Fusion proteins / peptides, methods and uses thereof

Abstract

Provided herein are fusion proteins / peptides and biopolymers comprising a keratin and a peptide sequence associated with the keratin. These fusion proteins and biopolymers may further comprise an elastin-like, silk-like, collagen or collagen-like, arthropod elastin-like, abexin-like peptide and / or linker-like sequences. These fusion proteins and biopolymers may exhibit functional properties derived from each peptide sequence contained therein, such as elasticity, hydration, restorability, texture or antimicrobial and UV protection, etc. In addition, provided herein are methods of obtaining and characterizing fusion proteins and biopolymers comprising keratin and peptide sequences associated with keratin. Fusion proteins and biopolymers comprising keratin and peptide sequences associated with keratin may be included in compositions for improving the properties of hair, skin and nails.

Description

Technical Field

[0001] This invention relates to fusion proteins / peptides with binding modules and biopolymers for hair and skin care, specifically fusion proteins and biopolymers with keratin binding modules.

[0002] This invention relates to fusion proteins and peptides for hair and skin applications, including cosmetic and skincare formulations. Background Technology

[0003] Fusion proteins, or chimeric proteins, are proteins formed by the fusion of two or more genes that originally encode different proteins, peptides, or modules. Translation of such a fusion gene produces one or more polypeptides with functional properties derived from each original sequence. Some fusion proteins bind the complete peptide, thus containing all the functional domains of the original protein. However, other fusion proteins, especially those that are naturally occurring, bind only a portion of the coding sequence and therefore cannot retain the original function of the parent genes that formed them. Novel recombinant technologies have made it possible to improve the design of fusion proteins for use in numerous fields such as biosensing, agriculture, the paper and food industries, and biopharmaceuticals. Summary of the Invention

[0004] Proteins, peptides, and peptide components or modules such as silk and collagen have been used as ingredients in compositions for hair, skin, and nail care. In some cases, they are used to strengthen and moisturize hair, skin, and nails. In others, they are used to improve the appearance of hair, skin, and nails and protect against environmental and chemical damage. However, when used in hair, skin, and nail care products, they do not always provide the expected functions. For example, many natural proteins have lower than expected isoelectric points, resulting in insufficient affinity for negatively charged hair, skin, and nails. Furthermore, many proteins have poor solubility due to their high molecular weight and hydrophobicity. Structural proteins such as silk and collagen can undergo chemical degradation, resulting in a mixture of molecular weight components / modules with diverse properties. To improve solubility for use in products, these proteins are often chemically modified. However, the chemical modification of these proteins can affect their desired characteristics. Typically, more than one protein may be needed to meet all the desired characteristics of a given composition, which can lead to increased product cost and production time. Therefore, there remains an unmet need for proteins designed to overcome the inherent limitations of natural products. These designed proteins can provide a better composition for hair, skin, and nail products.

[0005] These facts are disclosed to illustrate the technical problem addressed in this disclosure.

[0006] This disclosure relates to a novel class of fusion proteins and peptides specifically designed for optimal performance in hair, nail, and skin applications, including cosmetic and dermatological formulations. The fusion peptides / proteins of this disclosure exhibit significant efficacy in addressing a variety of cosmetic and skin-related problems. This disclosure relates to these novel fusion peptides / proteins, methods of synthesis, and their use in hair, nail, or skin care.

[0007] Other aspects and advantages of this disclosure will become apparent to those skilled in the art from the following description, which shows and describes only illustrative embodiments of this disclosure. As will be appreciated, this disclosure is capable of other and different embodiments, and certain details thereof can be modified in various obvious ways without departing from the content of this disclosure.

[0008] This disclosure relates to fusion proteins containing keratin peptides for hair and skin care, as well as biopolymers containing keratin peptides.

[0009] This disclosure relates to beauty and personal care compositions, and more specifically, to novel fusion proteins having binding molecules designed to improve the affinity, binding properties, and strength of hair, nails, and / or skin. In some embodiments, the effect of the fusion protein on strengthening hair is determined by Young's modulus. In some cases, the effect on strengthening hair is determined as the relative difference in Young's modulus between hair treated with and not treated with the fusion protein / peptide according to the invention or a composition containing the fusion protein / peptide. In some cases, treatment of hair with the fusion protein / peptide improves hair integrity. In some cases, hair integrity is determined by differential scanning calorimetry (DSC).

[0010] In some respects, this document discloses a fusion protein / peptide comprising: (a) a keratin or keratin-associated peptide module; and (b) at least one polypeptide module selected from: (i) an elastin or elastin-like polypeptide (ELP) module, (ii) a silk protein or silk protein-like polypeptide (SLP) module, (iii) a collagen or collagen-like polypeptide (CLP) module, (iv) an arthropod elastin or arthropod elastin-like polypeptide (RLP) module, (v) an abductin or abductin-like polypeptide (ALP) module, (vi) a spacer-like connector component, or (vii) a combination of two or more of (i) to (vi).

[0011] In this disclosure, the keratin or keratin-related peptide module may be selected from sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the following sequence list: SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 48, SEQ ID NO. 66, SEQ ID NO. 67, SEQ ID NO. 68, SEQ ID NO. 69, SEQ ID NO. 101, SEQ ID NO. 102, SEQ ID NO. 103, and SEQ ID NO. 70-81.

[0012] In this disclosure, the elastin or elastin-like polypeptide (ELP) module may be selected from sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the following sequence list: SEQ ID NO. 11-23, SEQ ID NO. 49-55, SEQ ID NO. 82-91, and SEQ ID NO. 104.

[0013] In this disclosure, the silk protein or silk protein-like polypeptide (SLP) module may be selected from sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the following sequence list: SEQ ID NO. 24-32, SEQ ID NO. 56, SEQ ID NO. 105, and SEQ ID NO. 92.

[0014] In this disclosure, the collagen or collagen-like polypeptide (CLP) module may be selected from sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the following sequence list: SEQ ID NO. 33, SEQ ID NO. 57-59, SEQ ID NO. 93-95, and SEQ ID NO. 106.

[0015] In this disclosure, the arthropod elastin or arthropod elastin-like polypeptide (RLP) module may be selected from sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the following sequence list: SEQ ID NO. 34-35, SEQ ID NO. 60, SEQ ID NO. 96, and SEQ ID NO. 107.

[0016] In this disclosure, the extensin or extensin-like polypeptide (ALP) module may be selected from sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the following sequence list: SEQ ID NO. 36-42, SEQ ID NO. 61, and SEQ ID NO. 108. In this disclosure, the adapter component as a spacer-like material may be selected from sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the following sequence list: SEQ ID NO. 97-100.

[0017] In some implementations, for better results, the peptide module may include an elastin or elastin-like peptide (ELP) module.

[0018] In some embodiments, for better results, the elastin or elastin-like polypeptide module contains 8 to 30 amino acids; preferably 10 to 30 amino acids, more preferably 15 to 30 amino acids.

[0019] In some implementations, to obtain better results, the peptide module may include a connector module as a spacer-like component.

[0020] In some embodiments, for better results, the connector module includes a hydrophilic module containing 1 to 75 amino acids; preferably 10 to 50 amino acids. In some embodiments, the hydrophilic module contains hydrophilic amino acids. In some embodiments, the hydrophilic amino acids include serine, threonine, glutamine, asparagine, glutamic acid, or aspartic acid.

[0021] In some embodiments, the adapter module is in an α-helical conformation to obtain better results. In some embodiments, the adapter module is in a random coil conformation. In some embodiments, the secondary structure of the fusion peptide is determined using circular dichroism (CD) spectroscopy.

[0022] In some implementations, to obtain better results, the adapter module contains 1 to 3 amino acids repeated 0 to 25 times, preferably 1 to 20 times.

[0023] In some implementations, for better results, the connector module comprises 1 to 2, or 10 to 25 times.

[0024] In some embodiments, for better results, the keratin or keratin-related peptide module comprises SEQ ID NO:8: GGVCGPSPPCITT, or comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:8.

[0025] In some embodiments, for better results, the keratin or keratin-related peptide module comprises SEQ ID NO:9: GGVCGPSPPC, or comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:9.

[0026] In some embodiments, for better results, the keratin or keratin-related peptide module comprises SEQ ID NO:10:CGPSPPCITT, or comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:10.

[0027] In some embodiments, for better results, the keratin or keratin-related peptide module comprises SEQ ID NO:68:CLPCLPAASC, or comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:68.

[0028] In some embodiments, for better results, the keratin or keratin-related peptide module comprises SEQ ID NO:73:CVSSCCKPQCC, or comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:73.

[0029] In some embodiments, for better results, the keratin or keratin-related peptide module comprises SEQ ID NO:74: PITCRRTCYH, or comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:74.

[0030] In some embodiments, for better results, the keratin or keratin-related peptide module comprises SEQ ID NO:75: DCKLPCNPCA, or comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:75.

[0031] In some embodiments, for better results, the keratin or keratin-related peptide module comprises SEQ ID NO:76:CLPCLPAASC, or comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:76.

[0032] In some embodiments, for better results, the keratin or keratin-related peptide module comprises SEQ ID NO:77: CEPAICEPSC, or comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:77.

[0033] In some embodiments, for better results, the fusion protein / peptide comprises SEQ ID NO:1: GGVCGPSPPCITTVPGVGVPGVGVPGLGVPGVGVPGVGR, or comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:1.

[0034] In some embodiments, for better results, the fusion protein comprises SEQ ID NO:2: GGVCGPSPPCITTVPGVGVPGVGVPGVGVPGVGVPGVGVPGVG, or comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:2.

[0035] In some embodiments, for better results, the fusion protein comprises SEQ ID NO:7: GGVCGPSPPCITTVPGVGVPGVGVPGLGVPGVGVPGVG, or comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:7.

[0036] In some implementations, for better results, the peptide module includes a silk protein or silk protein-like peptide (SLP) module.

[0037] In some embodiments, the fusion protein comprises SEQ ID NO:3: GGVCCGPSPPCITTGAGAGSGAGAGSGAGAGSGAGAGSGAGAGS, or comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:3.

[0038] In some implementations, the peptide module includes a collagen or collagen-like peptide (CLP) module.

[0039] In some embodiments, the fusion protein comprises SEQ ID NO:5: GGVCCGPSPPCITTGPTGPTGPAGPRGLQGLQGLQGERGEQGPT, or comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:5.

[0040] In some implementations, the polypeptide module comprises arthropod elastin or arthropod elastin-like polypeptide (RLP) modules.

[0041] In some embodiments, the fusion protein comprises SEQ ID NO:4: GGVCCGPSPPCITTGGRPSDSYGAPGGGN, or comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:4.

[0042] In some implementations, the peptide module includes an extensin or extensin-like peptide (ALP) module.

[0043] In some embodiments, the fusion protein comprises SEQ ID NO:6: GGVCGPSPPCITTGGFGGMGGGSGGFGGMGGGSGGFGGMGGGS, or comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:6.

[0044] In some embodiments, the sequence of the fusion protein / peptide is: GGVCGPSPPCITTGGFGGMGGGSGGFGGMGGGSGGFGGMGGGS (SEQ ID NO:6).

[0045] In some embodiments, the sequence of the fusion protein / peptide is: GVGVPGVGVPGLGVPGVGVPGVG (SEQ ID NO: 22).

[0046] In some embodiments, the sequence of the fusion protein / peptide is: GAGAGGSGAGAGSGAGAGSGAGAGSGAGAGS (SEQ ID NO:32).

[0047] In some embodiments, the sequence of the fusion protein / peptide is: GPTGPTGPAGPRGLQGLQGLQGERGEQGPT (SEQ ID NO:33).

[0048] In some embodiments, the sequence of the fusion protein / peptide is: GGVCGPSPPCITTGQGQGQGQGQGQGQGQGQGQGQGQGQGQGQGQ (SEQ ID NO:44).

[0049] In some embodiments, the sequence of the fusion protein / peptide is: GGVCGPSPPCITTAKAKAKAKAKAKAKAKAKAKAKAKAKAKAKAK (SEQ ID NO:45).

[0050] In some embodiments, the sequence of the fusion protein / peptide is: GGVCGPSPPCITTLKLKLKLKLKLKLKLKLKLKLKLKLKLKLKLKLKLK (SEQ ID NO:46).

[0051] In some implementations, the sequence of the fusion protein / peptide is as follows:

[0052] GGVCGPSPPCITTTGAGAGAGAGAGAGAGAGAGAGAGAGA (SEQ ID NO: 47).

[0053] In some embodiments, the adapter module includes a spacer-like repeat sequence module. The adapter may contain about 1 to about 3 amino acid repeats from 0 to 25 times. Alternatively, the adapter may contain 1-2 amino acid repeats from 10 to 25 times.

[0054] In some embodiments, this disclosure provides a fusion protein having at least 90% sequence identity with any one of the following sequences:

[0055] GGVCGPSPPCITTVPGVGVPGVGVPGLGVPGVGVPGVGR (SEQ ID NO: 1); GGVCGPSPPCITTVPGVGVPGVGVPGVGVPGVGVPGVGVPGVG (SEQ ID NO: 2);

[0056] GGVCGPSPPCITTGAGAGSGAGAGSGAGAGSGAGAGSGAGAGAGS(SEQ ID NO:3);

[0057] GGVCGPSPPCITTGGRPSDSYGAPGGGN(SEQ ID NO:4);

[0058] GGVCGPSPPCITTGPTGPTGPAGPRGLQGLQGLQGERGEQGPT(SEQ ID NO:5); GGVCGPSPPCITTGGFGGMGGGSGGFGGMGGGSGGFGGMGGGS(SEQ ID NO:6); or

[0059] GGVCGPSPPCITTVPGVGVPGVGVPGLGVPGVGVPGVG(SEQ ID NO:7);

[0060] GGVCGPSPPCITTGQGQGQGQGQGQGQGQGQGQGQGQGQGQGQ(SEQ ID NO:44);

[0061] GGVCGPSPPCITTAKAKAKAKAKAKAKAKAKAKAKAKAKAKAK(SEQ ID NO:45);

[0062] GGVCGPSPPCITTLKLKLKLKLKLKLKLKLKLKLKLKLKLKLK(SEQ ID NO:46);

[0063] GGVCGPSPPCITTGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA(SEQ ID NO:47).

[0064] The fusion protein according to embodiments of this disclosure comprises keratin or keratin-related peptide modules of 6 to 16 amino acids in length; silk protein or "SLP" comprising repeating sequences of 6 to 8 amino acids in length, and / or silk protein or SLP of 15 to 30 amino acids in length; collagen or "CLP" comprising repeating sequences of 3 amino acids in length, and / or collagen or CLP of 15 to 30 amino acids in length; elastin or "ELP" comprising repeating sequences of 5 amino acids in length, and / or elastin or ELP of 15 to 30 amino acids in length; arthropod elastin or "RLP" comprising repeating sequences of 11 to 15 amino acids in length, and / or arthropod elastin or RLP of 15 to 30 amino acids in length; and abductin or "ALP" comprising repeating sequences of 4 to 10 amino acids in length, and / or abductin or ALP of 15 to 30 amino acids in length.

[0065] In some embodiments, the average molecular weight of the fusion protein is from about 1 kDa to about 144 kDa. In some embodiments, the average molecular weight of the fusion protein is from about 10 kDa to about 144 kDa. In some embodiments, the average molecular weight of the fusion protein is from about 35 kDa to about 130 kDa.

[0066] In some embodiments, the average molecular weight of the fusion protein ranges as follows: about 1 kDa to about 5 kDa, about 5 kDa to about 10 kDa, about 6 kDa to about 17 kDa, about 10 kDa to about 15 kDa, about 14 kDa to about 30 kDa, about 15 kDa to about 20 kDa, about 17 kDa to about 39 kDa, about 20 kDa to about 25 kDa, about 25 kDa to about 30 kDa, about 30 kDa to about 35 kDa, about 35 kDa to about 40 kDa, about 39 kDa to about 54 kDa, about 39 kDa to about 80 kDa, about 40 kDa to about 45 kDa, about 45 kDa to about 50 kDa, about 50 kDa to about 55 kDa, about 55 kDa to about 60 kDa, about 60 kDa to about 100 kDa, and about 80 kDa to about 144 kDa.

[0067] In some embodiments, the fusion protein comprises a sequence having at least 80% sequence identity with any one of SEQ ID NO:1-7, 44-47 or 200-552.

[0068] In some embodiments, the fusion protein comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NO:1-7, 44-47, or 200-552.

[0069] In some embodiments, the fusion protein comprises a sequence identical to any one of SEQ ID NO:1-7, 44-47 or 200-552.

[0070] In some respects, this document provides a composition comprising a fusion protein according to embodiments of the present disclosure for improving the elasticity, resilience, hydration, integrity, hydrophobicity, texture, antibacterial properties, emulsifying properties, mechanical properties, thermal protection, or UV protection of hair, nails, or skin, or a combination of two or more of these.

[0071] In some embodiments, the composition improves skin elasticity, resilience, hydration, integrity, texture, antibacterial protection, or UV protection, or a combination of two or more of these.

[0072] In some embodiments, the composition improves the mechanical properties, color, texture, shape, hydration, integrity, resilience, hydrophobicity, or thermal protection of hair, or a combination of two or more of these.

[0073] In some embodiments, the composition improves the mechanical properties, integrity, antimicrobial properties, or ultraviolet protection of nails, or a combination of two or more of these.

[0074] In some embodiments, the composition further comprises urea, ammonium thioglycolate, thioanisole, benzyl alcohol, tris(hydroxymethyl)aminomethane, phosphate buffer, sodium chloride, citrate buffer, or ethanol, or a combination of two or more of these.

[0075] In some aspects, this document provides the use of a fusion protein composition comprising embodiments according to the present disclosure as an enhancer of one or more properties of hair, nails, or skin. In some embodiments, the use of the composition includes using the fusion protein as an agent to enhance the elasticity, hydration, resilience, texture, antimicrobial properties, and / or UV protection of hair, nails, and skin.

[0076] In some aspects, this document provides a fusion protein / peptide comprising: (a) at least one keratin or keratin-associated peptide module; and (b)(i) an elastin or elastin-like peptide (ELP) module, (ii) a silk protein or silk protein-like peptide (SLP) module, (iii) a collagen or collagen-like peptide (CLP) module, (iv) an arthropod elastin or arthropod elastin-like peptide (RLP) module, (v) an extensin or extensin-like peptide (ALP) module, or (vi) a combination of two or more selected from (i) to (v).

[0077] In some implementations, the fusion protein / peptide is represented by the following:

[0078] [Bn1 -A m -B n -C o -B n2 ] p

[0079] Equation (1); or

[0080] [B n1 -C o -B n -A m -B n2 ] p

[0081] Formula (II); or

[0082] [B n1 -B n -C o -A m -B n2 ] p

[0083] Formula (III); or

[0084] [B n1 -C o -A m -B n -B n2 ] p

[0085] Formula (IV)

[0086] in,

[0087] “A” contains a peptide sequence that contains at least one keratin or keratin-related peptide module, where “m” is an integer from 1 to 10;

[0088] "B" contains a peptide sequence of 1 to 40 amino acids, where "n", "n1" and "n2" are each an independent integer from 0 to 25;

[0089] “C” contains D p -E r -F s -G t -H u The peptide sequence of the structure;

[0090] Where “D” represents the silk protein or silk protein-like polypeptide (SLP) module;

[0091] The “E” in the designation includes a collagen or collagen-like polypeptide (CLP) module.

[0092] The “F” in the designation includes the elastin or elastin-like polypeptide (ELP) module.

[0093] The “G” in the designation contains arthropod elastin or arthropod elastin-like polypeptide (RLP) modules;

[0094] Where “H” contains the extensin or extensin-like peptide (ALP) module;

[0095] Where “q” is an integer from 0 to 100, “r” is an integer from 0 to 200, “s” is an integer from 0 to 220, “t” is an integer from 0 to 80, and “u” is an integer from 0 to 70;

[0096] Where “o” is an integer from 0 to 30; and

[0097] “p” is an integer from 1 to 40;

[0098] At least one of “n”, “n1”, “n2” or “o” is not 0.

[0099] In some implementations, each "n", "n1" and "n2" is an integer from 0 to 25 independently.

[0100] In some implementations, "o" is an integer from 1 to 30.

[0101] In some implementations, each “n”, “n1” and “n2” is an integer from 0 to 25, and “o” is an integer from 1 to 30.

[0102] In some implementations, "p" is an integer from 3 to 37. In some implementations, "p" is an integer from 8 to 15. In some implementations, "p" is an integer from 10 to 12.

[0103] In some embodiments, "B" comprises a peptide sequence of 1 to 40 amino acids. In some embodiments, "B" comprises a peptide sequence of 2 to 25 amino acids. In some embodiments, "B" comprises a peptide sequence of 4 to 25 amino acids.

[0104] In some implementations, "B" includes having X n Z c and / or Z c X n The peptide sequence; where "X" n "is proline, leucine, isoleucine, glutamic acid, aspartic acid, glutamine, lysine, alanine, arginine, or threonine; and among them, "Z" c "It contains glycine, alanine, lysine, proline, methionine, glutamine, arginine, threonine, valine, glutamic acid, aspartic acid, or asparagine."

