A recombinant fusion protein for scalp care and hair repair, its preparation method and application

Abstract

This invention discloses a recombinant fusion protein for scalp care and hair repair, its preparation method, and its applications. The recombinant fusion protein comprises a non-transmembrane functional fragment derived from the intracellular region of collagen type 17 (COL17A1), a cysteine-rich functional unit derived from metallothionein (MT), and a linker peptide connecting the two. Through modular targeted fusion design, this fusion protein integrates the scalp follicle microenvironment support function provided by the COL17A1 functional fragment with the antioxidant and damaged hair repair functions provided by the MT functional unit into a single molecule, achieving synergistic dual-function. Experimental data show that the fusion protein of this invention has significant DPPH free radical scavenging ability, effectively reduces the intracellular reactive oxygen species level of scalp-related cells under oxidative stress, and significantly improves the cuticle integrity and surface smoothness of damaged hair, while also exhibiting good cellular safety.

Description

Technical Field

[0001] This invention relates to the field of synthetic biology, specifically to a recombinant fusion protein for scalp care and hair repair, its preparation method, and its application. Background Technology

[0002] The scalp and hair, as coupled functional units, work together to maintain hair health and growth. The scalp, constantly exposed to the external environment, is susceptible to oxidative stress, UV radiation, dyeing and perming damage, and the degreasing effects of surfactants, leading to an imbalance in keratinocyte homeostasis, damage to the hair follicle microenvironment, and dysfunction of the sebum film. Hair fibers themselves lack metabolic activity; once damage occurs, repair relies primarily on exogenous active ingredients to adsorb, fill, cross-link, and strengthen the hair surface, as well as protect oxidative breakage sites. Currently, commercially available scalp and hair care protein raw materials generally suffer from the following core pain points: scalp care and hair repair functions are disconnected, with existing products typically designed for only a single functional dimension; there is a lack of integrated biomolecular raw materials that combine the ability to improve the hair follicle microenvironment and repair damaged hair shaft structures; protein raw materials lack clear target guidance and systematic modular design strategies, with functional screening heavily reliant on wet experiments, resulting in low development efficiency and high costs. Type XVII collagen (COL17A1) is a transmembrane protein located in the basement membrane region of the epidermis. It is a core component of hemidesmosomes and is mainly distributed in hair follicle stem cells and epidermal stem cells in the hair follicle bulb. It plays a key role in maintaining the stemness of hair follicle stem cells, regulating the periodic cycle of hair follicles, and promoting the regeneration of hair follicle tissue.

[0003] CN119264276A discloses a recombinant type XVII collagen-thymosin β4 fusion protein. By fusing the COL17A1 fragment with thymosin β4 protein, the problem of small molecular weight and difficult expression of thymosin β4 is solved. This fusion protein can promote hair follicle cell proliferation and hair regeneration. However, the fusion target of this protein is thymosin β4, a polypeptide composed of 43 amino acid residues. Its function is mainly focused on regulating actin polymerization and cell migration. Its sequence is not rich in cysteine ​​residues and it does not possess the metal ion coordination ability and free radical scavenging chemical mechanism unique to metallothioneins. Therefore, this fusion protein shows weak chemical antioxidant capacity and interface repair function against damaged hair shaft keratin. In addition, the functional positioning of this fusion protein is focused on promoting hair follicle cell proliferation and regulating hair follicle cycle. Essentially, it is still a single functional dimension (promoting hair follicle regeneration) and cannot achieve the dual function of improving the scalp microenvironment and repairing hair structure within the same molecule. Furthermore, this patent lacks a systematic consideration of modular design concepts and molecular weight window control. The full-length COL17A1 has a molecular weight of 180 kDa and a complex transmembrane protein structure. Although the patent uses a COL17A1 fragment, it fails to optimize the design of its functional module boundaries, linker peptide configuration, and the engineering feasibility of multiple copy functional units. This results in high difficulty for industrial expression and low purification yield, hindering its large-scale development as a cosmetic raw material. CN118496344A discloses a truncated recombinant human type XVII collagen protein with hair growth and conditioning effects. Its amino acid sequence is shown in SEQ ID NO.1 of this patent, corresponding to the extracellular region of COL17A1 from positions 482 to 698. This truncated protein promotes hair follicle cell proliferation and inhibits 5α-reductase activity. However, this protein is a single truncated protein, not a fusion design. Its functional dimensions are also concentrated on promoting hair follicle regeneration and preventing hair loss, lacking structural modules for repairing damaged hair shaft keratin networks and providing antioxidant protection. Therefore, scalp care and hair repair functions remain separate.

[0004] In summary, there is an urgent need to develop a recombinant fusion protein raw material that combines the dual functions of improving the scalp and hair follicle microenvironment and repairing damaged hair with antioxidant properties, achieves functional synergy through modular design, and has a suitable molecular weight for industrial expression, in order to meet the market demand for integrated scalp and hair care. Summary of the Invention

[0005] To address the aforementioned technical problems, this application provides a recombinant fusion protein, its preparation method, and its applications. By modularly and directionally fusing a non-transmembrane functional fragment of the intracellular region of COL17A1 with a cysteine-rich functional unit derived from metallothionein, a single protein molecule can simultaneously perform scalp follicle microenvironment support functions and damaged hair antioxidant repair functions, thus overcoming the shortcomings of the prior art.

[0006] This application provides a recombinant fusion protein comprising a functional fragment derived from type 17 collagen COL17A1, the functional fragment being selected from a non-transmembrane fragment of the intracellular region of COL17A1; a functional fragment derived from metallothionein MT, the functional fragment being a cysteine-rich MT functional unit; and a linker peptide connecting the COL17A1-derived functional fragment and the MT-derived functional fragment; the fusion direction of the recombinant fusion protein from the N-terminus to the C-terminus is as follows: COL17A1-derived functional fragment, linker peptide, and MT-derived functional fragment, to ensure that the N-terminal binding interface of the COL17A1 functional fragment remains free.

[0007] The COL17A1-derived functional fragment was localized to an intracellular functional region associated with the hemidesmosome adhesion complex, preserving its specific binding interface with proteins such as BP230, plectin, and integrin β4 in the hair follicle microenvironment. This endows the fusion protein with potential for scalp care and hair follicle homeostasis support. The N-terminus of this fragment is a BP230 / plectin binding hotspot and must be kept in a free state to ensure binding activity; therefore, it needs to be placed at the N-terminus of the recombinant fusion protein. Simultaneously, a cysteine-rich functional unit derived from MT was introduced. Utilizing the thiol chemical properties of cysteine, it provides an antioxidant active site and interfacial interaction ability with damaged hair shaft keratin. This achieves a dual-function synergy of scalp care and hair repair within a single molecule, overcoming the limitations of existing raw materials with only one function.

[0008] Preferably, the recombinant fusion protein structure is selected from: a structure composed of X, L1, and Y connected in sequence; a structure composed of X, L1, and n Y units connected in series; or a structure composed of X, L1, and n units formed by Y and L2 connected in series. The general formula is any one of X-L1-Y, X-L1-(Y)n, or X-L1-(Y-L2)n, where X is a non-transmembrane functional fragment derived from type 17 collagen COL17A1; Y is a cysteine-rich functional fragment derived from metallothionein MT; L1 is a linking peptide connecting the COL17A1-derived functional fragment and the MT-derived functional fragment; L2 is a linking peptide between adjacent MT-derived functional units; n is an integer from 1 to 8; and, based on the above general formula, the theoretical molecular weight of the recombinant fusion protein is 20 kDa to 60 kDa, more preferably 30 kDa to 55 kDa.

[0009] Controlling the molecular weight of recombinant fusion proteins within the aforementioned range offers several advantages: Firstly, it avoids the industrialization obstacles associated with using large-molecular-weight transmembrane proteins such as full-length COL17A1 (180 kDa), which present difficulties in expression, complex folding, and low purification yields. Secondly, it preserves sufficient functional module integrity, ensuring that neither the COL17A1 functional fragment nor the MT functional unit is excessively truncated and loses its activity. This molecular weight window balances the engineering feasibility of conventional expression systems such as E. coli, downstream purification efficiency, and formulation compatibility as a cosmetic ingredient.

[0010] Preferably, the length of the functional fragment derived from COL17A1 is 20 to 500 amino acids, more preferably 80 to 350 amino acids.

