A recombinant human-derived k25 keratin polypeptide, and a preparation method and use thereof

Abstract

The application provides a recombinant human K25 keratin polypeptide and a preparation method and application thereof, and relates to the technical field of genetic engineering. The recombinant human K25 keratin polypeptide comprises or consists of an amino acid sequence shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 6 or SEQ ID NO. 8. The recombinant human K25 keratin polypeptide is derived from human K25 keratin, retains a key fragment with biological activity, has cell adhesion activity, can promote cell adhesion and proliferation, and is suitable for applications such as tissue repair, cell culture and skin care. The selected length interval is helpful to improve expression efficiency and stability while maintaining activity, facilitates large-scale production and practical application, and realizes the unity of functionality and operability.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, and more specifically, to a recombinant human K25 keratin polypeptide, its preparation method, and its uses. Background Technology

[0002] Keratins are structural proteins widely found in mammalian epithelial tissues, primarily constituting keratinized tissues such as skin, hair, and nails. They are core components for maintaining the stability of the cytoskeleton and tissue structure. Based on their isoelectric points, keratins can be divided into two main categories: Type I (acidic) and Type II (basic to neutral). These two types of keratins co-form intermediate filaments in cells as heterodimers, imparting mechanical strength and elasticity to the cells. In the human genome, keratin genes are distributed in the regions of chromosome 17q21.2 (Type I) and 12q13.13 (Type II), with over 50 known keratin genes widely involved in the morphological maintenance and differentiation processes of various epithelial tissues.

[0003] In hair tissue, different keratin subtypes exhibit specific spatiotemporal expression patterns in various structural regions of the hair follicle. The hair follicle structure, from the outside in, includes multiple regions such as the outer root sheath, inner root sheath, and hair shaft. The inner root sheath, composed of the Henley layer, Huxley layer, and stratum corneum, tightly surrounds the hair shaft and is a crucial structural support unit during hair formation. The expression of some keratin subtypes is mainly concentrated in the inner root sheath region, regulating co-conformational changes with other proteins during hair shaft keratinization. Previous studies have identified several keratin proteins specifically expressed in the inner root sheath, including components expressed in pairs of types I and II, which exhibit a layered and zoned tissue distribution within the hair follicle structure.

[0004] At the molecular level, mutations or altered expression of some inner root sheath keratins have been observed to be associated with abnormal hair morphology, such as curliness, fragility, or sparseness. However, current research largely focuses on elucidating genetic mechanisms or conducting histopathological observations. While these studies provide fundamental data for understanding hair follicle development, systematic protein-level research is still lacking, particularly in the screening, construction, and functional validation of expression product domains; the technical pathways remain immature. In terms of functional applications, existing research has not fully explored the potential value of various hair-related keratins in tissue engineering, cell scaffolds, and skin barrier replacement.

[0005] Furthermore, existing protein engineering and functional protein expression platforms pose challenges in maintaining the stability, solubility, and activity of large structural proteins. Recombinant expression using eukaryotic keratin as a template often faces a series of problems, including difficulties in domain selection, low matching of expression systems, and a lack of standard models for functional validation. Especially in cases where standard expression systems or functional validation mechanisms have not yet been established for certain keratin subtypes, current technologies still need further improvement in protein construction, purification, and activity assessment. These technological gaps restrict the advancement of keratin molecules from basic research to product development.

[0006] In view of this, the present invention is hereby proposed. Summary of the Invention

[0007] The purpose of this invention is to provide a recombinant human K25 keratin polypeptide, its preparation method, and its uses. The recombinant human K25 keratin polypeptide is derived from a continuous fragment of human K25 keratin, possesses cell adhesion activity, and retains its biological functions while having a small molecular weight, facilitating expression, purification, and application. The defined length range enhances the flexibility and practicality of the polypeptide, making it suitable for applications such as cell adhesion, tissue repair, and skin care.

[0008] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0009] In a first aspect, the present invention provides a recombinant human K25 keratin polypeptide, wherein the polypeptide comprises or is composed of the amino acid sequence shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.6 or SEQ ID NO.8.

[0010] In a second aspect, the present invention provides a polynucleotide that encodes a recombinant human K25 keratin polypeptide as described in the foregoing embodiments.

[0011] In an optional embodiment, the nucleotide sequence of the polynucleotide includes any one of the nucleotide sequences shown in SEQ ID NO. 5, SEQ ID NO. 7 and SEQ ID NO. 9.

[0012] Thirdly, the present invention provides an expression vector comprising the polynucleotides described in the foregoing embodiments.

[0013] Fourthly, the present invention provides a host cell comprising the expression vector as described in the foregoing embodiments, or expressing the recombinant human K25 keratin polypeptide as described in the foregoing embodiments;

[0014] The host cell is either Escherichia coli or yeast.