[0105] In some embodiments, at least one keratin or keratin-associated peptide module is 6 to 16 amino acids in length, and "D" contains a polypeptide sequence including SEQ ID NO:24:GAGAGS, SEQ ID NO:25:GAGSGA, SEQ ID NO:26:GAGAGY, SEQ ID NO:27:GAGYGA, SEQ ID NO:28:GAGAGA, SEQ ID NO:29:GAGAGV, SEQ ID NO:30:GAGVGA, or SEQ ID NO:31:GAGAGVGY, or a combination of two or more thereof; "E" contains a polypeptide sequence GX. 1 Z, where "X" 1 " is proline, leucine, isoleucine, glutamic acid, aspartic acid, glutamine, lysine, alanine, arginine, or threonine; and Z is glycine, alanine, lysine, proline, methionine, glutamine, arginine, threonine, valine, glutamic acid, aspartic acid, or asparagine; "F" contains the polypeptide sequence including SEQ ID NO:21:VPGX 2 G and / or SEQ ID NO:13:VPAVG, where “X” 2 "G" represents arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan; "G" contains polypeptide sequences including SEQ ID NO:34: GGRPSDSYGAPGGGN and / or SEQ ID NO:35: GAPAQTPSSQY; "H" contains polypeptide sequences including SEQ ID NO:36: GGFGGMGGGS, SEQ ID NO:37: MGGG, SEQ ID NO:38: FGGMG, SEQ ID NO:39: FGGMGGG, SEQ ID NO:40: GGFGGMGGG or SEQ ID NO:41: FGGMGGGNAG, or a combination of two or more thereof. In some embodiments, keratin or keratin-related peptide modules include SEQ ID NO:34: GGRPSDSYGAPGGGN and / or SEQ ID NO:35: GAPAQTPSSQY. NO: 8, 9, 10, 66-81, 101-103, or fragments thereof, or combinations of two or more thereof.

[0106] In some implementations, "A" contains the peptide sequence X. 3 k CX 3 5CX 3 k , where “X 3"Contains glycine, alanine, valine, isoleucine, leucine, phenylalanine, proline, serine, threonine, asparagine, glutamine, cysteine, methionine, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, histidine, lysine, or arginine, and "k" is an integer from 0 to 3.

[0107] In some implementations, "A" includes SEQ ID NO:8:GGVCGPSPPCITT, SEQ ID NO:9:GGVCGPSPPC, SEQ ID NO:10:CGPSPPCITT, SEQ ID NO:66:CGPSPPC, or SEQ ID NO:67:GVCGPSPPC.

[0108] In some embodiments, the average molecular weight of the fusion protein / peptide is from about 1 kDa to about 144 kDa. In some embodiments, the average molecular weight of the fusion protein / peptide is from about 10 kDa to about 144 kDa. In some embodiments, the average molecular weight of the fusion protein / peptide is from about 35 kDa to about 130 kDa.

[0109] In some embodiments, the average molecular weight of the fusion protein / peptide is about 1 kDa to about 5 kDa, about 5 kDa to about 10 kDa, about 6 kDa to about 17 kDa, about 10 kDa to about 15 kDa, about 14 kDa to about 30 kDa, about 15 kDa to about 20 kDa, about 17 kDa to about 39 kDa, about 20 kDa to about 25 kDa, about 25 kDa to about 30 kDa, about 30 kDa to about 35 kDa, about 35 kDa to about 40 kDa, about 39 kDa to about 54 kDa, about 39 kDa to about 80 kDa, about 40 kDa to about 45 kDa, about 45 kDa to about 50 kDa, about 50 kDa to about 55 kDa, about 55 kDa to about 60 kDa, about 60 kDa to about 100 kDa, and about 80 kDa to about 144 kDa.

[0110] In some embodiments, the fusion protein has a secondary structure. In some embodiments, the secondary structure includes an alpha (α) helix, a 3... 10 Spiral, π-spiral, beta (β)-turn, β-fold, coil, or a combination thereof.

[0111] In some embodiments, silk fibroin or silk fibroin-like peptide (SLP) modules form α-helices, 3... 10 Helices, π-helices, β-turns, β-sheets, coils, or combinations thereof. In some embodiments, silk proteins or silk protein-like peptide (SLP) modules form β-sheet-rich structures.

[0112] In some embodiments, elastin or elastin-like peptide (ELP) modules form α-helices, 3... 10 Helices, π-helices, β-turns, β-sheets, coils, or combinations thereof. In some embodiments, elastin or elastin-like peptides (ELPs) form structures rich in β-turns.

[0113] In some implementations, the connector module forms an α-helix, 3 10 Spiral, π-spiral, β-turn, β-fold, coil, or combinations thereof. In some embodiments, the connector module forms an α-spiral. In some embodiments, the connector module forms a coil. In some embodiments, the connector forms a coil.

[0114] In some embodiments, the elastin-like peptide (ELP) module is thermoresponsive. In some embodiments, the ELP module may respond to changes in pH. In some embodiments, the ELP module may respond over a certain temperature range. In some embodiments, the ELP module may respond to salt.

[0115] In some embodiments, this document provides for the use of fusion proteins, fusion proteins / peptides, or compositions according to embodiments of this disclosure as cosmetic products, such as cosmetic products for hair, nails, or skin.

[0116] In some implementations, the beauty product is suitable for hair care or hair treatment.

[0117] In some respects, this document provides a method for obtaining the fusion protein described herein, comprising: inoculating a liquid culture medium with a microorganism containing a gene expressing the desired fusion protein; allowing the microorganism to proliferate over a certain culture period and inducing the expression under the action of a promoter; and extracting the desired fusion protein from the obtained microorganism by purifying the protein from endogenous contaminants.

[0118] In some embodiments, the microorganism is *Escherichia coli*. In some embodiments, the microorganism is *Pichia pastoris*.

[0119] In some implementations, the fusion protein is purified from endogenous contaminants.

[0120] On the other hand, it relates to a composition comprising the fusion protein / peptide of the present disclosure for improving the elasticity, resilience, hydration, integrity, hydrophobicity, texture, antibacterial properties, emulsifying properties, mechanical properties, thermal protection or UV protection of hair, nails or skin, or a combination of two or more of these.

[0121] In some embodiments, the composition improves skin elasticity, resilience, hydration, integrity, texture, antibacterial protection, or UV protection, or a combination of two or more of these.

[0122] In some embodiments, the content of the fusion protein / peptide is 0.0001% (w / w) to 20% (w / w) by weight; preferably, the content of the fusion protein / peptide is 0.001% (w / w) to 5% (w / w); from 0.1% (w / w) to about 1% (w / w).

[0123] In some embodiments, the composition can enhance the mechanical properties, color, texture, shape, hydration, integrity, resilience, hydrophobicity, or thermal protection of hair, or a combination of two or more of these.

[0124] In some embodiments, the composition improves the mechanical properties, integrity, antimicrobial properties, or ultraviolet protection of nails, or a combination of two or more of these.

[0125] In some embodiments, at least one excipient suitable for skin use is selected from: surfactants, emulsifiers, preservatives, thickeners, organic polymers, humectants, silicones, oils, fragrances, vitamins, buffers, antimicrobial agents, antibacterial agents, disinfectants, chelating agents, or mixtures thereof.

[0126] In some embodiments, the composition may contain at least one excipient suitable for skin applications.

[0127] In some embodiments, at least one excipient suitable for skin use is selected from: ethanol, benzyl alcohol, diol molecules, urea, ammonium thioacetate, anisole, tris(hydroxymethyl)aminomethane, phosphate buffer, sodium chloride, citrate buffer, or ethanol, or a combination of two or more of these.

[0128] In some embodiments, the diol molecule is selected from ethylene glycol, propylene glycol, butanediol, butene glycol, or mixtures thereof.

[0129] In some embodiments, the composition may comprise: 0-30% (w / v) ethanol, preferably 10-20% (w / v); 3% (w / v) benzyl alcohol, preferably 0.5-2% (w / v); and 0-20% (w / v) diol molecules, preferably 10-15% (w / v).

[0130] In some embodiments, the composition may further comprise a propellant, a fragrance, an oil, or a mixture thereof.

[0131] In some embodiments, the composition can be used for hair treatment.

[0132] The fusion proteins / peptides or compositions disclosed herein can be used as cosmetic products, i.e., cosmetic products for hair, nails or skin.

[0133] In some implementations, the beauty product is suitable for hair care or hair treatment.

[0134] On the other hand, it relates to a shampoo, lotion, serum, cream, conditioner, foam, elixir, oil, aerosol, or mask that contains the compositions / fusion proteins / peptides disclosed herein.

[0135] On the other hand, it relates to a composition comprising the fusion protein of the present disclosure as an enhancer of one or more properties of hair, nails, or skin, or as an agent that enhances the elasticity, hydration, resilience, texture, antimicrobial properties, and / or UV protection of hair, nails, and skin.

[0136] On the other hand, a method for obtaining the fusion protein / peptide disclosed herein is provided, comprising the following steps: inoculating a liquid culture medium with microorganisms including a gene expressing the desired fusion protein; allowing the microorganisms to proliferate over a certain culture period and inducing the expression under the action of a promoter; and extracting the desired fusion protein / peptide from the obtained microorganisms by purifying the protein from endogenous contaminants.

[0137] In one embodiment, the microorganism is Escherichia coli.

[0138] In one implementation, the fusion protein / peptide is separated and purified from endogenous contaminants.

[0139] On the other hand, a method for obtaining the fusion protein / peptide of this disclosure by solid-phase synthesis is disclosed, comprising the steps of: initiating solid-phase synthesis to bind a keratin or keratin-associated peptide module to at least one polypeptide module selected from: (i) an elastin or elastin-like polypeptide (ELP) module, (ii) a silk protein or silk protein-like polypeptide (SLP) module, (iii) a collagen or collagen-like polypeptide (CLP) module, (iv) an arthropod elastin or arthropod elastin-like polypeptide (RLP) module, (v) an extensin or extensin-like polypeptide (ALP) module, or (vi) a combination of two or more modules selected from (i) to (v); and performing a solid-phase reaction to obtain a stable peptide. Attached Figure Description

[0140] The following figures provide embodiments for illustrating the content of this disclosure and should not be construed as limiting the scope of this disclosure.

[0141] Figure 1A linear vector map of an expression vector according to an embodiment of the present disclosure is shown, the expression vector comprising a promoter, a target fusion protein sequence, an adapter sequence, a C-terminal affinity tag sequence, an AmpR gene, and a corresponding promoter.

[0142] Figure 2 A linear vector map of an expression vector according to an embodiment of the present disclosure is shown, the expression vector comprising a promoter, an N-terminal affinity tag sequence, an adapter sequence, a target fusion protein, and an AmpR gene and a corresponding promoter.

[0143] Figure 3 The permeation / location of FITC (fluorescent probe) labeled peptides applied to hair fibers is shown by fluorescence microscopy. Most peptides were able to penetrate the epidermis down to the cortex, especially peptides SEQ ID NO:2, SEQ ID NO:45, SEQ ID NO:46, and SEQ ID NO:47. Detailed Implementation

[0144] This disclosure relates to fusion proteins / peptides.

[0145] One aspect of this disclosure relates to fusion proteins / peptides comprising effective amounts of repetitive sequences of various proteins that can be used to improve the performance of hair, skin, and nails.

[0146] Another aspect of this disclosure relates to a composition comprising the fusion protein / peptide of this disclosure.

[0147] In some embodiments, the fusion protein / peptide can be used in hair care compositions, skin care compositions, nail care compositions, cosmetic compositions, or pharmaceutical compositions.

[0148] In some embodiments, the fusion protein / peptide and composition comprise effective amounts of repetitive sequences of various proteins / peptides, which is advantageous because the fusion protein / peptide can provide a composition with desired properties. Desired properties may include elasticity and stimulus-responsive behavior.

[0149] Keratin and keratin-related proteins are the main components of human hair, skin, and nails. Due to the presence of cysteine ​​amino acids, keratin and keratin-related proteins typically have a high sulfur content. The presence of sulfur is crucial for the structure of hair, skin, and nails because it enables the formation of disulfide bonds between peptide chains. The presence of disulfide bonds (i.e., bridging bonds) largely determines the structure and texture of hair, skin, and nails. Damage to disulfide bridging bonds (i.e., bonds) between keratin molecules can alter the appearance and texture of hair, skin, and nails. Maintaining the integrity of disulfide bonds between keratin molecules helps maintain the health of hair, skin, and nails and prevents breakage or brittleness.

[0150] Fusion protein

[0151] In some embodiments, the fusion protein is a keratin fusion protein, comprising keratin or keratin-associated peptide modules derived from the keratin protein moiety. The keratin and keratin-associated fusion proteins described herein can bind with high affinity to hair, skin, and nails, and repair keratin fiber damage by forming new disulfide bonds. The keratin or keratin-associated peptide modules of the fusion protein may have a sequence containing one or more cysteine ​​amino acids that can naturally form disulfide bonds with other free cysteine ​​amino acids in hair, skin, and nails. The formation of new disulfide bonds helps restore the mechanical properties of hair, skin, and nails, including elasticity, resilience, and appearance.

[0152] In some embodiments, the fusion proteins disclosed herein comprise amino acid sequences (the term "peptide" is used interchangeably to refer to an amino acid sequence) that are linked by covalent bonds or any other type of bond to create a structure that maintains the properties of the fusion proteins disclosed herein. These bonds may be selected from, but are not limited to, hydrogen bonds, ion pairs, hydrophobic associations, or inclusion complexes.

[0153] In some embodiments, keratin or a keratin-associated fusion protein / peptide comprises at least one keratin or keratin-associated peptide module.

[0154] In some embodiments, keratin, keratin peptides, or keratin-related peptides include wild-type keratin peptides, functional fragments of wild-type keratin peptides, or peptides having at least 90% identity with wild-type keratin peptides or functional fragments of wild-type keratin peptides. Wild-type keratin peptides may be keratin 85 having SEQ ID NO:48. Wild-type keratin peptides may include SEQ ID NO:8, 9, 10, 66, or 67. In some embodiments, keratin, keratin peptides, or keratin-related peptides form one or more disulfide bonds with hair. In some embodiments, keratin, keratin peptides, or keratin-related peptides form one or more disulfide bonds with skin. In some embodiments, keratin, keratin peptides, or keratin-related peptides form one or more disulfide bonds with nails.

[0155] In one non-limiting example embodiment, the functional fragment of the wild-type keratin peptide comprises at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive amino acids of the wild-type keratin peptide. In another non-limiting example embodiment, the functional fragment of the wild-type keratin peptide comprises at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids having at least 90% identity with the wild-type keratin peptide. In yet another non-limiting example embodiment, the functional fragment of the wild-type keratin peptide comprises at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids, wherein the at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids comprise one or more cysteine ​​amino acids. For example, one or more cysteine ​​amino acids may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 cysteine ​​amino acids. In a non-limiting example embodiment, the functional fragment of the wild-type keratin peptide comprises at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids, wherein at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, or 50% of the at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids are cysteine ​​amino acids. A non-limiting example embodiment of keratin or keratin peptide is provided in SEQ ID NO:48.

[0156] In some embodiments, the keratin-associated peptide (sometimes referred to as keratin-associated protein) comprises a peptide that forms a disulfide bond with wild-type keratin. In some embodiments, the keratin-associated peptide comprises at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids, and these at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids are at least 90% identical to the sequence of the wild-type keratin peptide. The wild-type keratin peptide may be keratin 85 having SEQ ID NO:48. The wild-type keratin peptide may comprise SEQ ID NO:8, 9, 10, 66, or 67.

[0157] In some embodiments, the number of amino acids in the keratin or keratin-related peptide components of the fusion protein is from about 6 residues to about 16 residues.

[0158] In some embodiments, the number of amino acids in the keratin or keratin-related peptide component of the fusion protein is about 6 residues.

[0159] In some embodiments, the number of amino acids in the keratin or keratin-related peptide component of the fusion protein is about 7 residues.

[0160] In some embodiments, the number of amino acids in the keratin or keratin-related peptide component of the fusion protein is about 8 residues.

[0161] In some embodiments, the number of amino acids in the keratin or keratin-related peptide component of the fusion protein is about 9 residues.

[0162] In some embodiments, the number of amino acids in the keratin or keratin-related peptide component of the fusion protein is about 10 residues.

[0163] In some embodiments, the number of amino acids in the keratin or keratin-related peptide component of the fusion protein is about 11 residues.

[0164] In some embodiments, the number of amino acids in the keratin or keratin-related peptide component of the fusion protein is about 12 residues.

[0165] In some embodiments, the number of amino acids in the keratin or keratin-related peptide component of the fusion protein is about 13 residues.

[0166] In some embodiments, the number of amino acids in the keratin or keratin-related peptide component of the fusion protein is about 14 residues.

[0167] In some embodiments, the number of amino acids in the keratin or keratin-related peptide component of the fusion protein is about 15 residues.

[0168] In some embodiments, the number of amino acids in the keratin or keratin-related peptide component of the fusion protein is about 16 residues.

[0169] In some embodiments, the keratin or keratin-associated peptide modules of the fusion protein have a high cysteine ​​content to enrich certain properties of the keratin or keratin-associated peptide modules, such as enhancing the formation of intermolecular crosslinks. The total cysteine ​​content accounts for 10% to 50% of the total number of amino acids in the keratin or keratin-associated peptide modules.

[0170] In some embodiments, the total cysteine ​​content of keratin or keratin-related peptides accounts for more than 15% of the total number of amino acids in the keratin or keratin-related peptide modules of the fusion protein / peptide.

[0171] In some implementations, the keratin or keratin-associated peptide module of the fusion protein / peptide contains two cysteine ​​residues.

[0172] In some implementations, the keratin or keratin-associated peptide module of the fusion protein contains three cysteine ​​residues.

[0173] In some implementations, the keratin or keratin-associated peptide module of the fusion protein contains four cysteine ​​residues.

[0174] In some implementations, the keratin or keratin-associated peptide module of the fusion protein contains five cysteine ​​residues.

[0175] In some embodiments, the keratin or keratin-associated peptide module of the fusion protein / peptide contains a peptide sequence (X). 3 ) k C(X 3 )5C(X 3 ) k .

[0176] In some implementations, each "X" 3 "Independently glycine, alanine, valine, isoleucine, leucine, phenylalanine, proline, serine, threonine, asparagine, glutamine, cysteine, methionine, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, histidine, lysine, or arginine."

[0177] In some implementations, each "k" is an independent integer from 0 to 3. If k is 2, then (X 3 ) k Having the first X 3 And the second X 3 Each X 3 Independently, it can be glycine, alanine, valine, isoleucine, leucine, phenylalanine, proline, serine, threonine, asparagine, glutamine, cysteine, methionine, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, histidine, lysine, or arginine. If k is 3, then (X 3 ) k Having the first X 3 The second X 3 And the third X 3 , where each X 3 Independently, it consists of glycine, alanine, valine, isoleucine, leucine, phenylalanine, proline, serine, threonine, asparagine, glutamine, cysteine, methionine, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, histidine, lysine, or arginine. (X) 3 )5 has the first X 3 The second X 3 The third X 3 The fourth X 3 And the fifth X3 , where each X 3 It can be independently glycine, alanine, valine, isoleucine, leucine, phenylalanine, proline, serine, threonine, asparagine, glutamine, cysteine, methionine, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, histidine, lysine, or arginine.

[0178] In some embodiments, the keratin or keratin-related peptide components of the fusion protein include sequences of GGVCGPSPPCITT (SEQ ID NO:8), GGVCGPSPPC (SEQ ID NO:9), CGPSPPCITT (SEQ ID NO:10), CGPSPPC (SEQ ID NO:66), GVCGPSPPC (SEQ ID NO:67), or SEQ ID NOS:70-81, or combinations of two or more of these.

[0179] In some embodiments, the keratin or keratin-related peptide sequence is GGVCGPSPPCITT (SEQ ID NO: 8). In some embodiments, the keratin or keratin-related peptide sequence is...

[0180] GGVCGPSPPC (SEQ ID NO:9). In some embodiments, the keratin or keratin-related peptide sequence is CGPSPPCITT (SEQ ID NO:10).

[0181] In some embodiments, keratin or keratin-related fusion proteins further comprise elastin or elastin-like peptide (ELP) modules, silk or silk-like peptide (SLP) modules, collagen or collagen-like peptide (CLP) modules, arthropod elastin or arthropod elastin-like peptide (RLP) modules, extensin or extensin-like peptide (ALP) modules, or combinations of two or more of these, such as a silk-elastin-like peptide (SELP) module.

[0182] In some embodiments, the keratin or keratin-related fusion proteins disclosed herein further comprise elastin or elastin-like polypeptide (ELP) modules. In some embodiments, an elastin-like polypeptide (ELP) refers to a peptide comprising an amino acid sequence, including synthetic peptides, said amino acid sequence having a secondary structure, tertiary structure, or amino acid sequence, or a combination of two or more, similar to that of wild-type elastin (e.g., SEQ ID NO: 55) or a fragment of wild-type elastin. In some embodiments, the fragment comprises a sequence having at least 4, 5, 6, 7, 8, 9, or 10 amino acids that is 100% identical to the sequence of wild-type elastin. Non-limiting examples of elastin and ELP include SEQ ID NOS: 11-23, 49-55, and 82-91.

[0183] ELP may comprise a polymeric or oligomeric repeating peptide sequence of elastin. In one example embodiment, the elastin or ELP comprises SEQ ID NO:49((VPGXG)n, where n is 1 to 10 and each X is independently any amino acid), or a sequence having no more than one amino acid substitution compared to SEQ ID NO:49. In some embodiments, each X is independently valine or leucine. In some embodiments, n is 5. In some embodiments, n is 6.