[0011] Functional fragments derived from COL17A1 with a length of 20-500 amino acids can remove the transmembrane region and large extracellular collagen repeat regions in the full-length COL17A1 sequence, significantly reducing the difficulty of engineering expression and the risk of protein aggregation, while retaining the core functional sequences in the intracellular region related to the hemidesmosome adhesion complex, thus achieving a balance between maintaining function and reducing development risks.

[0012] Preferably, the COL17A1-derived functional fragment includes a sequence segment with specific binding potential to hemidesmosome-associated adhesion interfaces, hair follicle adhesion microenvironment-related structures, or cytoskeleton connective complexes. More preferably, the sequence segment includes a sequence segment with binding activity to hemidesmosome protein BP230 and / or plectin.

[0013] The intracellular region of COL17A1 is a key area for interaction with hemispheric plate proteins such as BP230 and plectin, as well as the integrin β4 subunit, directly involved in the anchoring and homeostasis maintenance of hair follicle stem cells on the basement membrane. Retaining this region allows the fusion protein, when applied to the scalp, to support the structural integrity and microenvironmental homeostasis of hair follicles through specific interactions with corresponding proteins in the hair follicle microenvironment, thereby enhancing the targeting and biological rationality of scalp care efficacy.

[0014] Preferably, the functional fragment derived from COL17A1 is a polypeptide represented by amino acids 145 to 365 of COL17A1, or a functionally equivalent fragment obtained by truncation, elongation, conservative substitution, or repeated splicing.

[0015] This segment covers the core region where COL17A1 binds to BP230 / plectin, and is one of the sequences with the highest functional density in the intracellular region. Its theoretical molecular weight is approximately 23.25 kDa, and while preserving key functional interfaces, it remains within a molecular weight range favorable for engineered expression. This fragment differs from the truncated extracellular region (positions 482-698) selected in CN118496344A; its functional localization shifts from the extracellular matrix to the intracellular adhesion complex interface, making it crucial for supporting the hair follicle microenvironment.

[0016] Preferably, the functional fragment from which COL17A1 originates is the amino acid sequence shown in SEQ ID NO.1, or a derived sequence having at least 70%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO.1 and retaining scalp care and / or hair repair functions.

[0017] In protein engineering practice, to increase expression levels, improve solubility, or enhance activity, it is often necessary to perform conserved substitutions, boundary fine-tuning, or repetitive splicing of the original sequence. Including derivative sequences with high homology and functional equivalence within the protection scope is both a reasonable anticipation of functionally equivalent variants and an effective way to prevent others from circumventing the use of the core sequence through simple mutations. Promoting human dermal papillary cell adhesion activity is a unique function of the aa145-365 fragment in the intracellular region of COL17A1 and is a key indicator for evaluating the functional equivalence of recombinant fusion proteins in scalp care and / or hair repair.

[0018] Preferably, the MT-derived functional fragment exists in the form of 1 to 8 MT functional units; when the MT-derived functional fragment contains multiple MT functional units, adjacent MT functional units are directly connected or connected through linker peptides.

[0019] While a single MT domain contains a limited number of cysteine ​​residues, tandem copying can significantly increase the overall cysteine-rich site density of the fusion protein, thereby enhancing its antioxidant capacity and its potential for multivalent interfacial interactions with damaged hair shaft keratin. Furthermore, the variability in copy number provides design freedom to flexibly adjust the strength of the MT module according to target molecular weight and efficacy requirements.

[0020] Preferably, the MT functional unit is selected from at least one of the following: MT full-length functional module, MTβ domain functional unit, MTα domain functional unit, or engineered mini-MT functional unit.

[0021] Different MT domains exhibit variations in cysteine ​​arrangement, metal ion binding preference, and spatial conformation. Customized configurations of MT modules can be achieved by flexibly selecting single-type repeats or combinations of different types of repeats based on the target molecular weight, expression host, and functional emphasis.

[0022] Preferably, the MT functional unit is the MTβ domain functional unit shown in SEQ ID NO.2, or the MTα domain functional unit shown in SEQ ID NO.3.

[0023] These two are the β and α domains of metallothionein (MT), respectively. Both are known to be rich in cysteine, possess strong metal ion binding and free radical scavenging capabilities, have well-defined sequences and compact structures (approximately 30-31 amino acids), making them highly suitable as repetitive functional units for fusion proteins.

[0024] Preferably, the linker peptide has a length of 2 to 30 amino acids.

[0025] By controlling the length of the linker peptide to 2-30 amino acids, we can ensure sufficient spatial decoupling between modules while avoiding the introduction of unnecessary immunogenicity or degradation risks by excessively long linkers.

[0026] Preferably, the linker peptide is selected from GS, GPGGS, (GGGS)n, (GGS)n, EAAAK repeat sequences or combinations thereof, wherein n is 1 to 5.

[0027] GS and GPGGS are the shortest flexible linkers, suitable for module configurations requiring tight coupling, while minimizing the contribution of the linker peptide to the total molecular weight; (GGGS)n and (GGS)n offer adjustable flexibility and length gradients, suitable for different spatial requirements; EAAAK is a rigid α-helix linker, suitable for scenarios requiring a certain distance between modules. Providing these multiple options allows for flexible matching of the optimal linker scheme based on the specific configuration of the COL17A1 fragment and MT unit.

[0028] Preferably, the recombinant fusion protein further comprises a purified tag or a soluble enhancement tag selected from His tag, Strep tag, SUMO tag, Trx tag, MBP tag, GST tag and combinations thereof, and / or contains a TEV or 3C protease cleavage site.

[0029] His tags and other purification tags can significantly simplify downstream purification processes, allowing for the acquisition of high-purity target proteins through a single affinity chromatography step, thus reducing industrial production costs. Solubility-enhancing tags such as SUMO, Trx, and MBP can increase the soluble expression ratio of recombinant fusion proteins in prokaryotic expression systems such as E. coli, reducing inclusion body formation. The setting of TEV or 3C protease cleavage sites can achieve traceless or low-residue removal of tags when they affect efficacy verification or cosmetic applications.

[0030] Preferably, the recombinant fusion protein is any one of the following: (a) FP-1 is composed of the COL17A1 functional fragment shown in SEQ ID NO.1 and four MTβ domain functional units shown in SEQ ID NO.2 connected in series via GS linker peptides. Its amino acid sequence is shown in SEQ ID NO.6. The recombinant fusion protein FP-1 has DPPH free radical scavenging activity and / or activity that promotes the adhesion of human dermal papillary cells. (b) FP-2 is composed of the COL17A1 functional fragment shown in SEQ ID NO.1 and 7 MTα domain functional units shown in SEQ ID NO.3 connected in series via GPGGS linker peptides. Its amino acid sequence is shown in SEQ ID NO.7. The recombinant fusion protein FP-2 has DPPH free radical scavenging activity and / or activity that promotes the adhesion of human dermal papillary cells.

[0031] FP-1 (approximately 35.91 kDa) increases the density of cysteine-rich sites by tandemly connecting four MTβ domain units, thus controlling the total molecular weight and balancing expression feasibility and efficacy potential. FP-2 (approximately 46.76 kDa) further enhances the MT module density through the cascaded connection of 7 MTα structural domain units, exhibiting stronger DPPH free radical scavenging ability and better repair effect on damaged hair cuticles.

[0032] Both studies validated the technical feasibility of the modular design platform of "COL17A1 intracellular functional fragment and MT multiple copy module," providing an empirical basis for subsequent expansion of other fragment combinations. These studies differ substantially from existing technologies such as the COL17A1-thymosin β4 fusion protein (CN119264276A) and the truncated COL17A1 single protein (CN118496344A) in terms of fusion target selection, functional dimensions, and modular design strategy.

[0033] Preferably, the recombinant fusion protein further includes a 6×His tag at the N-terminus or C-terminus.

[0034] The 6×His tag contains only 6 histidine residues and has a very small molecular weight (approximately 0.84 kDa), so its impact on the overall molecular weight and functional conformation of the recombinant fusion protein is negligible. Simultaneously, this tag can bind with nickel ions (Ni2+). + Specific binding enables efficient one-step affinity purification, significantly reducing the complexity and cost of purification for industrial production. The C-terminal setting avoids interference with the native conformation and functional interface of the N-terminal COL17A1 functional fragment, and the N-terminal setting can be used in scenarios where the C-terminal functional module needs to be kept in a free state.

[0035] The present invention also provides a method for preparing the above-mentioned recombinant fusion protein, comprising the following steps: inserting a nucleic acid sequence encoding the fusion protein into an expression vector to obtain a recombinant expression vector; introducing the recombinant expression vector into a host cell; culturing the host cell into which the recombinant expression vector is introduced to induce expression of the recombinant fusion protein; and collecting and purifying the recombinant fusion protein.