[0015] Fifthly, the present invention provides a method for preparing recombinant human K25 keratin polypeptide as described in the foregoing embodiments, comprising:

[0016] The host cells were cultured in a culture medium; and the recombinant human K25 keratin polypeptide was isolated based on the host cells.

[0017] In a sixth aspect, the present invention provides a composition comprising the recombinant human K25 keratin polypeptide as described in the foregoing embodiments.

[0018] In a seventh aspect, the present invention provides an article comprising the recombinant human K25 keratin polypeptide as described in the foregoing embodiments, or the composition as described in the foregoing embodiments;

[0019] The product in question is a cosmetic.

[0020] Eighthly, the present invention provides the use of the recombinant human K25 keratin polypeptide as described in the foregoing embodiments in the preparation of cosmetics.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] This application provides a recombinant human K25 keratin polypeptide, its preparation method, and its uses. The recombinant human K25 keratin polypeptide is derived from SEQ ID NO.10, which corresponds to the full-length amino acid sequence of human K25 keratin. K25 is a type I keratin specifically expressed in the root sheath of hair follicles and plays an important role in maintaining hair shaft structure and hair follicle stability.

[0023] By constructing a polypeptide by extracting 36–91 consecutive amino acids from SEQ ID NO. 10, some helical structural segments of the natural protein can be retained. These structures are essential for the formation of intermediate filaments in keratin and help maintain its bioactive conformation. This source limitation helps ensure that the constructed recombinant polypeptide has intrinsic sequence and structural consistency and biological relevance to the original keratin, improving its functional compatibility in the tissue cellular environment.

[0024] Compared to expressing the full-length K25 protein (SEQ ID NO.10, 453 amino acids), the restricted peptide length of 36–91 residues is shorter, facilitating efficient expression and subsequent purification in microorganisms (such as E. coli or yeast). This shorter length reduces the expression burden, decreases the risk of protein folding errors, and helps obtain functional fragments with better water solubility and higher stability. Simultaneously, the truncated region possesses cell adhesion activity; therefore, retaining this sequence segment allows for the preservation of function while reducing sequence length, achieving synergistic optimization of protein function and expression feasibility.

[0025] A peptide derived from a specific fragment of SEQ ID NO. 10 exhibited good proliferative and adhesion-promoting abilities in L929 fibroblasts. This functional property allows the peptide to be used as an active ingredient in various biomedical applications such as tissue repair and cell culture medium additives. Therefore, this functional limitation means that the claim is not merely a structural limitation of the sequence, but a technology combined with function.

[0026] By defining the contiguous fragments of 36 to 91 amino acid residues in length within SEQ ID NO. 10, multiple potentially bioactive sequence regions can be covered, allowing for flexibility in the construction and application of functional fragments of different lengths. This interval-based length setting helps to preserve bioactivity while considering peptide expression efficiency and structural stability, providing a foundation for diverse functional exploration and application development. This setting also facilitates the selection of appropriate fragment lengths for different application scenarios to achieve specific biological effects or processing performance requirements. Attached Figure Description

[0027] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0028] Figure 1 This is a representative diagram of plasmid construction using the vector pET32a-K25A3 as an example in Example 1 of this application;

[0029] Figure 2 The image shows the protein electrophoresis diagrams of the three recombinant keratin peptides expressed in Escherichia coli in Example 2 of this application. The molecular weights of the recombinant proteins K25A3, K25B2, and K25C2, as determined by electrophoresis, are approximately 29 kDa, 38 kDa, 30 kDa, and 49 kDa, respectively.

[0030] Figure 3 This is an electrophoresis image of the K25B2 protein obtained after expression and purification in Example 3 of this application; the molecular weight of the K25B2 protein, as detected by electrophoresis, is approximately 38 kDa.

[0031] Figure 4 The results show the cell proliferation activity of the recombinant keratin and full-length K25 expressed protein in Example 4 of this application. Detailed Implementation

[0032] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0033] In this application embodiment, a recombinant human K25 keratin polypeptide is provided, which has cell adhesion activity and contains at least x consecutive amino acid residues as in SEQ ID NO.10; wherein, 450 > x ≥ 36.

[0034] K25, a type I keratin specific to the inner root sheath of human hair follicles, is expressed in a region that begins in the globular area of ​​the inner root sheath and extends to the three-layered terminal differentiation region consisting of the Henlein layer, Huxley layer, and stratum corneum. The inner root sheath tightly surrounds the hair shaft, playing a crucial supporting and constraining role in normal hair growth, morphology, and texture stability. Existing research indicates that mutations in certain keratin genes related to the inner root sheath can significantly impact hair condition. For example, mutations in the K25 gene may interfere with its heterodimer formation with type II keratin, thereby affecting the axial structure of the hair shaft and leading to phenotypic abnormalities such as hair curvature, fragility, and reduced density. Some studies also consider K25 gene mutations as a significant cause of human hypotrichosis (sparse hair). These findings suggest that K25 keratin plays a vital biological role in maintaining hair density and structural integrity. However, existing research mainly focuses on the molecular function and mutation mechanism of K25, and there is a lack of systematic exploration and application research on the recombinant K25 protein in hair follicle repair or dry hair improvement.

