY / / X where X=any amino acid, Φ=A, V, L, I, F, W, or Y, Z=any amino acid except V, and / / is the peptide bond to be cleaved.

14. The peptide adhesive of claim 13, selected from the group comprising:

15. A peptide adhesive comprising a plurality of adhesive hexapeptides according to any one of claims 1 to 3, said peptide adhesive having the following sequence: i)SGEGKK GYYGKK GYYGKK GYYGKK; ii) SGEGKK AYYAKK AYYAKK AYYAKK; iii)i･!!!,,YY66 7Y69KK yyyyylKyyyy 1.11.1.1.1.1.1.5; iv)i･Y,K ,YY 99K WYY9KK yyyyyｫ, y11.11.5.1.1.1.1.1.1.1.1.1.1.1.15.. The AYARR GKKYY W7666;;; viiSGEGKK AYYARR AYYARR ｡ｹ｡KK; vii)AKA GYYGRR WｹｹｧR ｡ｫ｡; viiiiAKA / YYGS7666SSA｡｡; ix)i･,KK,YﹹﹷKK,,YY9KK yyyyyv,,6666 1.11.1.1.1.1.1.1.1.1.1.1.1.1.1.

5. x)))(6!6, 9999KK 7Y69KK ,666!66 6!6! 1.11.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.0.. xii),E!,K ,9Y6 66 9999KK LEV6v,( 66 99 9 1,56,000,000,000,000,000,000,000 xiiii･･,661.151.11 1.56.6.0710 1.1.171.11.

11. xiiiiSGE9KK AYYAKK ARAAｹAAK; xivv)GE99K GYY9KK A2A yyyyv;; xv)SGEYKK GYY9KK E886666S9 xvi)FKG KKAYYA ARAAYY6KK WQQQ / ; xviiii･E!!K, ,YY96 799 99RR 0･6vy,,,99 111122 17.5; xviiiii)K9YY KK,YY MMMﷶVAｰ y y( y(   1.11.1.1.1.1.1.5; xixx) KK9YY KK,YY 9999 ﷷVAｰ yyyy y  1.11.1.1.1.1.1.5; xx)･!KK,Yﹹﹹ﹫K WYY9KK yyyyｦA. 1.676.. 11111.1.111.16; xxi)KKYYyy ,MMMMDS,999yyyｮ yyy xxiii)KK9YMMKKWTWWA xxiii) GKK GYYGKK GYYGKK GGRPSDSYGAPGGGGN GGRPSDSYGAPGGGGN GKKGYY GKKGYY GKK; xxiv) GKK GYYGKK GYYGKK KKWTWNPATGKWTWQE GKKGYY GKKGYY GKK; xxv) GKKGYY GKKGYY G GVGVAP LEVLFQGP GVGVAP G GYYGKK GYYGKK; xxvi) GKKGYY GKKGYY GVGVAP LEVLFQGP GVGVAP GYYGKK GYYGKK; xxvii) AKA GYYGSS GYYGSS GVGVAP LEVLFQGP GVGVAP GYYGSS GYYGSS AKA; xxviii) FKG GYYGRR GYYGRR GQQQLG; xxix) FKG GYYGSS GYYGSS GQQQLG; or xxx) FKG KKGYYG KKGYYG GQQQLG, 5. A peptide adhesive comprising at least one of, or a plurality of sequences comprising any combination of, 16. A peptide adhesive comprising at least one or more adhesive polypeptides according to claim 15.

17. A peptide adhesive formulation comprising the peptide adhesive of any one of claims 1 to 16 formulated in a 0.5 to 2% gelatin solution.

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