[0184] In one example embodiment, the elastin or ELP comprises SEQ ID NO:50 (VPGVG), or a sequence having no more than one amino acid substitution compared to SEQ ID NO:50. The elastin or ELP may comprise 1 to 10 sequences having SEQ ID NO:50, or 1 to 10 sequences, each independently having no more than one amino acid substitution compared to SEQ ID NO:50. These 1 to 10 sequences may be interconnected, for example, VPGVGVPGVGVPGVGVPGVGVPGVGVPGVG (SEQ ID NO:23), or separated by one or more amino acids, for example, separated by another ELP, as in...

[0185] Shown in VPGVGVPGVGVPGLGVPGVGVPGVG (SEQ ID NO:54).

[0186] In one example embodiment, the elastin or ELP comprises SEQ ID NO:51 (VPGLG), or a sequence having no more than one amino acid substitution compared to SEQ ID NO:51. The elastin or ELP may comprise 1 to 10 sequences having SEQ ID NO:51, or 1 to 10 sequences, each independently having no more than one amino acid substitution compared to SEQ ID NO:51. These 1 to 10 sequences may be interconnected or separated by one or more amino acids.

[0187] In one example embodiment, the elastin or ELP comprises SEQ ID NO:52 (VPGVGVPGL), or a sequence having no more than one amino acid substitution compared to SEQ ID NO:52. The elastin or ELP may comprise 1 to 10 sequences having SEQ ID NO:52, or 1 to 10 sequences, each independently having no more than one amino acid substitution compared to SEQ ID NO:52. These 1 to 10 sequences may be interconnected or separated by one or more amino acids.

[0188] In one example embodiment, the elastin or ELP comprises SEQ ID NO:53 (VPGVGVPGVGVPGLGVPGVGVPGVGR), or a sequence having no more than one amino acid substitution compared to SEQ ID NO:53. The elastin or ELP may comprise 1 to 10 sequences having SEQ ID NO:53, or 1 to 10 sequences, each independently having no more than one amino acid substitution compared to SEQ ID NO:53. These 1 to 10 sequences may be interconnected or separated by one or more amino acids.

[0189] In one example embodiment, the elastin or ELP comprises SEQ ID NO:23 (VPGVGVPGVGVPGVGVPGVGVPGVGVPGVG), or a sequence having no more than one amino acid substitution compared to SEQ ID NO:23. The elastin or ELP may comprise 1 to 10 sequences having SEQ ID NO:23, or 1 to 10 sequences, each independently having no more than one amino acid substitution compared to SEQ ID NO:23. These 1 to 10 sequences may be interconnected or separated by one or more amino acids.

[0190] In one example implementation, elastin or ELP comprises SEQ ID NO:54

[0191] (VPGVGVPGVGVPGLGVPGVGVPGVG), or a sequence having no more than one amino acid substitution compared to SEQ ID NO:54. Elastin or ELP may comprise 1 to 10 sequences having the sequence SEQ ID NO:54, or 1 to 10 sequences, each independently having no more than one amino acid substitution compared to SEQ ID NO:54. These 1 to 10 sequences may be interconnected or separated by one or more amino acids.

[0192] In one example embodiment, the elastin or ELP comprises SEQ ID NO:22 (GVGVPGVGVPGLGVPGVGVPGVG), or a sequence having no more than one amino acid substitution compared to SEQ ID NO:22. The elastin or ELP may comprise 1 to 10 sequences having SEQ ID NO:22, or 1 to 10 sequences, each independently having no more than one amino acid substitution compared to SEQ ID NO:22. These 1 to 10 sequences may be interconnected or separated by one or more amino acids.

[0193] In some embodiments, the elastin or ELP comprises polymeric or oligomeric repeating peptide sequences of elastin peptides. In some embodiments, each polymeric or oligomeric repeating peptide sequence comprises or has 4 amino acid residues.

[0194] In some embodiments, each polymeric or oligomeric repeating peptide sequence contains or has 5 amino acid residues. As a non-limiting example, SEQ ID NOS:49, 50, or 51.

[0195] In some implementations, each polymeric or oligomeric repeating peptide sequence contains or has 6 amino acid residues.

[0196] In some implementations, each polymeric or oligomeric repeating peptide sequence contains or has 7 amino acid residues.

[0197] In some implementations, each polymeric or oligomeric repeating peptide sequence contains or has 8 amino acid residues.

[0198] Non-limiting exemplary repeating peptide sequences include polytetrapeptides, polypentapeptides, polyhexapeptides, polyhepeptides, polyoctapeptides, and polynonapeptides, including but not limited to VPGG (SEQ ID NO:11), IPGG (SEQ ID NO:12), VPAVG (SEQ ID NO:13), AVGVP (SEQ ID NO:14), IPGVG (SEQ ID NO:15), LPGVG (SEQ ID NO:16), VAPGVG (SEQ ID NO:17), GVGVPGVG (SEQ ID NO:18), VPGFGVGAG (SEQ ID NO:19), VPGVGVPgg (SEQ ID NO:20), and VPGX. 2 G(SEQ ID NO:21),SEQ ID NO:49(((VPGXG) n, where n is 1 to 10, and each X is independently any amino acid), SEQ ID NO:50 (VPGVG), SEQ ID NO:51 (VPGLG) and SEQ ID NO:52 (VPGVGVPGL), and combinations of two or more of them.

[0199] In some implementations, "X" 2 "Amino acids are either naturally occurring or not naturally occurring."

[0200] In some embodiments, elastin or ELP is a polymeric or oligomeric repeating sequence of the pentapeptide VPAVG (SEQ ID NO:13).

[0201] In some implementations, elastin or ELP is the pentapeptide VPGX. 2 G is a polymeric or oligomeric repeating sequence, where "X" is a repeating sequence. 2 "X" is an amino acid selected from arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan. In some embodiments, "X" 2 "It can be leucine. In some embodiments, "X" 2 "It's asparagine."

[0202] In some implementations, "X" 2 It is valine.

[0203] In some embodiments, elastin or ELP is defined by the peptide sequence GVGVPGVGVPGLGVPGVGVPGVG (SEQ ID NO: 22).

[0204] In another embodiment, elastin or ELP is defined by the sequence VPGVGVPGVGVPGVGVPGVGVPGVGVPGVG (SEQ ID NO:23).

[0205] In some implementations, the elastin or elastin-like polypeptide (ELP) component forms a β-sheet-rich domain.

[0206] In some embodiments, the elastin or ELP component comprises amorphous domains. In some embodiments, the elastin or ELP component comprises crystalline domains. In some embodiments, the elastin or ELP component comprises both crystalline and amorphous domains.

[0207] In some embodiments, the elastin or ELP component is stimulus-responsive. In some embodiments, the ELP component is thermoresponsive. In some embodiments, the ELP component may respond to changes in pH. In some embodiments, the ELP component may respond over a certain temperature range. In some embodiments, the ELP component may respond to salt.

[0208] In some embodiments, the keratin or keratin-related fusion proteins / peptides disclosed herein comprise silk protein or silk protein-like polypeptide (SLP) components. In some embodiments, a silk protein-like polypeptide (SLP) refers to a peptide having a secondary structure, tertiary structure, or amino acid sequence, or a combination of two or more, similar to that of a wild-type silk protein (such as SEQ ID NO: 56) or a fragment of wild-type silk protein. In some embodiments, the fragment comprises a sequence having at least 4, 5, 6, 7, 8, 9, or 10 amino acids that is 100% identical to the sequence of wild-type silk protein. Wild-type silk protein may be filamentin. Non-limiting examples of silk proteins and SLPs include SEQ ID NOs: 24-32, 56, and 92.

[0209] The SLP may comprise a polymeric or oligomeric repeating peptide sequence of silk fibroin. In one example embodiment, the silk fibroin or SLP comprises SEQ ID NO:24 GAGAGS, or a sequence having no more than one amino acid substitution compared to SEQ ID NO:24. In another example embodiment, the silk fibroin or SLP comprises SEQ ID NO:24, or a sequence having no more than one amino acid substitution compared to SEQ ID NO:24. The silk fibroin or SLP may comprise 1 to 10 sequences having SEQ ID NO:24, or 1 to 10 sequences, each independently having no more than one amino acid substitution compared to SEQ ID NO:24. These 1 to 10 sequences may be interconnected or separated by one or more amino acids.

[0210] In some embodiments, the silk protein or SLP comprises polymeric or oligomeric repeating peptide sequences of silk protein peptides, wherein each polymeric or oligomeric repeating peptide sequence contains or has 6 amino acid residues.

[0211] In some embodiments, the silk protein or SLP comprises polymeric or oligomeric repeating peptide sequences of silk protein peptides, wherein each polymeric or oligomeric repeating peptide sequence contains or has 7 amino acid residues.

[0212] In some embodiments, the silk protein or SLP comprises polymeric or oligomeric repeating peptide sequences of silk protein peptides, wherein each polymeric or oligomeric repeating peptide sequence contains or has 8 amino acid residues.

[0213] Non-limiting examples of repeating peptide sequences include polytetrapeptides, polypentapeptides, polyhexapeptides, polyhepeptides, polyoctapeptides, and polynonapeptides, including but not limited to, GAGAGGS (SEQ ID NO:24), GAGSGA (SEQ ID NO:25), GAGAGY (SEQ ID NO:26), GAGYGA (SEQ ID NO:27), GAGGAGA (SEQ ID NO:28), GAGAGV (SEQ ID NO:29), GAGVGA (SEQ ID NO:30), and GAGAGVGY (SEQ ID NO:31), and combinations of two or more of them.

[0214] In some embodiments, silk protein or SLP is a polymeric or oligomeric repeating sequence of the pentapeptide GAGAGS (SEQ ID NO:24).

[0215] In some embodiments, the peptide sequence of silk fibroin or SLP is GAGAGGSGAGAGSGAGAGSGAGAGSGAGAGS (SEQ ID NO:32).

[0216] In some embodiments, the peptide sequence of silk fibroin or SLP is GAGX. 1 GX 2 (SEQ ID NO:92), where X 1 and X 2 Independently any amino acid, optionally wherein X 1 The variable can be A, S, Y, or V, with X being an optional variable. 2 It can be S, A, Y, or V.

[0217] In some embodiments, silk fibroin or SLP is a peptide sequence.

[0218] GYGAGAGSGAASGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSGAGAGSG

[0219] AGAGSGAGAGYGAGAGVGYGAGAGSGAASGAGAGSGAGAGSGAGAGSGA GAGS (SEQ ID NO: 56).

[0220] In some implementations, silk protein or silk protein-like (SLP) modules form β-sheet-rich domains.

[0221] In some embodiments, the silk fibroin or SLP component module includes crystalline domains. In some embodiments, the silk fibroin or SLP module includes amorphous domains. In some embodiments, the SLP module includes both crystalline and amorphous domains.

[0222] In some embodiments, the keratin or keratin-related fusion proteins disclosed herein comprise collagen or collagen-like polypeptide (CLP) modules. In some embodiments, a collagen-like polypeptide (CLP) refers to a peptide having a secondary structure, tertiary structure, or amino acid sequence, or a combination of two or more, similar to that of wild-type collagen (e.g., SEQ ID NO: 59) or a fragment of wild-type collagen, including synthetic peptides. In some embodiments, the fragment comprises a sequence having at least 3, 4, 5, 6, 7, 8, 9, or 10 amino acids that is 100% identical to the sequence of wild-type collagen. Non-limiting examples of collagen and CLPs include SEQ ID NOS: 33, 57-59, 93-95, and 106, and fragments having the amino acids “GTP” and / or “GLQ”.

[0223] CLPs may contain polymeric or oligomeric repeating peptide sequences of collagen peptides. In one example embodiment, the collagen or CLP contains the sequence GTP. The collagen or CLP may contain 1 to 10 sequences containing the sequence GTP. These 1 to 10 sequences may be interconnected or separated by one or more amino acids. These 1 to 10 sequences may be, for example, 2 or 3. For example, the collagen or CLP may contain GPTGPT (SEQ ID NO:57), GLQGLQ (SEQ ID NO:58), GPAGPA (SEQ ID NO:95), or GPTGPTGPAGPRGLQGLQGLQGERGEQGPT (SEQ ID NO:33). In one example embodiment, the collagen or CLP contains the sequence GLQ. The collagen or CLP may contain 1 to 10 sequences containing the sequence GLQ. These 1 to 10 sequences may be, for example, 2 or 3. These 1 to 10 sequences may be interconnected or separated by one or more amino acids. For example, collagen or CLP may contain GLQGLQ (SEQ ID NO:58) or

[0224] GPTGPTGPAGPRGLQGLQGLQGERGEQGPT (SEQ ID NO:33). In an exemplary embodiment, the collagen or CLP contains the sequence GPT. The collagen or CLP may contain 1 to 10 sequences containing the sequence GPT. These 1 to 10 sequences may be, for example, 2 or 3. These 1 to 10 sequences may be interconnected or separated by one or more amino acids. For example, the collagen or CLP may contain GPTGPT (SEQ ID NO:57) or GPTGPTGPAGPRGLQGLQGLQGERGEQGPT (SEQ ID NO:33). The collagen or CLP may contain 1 to 10 sequences containing the sequence GXP, where X is any amino acid. These 1 to 10 sequences may be, for example, 2 or 3. These 1 to 10 sequences may be interconnected or separated by one or more amino acids. For example, the collagen or CLP may contain GXPGXP (SEQ ID NO:93). The collagen or CLP may contain 1 to 10 sequences comprising the sequence GZP, where Z is 4-hydroxyproline. These 1 to 10 sequences may be, for example, 2 or 3. These 1 to 10 sequences may be linked together or separated by one or more amino acids. For example, the collagen or CLP may contain GLQGLQ (SEQ ID NO:94).

[0225] In some embodiments, each polymeric or oligomeric repeating peptide sequence of the collagen or collagen-like polypeptide (CLP) module comprises or has 3 amino acid residues. As a non-limiting example, these 3 amino acid residues may include GTP, GLQ, GXP (X being any amino acid), GZP (Z being 4-hydroxyproline), and GX. 1 H.

[0226] In some embodiments, the repeating peptide sequence includes polytetrapeptides, polypentapeptides, polyhexapeptides, polyhepeptides, polyoctapeptides, and polynonapeptides, including but not limited to GX. 1 H.

[0227] In some implementations, "X" 1 "Including proline, leucine, isoleucine, glutamic acid, aspartic acid, glutamine, lysine, alanine, arginine, and threonine; and where "H" includes amino acids selected from glycine, alanine, lysine, proline, methionine, glutamine, arginine, threonine, valine, glutamic acid, aspartic acid, or asparagine."

[0228] In some embodiments, collagen or CLP is defined by the peptide sequence GPTGPTGPAGPRGLQGLQGLQGERGEQGPT (SEQ ID NO:33).

[0229] In some implementations, collagen or CLP is defined by the peptide sequence SEQ ID NO:59.

[0230] In some embodiments, collagen or CLPs form alpha-helices (α-helices). In some embodiments, multiple collagen or CLPs form triple helices. In some embodiments, collagen or CLPs form 3.6... 13 Helical. In some embodiments, collagen or CLP forms a polyproline type II helix. In some embodiments, collagen or CLP forms at least one helical loop.

[0231] In some embodiments, the keratin or keratin-related fusion proteins disclosed herein comprise arthropod elastin or arthropod elastin-like polypeptide (RLP) modules. In some embodiments, an arthropod elastin-like polypeptide (RLP) refers to a peptide having a secondary structure, tertiary structure, or amino acid sequence, or a combination of two or more, similar to that of wild-type arthropod elastin (e.g., SEQ ID NO: 60) or a fragment of wild-type arthropod elastin, including synthetic peptides. In some embodiments, the fragment comprises a sequence having at least 4, 5, 6, 7, 8, 9, or 10 amino acids that is 100% identical to the wild-type filament protein sequence. Non-limiting examples of arthropod elastin and RLP include SEQ ID NOs: 34-35, 60, 96, and 107.

[0232] The RLP may comprise a polymeric or oligomeric repeating peptide sequence of arthropod elastin peptide. In an exemplary embodiment, the polymeric or oligomeric repeating peptide sequence is a fragment of a wild-type arthropod elastin peptide having about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.

[0233] In some embodiments, each polymeric or oligorepetitive peptide sequence of the arthropod elastin or arthropod elastin-like peptide (RLP) module contains or has 11 amino acid residues. In some embodiments, each polymeric or oligorepetitive peptide sequence of the arthropod elastin or arthropod elastin-like peptide (RLP) module contains or has 12 amino acid residues. In some embodiments, each polymeric or oligorepetitive peptide sequence of the arthropod elastin or arthropod elastin-like peptide (RLP) module contains or has 13 amino acid residues. In some embodiments, each polymeric or oligorepetitive peptide sequence of the arthropod elastin or arthropod elastin-like peptide (RLP) module contains or has 14 amino acid residues. In some embodiments, each polymeric or oligorepetitive peptide sequence of the arthropod elastin or arthropod elastin-like peptide (RLP) module contains or has 15 amino acid residues. In some embodiments, the repetitive peptide sequence includes polypentapeptides, polyhexapeptides, polyheptacapeptides, polyoctapeptides, and polynonapeptides, including but not limited to GGRPSDSYGAPGGGN (SEQ ID NO:34), GAPAQTPSSQY (SEQ ID NO:35), AQTPSSQYGAP (SEQ ID NO:96), and combinations thereof. In some embodiments, arthropod elastin or RLP is composed of peptide sequences.

[0234] GGRPSDSYGAPGGGN (SEQ ID NO:34) is defined. In some embodiments, arthropod elastin or RLP is defined by the peptide sequence SEQ ID NO:60. In some embodiments, arthropod elastin or RLP is defined by the peptide sequence SEQ ID NO:107. In some embodiments, arthropod elastin or RLP is defined by the peptide sequence GAPAQTPSSQY (SEQ ID NO:35). In some embodiments, arthropod elastin or RLP is defined by the peptide sequence AQTPSSQYGAP (SEQ ID NO:96).

[0235] In some embodiments, the keratin or keratin-related fusion proteins disclosed herein comprise extensin or extensin-like peptide (ALP) modules. In some embodiments, an extensin-like peptide (ALP) refers to a peptide comprising a secondary, tertiary, or amino acid sequence similar to, or a combination thereof, a fragment of wild-type extensin protein (such as SEQ ID NO: 61) or wild-type extensin, including synthetic peptides. In some embodiments, the fragment comprises a sequence having at least 4, 5, 6, 7, 8, 9, or 10 amino acids, which is 100% identical to the wild-type extensin sequence. Non-limiting examples of extensin and ALP include SEQ ID NOS: 36-42 and 61. An ALP may comprise a polymeric or oligomeric repeating peptide sequence of extensin.

[0236] In some embodiments, each polymeric or oligomeric repeating peptide sequence contains or has 4 expansin amino acid residues. In some embodiments, each polymeric or oligomeric repeating peptide sequence contains or has 5 expansin amino acid residues. In some embodiments, each polymeric or oligomeric repeating peptide sequence contains or has 6 expansin amino acid residues.

[0237] In some embodiments, each polymeric or oligomeric repeating peptide sequence comprises or has 7 exoside amino acid residues. In some embodiments, each polymeric or oligomeric repeating peptide sequence comprises or has 8 exoside amino acid residues. In some embodiments, each polymeric or oligomeric repeating peptide sequence comprises or has 9 exoside amino acid residues. In some embodiments, each polymeric or oligomeric repeating peptide sequence comprises or has 10 exoside amino acid residues. In some embodiments, the repeating peptide sequence is a variety of polytetrapeptides, polypentapeptides, polyhexapeptides, polyheptapeptides, polyoctapeptides, and polynonapeptides, including but not limited to GGFGGMGGGS (SEQ ID NO:36), MGGG (SEQ ID NO:37), FGGMG (SEQ ID NO:38), FGGMGGG (SEQ ID NO:39), GGFGGMG GGG (SEQ ID NO:40), FGGMGGGNAG (SEQ ID NO:41), and combinations thereof. In some embodiments, the exoside or ALP is derived from the peptide sequence.

[0238] GGFGGMGGGFGGMGGGSGGFGGMGGGSGGFGGMGGGS (SEQ ID NO:42) is defined. In some embodiments, the extensin or ALP is derived from a peptide sequence.

[0239] MNAYICLAACLIAAVSAAGYGGGAGSMGGTGGMGGGMNAGGFGGMGGMG

[0240] GGKGGFGGIGGFGGGMGGGPGGFGGMGGFGGMAAKGGFGGMGSGMGGFGGMGGGNAGFGGMGGGNAGFGGMGGQGGFGGKGY (SEQ ID NO: 61) is defined.

[0241] In an exemplary embodiment, the exoside or ALP comprises SEQ ID NO:36, or a sequence having no more than one amino acid substitution compared to SEQ ID NO:36. The exoside or ALP may comprise 1 to 10 sequences having SEQ ID NO:36, or 1 to 10 sequences, each independently having no more than one amino acid substitution compared to SEQ ID NO:36. These 1 to 10 sequences may be interconnected, as in SEQ ID NO:42, or may be separated by one or more amino acids.