[0035] In this embodiment, the human type 25 keratin K25 sequence was selected for screening and optimization. The sequence of the human type 25 keratin is the NCBI reference sequence: CAD91904.1 (SEQ ID NO.10), see https: / / www.ncbi.nlm.nih.gov / protein / 31074637.

[0036] The amino acid sequence of K25 is as follows (SEQ ID NO.10):

[0037] MSLRLSSASRRSCPRPTTGSLRLYGGGTSFGTGNSCGISGIGGSGFSSAFGGSSSGGNTGGGNPCAGFTVNERGLLSGN EKVTMQNLNDRLASYLDSVHALEEANADLEQKIKGW YEKFGPGSCRGLDHDYSRYFPI IDDLKNQI IASTTSNANAVLQIDNARLTADDFRLKYENELALHQSVEADVNGLRRVLDEITLCRTDLEIQYETLSEEMTYLKKN HKEEMQVL QCAAGGNVNVEMNAAPGVDLTVL LNNMRAEYEALAEQNRRDAEAWFNEKSASLQQQISEDVGATTSAR NELTEMKRTLQ TLEIELQSLLATKHSLECSLTETESNYCAQLAQIQAQIGALEEQLHQVRTETEGQKLEYEQLLDIKLHLEKEIETYCLLIGGDDGACKSGGYKSKDYGSGNVGSQVKDPAKAIVVKKVLEEVDQRSKILTTRLHSLEEKSQSN.

[0038] The bold underlined portion of the above sequence is the amino acid sequence selected in the embodiments of this application. Through extensive research, the applicant has found that the selected sequence exhibits high water solubility, high recombinant expression yield, simple purification process, and better hair follicle repair and hair regeneration effects than full-length K25 keratin, possessing a variety of excellent biomaterial properties. The polypeptide targeted in the embodiments of this application is not the full-length sequence of SEQ ID NO. 10.

[0039] The embodiments of this application are partly based on the following finding: peptides containing at least 36 consecutive amino acid residues as in SEQ ID NO. 10 can have better biomaterial properties than commercially available keratin, as demonstrated by the examples. Those skilled in the art can appropriately select the consecutive amino acid residues constituting the recombinant keratin. For example, the length of the consecutive amino acid residues can be 36, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 150, 200, 250, 300, 400, 410, 430, 440, 449, etc.

[0040] The recombinant human K25 keratin polypeptide provided in this embodiment is derived from the amino acid sequence of human keratin K25 (SEQ ID NO.10). This protein is a type I root sheath-specific keratin, expressed in the hair follicle structure from the bulbous region to the terminal differentiation point, and is one of the key components for maintaining the normal structure and growth of the hair shaft. Keratin K25 can form heterodimers with type II keratin to construct intermediate filament structures in cells, participating in the maintenance of cytoskeleton stability and tissue integrity.

[0041] As described above, in this embodiment, continuous amino acid residues with a length of 36 or more, or close to the full-length sequence, are selected from SEQ ID NO.10 to construct a recombinant polypeptide, with the aim of preserving the functionally active structural domains in the protein.

[0042] Experimental screening revealed that multiple fragments within this length range exhibited good expression and functional performance, particularly showing excellent effects in cell adhesion activity. For example, the enhanced adhesion and proliferation ability of L929 fibroblasts indicated that it has the activity of promoting cell adhesion and growth.

[0043] Compared to the full-length K25 protein, selecting a shorter functional fragment simplifies the expression and purification process and improves the protein's solubility and stability, making it suitable for recombinant production in common expression systems such as E. coli or yeast. This peptide can serve as a bioactive component with various applications, including cell scaffolds, skin repair, and hair care products.

[0044] In summary, this peptide, based on human K25 and combined with structural optimization and functional validation, exhibits good bioactivity and expression feasibility, and can meet the needs for functional protein fragments in tissue engineering and daily chemical applications.

[0045] In this application embodiment, a recombinant human K25 keratin polypeptide is provided, which has cell adhesion activity and includes:

[0046] The amino acid sequence shown in or composed of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.6 or SEQ ID NO.8.

[0047] Table 1. Sequence names and amino acid sequences of SEQ ID NO. 1~3

[0048]

[0049] The aforementioned polypeptide in this paper may be recombinant human keratin K25A3, which is a K25A sequence repeated three times, including 108 amino acids. The basic repeating unit is: EKVTMQNLNDRLASYLDSVHALEEANADLEQKIKGW (SEQ ID NO.1), which is a human keratin type 25 peptide.

[0050] The aforementioned recombinant human K25 keratin peptide is based on functional fragments in K25 keratin and uses repeating units to construct a modular structure.

[0051] The amino acid addition refers to the addition of an amino acid to the C-terminus or N-terminus of any one of the three keratin amino acid sequences (SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3), as long as the polypeptide has keratin characteristics and cell proliferation-promoting activity.