[0036] This invention also provides nucleic acids encoding the aforementioned recombinant fusion proteins, wherein the nucleic acids are codon-optimized to adapt to prokaryotic expression hosts. In one specific embodiment, a set of encoding nucleic acid sequences for FP-1 and FP-2 suitable for E. coli expression systems are shown in SEQ ID NO.8 and SEQ ID NO.9, respectively. The encoding sequences shown in SEQ ID NO.8 and SEQ ID NO.9 correspond to the coding regions of the FP-1 and FP-2 protein bodies, respectively.

[0037] The present invention also provides recombinant expression vectors containing the aforementioned nucleic acids. The vectors may contain regulatory sequences (such as transcription and translation initiation and termination codons) that are specific to the type of host to which the vector is to be introduced, taking into account whether the vector is DNA-based or RNA-based. In one specific embodiment, the expression vectors are pET-3C-FP-1 and pET-3C-FP-2.

[0038] The present invention also provides host cells into which the above-described expression vector is introduced. Host cells include *Escherichia coli*, *Lactobacillus*, *Bacillus subtilis*, *Saccharomyces cerevisiae*, *Candida*, *Pichia pastoris*, and tobacco cells. Further, *Escherichia coli* is preferred as the host cell.

[0039] The present invention also provides a scalp care or hair repair composition comprising the above-described recombinant fusion protein, and optionally cosmetically acceptable excipients. Preferably, the cosmetically acceptable excipients comprise thickeners, stabilizers, preservatives, pH adjusters, and polyols.

[0040] The present invention also provides the use of the above-mentioned recombinant fusion protein in the preparation of scalp care and / or hair repair products.

[0041] The beneficial effects are as follows: This application, through modular design, directionally fuses a non-transmembrane functional fragment derived from the intracellular region of COL17A1 with a cysteine-rich functional unit derived from metallothionein, constructing a bifunctional recombinant fusion protein that combines scalp and hair follicle microenvironment support with antioxidant repair of damaged hair. Specifically, the COL17A1 functional fragment is located in an intracellular functional region associated with the hemidesmosome adhesion complex, and can specifically bind to proteins such as BP230, plectin, and integrin β4 in the hair follicle microenvironment, endowing the fusion protein with the potential for scalp care and hair follicle homeostasis support; the directionally fused design (N-terminal COL17A1 fragment - C-terminal MT unit) ensures that the binding hotspot region at the N-terminus of the COL17A1 fragment is in a free state, which is the structural basis for its above-mentioned functions; The metallothionein functional unit is rich in cysteine, and its free thiol groups provide significant antioxidant activity and interfacial interaction with damaged hair shaft keratin. This enables the recombinant fusion protein to exhibit significant effects in scavenging DPPH free radicals, reducing intracellular reactive oxygen species levels in scalp-related cells under oxidative stress, and improving the integrity and surface smoothness of damaged hair cuticles. The multi-copy tandem MT functional unit design further enhances the antioxidant performance; FP-2 exhibits a higher DPPH scavenging rate and stronger intracellular ROS scavenging ability than FP-1. Simultaneously, experimental data show that this recombinant fusion protein has good cellular safety, exhibiting no significant cytotoxicity to human dermal papillary cells at effective concentrations. The molecular weight of the recombinant fusion protein of this invention is precisely controlled within the range of 20kDa to 60kDa, successfully overcoming the industrial expression challenges of large molecular weight proteins such as full-length COL17A1, and demonstrating engineering feasibility in prokaryotic expression systems. Compared with existing COL17A1-thymosin β4 fusion protein and COL17A1 truncated single protein, the recombinant fusion protein of this invention has substantial differences and significant progress in fusion target, functional dimensions and molecular design. It achieves integrated synergy of scalp care and hair repair functions, and the single-molecule fusion design is significantly superior to physical mixing methods in terms of thermal stability and freeze-thaw tolerance. It provides an innovative biomolecular raw material with both high efficiency and safety for the field of scalp and hair care. Attached Figure Description

[0042] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings: Figure 1 : Schematic diagram of the design of a recombinant fusion protein containing COL17A1 and MT functional fragments; Figure 2 Schematic diagram of the origin locations of candidate hotspot peptides P1 to P6 in the shared N-terminal region of FP-1 / FP-2; Figure 3 Schematic diagram of the KRT31-KRT85 receptor core complex model; Figure 4 Schematic diagram of representative candidate docking conformations of preferred candidate peptides P2 and P5; Figure 5 Figure showing the expression results of the FP-1 recombinant fusion protein; Figure 6 Figure showing the expression results of the FP-2 recombinant fusion protein; Figure 7 Figure: Purification results of FP-1 recombinant fusion protein; Figure 8 Figure: Purification results of FP-2 recombinant fusion protein; Figure 9 Figure: Effects of recombinant fusion proteins FP-1 and FP-2 on intracellular ROS levels in hDPC cells under oxidative stress. Figure 10 Scanning electron microscope (SEM) image of damaged hair repair.

[0043] Sequence List Description (Sequence list content provided separately): COL17-Fx, amino acids 145-365 of COL17A1, 221 amino acids, approximately 23.25 kDa; amino acid sequence as shown in SEQ ID NO.1.

[0044] MT-U1, MTβ domain functional unit, 30 aa, approximately 3.04 kDa; amino acid sequence as shown in SEQ ID NO.2.

[0045] MT-U2, MTα domain functional unit, 31aa, approximately 3.02kDa; amino acid sequence as shown in SEQ ID NO.3.

[0046] L1, a short, flexible linker peptide, 2aa; its amino acid sequence is shown in SEQ ID NO.4.

[0047] L2, a flexible linker peptide, 5 amino acids; its amino acid sequence is shown in SEQ ID NO.5.

[0048] The recombinant fusion protein FP-1 is composed of SEQ ID NO.1 and four SEQ ID NO.2 molecules linked by a GS linker peptide, with an amino acid sequence of 349 aa and approximately 35.91 kDa; the amino acid sequence is shown in SEQ ID NO.6.

[0049] The recombinant fusion protein FP-2 is composed of SEQ ID NO.1 and 7 SEQ ID NO.3 linked by GPGGS peptides, with a length of 473 aa and a length of approximately 46.76 kDa; the amino acid sequence is shown in SEQ ID NO.7.

[0050] The preferred nucleic acid sequence encoding the recombinant fusion protein FP-1 is shown in SEQ ID NO.8.

[0051] The preferred nucleic acid sequence encoding the recombinant fusion protein FP-2 is shown in SEQ ID NO.9.

[0052] The short peptide fragment P1 is shown in SEQ ID NO.10.

[0053] The short peptide fragment P2 is shown in SEQ ID NO.11.

[0054] The short peptide fragment P3 is shown in SEQ ID NO.12.

[0055] The short peptide fragment P4 is shown in SEQ ID NO.13.

[0056] The short peptide fragment P5 is shown in SEQ ID NO.14.

[0057] The short peptide fragment P6 is shown in SEQ ID NO.15.

[0058] The receptor KRT31 sequence is shown in SEQ ID NO.16.

[0059] The receptor KRT85 sequence is shown in SEQ ID NO.17. Detailed Implementation

[0060] The present invention will be further described in detail below through specific embodiments and comparative examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the invention. Unless otherwise specified, specific conditions in the embodiments were performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used, unless otherwise specified, are all commercially available products. In all embodiments and comparative examples of the present invention, experimental results are expressed as mean ± standard deviation. The features and performance of the present invention will be further described in detail below with reference to the embodiments.

[0061] Raw material source: Hydrolyzed keratin, CAS No. 169799-44-4, purity 99%, purchased from Wuhan Camick Technology Co., Ltd. Xanthan gum, CAS No. 11138-66-2, purchased from Jiangsu Weizhirun Biotechnology Co., Ltd. Flaxseed extract (flaxseed paste, active ingredients are linoleic acid, α-linolenic acid, etc.), brownish-yellow powder, 80-100 mesh, purchased from Baoji Kaiweikang Biotechnology Co., Ltd. All other raw and auxiliary materials are commercially available.