[0242] In some embodiments, the fusion protein includes a linker assembly. In some embodiments, the linker assembly is a spacer-like sample. In some embodiments, the linker assembly is spaced between keratin or keratin-associated peptide and polypeptide components. In some embodiments, the linker assembly is spaced between individual polypeptide components. In some embodiments, the linker or spacer-like sample contains (GQ). n , where n is an integer from 1 to 20. In some implementations, the connector or spacer sample includes (AK). n , where n is an integer from 1 to 20. In some embodiments, the connector or spacer sample is (LK). n , where n is an integer from 1 to 20. In some implementations, the connector or spacer sample includes (GA). n , where n is an integer from 1 to 20. In some embodiments, the connector or spacer sample comprises the sequence GQGQGQGQGQGQGQGQGQGQGQGQGQGQGQ (SEQ ID NO:97), AKAKAKAKAKAKAKAKAKAKAKAKAKAKAK (SEQ ID NO:98), LKLKLKLKLKLKLKLKLKLKLKLKLKLKLKLKLKLKLK (SEQ ID NO:99), GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA (SEQ ID NO:100), or combinations thereof. In some embodiments, the connector or spacer sample comprises fragments.

[0243] GQGQGQGQGQGQGQGQGQGQGQGQGQGQGQGQGQGQ (SEQ ID NO:97), AKAKAKAKAKAKAKAKAKAKAKAKAKAK (SEQ ID NO:98), LKLKLKLKLKLKLKLKLKLKLKLKLKLKLKLKLKLK (SEQ ID NO:99), GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGA (SEQ ID NO:100), or combinations thereof. In some embodiments, the connector or spacer sample contains (GQ). nWhere n is 1-20. In some embodiments, the connector or spacer sample includes (AK). n Where n is 1-20. In some embodiments, the connector or spacer sample includes (LK). n Where n is 1-20. In some embodiments, the connector or spacer sample includes (GA). n , where n is 1-20.

[0244] Table 1. Non-restrictive example peptide sequences

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253] Table 1 provides exemplary embodiments of elastin, elastin-like peptide (ELP), silk protein, silk protein-like peptide (SLP), collagen, collagen-like peptide (CLP), arthropod elastin, arthropod elastin-like peptide (RLP), extensin, and extensin-like peptide (ALP) peptides.

[0254] Table 2. Non-limiting exemplary peptide fusion sequences

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268] Table 2 provides example implementations of keratin and keratin-related fusion proteins.

[0269] In some embodiments, keratin or a keratin-related fusion protein / peptide comprises or is selected from the following sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7; SEQ ID NO:44; SEQ ID NO:45; SEQ ID NO:46; SEQ ID NO:47. In some embodiments, keratin or a keratin-related fusion protein comprises or is selected from any of SEQ ID NOs:200-551.

[0270] In some embodiments, keratin or keratin-related fusion proteins / peptides are SEQ ID NO:1.

[0271] In some embodiments, keratin or keratin-related fusion proteins / peptides are SEQ ID NO:2.

[0272] In some embodiments, keratin or keratin-related fusion proteins / peptides are SEQ ID NO:3.

[0273] In some embodiments, keratin or keratin-related fusion proteins / peptides are SEQ ID NO:4.

[0274] In some embodiments, keratin or keratin-related fusion proteins / peptides are SEQ ID NO:5.

[0275] In some embodiments, keratin or keratin-related fusion proteins / peptides are SEQ ID NO:6.

[0276] In some embodiments, keratin or keratin-related fusion proteins / peptides are SEQ ID NO:7.

[0277] In some embodiments, keratin or keratin-related fusion proteins / peptides are SEQ ID NO:44.

[0278] In some embodiments, keratin or keratin-related fusion proteins / peptides are SEQ ID NO:45.

[0279] In some embodiments, keratin or keratin-related fusion proteins / peptides are SEQ ID NO:46.

[0280] In some embodiments, keratin or keratin-related fusion proteins / peptides are SEQ ID NO:47.

[0281] In some embodiments, the keratin or keratin-associated fusion protein / peptide comprises a protein having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or higher sequence identity with any of the following sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46; SEQ ID NO:47, or SEQ ID NO:200-551.

[0282] In some embodiments, the keratin or keratin-associated fusion protein comprises a protein having at least 50% sequence identity with any of the following sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46; SEQ ID NO:47, or SEQ ID NO:200-551.

[0283] In some embodiments, the keratin or keratin-associated fusion protein comprises a protein having at least 55% sequence identity with any of the following sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46; SEQ ID NO:47, or SEQ ID NOS:200-551.

[0284] In some embodiments, the keratin or keratin-associated fusion protein comprises a protein having at least 60% sequence identity with any of the following sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46; SEQ ID NO:47, or SEQ ID NO:200-551.

[0285] In some embodiments, the keratin or keratin-associated fusion protein comprises a protein having at least 65% sequence identity with any of the following sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46; SEQ ID NO:47, or SEQ ID NOS:200-551.

[0286] In some embodiments, the keratin or keratin-associated fusion protein comprises a protein having at least 70% sequence identity with any of the following sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46; SEQ ID NO:47, or SEQ ID NOS:200-551.

[0287] In some embodiments, the keratin or keratin-associated fusion protein comprises a protein having at least 75% sequence identity with any of the following sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46; SEQ ID NO:47, or SEQ ID NOS:200-551.

[0288] In some embodiments, the keratin or keratin-associated fusion protein comprises a protein having at least 80% sequence identity with any of the following sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46; SEQ ID NO:47, or SEQ ID NOS:200-551.

[0289] In some embodiments, the keratin or keratin-associated fusion protein comprises a protein having at least 85% sequence identity with any of the following sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46; SEQ ID NO:47, or SEQ ID NOS:200-551.

[0290] In some embodiments, the keratin or keratin-related fusion protein comprises a protein having at least 90% sequence identity with any of the following sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46; SEQ ID NO:47, or SEQ ID NOS:200-551. In some embodiments, the keratin or keratin-related fusion protein comprises a protein having at least 91% sequence identity with any of the following sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46; SEQ ID NO:47, or SEQ ID NOS:200-551. In some embodiments, the keratin or keratin-related fusion protein comprises a protein having at least 92% sequence identity with any of the following sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46; SEQ ID NO:47, or SEQ ID NO:200-551. In some embodiments, the keratin or keratin-related fusion protein comprises a protein having at least 93% sequence identity with any of the following sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46; SEQ ID NO:47, or SEQ ID NO:200-551.In some embodiments, the keratin or keratin-associated fusion protein comprises a protein having at least 94% sequence identity with any of the following sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46; SEQ ID NO:47, or SEQ ID NOS:200-551.

[0291] In some embodiments, the keratin or keratin-related fusion protein comprises a protein having at least 95% sequence identity with any of the following sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46; SEQ ID NO:47, or SEQ ID NOS:200-551. In some embodiments, the keratin or keratin-related fusion protein comprises a protein having at least 96% sequence identity with any of the following sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46; SEQ ID NO:47, or SEQ ID NOS:200-551. In some embodiments, the keratin or keratin-related fusion protein comprises a protein having at least 97% sequence identity with any of the following sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46; SEQ ID NO:47, or SEQ ID NOS:200-551. In some embodiments, the keratin or keratin-related fusion protein comprises a protein having at least 98% sequence identity with any of the following sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46; SEQ ID NO:47, or SEQ ID NOS:200-551.

[0292] In some embodiments, the keratin or keratin-associated fusion protein comprises a protein having at least 99% sequence identity with any of the following sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 or SEQ ID NO:7, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46; SEQ ID NO:47, or SEQ ID NOS:200-551.

[0293] In some embodiments, the keratin or keratin-associated fusion protein comprises keratin or a keratin-associated peptide linked to elastin or ELP. This linking can be via a covalent bond. This linking can also be via a linker. The linker can be a peptide linker. The peptide linker can contain about 1 to about 10 amino acids. The fusion protein may contain GGVCGPSPPCITTVPGVG (SEQ ID NO: 62). The fusion protein may contain any one of SEQ ID NOs: 1, 2, 7, or 200-271.

[0294] In some embodiments, the keratin or keratin-associated fusion protein comprises keratin or a keratin-associated peptide linked to a silk protein or a silk protein-like polypeptide (SLP). This linking can be via a covalent bond. This linking can also be via a linker. The linker can be a peptide linker. The peptide linker may contain about 1 to about 10 amino acids. The fusion protein may contain GGVCGPSPPCITTGAGAGS (SEQ ID NO: 63). The fusion protein may contain any one of SEQ ID NOS: 3 or 272-315.

[0295] In some embodiments, the keratin or keratin-associated fusion protein comprises keratin or a keratin-associated peptide linked to arthropod elastin or an arthropod elastin-like polypeptide (RLP). This linkage can be covalently linked. This linkage can also be via a linker. The linker can be a peptide linker. The peptide linker may contain about 1 to about 10 amino acids. The fusion protein may contain GGVCGPSPPCITTGGRPSDSYGAPGGGN (SEQ ID NO:4). The fusion protein may contain any one of SEQ ID NO:4 or 375-418.

[0296] In some embodiments, the keratin or keratin-associated fusion protein comprises a keratin or keratin-associated peptide linked to collagen or a collagen-like polypeptide (CLP). This linkage can be covalently linked. This linkage can also be a linker. The linker can be a peptide linker, which may contain about 1 to about 10 amino acids. The fusion protein may contain GGVCGPSPPCITTGPT (SEQ ID NO: 64). The fusion protein may contain any one of SEQ ID NOS: 5 or 316-374.

[0297] In some embodiments, the keratin or keratin-associated fusion protein comprises keratin or a keratin-associated peptide linked to an extensin or an extensin-like peptide (ALP). This linkage can be covalently linked. This linkage can also be a linker. The linker can be a peptide linker. The peptide linker can contain about 1 to about 10 amino acids. The fusion protein may contain GGVCGPSPPCITTGGFGGMGGGS (SEQ ID NO: 65). The fusion protein may contain any one of SEQ ID NOS: 6 or 419-477.

[0298] Keratin and keratin-related fusion proteins / peptides

[0299] In other or additional aspects, this disclosure covers keratin and keratin-related fusion proteins / peptides. The keratin and keratin-related fusion proteins / peptides of this disclosure comprise copolymers containing different modules. Each module of the copolymer comprises a monomer of amino acid residues. For example, amino acids such as alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine, and combinations of two or more of these.

[0300] In some embodiments, the keratin and keratin-related fusion proteins / peptides disclosed herein comprise amino acid sequences (the term "peptide" is used interchangeably to mean an amino acid sequence) linked by covalent bonds or any other type of bond that forms a structure that maintains the properties of the fusion proteins disclosed herein. The bonds may be selected from, but are not limited to, hydrogen bonds, ionic pairing, hydrophobic association, or inclusion complex formation.

[0301] In some embodiments, keratin and keratin-associated fusion proteins have formula (I)[B n1 -A m -B n -C o -B n2 ] p .

[0302] In some embodiments, keratin and keratin-associated fusion proteins have formula (II) [B n1 -C o -B n -A m -B n2 ] p .

[0303] In some embodiments, keratin and keratin-associated fusion proteins have formula (III) [B n1 -B n -C o -A m -B n2 ] P .

[0304] In some embodiments, keratin and keratin-associated fusion proteins have formula (IV)[B n1 -C o -A m -B n -B n2 ] p .

[0305] In some embodiments, “A” comprises a peptide sequence containing keratin or a keratin-associated peptide module. In one example embodiment, the keratin or keratin-associated peptide module is a keratin or keratin-associated peptide component described elsewhere herein. In some embodiments, the keratin, keratin peptide, or keratin-associated peptide comprises a wild-type keratin peptide, a functional fragment of a wild-type keratin peptide, or a peptide having at least 90% identity with a wild-type keratin peptide or a functional fragment of a wild-type keratin peptide. The wild-type keratin peptide may be keratin 85 having SEQ ID NO:48. The wild-type keratin peptide may comprise SEQ ID NO:8, 9, 10, 66, or 67, or a combination of two or more of these. In some embodiments, the keratin, keratin peptide, or keratin-associated peptide forms one or more disulfide bonds with hair, skin, or nail, or a combination of two or more of these.

[0306] In one non-limiting example embodiment, the functional fragment of the wild-type keratin peptide comprises at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 consecutive amino acids of the wild-type keratin peptide. In another non-limiting example embodiment, the functional fragment of the wild-type keratin peptide comprises at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids having at least 90% identity with the wild-type keratin peptide. In yet another non-limiting example embodiment, the functional fragment of the wild-type keratin peptide comprises at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids, wherein the at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids comprise one or more cysteine ​​amino acids. For example, one or more cysteine ​​amino acids may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 cysteine ​​amino acids. In a non-limiting example embodiment, the functional fragment of the wild-type keratin peptide comprises at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids, wherein at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, or 50% of the at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids are cysteine ​​amino acids. Non-limiting example embodiments of keratin or keratin peptides are provided in SEQ ID NO:48.

[0307] In some embodiments, the keratin-associated peptide (sometimes referred to as the keratin-associated protein) comprises a peptide capable of forming a disulfide bond with wild-type keratin. In some embodiments, the keratin-associated peptide comprises at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids, and said at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids have at least 90% identity with the wild-type keratin peptide sequence. The wild-type keratin peptide may be keratin 85 having SEQ ID NO:48. The wild-type keratin peptide may comprise SEQ ID NO:8, 9, 10, 66, or 67, or a combination of two or more of these.

[0308] In some embodiments, the number of amino acids in the keratin or keratin-associated peptide module of the fusion protein / peptide is from about 6 residues to about 16 residues.

[0309] In some embodiments, the number of amino acids in the keratin or keratin-associated peptide module of the fusion protein / peptide is about 6 residues.

[0310] In some embodiments, the number of amino acids in the keratin or keratin-associated peptide module of the fusion protein / peptide is about 7 residues.

[0311] In some embodiments, the number of amino acids in the keratin or keratin-associated peptide module of the fusion protein / peptide is about 8 residues.

[0312] In some embodiments, the number of amino acids in the keratin or keratin-associated peptide module of the fusion protein / peptide is about 9 residues.

[0313] In some embodiments, the number of amino acids in the keratin or keratin-associated peptide module of the fusion protein / peptide is about 10 residues.

[0314] In some embodiments, the number of amino acids in the keratin or keratin-associated peptide module of the fusion protein / peptide is about 11 residues.

[0315] In some embodiments, the number of amino acids in the keratin or keratin-associated peptide module of the fusion protein / peptide is about 12 residues.

[0316] In some embodiments, the number of amino acids in the keratin or keratin-associated peptide module of the fusion protein / peptide is about 13 residues.

[0317] In some embodiments, the number of amino acids in the keratin or keratin-associated peptide module of the fusion protein / peptide is about 14 residues.

[0318] In some embodiments, the number of amino acids in the keratin or keratin-associated peptide module of the fusion protein / peptide is about 15 residues.

[0319] In some embodiments, the number of amino acids in the keratin or keratin-associated peptide module of the fusion protein / peptide is about 16 residues.

[0320] In some embodiments, the keratin or keratin-associated peptide module of the fusion protein / peptide has a relatively high cysteine ​​content to enrich certain properties of the keratin or keratin-associated peptide module, such as enhancing the formation of intermolecular crosslinks. The total cysteine ​​content accounts for 10% to 50% of the total amino acids in the keratin or keratin-associated peptide module. In some embodiments, the total cysteine ​​content accounts for at least 15% of the total amino acids in the keratin or keratin-associated peptide module.

[0321] In some implementations, the keratin or keratin-associated peptide module of the fusion protein contains two cysteine ​​residues.

[0322] In some implementations, the keratin or keratin-associated peptide module of the fusion protein contains three cysteine ​​residues.

[0323] In some implementations, the keratin or keratin-associated peptide module of the fusion protein contains four cysteine ​​residues.

[0324] In some implementations, the keratin or keratin-associated peptide module of the fusion protein contains five cysteine ​​residues.

[0325] In some embodiments, the keratin or keratin-associated peptide module of the fusion protein has a peptide sequence (X). 3 ) k C(X 3 )5C(X 3 ) k .

[0326] In some implementations, each "X" 3 "Independently glycine, alanine, valine, isoleucine, leucine, phenylalanine, proline, serine, threonine, asparagine, glutamine, cysteine, methionine, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, histidine, lysine, or arginine."

[0327] In some implementations, each "k" is an independent integer from 0 to 3. If k is 2, then (X 3 ) k Having the first X 3 And the second X 3 , where each X 3 Independently, it can be glycine, alanine, valine, isoleucine, leucine, phenylalanine, proline, serine, threonine, asparagine, glutamine, cysteine, methionine, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, histidine, lysine, or arginine. If k is 3, then (X 3 ) k Having the first X 3 The second X 3 And the third X 3 , where each X 3 Independently, it consists of glycine, alanine, valine, isoleucine, leucine, phenylalanine, proline, serine, threonine, asparagine, glutamine, cysteine, methionine, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, histidine, lysine, or arginine. (X) 3 )5 has the first X 3 The second X 3 The third X 3 The fourth X 3 And the fifth X3 , where each X 3 It can be independently glycine, alanine, valine, isoleucine, leucine, phenylalanine, proline, serine, threonine, asparagine, glutamine, cysteine, methionine, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, histidine, lysine, or arginine.

[0328] In some embodiments, the keratin or keratin-related peptide module of the fusion protein / peptide comprises GGVCGPSPPCITT (SEQ ID NO:8), GGVCGPSPPC (SEQ ID NO:9), CGPSPPCITT (SEQ ID NO:10), CGPSPPC (SEQ ID NO:66), GVCGPSPPC (SEQ ID NO:67), CLPCLPAASC (SEQ ID NO:68), CLPAASC (SEQ ID NO:69), YGSSSGGG (SEQ ID NO:70), FGGGS (SEQ ID NO:71), CCQSSCCKPSC (SEQ ID NO:72), CVSSCCKPQCC (SEQ ID NO:73), PITCRRTCYH (SEQ ID NO:74), DCKLPCNPCA (SEQ ID NO:75), CLPCLPAASC (SEQ ID NO:76), CEPAICEPSC (SEQ ID NO:77), CQCSCCKPYCS (SEQ ID NO:78), FCGFPSCTSSC (SEQ ID NO:79), GGVCGPSPPCITT (SEQ ID NO:8), GGVCGPSPPC (SEQ ID NO:9), CGPAASC (SEQ ID NO:70), CGPAASC (SEQ ID NO:71), CGPAASC (SEQ ID NO:72), CGPAASC (SEQ ID NO:73), CGPAASC (SEQ ID NO:74), CGPAASC (SEQ ID NO:75), CGPAASC (SEQ ID NO:76), CGPAASC (SEQ ID NO:77), CGPAASC (SEQ ID NO:78), CGPAASC (SEQ ID NO:79), CGPAASC (SEQ ID NO:70), CGPAASC (SEQ ID NO:71), CGPAASC (SEQ ID NO:72), CGPAASC (SEQ ID NO:73), CGPAASC (SEQ ID NO:74), CGPAASC (SEQ ID NO:75), CGPAASC (SEQ ID NO:76), CGPAASC (SEQ ID NO:77), CGPAASC (SEQ ID NO:79), CTPPSCCQLHHA (SEQ ID NO:80), SCCAPVYCCK (SEQ ID NO:81), or a combination of two or more of them.

[0329] In some embodiments, the keratin or keratin-related peptide module of the fusion protein / peptide comprises GGVCGPSPPCITT (SEQ ID NO:8), GGVCGPSPPC (SEQ ID NO:9), CGPSPPCITT (SEQ ID NO:10), CGPSPPC (SEQ ID NO:66), or GVCGPSPPC (SEQ ID NO:67), or a combination of two or more thereof.

[0330] In some embodiments, the keratin or keratin-related peptide module is GGVCGPSPPCITT (SEQ ID NO:8) or CGPSPPCITT (SEQ ID NO:10).

[0331] In some embodiments, the keratin or keratin-related peptide module of the fusion protein / peptide comprises YGSSSGGG (SEQ ID NO:70), FGGGS (SEQ ID NO:71), or a combination of two or more thereof.

[0332] In some embodiments, the keratin or keratin-related peptide module of the fusion protein / peptide comprises CCQSSCCKPSC (SEQ ID NO:72), CVSSCCKPQCC (SEQ ID NO:73), or a combination of two or more thereof.

[0333] In some embodiments, the keratin or keratin-associated peptide module of the fusion protein / peptide comprises PITCRRTCYH (SEQ ID NO:74).

[0334] In some embodiments, the keratin or keratin-associated peptide module of the fusion protein comprises DCKLPCNPCA (SEQ ID NO:75).

[0335] In some embodiments, the keratin or keratin-associated peptide module of the fusion protein comprises CLPCLPAASC (SEQ ID NO:76).

[0336] In some embodiments, the keratin or keratin-associated peptide module of the fusion protein comprises CEPAICEPSC (SEQ ID NO:77).

[0337] In some embodiments, the keratin or keratin-associated peptide module of the fusion protein comprises CQCSCCKPYCS (SEQ ID NO:78).

[0338] In some embodiments, the keratin or keratin-associated peptide module of the fusion protein comprises FCGFPSCTSSC (SEQ ID NO:79).

[0339] In some embodiments, the keratin or keratin-associated peptide module of the fusion protein comprises CTPPSCCQLHHA (SEQ ID NO:80).

[0340] In some embodiments, the keratin or keratin-associated peptide module of the fusion protein includes SCCAPVYCCK (SEQ ID NO:81).

[0341] In some implementations, "m" is an integer from 1 to 10.

[0342] In some implementations, "m" is approximately 1.

[0343] In some implementations, "m" is approximately 2.

[0344] In some implementations, "m" is approximately 3.