[0052] The amino acid substitution refers to the replacement of an amino acid residue at any position in any of the three keratin amino acid sequences (SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3) with other amino acid residues, as long as the polypeptide has keratin characteristics and cell proliferation-promoting activity.

[0053] The amino acid insertion refers to inserting an amino acid residue at an appropriate position in any of the three keratin amino acid sequences (SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3). The inserted amino acid residues may be all or partly adjacent to each other, or the inserted amino acids may not be adjacent to each other, as long as the polypeptide has keratin characteristics and cell proliferation-promoting activity.

[0054] The amino acid deletion refers to the deletion of one, two, or three or more amino acids from any of the three keratin amino acid sequences (SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3), provided that the polypeptide has keratin characteristics and cell proliferation-promoting activity.

[0055] In this invention, substitution can be a conserved amino acid substitution, meaning that compared to any of the three keratin amino acid sequences, three, more preferably two, or one amino acid is replaced by an amino acid with similar or related properties to form a peptide. These conserved variant peptides can be generated by amino acid substitution according to Table 1.

[0056] SEQ ID NO.1, NO.2, and NO.3 correspond to specific domains of the K25 protein that have been screened and exhibited good cell adhesion activity in in vitro experiments. For example, K25A (SEQ ID NO.1) is a 37-amino acid fragment, K25B (SEQ ID NO.2) is a 122-amino acid domain, and K25C (SEQ ID NO.3) is a 63-amino acid domain, all located within the helical region of K25, which are core functional regions constituting the intermediate filament structure of keratin. Experimental results show that peptides constructed using these sequences as basic units (such as K25A3 and K25B2) have good water solubility and expression yield, and exhibit significant adhesion-promoting and proliferation-promoting effects in L929 cells, demonstrating clear cell adhesion activity.

[0057] By using (A)n structural combinations, not only can the key functional regions of K25 keratin be preserved, but functional enhancement can also be achieved through unit repetition. For example, K25A3, composed of three repetitions of SEQ ID NO.1, exhibits stronger cell adhesion than a single fragment. This repetitive structure also helps optimize the spatial conformation of the peptide, enhancing its binding ability to cell surface receptors. By setting an adjustable range of n from 1 to 16, this structure offers greater construction freedom, facilitating optimization of length, activity, and stability for different application requirements.

[0058] In summary, this peptide, through modular fragment arrangement and variant-tolerant design, can maintain biological activity while improving expression feasibility and structural diversity, laying the foundation for its functional realization in applications such as tissue repair, cell adhesion promotion, and skin and hair care.

[0059] The recombinant human K25 keratin polypeptide, in some embodiments, can be recombinant human keratin K25A3, that is, the K25A sequence is repeated three times, including 108 amino acids, and the basic repeating unit is: EKVTMQNLNDRLASYLDSVHALEEANADLEQKIKGW (SEQ ID NO.1), which is a human keratin type 25 peptide.

[0060] The amino acid sequence of K25A3 is as follows:

[0061] EKVTMQNLNDRLASYLDSVHALEEANADLEQKIKGWEKVTMQNLNDRLASYLDSVHALEEANADLEQKIKGWEKVTMQNLNDRLASYLDSVHALEEANADLEQKIKGW (SEQ ID NO. 4).

[0062] In some embodiments, the recombinant human K25 keratin polypeptide can be recombinant human keratin K25B2, which is a double repetition of the K25B sequence, comprising 254 amino acids, with the basic repeating unit being:

[0063] IDDLKNQIIASTTSNANAVLQIDNARLTADDFRLKYENELALHQSVEADVNGLRRVLDEITLCRTDLEIQYETLSEEMTYLKKNHKEEMQVL (SEQ ID NO.2). This is a human keratin type 25 peptide.

[0064] The amino acid sequence of K25B2 is shown below:

[0065] IDDLKNQIIASTTSNANAVLQIDNARLTADDFRLKYENELALHQSVEADVNGLRRVLDEITLCRTDLEIQYETLSEEMTYLKKNHKEEMQVLIDDLKNQIIASTTSNANAVLQIDNARLTADDFRLKYENELALHQSVEADVNGLRRVLDEITLCRTDLEIQYETLSEEMTYLKKNHKEEMQVL (SEQ ID NO.6).

[0066] In some embodiments, the recombinant human K25 keratin polypeptide can be recombinant human keratin K25C2, which is a double repetition of the K25C sequence, comprising 278 amino acids, and a basic repeating unit:

[0067] LNNMRAEYEALAEQNRRDAEAWFNEKSASLQQQISEDVGATTSARNELTEMKRTLQ (SEQ ID NO.3) is a human keratin type 25 peptide.