[0062] This invention introduces a computer-aided candidate sequence screening method in the functional fragment screening and sequence design stage of recombinant fusion proteins, which significantly improves the targeting and rationality of molecular design. The specific screening steps are as follows: S1 candidate sequence preparation: Provide the original sequences of MT-U1, MT-U2, FP-1 and FP-2, and identify the shared N-terminal segment of FP-1 and FP-2 as the source of local hotspot screening; S2 candidate hotspot identification: The IUPred2A / ANCHOR2 tool (https: / / iupred2a.elte.hu) was used to analyze the disordered regions and candidate binding hotspots of the original sequence, and the shared N-terminal region of FP-1 / FP-2 was identified as a local region that is more likely to perform contact function of candidate hair-related proteins. S3 Candidate Peptide Extraction: Based on the shared N-terminal candidate hotspot region identified in step S2, six candidate short peptide fragments P1 to P6, each 20 aa in length, are extracted (e.g., ...). Figure 2 As shown, Figure 2 (This is a schematic diagram showing the origin of candidate hotspot peptides P1 to P6 in the shared N-terminal region of FP-1 / FP-2). The sequences of P1 to P6 are shown in SEQ ID NO.10 to 15, respectively. S4 receptor model construction: A hair keratin KRT31-KRT85 heterodimer core complex model was constructed using the AlphaFoldServer tool (https: / / alphafoldserver.com) as a receptor model for candidate contact conformation analysis (e.g., Figure 3 As shown, Figure 3 (A schematic diagram of the KRT31-KRT85 receptor core complex model), the KRT31 and KRT85 receptor sequences are shown in SEQ ID NO.16~17, respectively; S5 Flexible docking screening: The CABS-dock tool (https: / / biocomp.chem.uw.edu.pl / CABSdock) was used to perform flexible protein-peptide docking (e.g., the candidate short peptide fragments P1-P6 obtained in step S3) with the KRT31-KRT85 receptor model constructed in step 4. Figure 4 As shown, Figure 4To optimize the candidate peptides P2 and P5, a schematic diagram of representative candidate docking conformations is shown. The candidate contact conformations and clustering parameters of each candidate short peptide on the receptor surface are obtained. The clustering parameters include principal cluster density, average principal cluster RMSD, and number of principal cluster elements. The higher the principal cluster density, the lower the average principal cluster RMSD, and the more principal cluster elements, the more stable and concentrated the candidate binding mode of the candidate peptide on the current receptor surface is. S6 Priority Ranking and Candidate Retention: Based on the clustering parameters, P2 and P5 showed the best binding stability and conformational convergence. Therefore, the functional fragment of the COL17A1 intracellular region corresponding to the shared N-terminal segment of FP-1 / FP-2 was retained as the front functional module of the recombinant fusion protein.

[0063] The aforementioned computer-aided screening method can verify the non-obviousness of the selection of the COL17A1 functional fragment in this application at the molecular level, providing a precise theoretical basis for the modular design of fusion proteins, avoiding the blindness of traditional wet experimental screening, and significantly improving the efficiency and functional targeting of molecular design.

[0064] Example 1: Expression and purification of recombinant fusion proteins FP-1 and FP-2 (1) Gene design and synthesis like Figure 1 As shown, Figure 1 This diagram illustrates the design of a recombinant fusion protein containing functional fragments of COL17A1 and MT. Based on the amino acid sequences of FP-1 (SEQ ID NO.6) and FP-2 (SEQ ID NO.7), codon optimization was performed according to the codon preference of *E. coli*, yielding the coding nucleic acid sequences SEQ ID NO.8 and SEQ ID NO.9, respectively. A nucleotide sequence encoding a 6×His tag (CACCACCACCACCACC) was introduced at the 3' end of the coding sequence, and NdeI and XhoI restriction enzyme sites were introduced at the 5' and 3' ends, respectively. The optimized gene sequence was then commissioned to a commercial gene synthesis company for whole-genome synthesis.

[0065] (2) Construction of expression carrier The synthesized target gene fragment was double-digested with NdeI and XhoI, then inserted into the pET-3C expression vector digested with the same enzymes. The ligation product was transformed into E. coli DH5α competent cells and plated on LB agar plates containing ampicillin (100 μg / mL) and incubated overnight at 37°C. Single clones were picked, plasmids were extracted, and identified by enzyme digestion and sequencing to obtain the recombinant expression vectors pET-3C-FP-1 and pET-3C-FP-2.

[0066] (3) Induced expression of recombinant proteins The correctly sequenced recombinant expression vectors were transformed into *E. coli* BL21(DE3)pLysS competent cells and plated on LB agar plates containing ampicillin (100 μg / mL), and cultured overnight at 37°C. Single colonies were picked and inoculated into 50 mL of LB liquid medium containing ampicillin, and cultured overnight at 37°C with shaking at 220 rpm to prepare the seed culture. The seed culture was then transferred to 1 L of LB liquid medium containing ampicillin at a 2% (v / v) inoculation rate and cultured at 37°C with 220 rpm until the OD600 reached approximately 0.6–0.8. IPTG was then added to a final concentration of 1 mM, and expression was induced at 30°C for 6 h.

[0067] (4) Purification of recombinant fusion protein After induction, bacterial cells were collected by centrifugation at 4°C and 8000 rpm for 10 min. The cells were resuspended in lysis buffer (50 mMPB, 0.5 M NaCl, 8 M urea, pH 8.0) and homogenized by high-pressure homogenization at 800 bar for 3 cycles. The lysis buffer was centrifuged at 4°C and 13000 rpm for 20 min, and the supernatant was collected.

[0068] The supernatant was loaded at a flow rate of 4 mL / min onto a Ni-NTA affinity chromatography column (100 mL column volume) pre-equilibrated with equilibration buffer (50 mM PB, 0.5 M NaCl, 8 M urea, pH 8.0). Gradient renaturation was performed sequentially with renaturation buffer containing 4 M urea and 2 M urea (50 mM PB, 0.5 M NaCl, pH 8.0), followed by elution with elution buffer (50 mM MPB, 0.5 M NaCl, 400 mM imidazole, 2 M urea, pH 8.0). The elution peak was collected and the buffer was replaced with storage buffer (50 mM PB, 0.5 M NaCl, pH 6.0) using a G25 desalting column to obtain purified recombinant fusion proteins FP-1 and FP-2. The expression results and purification purity of the recombinant fusion proteins FP-1 and FP-2 were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results are shown in the appendix. Figures 5-8 .

[0069] Experimental Results: FP-1 with a theoretical molecular weight of approximately 35.91 kDa and FP-2 with a molecular weight of approximately 46.76 kDa were obtained, with SDS-PAGE purity ≥90%. Protein concentration was determined using the Bradford method, and the purified protein yield per liter of fermentation broth was calculated. The purification yield of FP-1 was approximately 58 mg / L, and the purification yield of FP-2 was approximately 65 mg / L. After on-column gradient refolding and G25 desalting, the protein purity reached over 95% (HPLC-SEC detection).

[0070] The results show that the recombinant fusion proteins FP-1 and FP-2 prepared by this invention can be expressed in a soluble and efficient manner in the Escherichia coli expression system, and the target proteins with high purity and high yield can be obtained by one-step affinity chromatography with Ni-NTA, which shows that it is feasible for industrial-scale production.

[0071] Example 2: Determination of DPPH free radical scavenging ability of recombinant fusion protein Experimental methods The purified FP-1 and FP-2 proteins were prepared into a series of concentration solutions of 0.05, 0.10, 0.25, 0.50, and 1.00 mg / mL using PBS buffer (pH 7.4). Vitamin C (VC) was used as a positive control, and solutions with the same concentration gradient were prepared.

[0072] Take 100 μL each of the protein sample and VC solution at various concentrations and add them to each well of a 96-well plate. Add 100 μL of 0.1 mM DPPPH anhydrous ethanol solution to each well and incubate at room temperature in the dark for 30 min. A blank control group (100 μL PBS + 100 μL DPPH solution) and a background control group (100 μL sample + 100 μL anhydrous ethanol) are also provided. The absorbance of each well is measured at 517 nm. The DPPH scavenging rate is calculated using the following formula: DPPH scavenging rate (%) = [1 - (A sample - A background) / A blank] × 100% Each concentration was set up with 3 replicates, and the experiment was independently repeated 3 times. The experimental results are shown in Table 1.

[0073] Table 1. DPPH radical scavenging rates of FP-1 and FP-2 (%, x±s, n=3)

[0074] Note: Data are expressed as mean ± standard deviation (x ± s), n = 3 (number of independent experiments). One-way ANOVA followed by Tukey's multiple comparison test (with VC as the positive control) was used for comparisons between groups. * indicates P < 0.05, ** indicates P < 0.01.