[0345] In some implementations, "m" is approximately 4.

[0346] In some implementations, "m" is approximately 5.

[0347] In some implementations, "m" is approximately 6.

[0348] In some implementations, "m" is approximately 7.

[0349] In some implementations, "m" is approximately 8.

[0350] In some implementations, "m" is approximately 9.

[0351] In some implementations, "m" is approximately 10.

[0352] In some implementations, "B" comprises a linker or terminator. Linkers facilitate fusion protein design by providing appropriate spacing between domains and support correct protein folding where N-terminal or C-terminal interactions are crucial for folding. Typically, linkers allow important domain interactions, enhancing stability, fluidity, and reducing steric hindrance, making them suitable for protein / peptide design even if N-terminal and C-terminal fusion is possible. The three main types of linkers are flexible, rigid, and in vivo cleavable. Terminating (N-terminal or C-terminal) peptides contribute to the binding process, synthetic stability, and correct folding.

[0353] In some embodiments, "B" comprises a peptide sequence containing or having 1 to 3 amino acids. In some embodiments, "B" comprises a peptide sequence containing or having 1 to 3 amino acids repeated 0 to about 25 times. In some embodiments, "B" comprises a peptide sequence containing 1 or 2 amino acids repeated about 10 to about 25 times. In some embodiments, each amino acid in the peptide sequence is independently arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, selenocysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan. In some embodiments, B has 1 to 40 amino acids.

[0354] In some implementations, each “n”, “n1” and “n2” is an integer from 0 to 25 independently.

[0355] In some implementations, "n, n1, and n2" are 0.

[0356] In some implementations, "n, n1, and n2" are approximately 1.

[0357] In some implementations, "n, n1, and n2" is approximately 2.

[0358] In some implementations, "n, n1, and n2" is approximately 3.

[0359] In some implementations, "n, n1, and n2" is approximately 4.

[0360] In some implementations, "n, n1, and n2" are approximately 5.

[0361] In some implementations, "n, n1, and n2" is approximately 6.

[0362] In some implementations, "n, n1, and n2" is approximately 7.

[0363] In some implementations, "n, n1, and n2" is approximately 8.

[0364] In some implementations, "n, n1, and n2" is approximately 9.

[0365] In some implementations, "n, n1, and n2" are approximately 10.

[0366] In some implementations, “n, n1, and n2” is approximately 11.

[0367] In some implementations, "n, n1, and n2" is approximately 12.

[0368] In some implementations, "n, n1, and n2" is approximately 13.

[0369] In some implementations, "n, n1, and n2" is approximately 14.

[0370] In some implementations, "n, n1, and n2" is approximately 15.

[0371] In some implementations, "n, n1, and n2" is approximately 16.

[0372] In some implementations, "n, n1, and n2" is approximately 17.

[0373] In some implementations, "n, n1, and n2" is approximately 18.

[0374] In some implementations, "n, n1, and n2" is approximately 19.

[0375] In some implementations, "n, n1, and n2" is approximately 20.

[0376] In some implementations, “n, n1, and n2” is approximately 21.

[0377] In some implementations, “n, n1, and n2” is approximately 22.

[0378] In some implementations, “n, n1 and n2” is approximately 23.

[0379] In some implementations, "n, n1, and n2" is approximately 24.

[0380] In some implementations, "n, n1, and n2" is approximately 25.

[0381] In some implementations, "C" comprises a peptide sequence having a Dq-Er-Fs-Gt-Hu structure.

[0382] In some implementations, modules “D”, “E”, “F”, “G” and “H” can be provided in any order.

[0383] In some implementations, "D" includes a silk protein or silk protein-like peptide (SLP) module.

[0384] In some implementations, "E" includes a collagen or collagen-like peptide (CLP) module.

[0385] In some implementations, "F" includes an elastin or elastin-like peptide (ELP) module.

[0386] In some implementations, "G" includes arthropod elastin or arthropod elastin-like peptide (RLP) modules.

[0387] In some implementations, "H" includes an expansin or expansin-like peptide (ALP) module.

[0388] In some embodiments, the keratin or keratin-related fusion proteins disclosed herein comprise elastin or elastin-like polypeptide (ELP) modules (“F”). In some embodiments, an elastin-like polypeptide (ELP) refers to a peptide comprising a secondary structure, tertiary structure, or amino acid sequence similar to, or a combination thereof, that of wild-type elastin (e.g., SEQ ID NO: 55) or a fragment of wild-type elastin, including synthetic peptides. In some embodiments, the fragment comprises a sequence having at least 4, 5, 6, 7, 8, 9, or 10 amino acids that is 100% identical to the sequence of wild-type elastin. Non-limiting exemplary elastins and ELPs include SEQ ID NOS: 11-23, 49-55, 82-91, and 104.

[0389] ELPs may comprise polymeric or oligomeric repeating peptide sequences of elastin. In an example embodiment, the elastin or elastin-like polypeptide (ELP) comprises SEQ ID NO:49((VPGXG)). nThe sequence X is defined as follows: where n is 1 to 10, and each X is independently any amino acid, or a sequence having no more than one amino acid substitution compared to SEQ ID NO:49. In some embodiments, each X is independently valine or leucine. In some embodiments, n is 5. In some embodiments, n is 6.

[0390] In an exemplary embodiment, the elastin or elastin-like polypeptide (ELP) comprises SEQ ID NO:50 (VPGVG), or a sequence having no more than one amino acid substitution compared to SEQ ID NO:50. The elastin or ELP may comprise 1 to 10 sequences having SEQ ID NO:50, or 1 to 10 sequences, each independently having no more than one amino acid substitution compared to SEQ ID NO:50. These 1 to 10 sequences may be linked together, for example, VPGVGVPGVGVPGVGVPGVGVPGVGVPGVG (SEQ ID NO:23), or may be separated by one or more amino acids, for example, separated by another ELP, such as VPGVGVPGVGVPGLGVPGVGVPGVG (SEQ ID NO:54).

[0391] In one example embodiment, the elastin or ELP comprises SEQ ID NO:51 (VPGLG), or a sequence having no more than one amino acid substitution compared to SEQ ID NO:51. The elastin or ELP may comprise 1 to 10 sequences having SEQ ID NO:51, or 1 to 10 sequences, each independently having no more than one amino acid substitution compared to SEQ ID NO:51. These 1 to 10 sequences may be linked together or separated by one or more amino acids.

[0392] In an exemplary embodiment, the elastin or ELP comprises SEQ ID NO:52 (VPGVGVPGL), or a sequence having no more than one amino acid substitution compared to SEQ ID NO:52. The elastin or ELP may comprise 1 to 10 sequences having SEQ ID NO:52, or 1 to 10 sequences, each independently having no more than one amino acid substitution compared to SEQ ID NO:52. These 1 to 10 sequences may be linked together or separated by one or more amino acids.

[0393] In an example embodiment, the elastin or ELP comprises SEQ ID NO:53 (VPGVGVPGVGVPGLGVPGVGVPGVGR), or a sequence having no more than one amino acid substitution compared to SEQ ID NO:53. The elastin or ELP may comprise 1 to 10 sequences having SEQ ID NO:53, or 1 to 10 sequences, each independently having no more than one amino acid substitution compared to SEQ ID NO:53. These 1 to 10 sequences may be linked together or separated by one or more amino acids.

[0394] In an example embodiment, the elastin or ELP comprises SEQ ID NO:23 (VPGVGVPGVGVPGVGVPGVGVPGVGVPGVG), or a sequence having no more than one amino acid substitution compared to SEQ ID NO:23. The elastin or ELP may comprise 1 to 10 sequences having SEQ ID NO:23, or 1 to 10 sequences, each independently having no more than one amino acid substitution compared to SEQ ID NO:23. These 1 to 10 sequences may be linked together or separated by one or more amino acids.

[0395] In an example embodiment, the elastin or ELP comprises SEQ ID NO:54 (VPGVGVPGVGVPGLGVPGVGVPGVG), or a sequence having no more than one amino acid substitution compared to SEQ ID NO:54. The elastin or ELP may comprise 1 to 10 sequences having SEQ ID NO:54, or 1 to 10 sequences, each independently having no more than one amino acid substitution compared to SEQ ID NO:54. These 1 to 10 sequences may be linked together or separated by one or more amino acids.

[0396] In an example embodiment, the elastin or ELP comprises SEQ ID NO:22 (GVGVPGVGVPGLGVPGVGVPGVG), or a sequence having no more than one amino acid substitution compared to SEQ ID NO:22. The elastin or ELP may comprise 1 to 10 sequences having SEQ ID NO:22, or 1 to 10 sequences, each independently having no more than one amino acid substitution compared to SEQ ID NO:22. These 1 to 10 sequences may be linked together or separated by one or more amino acids.

[0397] In an example embodiment, the elastin or ELP comprises SEQ ID NO:82 (IPGLG), or a sequence having no more than one amino acid substitution compared to SEQ ID NO:82. The elastin or ELP may comprise 1 to 10 sequences having SEQ ID NO:82, or 1 to 10 sequences, each independently having no more than one amino acid substitution compared to SEQ ID NO:82. These 1 to 10 sequences may be linked together or separated by one or more amino acids. In some embodiments, the elastin or ELP comprises SEQ ID NO:85 (IPGVGIPGLG), SEQ ID NO:89 (VPGVGIPGLG), or a combination thereof.

[0398] In some embodiments, the elastin or ELP comprises polymeric or oligomeric repeating peptide sequences of elastin peptides. In some embodiments, each polymeric or oligomeric repeating peptide sequence comprises or has 4 amino acid residues.

[0399] In some embodiments, each polymeric or oligomeric repeating peptide sequence contains or has 5 amino acid residues. As non-limiting examples, such as SEQ ID NOS:49, 50, or 51.

[0400] In some implementations, each polymeric or oligomeric repeating peptide sequence contains or has 6 amino acid residues.

[0401] In some implementations, each polymeric or oligomeric repeating peptide sequence contains or has 7 amino acid residues.

[0402] In some implementations, each polymeric or oligomeric repeating peptide sequence contains or has 8 amino acid residues.

[0403] Non-limiting exemplary repeating peptide sequences include polytetrapeptides, polypentapeptides, polyhexapeptides, polyhepeptides, polyoctapeptides, and polynonapeptides, including but not limited to VPGG (SEQ ID NO:11), IPGG (SEQ ID NO:12), VPAVG (SEQ ID NO:13), AVGVP (SEQ ID NO:14), IPGVG (SEQ ID NO:15), LPGVG (SEQ ID NO:16), VAPGVG (SEQ ID NO:17), GVGVPGVG (SEQ ID NO:18), VPFGFVGAG (SEQ ID NO:19), VPGVG VPGG (SEQ ID NO:20), and VPG X. 2 G(SEQ ID NO:21), SEQ ID NO:49((VPGXG) n, where n is 1 to 10, and each X is independently any amino acid), SEQ ID NO:50 (VPGVG), SEQ ID NO:51 (VPGLG), and SEQ ID NO:52 (VPGVGVPGL), and combinations of two or more of them.

[0404] In some implementations, "X" 2 It contains amino acids, whether naturally occurring or not.

[0405] In some implementations, elastin or ELP is a polymer or oligomeric repeat of the pentapeptide VPAVG.

[0406] In some implementations, elastin or ELP is the pentapeptide VPGX. 2 Polymers or oligomeric repeaters of G, wherein "X" 2 "X" is selected from the following amino acids: arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan. In some embodiments, "X" 2 "It is leucine. In some embodiments, "X" 2 "It's asparagine."

[0407] In some implementations, "X" 2 It is valine.

[0408] In some embodiments, elastin or ELP is defined by the peptide sequence GVGVPGVGVPGLGVPGVGVPGVG (SEQ ID NO: 22).

[0409] In some embodiments, elastin or ELP is defined by the sequence VPGVGVPGVGVPGVGVPGVGVPGVGVPGVG (SEQ ID NO:23).

[0410] In some implementations, elastin or elastin-like polypeptide (ELP) modules form β-sheet-rich domains.

[0411] In some embodiments, the elastin or ELP module includes amorphous domains. In some embodiments, the elastin or ELP module includes crystalline domains. In some embodiments, the elastin or ELP module includes both crystalline and amorphous domains.

[0412] In some embodiments, the elastin or ELP module is stimulus-responsive. In some embodiments, the ELP module is thermoresponsive. In some embodiments, the ELP component may respond to pH changes. In some embodiments, the ELP component may respond over a certain temperature range. In some embodiments, the ELP component may respond to salt.

[0413] In some implementations, "s" is an integer from 0 to approximately 220.

[0414] In some implementations, "s" is 0.

[0415] In some implementations, "s" is approximately 1.

[0416] In some implementations, "s" is approximately 2.

[0417] In some implementations, "s" is approximately 3.

[0418] In some implementations, "s" is approximately 4.

[0419] In some implementations, "s" is approximately 5.

[0420] In some implementations, "s" is approximately 6.

[0421] In some implementations, "s" is approximately 7.

[0422] In some implementations, "s" is approximately 8.

[0423] In some implementations, "s" is approximately 9.

[0424] In some implementations, "s" is approximately 10.

[0425] In some implementations, "s" is approximately 20.

[0426] In some implementations, "s" is approximately 30.

[0427] In some implementations, "s" is approximately 40.

[0428] In some implementations, "s" is approximately 50.

[0429] In some implementations, "s" is approximately 100.

[0430] In some embodiments, the keratin or keratin-related fusion proteins / peptides disclosed herein comprise a silk protein or silk protein-like polypeptide (SLP) module (“D”). In some embodiments, a silk protein-like polypeptide (SLP) refers to a peptide having a secondary structure, tertiary structure, or amino acid sequence similar to, or a combination thereof, with or similar to, a wild-type silk protein (e.g., SEQ ID NO: 56) or a fragment of wild-type silk protein, including synthetic peptides. In some embodiments, the fragment comprises a sequence having at least 4, 5, 6, 7, 8, 9, or 10 amino acids that is 100% identical to the wild-type silk protein sequence. Wild-type silk protein may be filamentin. Non-limiting exemplary silk proteins and SLPs include SEQ ID NOs: 24-32, 92, and 56.

[0431] The SLP may comprise a polymeric or oligomeric repeating peptide sequence of silk fibroin. In one example embodiment, the silk fibroin or SLP comprises SEQ ID NO:24 (GAGAGS), or a sequence having no more than one amino acid substitution compared to SEQ ID NO:24. In another example embodiment, the silk fibroin or SLP comprises SEQ ID NO:24, or a sequence having no more than one amino acid substitution compared to SEQ ID NO:24. The silk fibroin or SLP may comprise 1 to 10 sequences having SEQ ID NO:24, or 1 to 10 sequences, each independently having no more than one amino acid substitution compared to SEQ ID NO:24. These 1 to 10 sequences may be linked together or separated by one or more amino acids.

[0432] In some embodiments, the silk protein or SLP comprises polymeric or oligomeric repeating peptide sequences of silk protein peptides, wherein each polymeric or oligomeric repeating peptide sequence contains or has 6 amino acid residues.

[0433] In some embodiments, the silk protein or SLP comprises polymeric or oligomeric repeating peptide sequences of silk protein peptides, wherein each polymeric or oligomeric repeating peptide sequence contains or has 7 amino acid residues.

[0434] In some embodiments, the silk protein or SLP comprises polymeric or oligomeric repeating peptide sequences of silk protein peptides, wherein each polymeric or oligomeric repeating peptide sequence contains or has 8 amino acid residues.

[0435] Non-limiting exemplary repeating peptide sequences include polytetrapeptides, polypentapeptides, polyhexapeptides, polyhepeptides, polyoctapeptides, and polynonapeptides, including but not limited to GAGAGGS (SEQ ID NO:24), GAGSGA (SEQ ID NO:25), GAGAGY (SEQ ID NO:26), GAGYGA (SEQ ID NO:27), GAGGAGA (SEQ ID NO:28), GAGAGV (SEQ ID NO:29), GAGVGA (SEQ ID NO:30), and GAGAGVGY (SEQ ID NO:31), and combinations of two or more of them.

[0436] In some embodiments, silk fibroin or SLP is a polymer or oligomeric repeat of the pentapeptide GAGAGS (SEQ ID NO:24).

[0437] In some embodiments, the silk fibroin or SLP is defined by the peptide sequence GAGAGGSGAGAGSGAGAGSGAGAGSGAGAGS (SEQ ID NO:32).

[0438] In some implementations, silk protein or silk protein-like (SLP) modules form β-sheet-rich domains.

[0439] In some embodiments, the silk fibroin or SLP module includes crystalline domains. In some embodiments, the silk fibroin or SLP module includes amorphous domains. In some embodiments, the silk fibroin or SLP module includes both amorphous and crystalline domains.

[0440] In some implementations, "q" is an integer from 0 to 100.

[0441] In some implementations, "q" is 0.

[0442] In some implementations, "q" is approximately 1.

[0443] In some implementations, "q" is approximately 2.

[0444] In some implementations, "q" is approximately 3.

[0445] In some implementations, "q" is approximately 4.

[0446] In some implementations, "q" is approximately 5.

[0447] In some implementations, "q" is approximately 6.

[0448] In some implementations, "q" is approximately 7.

[0449] In some implementations, "q" is approximately 8.

[0450] In some implementations, "q" is approximately 9.

[0451] In some implementations, "q" is approximately 10.

[0452] In some implementations, "q" is approximately 20.

[0453] In some implementations, "q" is approximately 30.

[0454] In some implementations, "q" is approximately 40.

[0455] In some implementations, "q" is approximately 50.

[0456] In some implementations, "q" is approximately 100.

[0457] In some embodiments, the keratin or keratin-related fusion proteins / peptides disclosed herein comprise collagen or collagen-like polypeptide (CLP) modules (“E”). In some embodiments, a collagen-like polypeptide (CLP) refers to a peptide having a secondary structure, tertiary structure, or amino acid sequence similar to, or a combination thereof, that of wild-type collagen (e.g., SEQ ID NO: 59) or a fragment of wild-type collagen, including synthetic peptides. In some embodiments, the fragment comprises a sequence having at least 3, 4, 5, 6, 7, 8, 9, or 10 amino acids that is 100% identical to the sequence of wild-type collagen. Non-limiting exemplary collagen and CLPs include SEQ ID NOS: 33, 58, 59, 93, 94, 95, and 57, and fragments having the amino acids “GTP” and / or “GLQ”.

[0458] CLPs may contain polymeric or oligomeric repeating peptide sequences of collagen peptides. In one example embodiment, the collagen or CLP contains the sequence GTP. The collagen or CLP may contain 1 to 10 sequences containing the sequence GTP. These 1 to 10 sequences may be linked together or separated by one or more amino acids. These 1 to 10 sequences may be, for example, 2 or 3. For example, the collagen or CLP may contain GPTGPT (SEQ ID NO:57) or GPTGPTGPAGPRGLQGLQGLQGERGEQGPT (SEQ ID NO:33). In one example embodiment, the collagen or CLP contains the sequence GLQ. The collagen or CLP may contain 1 to 10 sequences containing the sequence GLQ. These 1 to 10 sequences may be, for example, 2 or 3. These 1 to 10 sequences may be linked together or separated by one or more amino acids. For example, collagen or CLP may contain GLQGLQ (SEQ ID NO:58) or GPTGPTGPAGPRGLQGLQGLQGERGEQGPT (SEQ ID NO:33).

[0459] In some embodiments, each polymeric or oligomeric repeating peptide sequence of the collagen or collagen-like polypeptide (CLP) module comprises or has 3 amino acid residues. As a non-limiting example, these 3 amino acid residues may include GTP, GLQ, and GX. 1 H.

[0460] In some embodiments, the repeating peptide sequence includes polytetrapeptides, polypentapeptides, polyhexapeptides, polyhepeptides, polyoctapeptides, and polynonapeptides, including but not limited to GX. 1 H.

[0461] In some implementations, "X" 1 "Contains proline, leucine, isoleucine, glutamic acid, aspartic acid, glutamine, lysine, alanine, arginine, and threonine; and "H" contains an amino acid selected from glycine, alanine, lysine, proline, methionine, glutamine, arginine, threonine, valine, glutamic acid, aspartic acid, or asparagine."

[0462] In some embodiments, collagen or CLP is defined by the peptide sequence GPTGPTGPAGPRGLQGLQGLQGERGEQGPT (SEQ ID NO:33).

[0463] In some implementations, "r" is an integer from 0 to approximately 200.

[0464] In some implementations, "r" is 0.

[0465] In some implementations, "r" is approximately 1.

[0466] In some implementations, "r" is approximately 2.

[0467] In some implementations, "r" is approximately 3.

[0468] In some implementations, "r" is approximately 4.

[0469] In some implementations, "r" is approximately 4.

[0470] In some implementations, "r" is approximately 5.

[0471] In some implementations, "r" is approximately 6.

[0472] In some implementations, "r" is approximately 7.

[0473] In some implementations, "r" is approximately 8.

[0474] In some implementations, "r" is approximately 9.

[0475] In some implementations, "r" is approximately 10.

[0476] In some implementations, "r" is approximately 20.

[0477] In some implementations, "r" is approximately 30.

[0478] In some implementations, "r" is approximately 40.

[0479] In some implementations, "r" is approximately 50.

[0480] In some implementations, "r" is approximately 100.