[0068] The amino acid sequence of K25C2 is as follows:

[0069] LNNMRAEYEALAEQNRRDAEAWFNEKSASLQQQISEDVGATTSARNELTEMKRTLQLNNMRAEYEALAEQNRRDAEAWFNEKSASLQQQISEDVGATTSARNELTEMKRTLQ (SEQ ID NO. 8).

[0070] In this application embodiment, a polynucleotide is provided, which encodes the recombinant human K25 keratin polypeptide as described in the foregoing embodiments.

[0071] In some embodiments, the nucleotide sequence of the polynucleotide includes any one of the nucleotide sequences shown in SEQ ID NO. 5, SEQ ID NO. 7 and SEQ ID NO. 9.

[0072] Specifically, it can be any one of the nucleotide sequences corresponding to K25A3, K25B2, and K25C2.

[0073] The DNA sequence of K25A3 is as follows:

[0074] GAAAAAGTAACAATGCAAAACCTAAATGATCGCCTGGCGTCTTACCTGGATAGCGTTCATGCGCTGGAGGAAGCAAATGCGGACTTGGAGCAGAAGATCAAGGGCTGGGAAAAGGTGACGATGCAAAACCTGAATGATCGTCTGGCGAGCTATCTGGACAGCGTCCACGCCTTAGAGGAAGCGAACGCTGACCTGGAGCAGAAAATCAAAGGTTGGGAAAAAGTTACCATGCAGAATCTGAACGACCGTCTGGCTTCGTACCTGGATTCCGTGCACGCATTGGAAGAGGCCAACGCGGATTTGGAGCAAAAAATTAAGGGTTGG (SEQ ID NO.5)

[0075] The DNA sequence of K25B2 is shown below:

[0076] ATAGACGATCTAAAAAATCAAATTATAGCTTCGACCACCTCCAATGCCAATGCGGTGTTACAAATTGACAACGCTCGCCTGACGGCAGACGACTTCCGCCTGAAATACGAGAATGAACTGGCACTGCATCAGTCTGTTGAGGCTGACGTGAACGGTCTGCGTCGCGTGCTGGATGAGATCACCCTGTGTCGTACCGATCTGGAAATTCAGTACGAAACTCTGAGCGAAGAAATGACCTATCTGAAGAAAAACCATAAGGAAGAGATGCAGGTGCTGATTGATGATTTGAAGAACCAGATCATCGCCAGCACCACGAGCAACGCGAATGCGGTTTTGCAAATTGATAATGCGCGTTTGACCGCAGACGACTTTAGACTTAAGTATGAGAACGAGCTGGCGCTGCACCAAAGCGTAGAAGCGGATGTCAACGGCCTCCGTCGTGTTTTGGACGAAATCACTCTGTGCCGTACCGATCTGGAGATCCAGTATGAAACCTTGTCCGAGGAGATGACGTACCTGAAAAAGAACCACAAAGAAGAGATGCAAGTTTTG(SEQ ID NO.7)。

[0077] The DNA sequence of K25C2 is as follows:

[0078] CTAAATAACATGAGGGCTGAATATGAGGCATTGGCGGAGCAGAATAGACGTGATGCGGAGGCCTGGTTTAACGAAAAGTCCGCGAGCCTGCAACAGCAGATTAGCGAAGATGTTGGTGCAACTACGAGCGCTCGCAACGAGCTGACCGAAATGAAACGTACCTTACAG CTGAATAACATGCGTGCAGAGTACGAAGCGCTGGCGGAACAAAACCGCCGTGACGCCGAGGCGTGGTTCAACGAAAAATCTGCGTCCCTGCACAAATCTCGGAGGACGTGGGCGCAACCACCAGCGCTCGTAATGAGTTGACGGAAATGAAGCGCACCCTGCAG (SEQ ID NO. 9).

[0079] In this application embodiment, an expression vector is provided, the expression vector comprising the polynucleotides described in the foregoing embodiments.

[0080] In this embodiment of the application, a host cell is provided, wherein the host cell includes the expression vector as described in the foregoing embodiments, or expresses the recombinant human K25 keratin polypeptide as described in the foregoing embodiments;

[0081] The host cell is either Escherichia coli or yeast.

[0082] In this embodiment, a method for preparing recombinant human K25 keratin polypeptide as described in the foregoing embodiments is provided, comprising:

[0083] The host cells were cultured in a culture medium; and the recombinant human K25 keratin polypeptide was isolated based on the host cells.

[0084] In this application embodiment, a composition is provided, comprising the recombinant human K25 keratin polypeptide as described in the foregoing embodiments.

[0085] In this application embodiment, an article is provided, comprising the recombinant human K25 keratin polypeptide as described in the foregoing embodiments, or the composition as described in the foregoing embodiments;

[0086] The product in question is a cosmetic.

[0087] In this application embodiment, the use of the recombinant human K25 keratin polypeptide as described in the foregoing embodiments in the preparation of cosmetics is provided.