[0075] The results showed that both recombinant fusion proteins FP-1 and FP-2 exhibited concentration-dependent DPPH radical scavenging capabilities, confirming that the MT-derived functional module endowed the recombinant fusion proteins FP-1 and FP-2 with significant chemical antioxidant activity. Under the same concentration conditions, the DPPH scavenging rate of FP-2 was significantly higher than that of FP-1 (e.g., 68.9% and 56.2%, respectively, at 1.00 mg / mL), indicating that the increase in the copy number of MT functional units (FP-2 contains 7 MTα units, and FP-1 contains 4 MTβ units) can significantly enhance the antioxidant capacity of the recombinant fusion proteins.

[0076] Example 3: Evaluation of the protective effect of recombinant fusion protein against oxidative stress in scalp-related cells Experimental methods Human dermal papillary cells (hDPCs) were used as a scalp-related cell model. hDPC cells were cultured at a rate of 1 × 10⁻⁶. 4 Inoculate the cells at a density of 1 cell / well in 96-well plates and incubate overnight at 37°C and 5% CO2 until adherent.

[0077] Cell safety evaluation: Discard the old culture medium and add fresh culture medium containing different concentrations of FP-1 or FP-2 (0.05, 0.10, 0.25, 0.50, 1.00 mg / mL). A blank control group (culture medium only) was also included. Each group had 6 replicates. After culturing for 24 h, 10 μL of LCK-8 reagent was added to each well, and the cells were incubated at 37°C in the dark for 2 h. The absorbance was measured at 450 nm. Cell viability (%) = (A sample / A blank) × 100%.

[0078] Evaluation of protection against oxidative stress: Adhered hDPC cells were divided into four groups: ① Blank control group (culture medium only); ② Model group (H2O2 added to a final concentration of 0.4 mmol / L); ③ FP-1 treatment group (pretreated with 0.50 mg / mL FP-1 for 2 h, then H2O2 added to a final concentration of 0.4 mmol / L); ④ FP-2 treatment group (pretreated with 0.50 mg / mL FP-2 for 2 h, then H2O2 added to a final concentration of 0.4 mmol / L). After 6 h of H2O2 treatment, the culture medium was discarded, the cells were washed twice with PBS, and serum-free culture medium containing 10 μM DCFH-DA fluorescent probe was added. The cells were incubated at 37°C in the dark for 30 min. After washing with PBS, the intracellular ROS level was detected using a fluorescence microplate reader (excitation wavelength 488 nm, emission wavelength 525 nm). The relative ROS level of the model group was normalized to 100%. The experimental results are shown in Tables 2 and 3.

[0079] Table 2 Effects of FP-1 and FP-2 on cell viability of hDPC cells (%, x±s, n=6)

[0080] Note: Data are expressed as mean ± standard deviation (x ± s), n = 6 (number of independent experiments). One-way ANOVA followed by Dunnett's multiple comparison test was used to compare cell viability in each group with the blank control group; no statistically significant differences were found (P > 0.05).

[0081] Table 3 Effects of FP-1 and FP-2 on intracellular ROS levels in oxidative stress hDPC cells (%, x±s, n=6)

[0082] Note: Data are expressed as mean ± standard deviation (x ± s), n = 6 (number of independent experiments). One-way ANOVA followed by Dunnett's multiple comparison test (with the model group as the control) was used for comparisons between groups. ** indicates P < 0.01 compared to the model group.

[0083] As shown in Table 2, within the test concentration range (0.05~1.00 mg / mL), the cell viability of both FP-1 and FP-2 treatment groups remained above 95%, indicating that neither of the two recombinant fusion proteins was cytotoxic to hDPC cells.

[0084] Table 3 shows that the relative ROS level in the model group significantly increased to 100.00%, while it decreased to 50.56% in the FP-1 treatment group (P<0.01, compared with the model group), and further decreased to 29.54% in the FP-2 treatment group (P<0.01, compared with the model group), approaching the level of the blank control group (29.09%). This conclusion is valid. Figure 9 The effect of recombinant fusion proteins FP-1 and FP-2 on intracellular ROS levels in hDPC cells under oxidative stress is also clearly demonstrated in the figure. FP-2 showed significantly better ROS scavenging than FP-1, consistent with the DPPH antioxidant assay results, further validating the positive contribution of increased MT functional unit copy number to cellular antioxidant protection. These results confirm that the recombinant fusion protein of this invention possesses both basic safety and antioxidant stress protection functions at the cellular level.

[0085] Example 4: Systematic evaluation of the effect of repairing damaged hair Experimental methods Establishment of the damaged hair model: Healthy, naturally straight, black hair bundles from healthy Asian individuals of consistent origin and without significant chemical treatment were selected and grouped according to uniform length (approximately 10 cm) and weight (approximately 2 g). A thermal damage model was established by treating the hair bundles at 180-200℃ for 10-15 seconds per treatment, for a total of 3 treatments. The groups were as follows: normal hair bundle group, thermally damaged hair bundle group (negative control), FP-1 treatment group, and FP-2 treatment group, with 5 bundles in each group (n=5).

[0086] Protein treatment method: Prepare treatment solutions of FP-1 and FP-2 proteins at 2 mg / mL using deionized water. Immerse the heat-damaged hair strands in the treatment solutions at room temperature for 15 minutes, then rinse gently with deionized water for 30 seconds and allow to air dry at room temperature.

[0087] SEM Observation and Scoring: Three segments were randomly selected from each treated hair bundle, pasted onto the sample stage, sputter-coated with gold, and then the surface morphology of the hair was observed using a scanning electron microscope (SEM). Three trained technicians, completely unaware of the sample grouping information (double-blind), scored the cuticle integrity and surface smoothness of each SEM image independently. The final score was the average of the three scores for statistical analysis. The scoring criteria are as follows: Table 4 Scoring Criteria for Cuticle Integrity and Surface Smoothness

[0088] Table 5. SEM observation results of capillary surface morphology in each group (x±s, n=15)

[0089] Note: Data are expressed as mean ± standard deviation (x ± s), n = 15 (5 bundles × 3 regions, the mean of 3 regions in each bundle is taken for statistical analysis). One-way ANOVA followed by Tukey multiple comparison test was used for comparisons between groups. * indicates that P < 0.05 between the FP-1 group, the FP-2 group and the thermal damage group.

[0090] As shown in Tables 4 and 5, heat damage treatment caused large-scale lifting, breakage, and even peeling of the hair cuticles, resulting in extremely poor surface smoothness (both scores were approximately 1.2-1.3). After FP-1 treatment, the cuticle integrity and surface smoothness recovered to 3.5 and 3.3 points respectively, and after FP-2 treatment, they further recovered to 4.1 and 4.0 points, approaching the level of normal hair strands. These results... Figure 10 This has been confirmed. Figure 10 This is a scanning electron microscope (SEM) image of damaged hair repair. The recombinant fusion protein of this invention can effectively adsorb and deposit on the surface of damaged hair, significantly improving the integrity of the hair cuticle and surface smoothness. Among them, FP-2 is more effective than FP-1, consistent with the increasing trend of MT functional unit density. Combined with the data from Examples 2 and 3, this verifies that the recombinant fusion protein has dual functional properties of both scalp cell antioxidant protection and damaged hair structure repair.

[0091] Example 5: Comparative Verification of the Selection Criteria for Intracellular Fragments of COL17A1 Experimental methods Following the method in Example 1, three fusion proteins were constructed (all with 7 MTα functional units fused to the back end, GPGGS linkage, and a 6×His tag at the C-terminus), differing only in the front end COL17A1-derived functional fragment: Fusion protein A: Utilizes the intracellular region aa145-365 of COL17A1 of this invention (i.e., the COL17A1 module of FP-2). Fusion protein B: The extracellular region aa482-698 (corresponding segment of XVII-NT-1) disclosed in CN118496344A was used. Fusion protein C: The extracellular region aa439-698 (corresponding segment of XVII-NT-2) disclosed in CN118496344A was used.

[0092] All three fusion proteins were expressed and purified under the same conditions (BL21(DE3)pLysS, 30℃, IPTG 1mM induction for 6h).

[0093] hDPC cell adhesion activity assay: hDPC cells were sputtered at 5 × 10⁻⁶ m³ / hDPC cells. 4 Cells were seeded at a density of 1 cell / well in 96-well plates pre-coated with each fusion protein (50 μg / mL, overnight at 4°C). After incubation at 37°C and 5% CO2 for 4 h, unattached cells were gently washed away with PBS. The number of adherent cells was detected by CCK-8 assay, with the number of adherent cells in the blank control group (uncoated protein) considered as 100%.