[0481] In some embodiments, the keratin or keratin-related fusion protein / peptide disclosed herein comprises arthropod elastin or arthropod elastin-like polypeptide (RLP) modules (“G”). In some embodiments, an arthropod elastin-like polypeptide (RLP) refers to a peptide having a secondary structure, tertiary structure, or amino acid sequence similar to, or a combination thereof, with or similar to, a fragment of wild-type arthropod elastin (e.g., SEQ ID NO: 60) or a fragment of wild-type arthropod elastin, including synthetic peptides. In some embodiments, the fragment comprises a sequence having at least 4, 5, 6, 7, 8, 9, or 10 amino acids that is 100% identical to the sequence of wild-type arthropod elastin. Non-limiting exemplary arthropod elastin and arthropod elastin-like polypeptide (RLP) include SEQ ID NOs: 34-35, 96, and 60.

[0482] RLPs comprise polymeric or oligomeric repeating peptide sequences of arthropod elastin. In an example embodiment, the polymeric or oligomeric repeating peptide sequence is a fragment of a wild-type arthropod elastin peptide having about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.

[0483] In some embodiments, each polymeric or oligomeric repeating peptide sequence of arthropod elastin or arthropod elastin-like polypeptide (RLP) component contains or has 11 amino acid residues.

[0484] In some embodiments, each polymeric or oligomeric repeating peptide sequence of arthropod elastin or arthropod elastin-like polypeptide (RLP) component contains or has 12 amino acid residues.

[0485] In some embodiments, each polymeric or oligomeric repeating peptide sequence of arthropod elastin or arthropod elastin-like polypeptide (RLP) component contains or has 13 amino acid residues.

[0486] In some embodiments, each polymeric or oligomeric repeating peptide sequence of arthropod elastin or arthropod elastin-like polypeptide (RLP) component contains or has 14 amino acid residues.

[0487] In some embodiments, each polymeric or oligomeric repeating peptide sequence of arthropod elastin or arthropod elastin-like polypeptide (RLP) component contains or has 15 amino acid residues.

[0488] In some embodiments, the repeating peptide sequence includes polytetrapeptides, polypentapeptides, polyhexapeptides, polyhepeptides, polyoctapeptides, and polynonapeptides, including but not limited to GGRPSDSYGAPGGGN (SEQ ID NO:34), GAPAQTSSQY (SEQ ID NO:35), AQTSSQYGAP (SEQ ID NO:96), and combinations thereof.

[0489] In some embodiments, arthropod elastin or RLP is defined by the peptide sequence GGRPSDSYGAPGGGN (SEQ ID NO:34).

[0490] In some implementations, "t" is an integer from 0 to 80.

[0491] In some implementations, "t" is 0.

[0492] In some implementations, "t" is approximately 1.

[0493] In some implementations, "t" is approximately 2.

[0494] In some implementations, "t" is approximately 3.

[0495] In some implementations, "t" is approximately 4.

[0496] In some implementations, "t" is approximately 5.

[0497] In some implementations, "t" is approximately 6.

[0498] In some implementations, "t" is approximately 7.

[0499] In some implementations, "t" is approximately 8.

[0500] In some implementations, "t" is approximately 9.

[0501] In some implementations, "t" is approximately 10.

[0502] In some implementations, "t" is approximately 20.

[0503] In some implementations, "t" is approximately 30.

[0504] In some implementations, "t" is approximately 40.

[0505] In some implementations, "t" is approximately 50.

[0506] In some implementations, "t" is approximately 80.

[0507] In some implementations, "t" is approximately 100.

[0508] In some embodiments, the keratin or keratin-related components disclosed herein comprise an extensin or an extensin-like peptide (ALP) module (“H”). In some embodiments, an extensin-like peptide (ALP) refers to a peptide comprising a secondary structure, tertiary structure, or amino acid sequence, or a combination thereof, similar to that of a wild-type extensin (e.g., SEQ ID NO: 61) or a fragment of wild-type extensin, including synthetic peptides. In some embodiments, the fragment comprises a sequence having at least 4, 5, 6, 7, 8, 9, or 10 amino acids that is 100% identical to the sequence of wild-type extensin. Non-limiting exemplary extensins and ALPs include SEQ ID NOs: 36-42, 61, and 108. An ALP comprises a polymeric or oligomeric repeating peptide sequence of an extensin protein.

[0509] In some implementations, each polymeric or oligomeric repeating peptide sequence contains or has four amino acid residues of an expansin protein.

[0510] In some implementations, each polymeric or oligomeric repeating peptide sequence contains or has 5 amino acid residues of an expansin protein.

[0511] In some implementations, each polymeric or oligomeric repeating peptide sequence contains or has 6 amino acid residues of an expansinin protein.

[0512] In some implementations, each polymeric or oligomeric repeating peptide sequence contains or has 7 amino acid residues of an expansinin protein.

[0513] In some implementations, each polymeric or oligomeric repeating peptide sequence contains or has 8 amino acid residues of an expansin protein.

[0514] In some implementations, each polymeric or oligomeric repeating peptide sequence contains or has 9 amino acid residues of an expansin protein.

[0515] In some embodiments, each polymeric or oligomeric repeating peptide sequence contains or has 10 amino acid residues of an expansin protein.

[0516] In some embodiments, the repeating peptide sequence includes polytetrapeptides, polypentapeptides, polyhexapeptides, polyhepeptides, polyoctapeptides, and polynonapeptides, including but not limited to GGFGGMGGGS (SEQ ID NO:36), MGGG (SEQ ID NO:37), FGGMG (SEQ ID NO:38), FGGMGGG (SEQ ID NO:39), GGFGGMGGG (SEQ ID NO:40), FGGMGGGNAG (SEQ ID NO:41), MNAYICLAACLIAAVSAAGYGGGAGSMGGTGGMGGGMNAGGFGGMGGMGG GGKGGFGGIGGFGGGMGGGPGGFGGMGGFGGMAAKGGFGGMGSGMGGFG GMGGGNAGFGGMGGGNAGFGGMGGGAQGGFGGKGY (SEQ ID NO:36), MGGG (SEQ ID NO:37), FGGMG (SEQ ID NO:38), FGGMGGG (SEQ ID NO:39), GGFGGMGGG (SEQ ID NO:40), FGGMGGGNAG (SEQ ID NO:41), MNAYICLAACLIAAVSAAGYGGGAGSMGGTGGMGGGMNAGGFGGMGGMGGGGMAAKGGFGGMGSGMGGFG GMGGGNAGFGGMGGGNAGFGGMGGQGGFGGKGY (SEQ ID NO:36), MGGG (SEQ ID NO:37), FGGMG (SEQ ID NO:38), FGGMGGG (SEQ ID NO:39), FGGMGGG (SEQ ID NO:39), FGGMGGG (SEQ ID NO:40), FGGMGGGGA (SEQ ID NO:41), FGGMGGGA (SEQ ID NO:41), FGGMGGGA (SEQ ID NO:39), FGGMGGGA (SEQ ID NO:41), FGGMGGGA NO:61), GGRPSDSYGAPGGGNGGRPSDSYGAPGGGNGGRPSDSYGAPGGGNGGRPSDSYGAPGGGNGGRPSDSYGAPGGGNGGRPSDSYGAPGGGNGGRPSDSYGAPGGGNGGRPS DSYGAPGGGNGGRPSDSYGAPGGGNGGRPSDSYGAPGGGNGQGQGQGQGQGQGQGQGQGQGGVCGPSPPCITTGQGQGQGQGQGQGQGQGQGQVPAVGVPAVGVPAVGVPAVGVPAV GVPAVGVPAVGVPAVGVPAVGVPAVGGQGQGQGQGQGQGQGQGQGQGGVCGPSPPCITTGQGQGQGQGQGQGQGQGQGQGGRPSDSYGAPGGGNGGRPSDSYGAPGGGNGGRPSDSY GAPGGGNGGRPSDSYGAPGGGNGGRPSDSYGAPGGGNGGRPSDSYGAPGGGNGGRPSDSYGAPGGGNGGRPSDSYGAPGGGNGGRPSDSYGAPGGGNGGRPSDSYGAPGGGN(SEQID NO:108), and their combinations.

[0517] In some embodiments, the expansin or ALP is defined by the peptide sequence GGFGGMGGGSGGFGGMGGGSGGFGGMGGGS (SEQ ID NO:42). In one example embodiment, the expansin or ALP comprises SEQ ID NO:36, or a sequence having no more than one amino acid substitution compared to SEQ ID NO:36. The expansin or ALP may comprise 1 to 10 sequences having SEQ ID NO:36, or 1 to 10 sequences, each independently having no more than one amino acid substitution compared to SEQ ID NO:36. These 1 to 10 sequences may be linked together, for example, SEQ ID NO:42, or may be separated by one or more amino acids.

[0518] In some implementations, "u" is an integer from 0 to 100.

[0519] In some implementations, "u" is between 0 and 70.

[0520] In some implementations, "u" is 0.

[0521] In some implementations, "u" is approximately 1.

[0522] In some implementations, "u" is approximately 2.

[0523] In some implementations, "u" is approximately 3.

[0524] In some implementations, "u" is approximately 4.

[0525] In some implementations, "u" is approximately 5.

[0526] In some implementations, "u" is approximately 6.

[0527] In some implementations, "u" is approximately 7.

[0528] In some implementations, "u" is approximately 8.

[0529] In some implementations, "u" is approximately 9.

[0530] In some implementations, "u" is approximately 10.

[0531] In some implementations, "u" is approximately 20.

[0532] In some implementations, "u" is approximately 30.

[0533] In some implementations, "u" is approximately 40.

[0534] In some implementations, "u" is approximately 50.

[0535] In some implementations, "u" is approximately 70.

[0536] In some implementations, "o" is an integer from 0 to 30.

[0537] In some implementations, "o" is 0.

[0538] In some implementations, "o" is approximately 1.

[0539] In some implementations, "o" is approximately 2.

[0540] In some implementations, "o" is approximately 3.

[0541] In some implementations, "o" is approximately 4.

[0542] In some implementations, "o" is approximately 5.

[0543] In some implementations, "o" is approximately 6.

[0544] In some implementations, "o" is approximately 7.

[0545] In some implementations, "o" is approximately 8.

[0546] In some implementations, "o" is approximately 9.

[0547] In some implementations, "o" is approximately 10.

[0548] In some implementations, "o" is approximately 15.

[0549] In some implementations, "o" is approximately 20.

[0550] In some implementations, "o" is approximately 25.

[0551] In some implementations, "o" is approximately 30.

[0552] In some implementations, "p" is an integer from 1 to 30.

[0553] In some implementations, "p" is approximately 1.

[0554] In some implementations, "p" is approximately 2.

[0555] In some implementations, "p" is approximately 3.

[0556] In some implementations, "p" is approximately 4.

[0557] In some implementations, "p" is approximately 5.

[0558] In some implementations, "p" is approximately 6.

[0559] In some implementations, "p" is approximately 7.

[0560] In some implementations, "p" is approximately 8.

[0561] In some implementations, "p" is approximately 9.

[0562] In some implementations, "p" is approximately 10.

[0563] In some implementations, "p" is approximately 15.

[0564] In some implementations, "p" is approximately 20.

[0565] In some implementations, "p" is approximately 25.

[0566] In some implementations, "p" is approximately 30.

[0567] In some embodiments, the average molecular weight of keratin or keratin-related fusion proteins / peptides is in the range of about 1 kDa to about 144 kDa.

[0568] In some embodiments, the average molecular weight of keratin or keratin-related fusion proteins / peptides is in the range of about 1 kDa to about 5 kDa.

[0569] In some embodiments, the average molecular weight of keratin or keratin-related fusion proteins / peptides is in the range of about 5 kDa to about 10 kDa.

[0570] In some embodiments, the average molecular weight of keratin or keratin-related fusion proteins / peptides is in the range of about 6 kDa to about 17 kDa.

[0571] In some embodiments, the average molecular weight of keratin or keratin-related fusion proteins / peptides is in the range of about 10 kDa to about 15 kDa.

[0572] In some embodiments, the average molecular weight of keratin or keratin-related fusion proteins / peptides is in the range of about 14 kDa to about 30 kDa.

[0573] In some embodiments, the average molecular weight of keratin or keratin-related fusion proteins / peptides is in the range of about 15 kDa to about 20 kDa.

[0574] In some embodiments, the average molecular weight of keratin or keratin-related fusion proteins / peptides is in the range of about 17 kDa to about 39 kDa.

[0575] In some implementations, the average molecular weight of keratin or keratin-related fusion proteins / peptides may be in the range of about 20 kDa to about 25 kDa.

[0576] In some embodiments, the average molecular weight of keratin or keratin-related fusion proteins / peptides is in the range of about 25 kDa to about 30 kDa.

[0577] In some embodiments, the average molecular weight of keratin or keratin-related fusion proteins / peptides is in the range of about 30 kDa to about 35 kDa.

[0578] In some embodiments, the average molecular weight of keratin or keratin-related fusion proteins / peptides is in the range of about 35 kDa to about 40 kDa.

[0579] In some embodiments, the average molecular weight of keratin or keratin-related fusion proteins / peptides is in the range of about 39 kDa to about 54 kDa.

[0580] In some embodiments, the average molecular weight of keratin or keratin-related fusion proteins / peptides is in the range of about 39 kDa to about 80 kDa.

[0581] In some embodiments, the average molecular weight of keratin or keratin-related fusion proteins / peptides is in the range of about 40 kDa to about 45 kDa.

[0582] In some embodiments, the average molecular weight of keratin or keratin-related fusion proteins / peptides is in the range of about 45 kDa to about 50 kDa.

[0583] In some embodiments, the average molecular weight of keratin or keratin-related fusion proteins / peptides is in the range of about 50 kDa to about 55 kDa.

[0584] In some embodiments, the average molecular weight of keratin or keratin-related fusion proteins / peptides is in the range of about 55 kDa to about 60 kDa.

[0585] In some embodiments, the average molecular weight of keratin or keratin-related fusion proteins / peptides is in the range of about 60 kDa to about 100 kDa.

[0586] In some embodiments, the average molecular weight of keratin or keratin-related fusion proteins / peptides is in the range of about 80 kDa to about 144 kDa.

[0587] In another aspect or another, this disclosure relates to compositions comprising the fusion proteins / peptides disclosed herein.

[0588] In some embodiments, the composition is used to improve one or more properties of hair, nails, or skin, or combinations thereof. For example, the composition improves skin elasticity, hydration, or texture, or provides ultraviolet (UV) protection to the skin, or a combination of two or more of these.

[0589] In some embodiments, the composition improves the mechanical properties, color, texture, shape, or hydration of hair, or provides better thermal protection for hair, or a combination of two or more of these.

[0590] In some embodiments, the composition improves the mechanical or antimicrobial properties of nails, or provides better UV protection for nails, or a combination of two or more of these.

[0591] In some embodiments, the compositions disclosed herein further comprise at least one additive selected from: water, preservatives, antioxidants, chelating agents, sunscreens, vitamins, dyes, hair dyeing preparations, surfactants, detergents, emulsifiers, sunscreens, volatile substances, propellants, liquid media, carriers, salts, pH adjusters, neutralizers, buffers, hair conditioners, antistatic agents, anti-frizz agents, anti-dandruff agents, natural extracts, wetting agents, fragrances, perfumes, oils, softeners, lubricants, lipids, penetrants, thickeners, viscosity modifiers, polymers, resins, hair styling agents, film-forming agents, absorbents, and combinations thereof, to achieve approximation to commercially available product forms.

[0592] In some embodiments, the compositions disclosed herein are formulated into soaps, shampoos, conditioners, sprays, gels, low to medium viscosity liquids, lotions, emulsions, mousses, or creams containing the fusion proteins / peptides disclosed herein.

[0593] In some embodiments, the additive is included in or excluded from the composition, depending on its form of use.

[0594] Another aspect or additional aspect of this disclosure relates to methods for obtaining the fusion proteins / peptides disclosed herein.

[0595] In some embodiments, the method for obtaining the fusion protein of this disclosure includes the following steps:

[0596] a) Inoculate liquid culture medium with microorganisms containing genes expressing the desired fusion protein / peptide;

[0597] b) Allow the microorganisms to multiply within a certain culture period and induce the expression of the desired fusion protein / peptide under the action of the promoter;

[0598] c) Extract the desired fusion protein / peptide from the obtained microorganism by purifying the protein from endogenous contaminants.

[0599] In some embodiments, the method for obtaining a fusion protein / peptide includes the step of inoculating a liquid culture medium with a microorganism, particularly an Escherichia coli strain, that includes a gene expressing the desired fusion protein / peptide.

[0600] In some embodiments, the method for obtaining the fusion protein includes the step of extracting the desired fusion protein / peptide from microorganisms by purifying the fusion protein / peptide from endogenous contaminants.

[0601] In some respects, this article provides a method for providing fusion peptides described elsewhere.

[0602] definition

[0603] Unless otherwise defined, all technical terms, symbols, and other technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms having a commonly understood meaning are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as indicating a material difference from the commonly understood meaning in the art.

[0604] Throughout this disclosure, various embodiments may be presented in the form of ranges. It should be understood that descriptions in the form of ranges are merely for convenience and brevity and should not be construed as a rigid limitation on the scope of this disclosure. Accordingly, a description of a range should be considered as specifically disclosing all possible subranges within that range and the individual values ​​therein. For example, a description of a range such as 1 to 6 should be considered as specifically disclosing subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and the individual numbers within that range, such as 1, 2, 3, 4, 5, and 6. This applies regardless of the magnitude of the range.

[0605] As used in the specification and claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise. For example, the term “a sample” includes multiple samples, including mixtures thereof.

[0606] Whenever the terms "at least," "greater than," or "greater than or equal to" appear before the first value in a series of two or more values, the terms "at least," "greater than," or "greater than or equal to" apply to each value in that series. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0607] The terms “determine,” “measure,” “assess,” “evaluate,” “appraise,” “analyze,” and “analyze” are often used interchangeably in this document and all refer to a form of measurement. These terms include determining the presence of an element (e.g., detecting). These terms may cover quantitative, qualitative, or a combination of quantitative and qualitative determinations. Assessments can be relative or absolute. “Detecting the presence of…” may include, in addition to determining the presence of something, determining the quantity of something present, depending on the specific circumstances.

[0608] In this article, the term "approximately" refers to a number plus or minus 10%. The term "approximately" refers to a range minus 10% of its minimum value and plus 10% of its maximum value.

[0609] In this document, the terms "pharmaceutically acceptable" and "cosmetically acceptable" are used interchangeably and refer to compounds, materials, compositions, and / or dosage forms that, to a reasonable extent of medical judgment, are suitable for use in contact with human and animal tissues without causing excessive toxicity, irritation, allergic reactions, or other problems or complications commensurate with a reasonable benefit / risk ratio. More specifically, pharmaceutically acceptable means a material, compound, or composition suitable for use in contact with skin, scalp, or hair. Pharmaceutically acceptable materials are well known to those skilled in the art.

[0610] In this document, the terms “treatment” or “curing” refer to pharmaceutical or other interventions used to obtain a beneficial or anticipated outcome in a recipient. Beneficial or anticipated outcomes include, but are not limited to, therapeutic benefits and / or preventative benefits. A therapeutic benefit may refer to the eradication or improvement of the underlying condition or symptom being treated. Furthermore, a therapeutic benefit may be obtained when one or more physical symptoms associated with the underlying condition are eradicated or improved, resulting in an observed improvement in the subject, even if the subject may still be troubled by the underlying condition. Preventative effects include delaying, preventing, or eliminating the onset of a disease or symptom, delaying or eliminating the onset of symptoms of a disease or symptom, slowing, halting, or reversing the progression of a disease or symptom, or any combination thereof. To obtain a preventative benefit, a subject at risk of developing a specific disease, or a subject exhibiting physical symptoms of one or more diseases, may receive treatment, even if the disease may not yet be diagnosed.

[0611] In this document, the terms "hair care" or "hair treatment" are used to refer to compositions used to improve a particular property of hair. This property includes, but is not limited to, strength, elasticity, shine, smoothness, volume, or a combination thereof. "Hair care" or "hair treatment" can also be used to describe the process of providing a composition to previously treated or damaged hair, such as bleached or heat-treated hair.

[0612] Whenever "at least," "greater than," or "greater than or equal to" appears before the first value in a series of two or more values, "at least," "greater than," or "greater than or equal to" applies to every value in that series. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0613] Whenever "no more than," "less than," or "less than or equal to" appears before the first value in a series of two or more values, "no more than," "less than," or "less than or equal to" applies to every value in that series. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0614] The expressions "at least one of A and B" and "at least one of A or B" can be understood as referring to at least A, at least B, or at least A and B (i.e., a set containing A and B, which may contain one or more other elements). The term "A and / or B" can be understood as referring to only A, only B, or both A and B.

[0615] The expressions "at least about A, B, and C" and "at least about A, B, or C" can be understood as referring to at least about A, at least about B, or at least about C. The expressions "at most about A, B, and C" and "at most about A, B, or C" can be understood as referring to at most about A, at most about B, or at most about C.

[0616] The expression "between about A and B, between about C and D, and between about E and F" can be understood as referring to the period between about A and about B, between about C and about D, and between about E and about F. The expression "between about A and B, between about C and about D, or between about E and about F" can be understood as referring to the period between about A and about B, between about C and about D, or between about E and about F.

[0617] The expression "about A to B and C to D" can be understood as referring to the period between about A and about B, and between about C and about D. The expression "about A to B or C to D" can be understood as referring to the period between about A and about B, or between about C and about D.