[0088] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0089] Unless otherwise specified, all biological materials, reagents, and devices used in the following examples are available from conventional commercial sources or obtained by existing known methods. Molecular biology experimental methods not specifically described in the following examples were performed in accordance with the specific methods listed in J. Sambrook's *Molecular Cloning: A Laboratory Manual* (3rd Edition), or according to the kit and product instructions.

[0090] The experimental materials and reagents used in the following examples are as follows:

[0091] 1. Strains, cells and vectors: Strains containing the target gene and expression plasmid, such as Escherichia coli DH5a, TOP10, DE3, Rosetta(DE3), BL21(DE3) PLysE, and BL21(DE3) PLysS, were purchased from Beyotime and Pichia pastoris X33 (Miaoling Biotechnology). Vectors pet28a, pPICzalpha, and pGAPzalpha, and the antibiotic Zeocin were purchased from Invitrogen.

[0092] 2. Reagent kits and enzymes: LDH detection kit (Roche 04744926001), modified Bradford protein assay kit (Sangon Biotech), seamless cloning kit In-Fusion Snap Assembly Master Mix (TaKaRa), plasmid extraction kit (Universal Biotech), universal gel extraction kit (Universal Biotech), restriction endonucleases, Q5 ultra-fidelity 2x Master Mix PCR polymerase, etc. were purchased from NEB.

[0093] 3. Culture medium:

[0094] The culture medium for Escherichia coli was LB medium (1% peptone, 0.5% yeast extract, 1% sodium chloride, pH 7.0), LB+AMP medium was LB medium with ampicillin added to a final concentration of 100 ug / mL, LB+Kana medium was LB medium with kanamycin added to a final concentration of 25 ug / mL, and LB+Zeo medium was LB medium with zeocin added to a final concentration of 100 ug / mL.

[0095] The induction medium for E. coli was TB medium (2% yeast extract, 1% peptone, 1.5% dipotassium hydrogen phosphate, 2% potassium dihydrogen phosphate, adjusted to pH 7.0).

[0096] The high-density fermentation medium for *E. coli* was prepared by adding 0.1%-0.2% antifoaming agent to TB, with a pH of 7.0, and feeding with 400 g / L of glycerol.

[0097] 4. Protein purification materials: Ni-NTA packing material and nickel column were purchased from Sanji Biotechnology.

[0098] Example 1: Gene Synthesis of Recombinant Keratin Peptides

[0099] Genetic analysis: The amino acid sequence of natural human hair keratin K25 (Genebank: CAD91904.1) was selected and analyzed for physicochemical properties such as hydrophilicity / hydrophobicity and isoelectric point. Helical regions in human keratin K25 were selected for recombination. The recombinant sequences were codon-optimized against E. coli codons. The resulting new genes are the genes K25A3, K25B2, and K25C2 of this invention, with amino acid sequences as shown in SEQ ID NO.4, SEQ ID NO.6, or SEQ ID NO.8.

[0100] Gene synthesis: The full lengths of the K25A3, K25B2, K25C2, and K25 genes are 324 bp, 552 bp, 363 bp, and 1353 bp, respectively. The optimized sequences based on the codons are SEQ ID NO.5, SEQ ID NO.7, and SEQ ID NO.9, respectively. Genes were synthesized using the optimized sequences. The gene fragments were provided by Universal Gene Biotechnology Co., Ltd. The synthesized K25A3, K25B2, K25C2, and K25 gene fragments were inserted into the pET32a expression vector using NcoI and XhoI, respectively. The vectors were then transformed into *E. coli* TOP10 and preserved.

[0101] The vector maps constructed using the K25A3 gene as an example are shown below. Figure 1 .

[0102] Example 2: Construction and Induced Expression of Recombinant Keratin Peptides

[0103] (1) Construction of genetically engineered Escherichia coli:

[0104] Plasmid extraction: Referring to Example 1, different bacterial strains containing the target gene were streaked onto LB+A or LB+K plates containing the corresponding antibiotic resistance and incubated overnight at 37°C. Subsequently, single colonies were selected from the overnight culture plates and inoculated into 10 mL of LB+A or LB+K liquid medium, and incubated overnight at 37°C. An appropriate amount of the overnight culture was centrifuged at 5000 rpm for 5 minutes, and the supernatant was removed to collect the bacterial pellet. Plasmid extraction was performed using the Tiangen Plasmid Extraction Kit. The concentration of the extracted plasmids was determined using NanoDrop, and the plasmids were either directly used for transformation of the expression host or stored at -20°C as needed.

[0105] Strain construction: Add 2 μL of the plasmid containing the target gene to 100 μL of competent *E. coli* cells and incubate on ice for 15-30 minutes. Heat shock the mixture in a 42°C water bath for 45 seconds, incubate on ice for 2-3 minutes, add 500 μL of antibiotic-free LB liquid medium, and incubate at 37°C and 190 rpm for 20 minutes. Spread 100 μL of this bacterial culture evenly onto LB+K or LB+Amp plates. Incubate overnight at 37°C until clearly visible colonies appear.