[0094] Table 6. Performance comparison of fusion proteins derived from different COL17A1 fragments.

[0095] Note: The data processing for DPPH, ROS, and cuticle integrity scores was consistent with the previous table. Adhesion rate comparisons were performed using one-way ANOVA followed by Tukey's multiple comparison test. ** indicates that fusion protein A was significantly different from fusion proteins B and C (P < 0.01). No statistically significant differences were found among the groups for the other indicators (P > 0.05).

[0096] As shown in the table above, there were no significant differences in DPPH and ROS clearance rates among the three fusion proteins (due to the identical back-end MT module). However, fusion protein A significantly outperformed fusion proteins B and C in hDPC cell adhesion activity (relative adhesion rates of 185.2%, 135.8%, and 140.3%, respectively, P < 0.01), and also showed a certain advantage in SEM cuticle repair scores. These results confirm that the intracellular region fragment (aa145-365) of COL17A1 has a clear advantage over the extracellular region fragment in the hair follicle-related cell adhesion function dimension. Selecting the intracellular region aa145-365 is not an equivalent result that can be obtained by arbitrarily replacing existing truncated protein sequences. This functional difference stems from the fact that the intracellular region fragment contains a core sequence segment that interacts with the hemidesmosome adhesion complex (BP230 / plectin / integrin β4). This is consistent with the computer-aided screening results of this invention, further confirming that this fragment is the optimal functional fragment, demonstrating the non-obviousness and functional technical effectiveness of fragment selection.

[0097] Example 6: Hair repair composition containing recombinant fusion protein and its efficacy evaluation Serum formulation: Prepare hair repair compositions containing FP-2 according to the formulations in the table below (Formula A and Formula B), with a protein-free matrix as a control (Formula C).

[0098] Table 7 Hair Repair Composition Formulation Table

[0099] Preparation process: Step S1: Add xanthan gum to glycerin and stir at 800 rpm until a uniform, particle-free dispersion is formed. Set aside. Step S2: Mix deionized water, butylene glycol, 1,2-hexanediol, p-hydroxyacetophenone, phenoxyethanol, and flaxseed extract, and heat to 65±2℃ with stirring at 600 rpm. Stir at this temperature for 10-15 minutes until all solids are dissolved. Stop heating and turn on the cooling water circulation to lower the system temperature to 50±2℃. At this temperature, add hydrolyzed keratin and stir at 300 rpm until the hydrolyzed keratin is completely dissolved to obtain mixture a. Step S3: Add the dispersion from step S1 to mixture a and continue stirring at 50±2℃ for 15~20min. Continue cooling to 20~40℃, add the pre-dissolved FP-2 protein solution (dissolved in a small amount of deionized water), gently mix evenly, and adjust the pH to 5.5-6.0 to obtain the hair repair composition.

[0100] Formulation stability test: Formulation A was placed at 4℃, 25℃ (room temperature), and 40℃ for 4 weeks, and samples were taken at 0, 1, 2, and 4 weeks. The DPPH scavenging method (refer to Example 2) was used to detect the retention rate of protein antioxidant activity. The results showed that after 4 weeks of placement at 4℃ and 25℃, the activity retention rate was >90%; after 4 weeks of placement at 40℃, the activity retention rate was 85.2%, indicating that FP-2 has good formulation stability in the serum matrix.

[0101] The strength and anti-breakage efficacy of detached real hair were tested using a tensile test. According to the industry standard for cosmetic efficacy evaluation (QB / T 5108-2017 "Evaluation Method for Hair Damage Repair Efficacy of Cosmetics"), high-temperature damaged detached hair strands with a diameter of 65-75 μm were selected and grouped, with 37 strands tested in each group. The blank control group received no treatment. The sample group hair strands were treated with a hair repair composition sample, residual liquid was removed, and the strands were dried. The tensile strength of the hair strands from the start of stretching to breakage was then measured. A higher tensile strength value indicates better hair toughness and stronger anti-breakage ability. The results are shown in Table 8.

[0102] Table 8 Results of tensile testing on isolated real hair (x±s, n=37)

[0103] Note: Data are expressed as mean ± standard deviation (x ± s), n = 37 (each hair strand is an independent sample), and comparisons between groups were performed using one-way ANOVA followed by Dunnett's multiple comparison test (with the blank control group as the control). * indicates P < 0.05, ** indicates P < 0.01.

[0104] As shown in Table 8, all sample groups containing recombinant fusion proteins (sample groups 1-4) significantly improved the average breaking strength of high-temperature damaged hair compared to the blank control group, and the improvement was significantly higher than that of sample group 5, which only contained basic repair ingredients such as hydrolyzed keratin. Among them: Compared with the blank control group, sample group 5 showed a 33.39% increase in hair breakage capacity, confirming that components such as hydrolyzed keratin can play a certain role in strengthening hair by adsorbing and filling defects on the hair surface. However, the increase was limited and could not meet the repair needs of severely damaged hair.

[0105] Comparing sample groups 1 and 2, it was found that when 8% and 15% of FP-1 protein were added to the basic formula, respectively, the hair breakage resistance increased from 51.77% to 65.65%. Comparing sample groups 3 and 4, when 8% and 15% of FP-2 protein were added to the basic formula, respectively, the increase increased from 71.13% to 84.03%. This indicates that the strengthening and repairing effects of FP-1 and FP-2 on damaged hair exhibit a clear dose-dependent effect; within the experimental concentration range, the higher the protein addition, the more significant the improvement in hair breakage resistance.

[0106] At the same protein addition level, the formulations containing FP-2 (sample groups 3 and 4) showed significantly better results than the formulations containing FP-1 (sample groups 1 and 2). This result is completely consistent with the stronger DPPH free radical scavenging ability, intracellular ROS scavenging ability, and hair cuticle repair effect shown by FP-2 in Examples 2-4, further confirming that the increase in the copy number of MT functional units can simultaneously enhance the hair strengthening and repair efficacy of the fusion protein. The mechanism lies in the fact that more cysteine-rich sites can form denser disulfide bond crosslinks with damaged hair shaft keratin, while more comprehensively repairing the hair cuticle structure and enhancing the mechanical strength of the hair shaft from the inside out.

[0107] Comparing sample groups 3 and 5, it is evident that the combined effect of both is significantly higher than that of hydrolyzed keratin alone. This indicates a significant synergistic repair effect between the recombinant fusion protein and hydrolyzed keratin of this application. Hydrolyzed keratin can rapidly fill macroscopic cracks on the hair surface, while the recombinant fusion protein repairs the broken disulfide bonds between keratin molecules through thiol chemistry. Simultaneously, the COL17A1 functional fragment enhances the adsorption stability of the protein on the hair surface. Together, they achieve deep repair and strengthening of damaged hair structure. Therefore, hair repair compositions containing the recombinant fusion protein (especially FP-2) of this application can significantly repair damaged hair and improve the breakage resistance of hair strands, demonstrating promising application prospects.

[0108] Comparative Example 1: Monofunctional protein containing only the COL17A1 fragment and lacking the MT module Preparation method: Following the method in Example 1, only the COL17-Fx fragment (SEQ ID NO.1, without any MT functional unit) was expressed and purified to obtain a monofunctional protein with a theoretical molecular weight of approximately 23.25 kDa.

[0109] Experimental methods: The DPPH clearance experiment in Example 2 and the hDPC cell oxidative stress protection experiment in Example 3 were followed. The results are as follows.

[0110] Table 9 Comparison of DPPH radical scavenging rates between Comparative Example 1 and FP-1 and FP-2 (%, x±s, n=3)

[0111] Note: Data are expressed as mean ± standard deviation (x ± s), n = 3 (number of independent experiments). One-way ANOVA followed by Tukey's multiple comparison test was used for comparisons between groups. ** indicates that all values ​​are P < 0.01 compared to Comparative Example 1.

[0112] Table 10 Comparison of intracellular ROS levels in hDPCs of Comparative Example 1 with FP-1 and FP-2 (%, x±s, n=6)

[0113] Note: Data are expressed as mean ± standard deviation (x ± s), n = 6 (number of independent experiments). One-way ANOVA followed by Tukey's multiple comparison test (with the model group as the control) was used for comparisons between groups. * indicates P < 0.05, ** indicates P < 0.01. There was no statistically significant difference between the control group and the model group (P > 0.05).