[0618] The term "comprising" as used herein is intended to indicate the presence of the stated feature, integer, step, or component, but does not preclude the presence or addition of one or more other features, integers, steps, components, or groups thereof.

[0619] Example

[0620] This disclosure can be further understood by referring to the following embodiments. The scope of this disclosure is not limited to the specific implementations described in the embodiments, which are intended only as examples of a single aspect of this disclosure. Any functionally equivalent methods are within the scope of this disclosure. Various modifications to this disclosure, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and drawings. Such modifications fall within the scope of the appended claims.

[0621] Carrier construction

[0622] The target nucleic acid sequence encoding a fusion protein is inserted into a suitable expression vector, bacteriophage, or directly integrated into the microbial genome or its derivatives to obtain a polynucleotide composition. The target nucleic acid sequence encodes a fusion protein / peptide of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:44-47 or 200-552, or any combination thereof, or any nucleic acid sequence encoding a fusion protein having at least 60% homology with any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:44-47 or 200-552. The target nucleic acid sequence comprises at least one keratin or keratin-related peptide component of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:66, SEQ ID NO:67 or SEQ IDNOS:70-81, and at least one of the following: ELP component (SEQ ID NO:11-23, 49-55, 82-91), SLP component (SEQ ID NO:24-32, 56, 92), CLP component (SEQ ID NO:33, 57-59, 93-95), RLP component (SEQ ID NO:34-35, 60, 96), ALP component (SEQ ID NO:36-42, 61) or any combination thereof.

[0623] The expression vector contains a promoter and a target nucleic acid sequence encoding a target fusion protein / peptide. The vector optionally contains a polynucleotide encoding: antibiotic resistance (e.g., ampR gene, camR gene, kmR gene, tetR gene, etc.), affinity tags (e.g., C-terminal or N-terminal tags), a reporter gene, a cleavable adapter, a terminator (e.g., T7 terminator, etc.), a ribozyme, or any combination thereof.

[0624] The vector backbone contains a promoter located upstream of the start codon. The promoter may be the T7 promoter, lac, T7lac, araBAD, Sp6, trp, or any combination thereof. The promoter may be an inducible or constitutive promoter, respectively inducing overexpression continuously or in the presence of an inducer. For example, but not limited to, integrating the lac promoter into the vector to induce overexpression in the presence of isopropyl-β-D-thiogalactopyranoside (IPTG).

[0625] The vector contains an affinity tag, which can be a C-terminal or N-terminal affinity tag. The affinity tag can include a polyhistidine tag (3xHis, 4xHis, 5xHis, 6xHis, 7xHis, 8xHis, or 9xHis), a GST tag, a FLAG tag, a MBP tag, a streptavidin (Strep) tag or a derivative of a streptavidin tag (e.g., Strep-tagII, Twin-Strep), a calmodulin tag, a chitin-binding tag, or any combination or derivative thereof. If an N-terminal affinity tag is used, an adapter sequence is placed between the polynucleotide encoding the target fusion protein and the N-terminal affinity tag. If a C-terminal affinity tag is used, a cleavable adapter sequence is placed between the 3' end of the nucleic acid sequence encoding the C-terminal tag and the 5' start codon of the polynucleotide encoding the target fusion protein. The adapter sequence encodes a cleavable adapter motif that is a known substrate of a protease that selectively cleaves the cleavable adapter without cleaving the target fusion protein. For example, a cleavable adapter sequence containing the IEGR sequence is a known substrate of factor Xa. Introducing a nucleic acid sequence encoding IEGR-(His)6 allows for the expression of both the affinity tag (6xHis) and the cleavable adapter sequence IEGR, enabling the affinity tag to be removed after purification. Optionally, a spacer sequence can be placed between the cleavable adapter sequence and the affinity tag sequence.

[0626] An exemplary vector map of the expression vector used is shown below. Figure 1 and Figure 2 As shown. In Figure 1 In this expression vector, the promoter is located upstream of the N-terminal affinity tag sequence, followed by the adapter sequence and the target nucleic acid sequence. The expression vector contains antibiotic resistance genes, such as equipping host bacteria with ampicillin resistance via the AmpR gene and AmpR promoter. Figure 2 In this expression vector, the promoter is located upstream of the target nucleic acid sequence, the adapter sequence, and the C-terminal affinity tag sequence. The expression vector can contain antibiotic resistance, such as equipping host bacteria with ampicillin resistance via the AmpR gene and the AmpR promoter.

[0627] The target nucleic acid sequence encoding the fusion protein is inserted into the vector backbone via conventional assembly or PCR-based Gibson assembly. For conventional assembly (TA), primers are designed to contain restriction sites and the target nucleic acid sequence to be inserted into the vector backbone. For Gibson assembly of nucleic acid fragments, Gibson primers are designed to be complementary to (1) the region of the vector backbone and the nucleic acid sequence encoding the target fusion protein / peptide, or (2) two different regions of the target nucleic acid sequence. For Gibson assembly cloning, the target nucleic acid sequence is inserted into the vector backbone via conventional assembly or PCR-based Gibson assembly. Cloning kit (New England Biolabs, Inc.)

[0628] Transformation

[0629] After the target nucleic acid sequence is cloned into the vector, the insertion is verified using a DNA gel (e.g., agarose gel). Once confirmed, the vector is transformed into microbial cells via heat shock transformation or electroporation. In one embodiment, the *E. coli* strain is selected from K-12, B, or derivatives thereof. In another embodiment, the vector carrying the target sequence is inserted into *Komagataella phaffii* via electroporation, and the gene is integrated into the genome via homologous recombination. The *Komagataella phaffii* strain is selected from GS115, X-33, or derivatives thereof.

[0630] Heat shock transformation: Thaw the stock of competent cells on ice, mix with the ligation mixture (e.g., T4 ligase), and then further mix with an appropriate amount of a vector encoding the target fusion protein / peptide. Gently mix the mixture and then transfer it to a hot water bath or preheated module at approximately 42°C for about 30 seconds. Immediately place the heated mixture on ice to cool and let it stand for about 2 minutes. Mix the cooled mixture with LB medium. Incubate the cooled mixture at 37°C for about 30 to 60 minutes before plating. Plate aliquots of the mixture and incubate overnight at 37°C.

[0631] Electroporation: Allow the round-bottom culture tubes to reach room temperature. Preheat the resuscitation medium in a 37°C water bath. Cool the electroporation cuvettes on ice. Cool the source of competent *E. coli* cells on ice and mix with the expression vector containing the target nucleic acid sequence. Pipette the solution containing competent cells to vector at a ratio of 25:1 into a microcentrifuge tube and mix gently. Electroporate the microcentrifuge tube containing the cell / DNA mixture on an electroporator (Gene-Pulser, Bio-Rad) at 2.1 kV, 100 Ω, and 25 μF using a time constant of 2.6 ms. Add the preheated resuscitation medium to the electroporated cell / DNA mixture and shake vigorously at 37°C for approximately 1 hour. Dilute the cells, plate them, and incubate overnight at 37°C.

[0632] Transformation successful. Successful transformation is achieved by obtaining aliquots of bacterial culture (as described below), lysing the culture, and extracting plasmids (e.g., using a DNA extraction kit (QIAamp DNA Kit)). The relative molecular weights of plasmids extracted from cultures and empty vectors are compared on DNA gels (e.g., agarose gels) to determine the molecular weight. DNA extracted from cultures has a larger molecular weight than that of empty vectors. If the target nucleic acid sequence is inserted using conventional assembly methods, digestion with the appropriate restriction enzymes is required before DNA gelation.

[0633] Cell culture

[0634] Colonies are inoculated into growth medium (e.g., LB medium). The inoculated medium is mixed with or without the addition of a selection marker. The culture is incubated on a shaker at 37°C for several hours. Aliquots are obtained, and the growth stage is examined by measuring the absorbance (or optical density) of the culture at 600 nm. When the optical density (OD) of the culture at 600 nm... 600 When the exponential or logarithmic phase occurs, remove the culture from the shaker.

[0635] Small-scale. Add approximately 25 mL to approximately 100 mL of culture to several sterile baffled flasks containing culture medium, and incubate the culture overnight on a shaker at 37°C. When OD... 600 At the corresponding lag phase, add IPTG (or other promoter inducer) to the culture, and then incubate with shaking at 37°C until OD (Occurrence Discharge) is reached. 600 This corresponds to either the exponential or logarithmic phase. The cells were then granulated by centrifugation at approximately 1800 × g for about 30 minutes at 4°C. The supernatant was discarded, and the pellets were stored at -20°C for later use.

[0636] Large-scale. Bioreactors (e.g., The bioreactor (Eppendorf) is equipped with a stirring device (e.g., a stirrer with baffles), sterilized, and filled at least halfway with fresh culture medium. The culture medium optionally contains an antifoaming agent. The culture medium is stirred at approximately 300 rpm to approximately 1200 rpm. The bioreactor is maintained at an internal temperature of approximately 37°C. The stirring rate can be automatically adjusted according to other parameters (e.g., carbon dioxide (CO2) content). A certain amount of transformed cells is slowly introduced into the bioreactor (e.g., via a peristaltic pump). The air composition in the headspace within the bioreactor is monitored, such as the CO2 content. The dissolved oxygen (DO) content is monitored and maintained at approximately 35% DO. The pH of the culture is also monitored. A pH maintenance feed, comprising an acidic buffer feed and an alkaline buffer feed, is fluidly connected to the bioreactor. The pH of the culture in the bioreactor is maintained between approximately 7.2 and approximately 7.6. A nutrient feed, comprising glucose (e.g., 20 g / L), is fluidly connected to the bioreactor. The nutrient feed is slowly introduced into the bioreactor. When the dissolved oxygen (DO) content exceeds a preset threshold, glucose is introduced automatically. Aliquots of the culture are obtained, and OD is measured. 600 To monitor the growth stage of the culture. When OD 600 At the corresponding lag phase or late lag phase, add an appropriate amount of IPTG or other inducers to induce expression. Reporter proteins, such as green fluorescent protein (GFP), can be expressed, and the green fluorescent hue of the culture can be used to approximate the expression of the target fusion protein. When OD... 600 At the corresponding exponential or logarithmic phase, the culture was pumped out of the bioreactor and centrifuged at approximately 1800 × g for approximately 20 to 30 minutes at 4°C to granulate the culture. The supernatant was discarded, and the pellets were stored at -30°C.

[0637] Protein purification

[0638] Cell pellets are reconstituted in buffer (e.g., PBS buffer, pH 7.4). Add an appropriate amount of buffer to the pellets to form a suspension. Mix the pellets and buffer until homogeneous. A vortex mixer or homogenizer can be used. After homogenization, lyse the suspension by freezing / thawing, sonication, high-pressure homogenization, mixing with glass beads, or through multiple freeze / thawing cycles. Add a protease inhibitor to the suspension of lysed cells. Centrifuge the lysed cells at 4°C at at least 2000 × g for approximately 5 to approximately 30 minutes. Collect the supernatant and discard the pellets. Purify the supernatant by dialysis using size exclusion chromatography, affinity chromatography, or direct drying. Dialyze the purified fraction with buffer or water, or perform percolation.

[0639] Chromatography

[0640] Size exclusion. The concentrated supernatant is passed through a size exclusion column, selected according to the desired molecular weight resolution. For example, but not limited to, when the fusion protein exhibits a molecular weight of approximately 35 kDa, a size exclusion resin with resolution for proteins between 5 kDa and 250 kDa (e.g., HiPrep Sephacryl S-200HR, Siter Life Sciences) is used.

[0641] Affinity chromatography. The affinity chromatography medium is selected based on the affinity tag introduced in the expressed fusion protein. When a 6xHis tag is introduced in the expression vector, the expressed 6xHis tag is suitable for affinity chromatography on Ni(II)-NTA resin. A high-performance liquid chromatography (HPLC) apparatus equipped with buffer A and buffer B is used. The Pure protein purification system (Stopfan Life Sciences) uses buffers A and B in fluid communication with an affinity chromatography medium (e.g., a Ni(II)-NTA column). Buffer A contains 0 mM imidazole. Buffer B contains approximately 200 mM imidazole. The column is perfused with several column volumes of buffer A, followed by several volumes of buffer B diluted in buffer A. The concentrated supernatant is passed through the column and incubated for several minutes. Unwanted components are eluted using a gradient from 0 mM imidazole to approximately 150 mM imidazole. A final wash of several column volumes of 100% buffer B (200 mM imidazole) is used to elute the purified target fusion protein. The purified target fusion protein / peptide is collected for further processing.

[0642] Further purification—cutting affinity tags

[0643] The purified His-tagged target fusion protein / peptide was further processed with a protease that selectively cleaves the linker to obtain a purified, untagged target fusion protein / peptide. The untagged target fusion protein / peptide was then passed through a spin column with an appropriate molecular weight cutoff using a benchtop centrifuge to obtain a concentrated target fusion protein / peptide.

[0644] Characterization

[0645] Purified and concentrated fusion proteins / peptides can be characterized by SDS-PAGE, circular dichroism (CD) spectroscopy, nuclear magnetic resonance (NMR), and mass spectrometry (MS).

[0646] SDS-PAGE. A series of concentrated target fusion protein / peptide dilutions are subjected to protein gel electrophoresis (e.g., SDS-PAGE) relative to a gradient. The protein gels will show bands corresponding to the expected molecular weight of the target fusion protein / peptide, which does not contain cleavable linker motifs and affinity tags. The concentrated target fusion protein / peptide is pre-digested with an enzyme to obtain digestion products, which are then evaluated by SDS-PAGE.

[0647] Circular dichroism spectroscopy. The concentrated target fusion protein / peptide was analyzed using a circular dichroism spectrometer (CD; J-1000 series circular dichroism spectrometer, Japan Spectrophotometer) to study its secondary structure. A maximum peak at 195 nm and minimum peaks at 208 nm and 222 nm were observed, corresponding to the α-helix of the target fusion protein / peptide. A maximum peak at 195 nm and a minimum peak at 218 nm were observed, corresponding to the β-sheet of the target fusion protein. A minimum peak at 198 nm was observed, corresponding to the random coil of the target fusion protein.

[0648] Mass spectrometry. This involves using mass spectrometry (MS), such as liquid chromatography-mass spectrometry (LC-MS). 2 The concentrated target fusion protein / peptide was analyzed. The concentrated target fusion protein / peptide was pretreated with dithiothreitol (DTT) and optionally digested in the presence of a protease such as chymotrypsin, trypsin, LysC, or AspN. The concentrated target fusion protein / peptide was mixed with 1.0 v / v trifluoroacetic acid (TFA) before fragmentation by ESI–MS (Orbitrap, ThermoFisher). Data were collected and processed using Proteome Discoverer (ThermoFisher). Predicted fragment sizes for various fragmentation and charge states were estimated using Proteome Discoverer or ExPasy.

[0649] A fusion protein / peptide (0.01%) solution in an ethanol formulation was applied to Asian bleached hair strands (300 mg). Several properties were evaluated: emulsification properties, antimicrobial properties, fiber integrity, elasticity, Young's modulus, hydrophobicity, etc.

[0650] Emulsifying properties. The emulsifying properties of each peptide were assessed by adding 0.1% (m / v) of each peptide to a mixture containing 15% edible oil and water. The pH of the sample was adjusted to 7. To promote emulsification, the sample was sonicated for 20 seconds. The sample was examined immediately after sonication and again after 7 days. If the peptide exhibited emulsifying properties, the assessment result was classified as "yes"; if the peptide did not exhibit emulsifying properties, the assessment result was classified as "no". The results are listed in Table 3.

[0651] Table 3. Emulsifying properties of peptides in this disclosure

[0652] SEQ ID NO: Emulsifying properties control group no 2 no 3 no 4 yes 5 yes 6 no 44 no 45 no 46 no 47 no

[0653] Antibacterial agents – pharmaceutical or cosmetic. The antibacterial susceptibility testing of the peptides was performed according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST). The test was prepared in lysogenic broth. Pre-cultures of *Pseudomonas aeruginosa* and *Staphylococcus aureus* were prepared and adjusted to a final count of 1 × 10⁻⁶ using previously prepared calibration curves. 6 CFU / mL. Minimum inhibition concentration (MIC) assays were prepared in 96-well plates, ranging from 0.78 to 400 μM. If the peptide exhibited antibacterial activity against both bacteria, the assessment result was classified as "Yes"; if the peptide showed no antibacterial activity, the assessment result was classified as "No". The results are listed in Table 4.

[0654] Table 4. Antibacterial properties of the peptides in this disclosure

[0655] SEQ ID NO: Antibacterial properties control group no 2 no 3 no 4 no 5 no 6 no 44 no 45 yes 46 yes 47 no

[0656] Fiber Integrity – The fiber integrity of hair samples containing the peptide (2 ± 0.1 mg) was analyzed using differential scanning calorimetry (DSC) on a Netzsch DSC 3500 Sirius. The thermal effects of the selected peptide on hair were studied using a power-compensated DSC apparatus and an aluminum disc (maximum pressure: 1 bar) at temperatures ranging from 25 °C to 150 °C (heating rate: 10 °C / min), followed by a 5-minute isothermal phase at 150 °C, and then further heating to 300 °C (heating rate: 10 °C / min). Data are presented as mean ± standard error of replicates and as a percentage of hair integrity compared to water-treated hair (enthalpy = 16.4 ± 0.6). Results are listed in Table 5.

[0657] Table 5. Fiber integrity of hair samples using the peptides of this disclosure

[0658]

[0659]

[0660] Young's modulus. The mechanical properties (Young's modulus) of hair fibers were evaluated using a texture analyzer (TA.XTplusC, Stable Micro Systems) to investigate the effects of treatment with selected peptides. Differences in mechanical properties were measured for 30 individual hair fibers with small diameter variability. These fibers were individually fixed to a tension jig (a paper template with a fixed gauge length of 55 mm). Test samples were kept in a desiccator prior to testing to avoid excessive moisture. Tensile measurements were performed at a rate of 0.03 mm / s. This parameter was assessed as the percentage of hair recovery from peptide-treated hair compared to damaged bleached hair (untreated). Data are presented as mean ± standard error. The results are listed in Table 6.

[0661] Table 6. Mechanical properties (Young's modulus) of hair fibers using the peptides of this disclosure

[0662] SEQ ID NO: Hair recovery status control group 0 2 +18±4％ 3 +18±5％ 4 -1±3％ 5 +23±6％ 6 +13±5％ 44 +4±4％ 45 +13±5％ 46 -5±4％ 47 -11±4％

[0663] Hydrophobicity. The forward contact angle was measured using a Force Tensiometer K100C (KRUSS). The contact angle in water was calculated using force and an estimated fiber diameter. During the measurement, 10 individual hair fibers were immersed to a certain depth at a rate of 5 mm / min. The results are shown in Table 7.

[0664] Table 7. Hydrophobicity of the peptides disclosed herein

[0665] SEQ ID NO: hydrophobic control group 32.8±16.9 2 30.7±16.6 3 39.6±10.0 4 37.9±9.4 5 28.2±10.7 6 26.0±23.7 44 21.2±23.6 45 34.2±16.8 46 43.5±6.7 47 38.8±12.2

[0666] Peptides were labeled with fluorescein isothiocyanate (FITC, a fluorescent probe) and observed under a fluorescence microscope. Figure 3 To observe the penetration / localization of peptides in hair fibers.

[0667] Computational simulations of the interaction between the fusion protein and the model keratin. The free energy of the binding between the fusion peptide and the model keratin was modeled. The calculated free energies and the number of hydrogen bonds are listed in Table 8. [[More experimental details here?]]

[0668] Table 8. Affinity ΔG (kcal / mol) and hydrogen bonding (HB) of the peptides disclosed herein

[0669]

[0670]

[0671] A more negative ΔG indicates a better interaction between the peptide and the keratin model. The calculated number of hydrogen atoms between the peptide and the keratin model is also a direct measure of their interaction. These parameters illustrate how the fusion peptide interacts with keratin.

[0672] Methods for sequence alignment for comparison are well known in the art, including GAP, BESTFIT, BLAST, FASTA, and TFASTA. GAP employs the algorithm of Needleman and Wunsch ((1970) J Mol Biol, 48:443-453) to find a global (overall sequence) alignment of two sequences that maximizes the number of matches and minimizes the number of gaps. The BLAST algorithm (Altschul et al. (1990) J Mol Biol 215:403-10) calculates the sequence similarity rate and performs a statistical analysis of the similarity between two sequences. Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information (NCBI). Global similarity and the percentage of similarity can also be determined using one of the methods available in the MatGAT software package (Campanella et al., BMC Bioinformatics, 2003 Jul 10; 4:29. MatGAT: an application that generates similarity / consistency matrices using protein or DNA sequences). Minor manual edits can be made to optimize alignments between conserved motifs, which will be apparent to those skilled in the art. The sequence consistency percentages expressed as a percentage in this subject matter were determined using BLAST with default parameters for the entire amino acid sequence.

[0673] When an element or feature is used in the description of a claim in the singular form, the plural form is also included, and vice versa, unless specifically excluded. For example, the terms "a peptide" or "the peptide" also include the plural forms "peptide" or "these peptides," and vice versa. In a claim, articles such as "an," "a," and "the" may indicate one or more, unless the context indicates otherwise or expressly states otherwise. If a claim or description includes "or" among one or more members of a group, then the claim or description is deemed satisfied when one, more than one, or all members of that group are present, used in, or associated with a given product or process, unless the context indicates otherwise or expressly states otherwise. This disclosure includes embodiments in which exactly one member of the group is present, used in, or associated with a given product or process. This disclosure also includes embodiments in which more than one or all members of the group are present, used in, or associated with a given product or process.