[0106] Induction of target protein expression: Single colonies were picked from overnight culture plates and cultured overnight in 10 mL of LB+A or LB+K liquid medium. Then, they were transferred to TB medium at a ratio of 1% for expansion culture. The culture was carried out at 37℃ and 190 rpm until the OD600 of the bacterial culture reached 0.5 to 0.8. Then, 0.1 to 0.5 mM IPTG was added to induce expression. The induction conditions were 16 to 26℃ and 190 rpm for 16-20 h. The bacterial cells were collected by centrifugation and washed three times with phosphate buffer. The washed bacterial cells could be frozen at -20℃ or directly lysed using an autoclave, followed by centrifugation at 10,000 rpm for 30 minutes. The supernatant was then collected and stored.

[0107] SDS-PAGE detection of the target protein: Take 20 μL of the supernatant after centrifugation, add 10 μL of 3× protein loading buffer, boil in 100℃ water for 10 minutes, then add 20 μL to each well of the SDS-PAGE protein gel, run at 80V for 1 hour, then switch to 120V until the bands are completely separated. Stain the protein with Coomassie Brilliant Blue staining solution (0.1% Coomassie Brilliant Blue R-250, 25% isopropanol, 10% glacial acetic acid) for 30 minutes, and then destain overnight with protein destaining solution (10% acetic acid, 5% ethanol).

[0108] See results Figure 2 The recombinant proteins K25A3, K25B2, K25C2, and K25 were found to have molecular weights of approximately 29 kDa, 38 kDa, 30 kDa, and 49 kDa, respectively, as determined by electrophoresis.

[0109] Example 3: Purification of recombinant keratin peptides

[0110] Crude protein treatment: The frozen bacterial cells, washed with phosphate buffer, were weighed and resuspended in 8 times their volume of lysis buffer (50 mM PPB, 10 mM imidazole, 0.5 M NaCl, pH 7.0). The cells were then homogenized using a high-pressure homogenizer at low temperature. The resulting mixture was centrifuged at 4°C and 10,000 rpm for 25 minutes. The precipitate was discarded, and the supernatant was collected and its volume recorded.

[0111] Ni-NTA nickel column purification of proteins: Wash 5 column volumes with (50 mM Pb, 0.5 M NaCl, pH 7.0) to equilibrate the nickel column. Load pretreated protein onto the column. Wash 3-5 column volumes with low-concentration imidazole (10 mM~50 mM) buffer to remove contaminating proteins, followed by gradient elution with 50 mM~500 mM imidazole. Collect protein samples eluted with different concentrations of imidazole and analyze purity using SDS-PAGE. After collection, wash the nickel column with 20% ethanol, fill with ethanol, and store. For nickel columns used multiple times, regeneration is required. The regeneration steps are as follows: Wash 5 column volumes with ddH2O, 5 column volumes with EDTA solution, 5 column volumes with NaOH solution, fill the affinity column with NiSO4 solution, and wash 5 column volumes. Analyze the purified protein from different receiving samples using SDS-PAGE to determine the purity and size of the target protein.

[0112] See results Figure 3 The molecular weight of K25B2, as determined by protein electrophoresis, is approximately 38 kDa.

[0113] Example 4: Cell proliferation activity test of recombinant keratin

[0114] L929 Cell Culture: L929 cells were placed in DMEM medium containing 10% fetal bovine serum and antibiotics, and cultured in a CO2 incubator at 5% concentration, 95% relative humidity, and 37°C. The medium was changed every two days, and after the cells reached basic confluence on day 5, they were passaged. First, the old medium was aspirated, and the cells were washed twice with PBS solution. Then, 0.5 mL of 0.25% trypsin was added for digestion. Observation was performed under an inverted microscope. After the cells shrank and became rounded, 10 mL of DMEM medium was added to stop digestion. The cells were gently pipetted to suspend them, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, and an appropriate amount of serum-containing medium was added. The cells were gently pipetted again to prepare a cell suspension, and 50 μL was collected for cell counting. For L929 cells in the logarithmic growth phase, the supernatant was aspirated, the cells were washed twice with PBS solution, and digested with 0.5 mL of 5% DMEM-0.25% trypsin to prepare a single-cell suspension. The cells were washed twice with PBS solution again, and the suspended cells were collected directly.