[0114] As shown in Table 9, Comparative Example 1 (a monofunctional protein without the MT module) had a DPPH clearance rate of only 5.2% at a concentration of 1.00 mg / mL.

[0115] Table 10 shows that Comparative Example 1, at a concentration of 0.50 mg / mL, had almost no effect on reducing intracellular ROS levels in hDPC cells (relative ROS was 92.35%, not significantly different from the model group). This indicates that the COL17-Fx fragment itself does not have significant antioxidant function. In contrast, FP-1 and FP-2, after the introduction of the MT functional module, exhibited significant antioxidant capacity. This comparative example confirms that the MT-derived functional module is the main contributor to the antioxidant activity of the fusion protein, and it is indispensable for the formation of a dual-function fusion protein of "scalp care and antioxidant repair".

[0116] Comparative Example 2: Monofunctional protein containing only the MT module and lacking the COL17A1 fragment Preparation method: Following the method in Example 1, only the protein consisting of 7 MTα domain functional units linked by GPGGS linker peptides (excluding the functional fragment derived from COL17A1) was expressed and purified to obtain a multicopy MT protein (denoted as MT-7α) with a theoretical molecular weight of approximately 23.50 kDa.

[0117] Experimental methods: The safety evaluation and oxidative stress protection experiments of hDPC cells were conducted according to Example 3, and the SEM hair repair evaluation method was conducted according to Example 4. The results are as follows.

[0118] Table 11 Comparison of intracellular ROS levels in hDPCs of Comparative Example 2 with FP-1 and FP-2 (%, x±s, n=6)

[0119] Note: Data are expressed as mean ± standard deviation (x ± s), n = 6 (number of independent trials). One-way ANOVA followed by Tukey's multiple comparison test (with the model group as the control) was used for comparisons between groups. ** indicates P < 0.01. There was no statistically significant difference between Comparative Example 2 and FP-2 (P > 0.05).

[0120] Table 12 SEM observation results of capillary surface morphology in each group (x±s, n=5 bundles×3 regions=15)

[0121] Note: Data are expressed as mean ± standard deviation (x ± s). One-way ANOVA followed by Tukey's multiple comparison test (with the thermal damage group as the control) was used for comparisons between groups. * indicates P < 0.05.

[0122] As shown in Table 11, Comparative Example 2 (simple MT multicopy protein) showed no significant difference from FP-2 in terms of antioxidant capacity (relative ROS were 31.18% and 29.54%, respectively), indicating that the MT module is the main source of antioxidant function, consistent with the conclusion of Comparative Example 1.

[0123] Table 12 shows that in the SEM hair repair evaluation, the cuticle integrity score of the Comparative Example 2 treatment group was 3.1±0.7, and the surface smoothness score was 2.9±0.6, both lower than those of the FP-2 treatment group (4.1±0.6 and 4.0±0.5). This indicates that the COL17A1-derived functional fragment enhances or synergistically affects the adsorption of hair surface proteins and interfacial interactions. This comparative example confirms that the COL17A1 module and the MT module in the recombinant fusion protein have a synergistic effect on damaged hair repair, and proteins containing only a single functional module cannot achieve optimal repair results.

[0124] Comparative Example 3: Simple Hybrid Use of Non-Modular Integration Preparation method: COL17-Fx fragment (Comparative Example 1) and MT-7α protein (Comparative Example 2) were prepared according to the methods of Comparative Example 1 and Example 1, respectively, and were mixed in equimolar ratios by physical mixing.

[0125] Experimental methods: The safety evaluation and oxidative stress protection experiments of hDPC cells were performed according to Example 3, and the DPPH scavenging experiment was performed according to Example 2. The FP-2 treatment group prepared in this application was used as a control. Stability testing was also performed: the above samples were treated at 60℃ for 30 min or subjected to 5 freeze-thaw cycles, and the DPPH scavenging rate was then measured, and the activity retention rate was calculated.

[0126] Table 13 Comparative Study of Example 3 and FP-2 (n=3~6)

[0127] Note: Data are expressed as mean ± standard deviation (x ± s). Independent samples t-tests were used for comparisons between groups (FP-2 vs. 3 for each indicator), * indicates P < 0.05. There were no statistically significant differences in DPPH clearance rate and ROS level between the two groups (P > 0.05).

[0128] As shown in the table above, there was no statistically significant difference in DPPH scavenging rate and cellular ROS scavenging activity between the Comparative Example 3 treatment group and the FP-2 treatment group of this application. This is because both groups contain the same total copy number of the MT functional module, further confirming that the MT module is the main source of antioxidant function. However, after heat treatment at 60℃ for 30 min, the DPPH free radical scavenging rate retention rate of the physically mixed group was only 78.2%, while that of the FP-2 treatment group was as high as 94.6%. After repeated freeze-thaw cycles 5 times, the activity retention rate of the Comparative Example 3 treatment group was 71.3%, while that of FP-2 was 92.8%. These results indicate that the activity of the two physically mixed proteins decreased significantly under thermal stress and freeze-thaw conditions, suggesting that the two proteins exist independently in the mixed system, lacking the intramolecular stabilization effect brought about by the linker peptide. In contrast, the FP-2 prepared in this application showed higher structural stability and functional retention rate under the same conditions. This comparative example confirms that the single-molecule design of fusing two functional modules through linker peptides has significant advantages in terms of formulation stability and process tolerance compared to simple physical mixing.

[0129] Comparative Example 4: The COL17-Tβ4 fusion protein disclosed in CN119264276A Preparation method: Recombinant type XVII collagen-thymosin β4 fusion protein (COL17-Tβ4, amino acid sequence as shown in SEQ ID NO.1 of the patent) was expressed and purified in Pichia pastoris according to the method disclosed in CN119264276A.

[0130] Experimental methods: The DPPH clearance experiment in Example 2 and the hDPC cell oxidative stress protection experiment in Example 3 were followed. The results are as follows.

[0131] Table 14 Comparison of DPPH radical scavenging rates between Comparative Example 4 and FP-1 and FP-2 (%, x±s, n=3)

[0132] Note: Data are expressed as mean ± standard deviation (x ± s). Intergroup comparisons were performed using one-way ANOVA followed by Tukey's multiple comparison test (with Comparative Example 4 as the control). ** indicates P < 0.01.

[0133] Table 15 Comparison of intracellular ROS levels in hDPCs of Comparative Example 4 with FP-1 and FP-2 (%, x±s, n=6)

[0134] Note: Data are expressed as mean ± standard deviation (x ± s). One-way ANOVA followed by Dunnett's multiple comparison test (with the model group as the control) was used for comparisons between groups. ** indicates P < 0.01, * indicates P < 0.05.

[0135] Table 14 shows that Comparative Example 4 exhibited a DPPH scavenging rate of only 11.5% at a concentration of 1.00 mg / mL; Table 15 shows that Comparative Example 4 had limited ROS scavenging effect on hDPC cells at a concentration of 0.50 mg / mL (relative ROS scavenging rate of 78.63%); both indicators are significantly weaker than FP-1 and FP-2 of this invention. The fundamental reason for this difference is that the fusion partner of COL17-Tβ4, thymosin β4, does not contain a cysteine-rich region and lacks the metal ion binding and free radical scavenging chemical mechanisms unique to the MT module. This comparative example confirms that, compared to the COL17-thymosin β4 fusion concept in CN119264276A, this invention selects an MT-derived functional fragment as a fusion partner, which can endow the fusion protein with significant antioxidant functions that thymosin β4 cannot provide, reflecting the non-obviousness of the fusion target selection and the unpredictability of the technical effect.

[0136] Comparative Example 5: Using the COL17A1 truncated single protein disclosed in CN118496344A Preparation method: Recombinant human type XVII collagen XVII-NT-1 (amino acid sequence as shown in SEQ ID NO.1 of the patent, corresponding to the extracellular segment of COL17A1 from position 482 to 698) was expressed and purified in Pichia pastoris according to the method disclosed in CN118496344A.

[0137] Experimental methods: The DPPH clearance experiment in Example 2 and the hDPC cell oxidative stress protection experiment in Example 3 were followed. The results are as follows.

[0138] Table 16 Comparison of DPPH radical scavenging rates between Comparative Example 5 and FP-1 and FP-2 (%, x±s, n=3)

[0139] Note: Data are expressed as mean ± standard deviation (x ± s). One-way ANOVA followed by Tukey's multiple comparison test was used for comparisons between groups. ** in the table indicates that P < 0.01 between FP-1 and FP-2 groups and Comparative Example 5.