[0674] Furthermore, it should be understood that this disclosure covers all variations, combinations, and arrangements in which one or more limitations, elements, clauses, descriptive terms, etc., derived from one or more claims or related parts of the specification are incorporated into another claim. For example, any claim dependent on another claim may be modified to include one or more limitations found in any other claim dependent on the same basic claim.

[0675] Furthermore, when a claim describes a composition, it should be understood that it includes methods of using the composition for any purpose disclosed herein, as well as methods of preparing the composition according to any preparation method disclosed herein or other methods known in the art, unless otherwise stated, or unless contradictions or inconsistencies would be obvious to a person skilled in the art.

[0676] When a range is given, the endpoints are included. Furthermore, it should be understood that, unless otherwise stated, or obvious from the context and / or to those skilled in the art, in different embodiments of this disclosure, values ​​expressed as ranges can take any particular value within the range, accurate to the tenths of the lower limit unit of the range, unless explicitly specified by the context. It should also be understood that, unless otherwise stated, or obvious from the context and / or to those skilled in the art, values ​​expressed as ranges can take any subrange within a given range, wherein the endpoints of the subrange are represented with the same precision as the tenths of the lower limit unit of the range.

[0677] This disclosure should not be construed in any way as limiting it to the described embodiments, and those skilled in the art will foresee many possibilities for modification thereto.

[0678] The implementation methods described above are composable.

[0679] The following claims further illustrate specific embodiments of this disclosure.

Claims

1. A fusion protein / peptide comprising: (a) Keratin or keratin-related peptide modules; and (b) at least one polypeptide module selected from the following: (i) elastin or elastin-like polypeptide (ELP) module, (ii) silk protein or silk protein-like polypeptide (SLP) module, (iii) collagen or collagen-like polypeptide (CLP) module, (iv) arthropod elastin or arthropod elastin-like polypeptide (RLP) module, (v) extensin or extensin-like polypeptide (ALP) module, or (vi) a combination of two or more modules selected from (i) to (v).

2. The fusion protein / peptide according to claim 1, wherein the polypeptide module comprises elastin or an elastin-like polypeptide module.

3. The fusion protein / peptide according to any one of the preceding claims, wherein the elastin or elastin-like polypeptide (ELP) module has a length of 3 to 30 amino acids; preferably 5 to 20 amino acids, more preferably 15 to 30 amino acids.

4. The fusion protein / peptide according to any one of claims 1 or 2, comprising a connector module as a spacer-like component.

5. The fusion protein / peptide according to the preceding claims, wherein the linker module has a repeating sequence of 2 to 8 amino acids in length.

6. The fusion protein / peptide according to any one of claims 4-5, wherein the linker module has a repeating sequence of 2 to 40 amino acids in length, preferably 8 to 30 amino acids in length.

7. The fusion protein / peptide according to any one of claims 4-6, wherein the linker module comprises a hydrophilic module having a length of 1 to 75 amino acids.

8. The fusion protein / peptide according to the preceding claims, wherein the hydrophilic module comprises a length of 10 to 50 amino acids.

9. The fusion protein / peptide according to any one of the preceding claims, wherein the linker module is in an α-helical conformation.

10. The fusion protein / peptide according to any one of the preceding claims, wherein the length of the linker module is 1 to 3 amino acids repeated 0 to 25 times, preferably 1 to 20 times.

11. The fusion protein / peptide according to the preceding claim, wherein the linker module comprises one or two amino acids repeated 10 to 25 times.

12. The fusion protein / peptide according to any one of the preceding claims, wherein the keratin or keratin-related peptide module comprises SEQ ID NO:8:GGVCGPSPPCITT, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:

8.

13. The fusion protein / peptide according to any one of the preceding claims, wherein the keratin or keratin-related peptide module comprises SEQ ID NO:8:GGVCGPSPPCITT, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:8:GGVCGPSPPCITT.

14. The fusion protein / peptide according to any one of the preceding claims, wherein the keratin or keratin-related peptide module comprises SEQ ID NO:9:GGVCGPSPPC, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:

9.

15. The fusion protein / peptide according to any one of the preceding claims, wherein the keratin or keratin-related peptide module comprises SEQ ID NO:10:CGPSPPCITT, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:

10.

16. The fusion protein / peptide according to any one of the preceding claims, wherein the keratin or keratin-related peptide module comprises SEQ ID NO:68:CLPCLPAASC, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:

68.

17. The fusion protein / peptide according to any one of the preceding claims, wherein the keratin or keratin-related peptide module comprises SEQ ID NO:73:CVSSCCKPQCC, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:

73.

18. The fusion protein / peptide according to any one of the preceding claims, wherein the keratin or keratin-related peptide module comprises SEQ ID NO:74:PITCRRTCYH, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:

74.

19. The fusion protein / peptide according to any one of the preceding claims, wherein the keratin or keratin-related peptide module comprises SEQ ID NO:75:DCKLPCNPCA, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:

75.

20. The fusion protein / peptide according to any one of the preceding claims, wherein the keratin or keratin-related peptide module comprises SEQ ID NO:76:CLPCLPAASC, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:

76.

21. The fusion protein / peptide according to any one of the preceding claims, wherein the keratin or keratin-related peptide module comprises SEQ ID NO:77:CEPAICEPSC, or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:

77.

22. The fusion protein / peptide according to any one of the preceding claims, wherein the fusion protein / peptide comprises SEQ ID NO. 1: GGVCGPSPPCITTVPGVGVPGVGVPGLGVPGVGVPGVGR, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

1.

23. The fusion protein / peptide according to any one of the preceding claims, wherein the fusion protein / peptide comprises SEQ ID NO. 2: GGVCGPSPPCITTVPGVGVPGVGVPGVGVPGVGVPGVGVPGVG, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

2.

24. The fusion protein / peptide according to any one of the preceding claims, wherein the fusion protein / peptide comprises SEQ ID NO: 7: GGVCGPSPPCITTVPGVGVPGVGVPGLGVPGVGVPGVG, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

7.

25. The fusion protein / peptide according to any one of the preceding claims, wherein the polypeptide module comprises a silk protein or a silk protein-like polypeptide (SLP) module.

26. The fusion protein / peptide according to any one of the preceding claims, wherein the fusion protein / peptide comprises SEQ ID NO:3: GGVCGPSPPCITTGAGAGSGAGAGSGAGAGSGAGAGSGAGAGS, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

3.

27. The fusion protein / peptide according to any one of the preceding claims, wherein the polypeptide module comprises a collagen or collagen-like polypeptide (CLP) module.

28. The fusion protein / peptide according to any one of the preceding claims, wherein the fusion protein comprises SEQ ID NO:5:GGVCGPSPPCITTGPTGPTGPAGPRGLQGLQGLQGERGEQGPT, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

5.

29. The fusion protein / peptide according to any one of the preceding claims, wherein the polypeptide component comprises arthropod elastin or arthropod elastin-like polypeptide (RLP) component.

30. The fusion protein / peptide according to any one of the preceding claims, wherein the fusion protein comprises SEQ ID NO:4:GGVCGPSPPCITTGGRPSDSYGAPGGGN, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

4.

31. The fusion protein / peptide according to any one of the preceding claims, wherein the polypeptide component comprises an extensin or an extensin-like polypeptide (ALP) component.

32. The fusion protein / peptide according to any one of the preceding claims, wherein the fusion protein comprises SEQ ID NO. 6: GGVCGPSPPCITTGGFGGMGGGSGGFGGMGGGSGGFGGMGGGS, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

6.

33. The fusion protein / peptide according to any one of the preceding claims, wherein: The keratin or keratin-related peptide component has a length of 6 to 16 amino acids; and / or Silk protein or "SLP" has a repeating sequence of 6 to 8 amino acids in length, and / or silk protein or SLP has a length of 10 to 30 amino acids, preferably 15 to 20 amino acids in length; and / or Collagen or "CLP" has a repeating sequence of 3 amino acids in length, and / or collagen or CLP has a length of 10 to 32 amino acids, preferably 15 to 30 amino acids in length; and / or Elastin or "ELP" has a repeating sequence of 5 amino acids in length, and / or elastin or ELP has a length of 3 to 30 amino acids; preferably 8 to 32 amino acids, more preferably 15 to 30 amino acids; and / or Arthropod elastin or "RLP" has a repeating sequence of 11 to 15 amino acids in length, and / or arthropod elastin or RLP has a length of 10 to 30 amino acids, preferably 15 to 20 amino acids in length; and / or The extensin or "ALP" has a repeating sequence of 4 to 10 amino acids in length, and / or the extensin or ALP has a length of 15 to 32, more preferably 20 to 30.

34. The fusion protein / peptide according to any one of the preceding claims, wherein the average molecular weight of the fusion protein is about 1 kDa to about 144 kDa, about 10 kDa to about 144 kDa, or about 35 kDa to about 130 kDa.

35. The fusion protein / peptide according to any one of the preceding claims, wherein the average molecular weight of the fusion protein is in the range of about 1 kDa to about 5 kDa, about 5 kDa to about 10 kDa, about 6 kDa to about 17 kDa, about 10 kDa to about 15 kDa, about 14 kDa to about 30 kDa, about 15 kDa to about 20 kDa, about 17 kDa to about 39 kDa, about 20 kDa to about 25 kDa, about 25 kDa to about 30 kDa, about 30 kDa to about 35 kDa, about 35 kDa to about 40 kDa, about 39 kDa to about 54 kDa, about 39 kDa to about 80 kDa, about 40 kDa to about 45 kDa, about 45 kDa to about 50 kDa, about 50 kDa to about 55 kDa, about 55 kDa to about 60 kDa, about 60 kDa to about 100 kDa, and about 80 kDa to about 144 kDa.

36. The fusion protein / peptide according to any one of the preceding claims, wherein the fusion protein / peptide comprises a sequence having at least 80% sequence identity with any one of SEQ ID NOS:1-7, SEQ ID NOS:43-47 or SEQ ID NOS:200-552.

37. The fusion protein / peptide according to any one of the preceding claims, wherein the fusion protein / peptide comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NOS:1-7, SEQ ID NOS:43-47, or SEQ ID NOS:200-552.

38. The fusion protein / peptide according to any one of the preceding claims, wherein the fusion protein / peptide comprises a sequence identical to any one of SEQ ID NOS:1-7, SEQ ID NOS:43-47 or SEQ ID NOS:200-552.

39. The fusion protein / peptide or composition according to any one of the preceding claims, wherein the beauty product is suitable for hair care or hair treatment.

40. The fusion protein / peptide according to claim 1, wherein the fusion protein / peptide is represented by formulas (I) to (IV): [B n1 -A m -B n -C o -B n2 ] p Equation (Ⅰ); or [B n1 -C o -B n -A m -B n2 ] p Formula (II); or [B n1 -B n -C o -A m -B n2 ] p Formula (Ⅲ); or [B n1 -C o -A m -B n -B n2 ] p Formula (Ⅳ) in: "A" contains a peptide sequence containing keratin or a keratin-related peptide module, where "m" is an integer from 1 to approximately 10; "B" contains a peptide sequence of 1 to 3 amino acids, where each "n", "n1" and "n2" is an integer from 0 to 25 independently; "C" contains structure D q -E r -F s -G t -H u The peptide sequence; Where "D" contains the silk protein or silk protein-like polypeptide (SLP) module; The "E" in the designation includes a collagen or collagen-like polypeptide (CLP) module. The "F" in the designation contains the elastin or elastin-like polypeptide (ELP) module. The "G" in the designation contains arthropod elastin or arthropod elastin-like polypeptide (RLP) modules; Where "H" contains the extensin or extensin-like polypeptide (ALP) module; Where "q" is an integer from 0 to 100, "r" is an integer from 0 to 200, "s" is an integer from 0 to 220, "t" is an integer from 0 to 80, and "u" is an integer from 0 to 70. Where "o" is an integer from 0 to 30; and "p" is an integer from approximately 1 to approximately 40; At least one of "n", "n1", "n2" or "o" is not 0. The fusion protein / peptide according to claim 33, wherein each "n", "n1" and "n2" is independently an integer from 0 to about 25.

41. The fusion protein / peptide according to claim 33 or 34, wherein "o" is an integer from about 1 to about 30.

42. The fusion protein / peptide according to any one of claims 33-35, wherein each "n", "n1" and "n2" is an integer from 0 to 25, and "o" is an integer from 1 to 30.

43. The fusion protein / peptide according to any one of claims 33-36, wherein "p" is an integer from 3 to 37, 8 to 15, or 10 to 12.

44. The fusion protein / peptide according to any one of claims 33-37, wherein "B" comprises a peptide sequence containing 1 to 40 amino acids, 2 to 25 amino acids, or 4 to 25 amino acids. The fusion protein / peptide according to any one of claims 33-38, wherein "B" comprises having an X n Zc and / or Z c X n The peptide sequence; where "X" n "It is proline, leucine, isoleucine, glutamic acid, aspartic acid, glutamine, lysine, alanine, arginine, or threonine; and among them, "Z" c "It contains glycine, alanine, lysine, proline, methionine, glutamine, arginine, threonine, valine, glutamic acid, aspartic acid, or asparagine." The fusion protein / peptide according to any one of claims 32-38, wherein the length of the at least one keratin or keratin-associated peptide module is 6 to 16 amino acids; "D" contains the following polypeptide sequences: SEQ ID NO:24: GAGAGS, SEQ ID NO:25: GAGSGA, SEQ ID NO:26: GAGAGY, SEQ ID NO:27: GAGYGA, SEQ ID NO:28: GAGAGGA, SEQ ID NO:29: GAGAGV, SEQ ID NO:30: GAGVGA or SEQ ID NO:31: GAGAGVGY, or a combination of two or more of them; "E" contains the polypeptide sequence GX1Z. Where "X1" is proline, leucine, isoleucine, glutamic acid, aspartic acid, glutamine, lysine, alanine, arginine, or threonine; and Where Z is glycine, alanine, lysine, proline, methionine, glutamine, arginine, threonine, valine, glutamic acid, aspartic acid, or asparagine; "F" contains a polypeptide sequence containing SEQ ID NO:21: VPGX2G and / or SEQ ID NO:13: VPAVG. Where "X2" is arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, glycine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, or tryptophan; "G" contains the following polypeptide sequences: SEQ ID NO:34: GGRPSDSYGAPGGGN and / or SEQ ID NO:35: GAPAQTPSSQY; "H" contains a polypeptide sequence containing the following: SEQ ID NO:36: GGFGGMGGGS, SEQ ID NO:37: MGGG, SEQ ID NO:38: FGGMG, SEQ ID NO:39: FGGMGGG, SEQ ID NO:40: GGFGGMGGG or SEQ ID NO:41: FGGMGGGNAG, or a combination of two or more of them.

45. The fusion protein / peptide according to any one of claims 32-39, wherein, "A" contains peptide sequence X 3 k CX 3 5CX 3 k ,in: X 3 "The following are the amino acids: glycine, alanine, valine, isoleucine, leucine, phenylalanine, proline, serine, threonine, asparagine, glutamine, cysteine, methionine, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, histidine, lysine, or arginine; and..." "k" is an integer from 0 to 3.

46. ​​The fusion protein / peptide according to any one of claims 32-40, wherein, "A" includes SEQ ID NO:8: GGVCGPSPPCITT, SEQ ID NO:9: GGVCGPSPPC, SEQ ID NO:10: CGPSPPCITT, CGPSPPC (SEQ ID NO:66) or GGVCGPSPPC (SEQ ID NO:67).

47. The fusion protein / peptide according to any one of claims 32-41, wherein the average molecular weight of the fusion protein is in the range of about 1 kDa to about 144 kDa, about 10 kDa to about 144 kDa, or about 35 kDa to about 130 kDa.

48. The fusion protein / peptide according to any one of claims 32-42, wherein the average molecular weight of the fusion protein is in the range of about 1 kDa to about 5 kDa, about 5 kDa to about 10 kDa, about 6 kDa to about 17 kDa, about 10 kDa to about 15 kDa, about 14 kDa to about 30 kDa, about 15 kDa to about 20 kDa, about 17 kDa to about 39 kDa, about 20 kDa to about 25 kDa, about 25 kDa to about 30 kDa, about 30 kDa to about 35 kDa, about 35 kDa to about 40 kDa, about 39 kDa to about 54 kDa, about 39 kDa to about 80 kDa, about 40 kDa to about 45 kDa, about 45 kDa to about 50 kDa, about 50 kDa to about 55 kDa, about 55 kDa to about 60 kDa, about 60 kDa to about 100 kDa, and about 80 kDa to about 144 kDa.

49. The fusion protein / peptide according to any one of claims 32-43, wherein the silk protein or silk protein-like peptide (SLP) module forms a structure rich in β-sheets.

50. The fusion protein / peptide according to any one of claims 32-44, wherein the elastin or elastin-like peptide (ELP) module forms a structure rich in β-turns.

51. The fusion protein / peptide according to any one of claims 32-45, wherein the elastin-like peptide (ELP) module is temperature-responsive.

52. A composition comprising the fusion protein / peptide according to any one of claims 1-43, for improving the elasticity, resilience, hydration, integrity, hydrophobicity, texture, antibacterial properties, emulsifying properties, mechanical properties, thermal protection or ultraviolet protection of hair, nails or skin, or a combination of two or more thereof.

53. The composition according to the preceding claim, wherein the composition improves the elasticity, resilience, hydration, integrity, texture, antibacterial protection, or UV protection of the skin, or a combination of two or more thereof.

54. The composition according to any one of claims 44-45, wherein the content of the fusion protein / peptide is 0.0001%-20% (w / w); preferably 0.001%-5% (w / w); or 0.1% to about 1% (w / w).

55. The composition according to any one of claims 43-46, wherein the composition improves the mechanical properties, color, texture, shape, hydration, integrity, resilience, hydrophobicity, or thermal protection of hair, or a combination of two or more thereof.

56. The composition according to any one of claims 44-47, wherein the composition improves the mechanical properties, integrity, antimicrobial properties, or ultraviolet protection of the nail, or a combination of two or more thereof.

57. The composition according to any one of claims 44-48, wherein at least one excipient suitable for use with the skin is selected from the list of surfactants, emulsifiers, preservatives, thickeners, organic polymers, humectants, silicones, oils, fragrances, vitamins, buffers, antimicrobial agents, antibacterial agents, disinfectants, chelating agents, or mixtures thereof.

58. The composition according to any one of claims 44-49, comprising at least one excipient suitable for use with the skin.

59. The composition according to the preceding claim, wherein at least one excipient suitable for use with the skin is selected from the following list: ethanol, benzyl alcohol, diol molecules, urea, ammonium thioacetate, anisole, tris(hydroxymethyl)aminomethane, phosphate buffer, sodium chloride, citrate buffer or ethanol, or a combination of two or more of these.

60. The composition according to any one of the preceding claims, wherein the diol molecule is selected from: ethylene glycol, propylene glycol, butanediol, butenediol, or mixtures thereof.

61. The composition according to any one of claims 44-52, comprising: Ethanol of 0-30% (w / v), preferably 10-20% (w / v); 0-3% (w / v) benzyl alcohol, preferably 0.5-2% (w / v); 0-20% (w / v) of diol molecules, preferably 10-15% (w / v).

62. The composition according to any one of claims 44-53 further comprises a propellant, a fragrance, an oil, or a mixture thereof.

63. The composition according to any one of claims 44-54, for use in hair treatment.

64. The fusion protein / peptide or composition according to any one of the preceding claims, used as a cosmetic product; that is, used as a cosmetic product for hair, nails or skin.

65. The fusion protein / peptide or composition according to any one of claims 44-56, wherein the cosmetic product is suitable for hair care or hair treatment.

66. Shampoos, lotions, serums, creams, conditioners, foams, elixirs, oils, aerosols, or masks comprising the composition / fusion protein / peptide of any one of the preceding claims.

67. Use of a composition comprising any one of the fusion protein / peptides according to claims 1-43, as an enhancer of one or more properties of hair, nails or skin, or as an agent for enhancing the elasticity, hydration, resilience, texture, antimicrobial properties and / or UV protection of hair, nails and skin.

68. A method for obtaining a fusion protein / peptide according to any one of claims 1-43, comprising the following steps: Inoculate the liquid culture medium with microorganisms containing the gene expressing the desired fusion protein; Microorganisms are allowed to multiply within a certain culture period and are induced to express their contents under the action of a promoter; Proteins are purified from endogenous contaminants to extract the desired fusion proteins / peptides from the obtained microorganisms.

69. The method according to the preceding claim, wherein the microorganism is Escherichia coli.

70. The method according to any one of claims 60-61, wherein the fusion protein / peptide is purified from endogenous contaminants.

71. A method for obtaining a fusion protein / peptide according to any one of claims 1-51 by solid-phase synthesis, comprising the following steps: Initiate solid-phase synthesis to combine a keratin or keratin-associated peptide module with at least one polypeptide module selected from: (i) an elastin or elastin-like polypeptide (ELP) module, (ii) a silk protein or silk protein-like polypeptide (SLP) module, (iii) a collagen or collagen-like polypeptide (CLP) module, (iv) an arthropod elastin or arthropod elastin-like polypeptide (RLP) module, (v) an extensin or extensin-like polypeptide (ALP) module, or (vi) a combination of two or more modules selected from (i) to (v); Multiple solid-phase reactions are carried out to obtain fusion proteins / peptides.

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