[0115] MTT assay: Select a 96-well plate, and inoculate 2 × 10⁶ cells per well. 4 Cells were collected and set up as follows: experimental group, blank control group (containing only PBS solution), and calibration / zeroing group (containing no cells). Two replicate wells were set up for each group. After 24 hours, microscopic observation showed that most cells had adhered and spread. At this point, unattached cells and liquid were removed from the wells. The peptide concentration in the experimental group was adjusted to 0.5 mg / mL, and 20 μL was added. The blank control group received 20 μL of PBS buffer, and the calibration / zeroing group received only DMEM medium. When adding samples, ensure the pipette tip is close to the well wall and inject slowly, gently pipetting to mix after each well. After incubation for 30 minutes, the plate was placed in a CO2 incubator. Cell proliferation was observed after 72 hours of culture. The absorbance (OD) value was measured using the MTT assay. 20 μL of MTT solution was added to each well of the 96-well plate, and the plate was incubated for another 4 hours to complete the colorimetric reaction. Afterwards, terminate the culture, aspirate the culture medium from the wells or cover the culture plate with a layer of filter paper, quickly invert the culture plate, add 150 μL LDMSO to each well, shake for 10 minutes to fully dissolve the formazan, and finally use an ELISA reader to measure the absorbance of each well at an A490 nm wavelength.

[0116] See results Figure 4 Compared with blank and full-length expressed K25, recombinant keratin K25A3, K25B2, and K25C2 exhibit better effects in promoting the proliferation and differentiation of L929 cells and enhancing cell activity. The human recombinant keratin of this invention has the function of promoting cell proliferation and can be used to prepare biomaterials or drugs that promote cell proliferation.

[0117] Example 5: Hair tensile strength and stretching performance test

[0118] Sample preparation: The hair tensile strength test method was based on NBFair and BSGupta: The use of multiple test methods in the analysis of the effect of cosmetic treatment on the surface properties of human hair, XIVth IFSCC Congress, Barcelona, ​​Vol II, pp. 113-1124 (1986) and modified from patent CN 110025552A. The specific process is as follows: Recombinant keratin K25A3, K25B2, K25C2 and full-length expressed K25 were respectively combined with glycerin, propylene glycol, xanthan gum and other ingredients to prepare a keratin hair essence with a keratin content of 0.075%. Hair from the same source was washed with 5% sodium lauryl sulfate, soaked in 5% sodium hydroxide for 30 minutes, rinsed with purified water, treated with keratin hair essence for 1 week, rinsed with purified water and air-dried. Hair samples were then taken to measure the tension reduction rate and elongation at break.

[0119] Hair tensile strength test: Determination of tensile reduction rate (%). The tensile reduction rate (%) was measured using a rheometer at 20℃±2℃ and relative humidity of 60±4%. The original hair length was 50mm, the elongation was 5cm / min, and the full scale was 200gf.

[0120] Calculate using the following formula:

[0121] ;

[0122] Where F0 represents the initial force required to elongate the fiber by 20%; F1 represents the force required to elongate the same fiber by another 20%. The tension reduction rate (%) of five hair samples in each group was tested, and the average value was taken. The test results are shown in Table 2 below.

[0123] Tensile property test: The test was conducted using an INSTRON-4466 tensile testing machine at 20℃ and 45% relative humidity, with a tensile speed of 15 mm / min. -1 The force resolution was 0.01 N. The breaking elongation (%) of 5 hair samples in each group was tested, and the average value was taken. The test results are shown in Table 2 below.

[0124] Table 2. Results of hair tensile strength and tensile properties test

[0125]

[0126] The test results show that the recombinant keratin K25A3, K25B2, and K25C2 keratin serums in this application embodiment have excellent performance in improving hair tensile properties and hair breakage elongation, with more significant effects than full-length expressed K25. Among them, the K25C2 serum has the most significant effect.

[0127] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant human-derived K25 keratin polypeptide, characterized in that, The polypeptide is an amino acid sequence as shown in SEQ ID NO. 4, SEQ ID NO. 6 or SEQ ID NO.

8.

2. A polynucleotide, comprising, The polynucleotide encodes the recombinant human K25 keratin polypeptide as claimed in claim 1.

3. The polynucleotide of claim 2, wherein The nucleotide sequence of the polynucleotide is any one of the nucleotide sequences as shown in SEQ ID NO. 5, SEQ ID NO. 7 and SEQ ID NO.

9.

4. An expression vector, characterized by, The expression vector comprises the polynucleotide as claimed in claim 2 or 3.

5. A host cell, characterized in that, The host cell comprises the expression vector as claimed in claim 4, or expresses the recombinant human K25 keratin polypeptide as claimed in claim 1. The host cell is E. coli or yeast.

6. A method for preparing the recombinant human K25 keratin polypeptide according to claim 1, wherein the method comprises the steps of: a) culturing the host cell of claim 5 in a suitable culture medium; b) collecting the culture medium; c) purifying the recombinant human K25 keratin polypeptide from the culture medium. The method comprises: culturing the host cell in a culture medium; and isolating the recombinant human K25 keratin polypeptide based on the host cell.

7. A composition characterized in that, The method comprises the recombinant human K25 keratin polypeptide as claimed in claim 1.

8. An article characterized by, The method comprises the recombinant human K25 keratin polypeptide as claimed in claim 1 or the composition as claimed in claim 7. The product is a cosmetic product.

9. Use of the recombinant human K25 keratin polypeptide as claimed in claim 1 in the preparation of a cosmetic product.

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