[0140] Table 17 Comparison of intracellular ROS levels in hDPCs of Comparative Example 5 with FP-1 and FP-2 (%, x±s, n=6)

[0141] Note: Data are expressed as mean ± standard deviation (x ± s). One-way ANOVA followed by Dunnett's multiple comparison test (with the model group as the control) was used for comparisons between groups. ** indicates P < 0.01, * indicates P < 0.05.

[0142] As shown in Table 16, Comparative Example 5 showed a DPPH scavenging rate of only 4.3% at a concentration of 1.00 mg / mL. Table 17 shows that at 0.50 mg / mL, Comparative Example 5 had no significant effect on reducing intracellular ROS levels in hDPC cells (relative ROS was 89.81%, not significantly different from the model group). This protein uses a truncated extracellular fragment (positions 482-698), does not contain a cysteine-rich module, and its functional localization is focused on promoting cell proliferation and hair follicle regeneration, lacking antioxidant function. This comparative example confirms that the truncated COL17A1 single protein disclosed in CN118496344A is a single-function protein, which differs substantially from the fusion protein of this invention, which has both scalp care and hair antioxidant repair functions, in terms of molecular design concept and efficacy. Furthermore, it should be noted that the core functional advantage of the intracellular functional fragment of COL17A1 in this application (as shown in SEQ ID NO.1) compared with the extracellular fragment used in Comparative Example 5 is not in terms of anti-oxidation, but in that it can significantly promote the adhesion activity of hair follicle-related cells (as demonstrated in Example 5), which has higher targeting and biological significance in supporting the homeostasis of the hair follicle microenvironment.

[0143] Comparative Example 6: Using a high molecular weight fusion protein Preparation method: Following the method in Example 1, a fusion protein containing 15 MTα repeat units (COL17-Fx+ 15×MTα, GPGGS linked) was constructed with a theoretical molecular weight of approximately 73.12 kDa.

[0144] Experimental methods: Expression and purification were carried out under the same conditions as in Example 1, and the differences between the soluble expression level and purification yield and FP-2 (theoretical molecular weight approximately 46.76 kDa) were compared.

[0145] Table 18 Comparison of engineering feasibility between Comparative Example 6 and FP-2

[0146] As shown in the table above, under the same expression conditions, the soluble expression level of Comparative Example 6 was significantly lower than that of FP-2, the proportion of inclusion bodies increased significantly, and the final purification yield was only about 30% of that of FP-2. This result directly confirms that the feasibility of engineered soluble expression and purification in E. coli expression systems decreases significantly when the molecular weight exceeds the optimal range.

[0147] In summary, this application successfully constructed a class of bifunctional recombinant fusion proteins that combine scalp follicle microenvironment support with antioxidant repair of damaged hair by modularly fusing functional fragments of the intracellular region of COL17A1 with cysteine-rich functional units derived from metallothionein. Example verification shows that the representative fusion proteins FP-1 and FP-2 can be expressed solublely and efficiently in a prokaryotic expression system, and high-purity target proteins can be obtained through one-step affinity chromatography. In terms of efficacy, FP-1 and FP-2 exhibit concentration-dependent DPPH free radical scavenging activity, significantly reducing ROS levels in human dermal papilla cells under oxidative stress, and effectively improving the cuticle integrity and surface smoothness of heat-damaged hair, demonstrating that the materials possess both antioxidant protection and damaged hair structure repair functions. Compared to control proteins that do not contain the MT module or contain only a single functional module, the fusion protein of this application exhibits superior performance in antioxidant and hair repair functions. Compared to physical mixing methods, the covalently fused single-molecule design demonstrates significant advantages in thermal stability and freeze-thaw tolerance, confirming the synergistic effect and formulation application value of the fusion design. Therefore, this invention provides a recombinant fusion protein with a well-defined structure, synergistic function, and feasible preparation process, offering a new solution for developing integrated products that combine scalp care and hair repair effects, and possessing high industrial application prospects.

[0148] The above are merely embodiments of the present invention and are not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of the present invention should be included within the scope of the claims of the present invention.

Claims

1. A recombinant fusion protein, characterized in that, Include: (i) A functional fragment derived from type 17 collagen COL17A1, wherein the functional fragment is selected from the non-transmembrane fragment of the intracellular region of COL17A1; (ii) A functional fragment derived from metallothionein (MT), wherein the functional fragment is a cysteine-rich MT functional unit; (iii) A linker peptide connecting the COL17A1-derived functional fragment to the MT-derived functional fragment; The fusion direction of the recombinant fusion protein, from the N-terminus to the C-terminus, is as follows: functional fragment derived from COL17A1, linker peptide, and functional fragment derived from MT.

2. The recombinant fusion protein according to claim 1, characterized in that, The recombinant fusion protein structure is selected from: composed of X, L1, and Y sequentially linked; composed of X, L1, and n Y units sequentially connected in series; or composed of X, L1, and n tandem units formed by Y and L2 sequentially linked; wherein... X is a non-transmembrane functional fragment derived from the intracellular region of type 17 collagen COL17A1; Y is a cysteine-rich functional fragment derived from metallothionein MT. L1 is a linker peptide that connects the COL17A1-derived functional fragment to the MT-derived functional fragment; L2 is a linking peptide between adjacent MT-derived functional units; n is an integer from 1 to 8; Furthermore, based on the general formula, the theoretical molecular weight of the recombinant fusion protein is 20kDa to 60kDa.

3. The recombinant fusion protein according to claim 1 or 2, characterized in that, The COL17A1-derived functional fragment includes a sequence segment that has binding activity with hemidesmosome protein BP230 and / or plectin.

4. The recombinant fusion protein according to claim 3, characterized in that, The length of the functional fragment derived from COL17A1 is 20 to 500 amino acids.

5. The recombinant fusion protein according to claim 4, characterized in that, The COL17A1-derived functional fragment is a polypeptide represented by amino acids 145 to 365 of COL17A1, or a functionally equivalent fragment obtained by truncation, elongation, conservative substitution, or repeated splicing.

6. The recombinant fusion protein according to claim 5, characterized in that, The COL17A1 source functional fragment is the amino acid sequence shown in SEQ ID NO.1, or a derived sequence that has at least 70%, 80%, 85%, 90% or 95% sequence identity with SEQ ID NO.1 and retains scalp care and / or hair repair functions.

7. The recombinant fusion protein according to claim 1, characterized in that, The MT-derived functional fragment exists in the form of 1 to 8 MT functional units; when the MT-derived functional fragment contains multiple MT functional units, adjacent MT functional units are directly connected or connected through linker peptides.

8. The recombinant fusion protein according to claim 7, characterized in that, The MT functional unit is selected from at least one of the following: MT full-length functional module, MTβ structural domain functional unit, MTα structural domain functional unit, or engineered mini-MT functional unit.

9. The recombinant fusion protein according to claim 8, characterized in that, The MT functional unit is the MTβ structural domain functional unit shown in SEQ ID NO.2, or the MTα structural domain functional unit shown in SEQ ID NO.

3.

10. The recombinant fusion protein according to claim 1, characterized in that, The linker peptide has a length of 2 to 30 amino acids and is selected from GS, GPGGS, (GGGS)n, (GGS)n, EAAAK repeat sequences or combinations thereof, where n is 1 to 5.

11. The recombinant fusion protein according to claim 1, characterized in that, The fusion protein is any one of the following: (a) It is composed of the COL17A1-derived functional fragment shown in SEQ ID NO.1 and four MTβ domain functional units shown in SEQ ID NO.2 connected in series via GS linker peptides, and its amino acid sequence is shown in SEQ ID NO.6; (b) It is composed of the functional fragment derived from COL17A1 shown in SEQ ID NO.1 and 7 functional units of MTα domain shown in SEQ ID NO.3 connected in series via GPGGS linker peptides, and its amino acid sequence is shown in SEQ ID NO.

7.

12. A method for preparing the recombinant fusion protein as described in claim 1, comprising the following steps: The nucleic acid sequence encoding the fusion protein is inserted into an expression vector to obtain a recombinant expression vector; the recombinant expression vector is then introduced into a host cell. Host cells in which the recombinant expression vector was introduced were cultured to induce the expression of the recombinant fusion protein; the recombinant fusion protein was collected and purified.

13. A nucleic acid encoding the recombinant fusion protein as described in claim 1, characterized in that, The sequence of the nucleic acid is shown in either SEQ ID NO.8 or SEQ ID NO.

9.

14. A recombinant expression vector comprising the nucleic acid as described in claim 13.

15. A host cell into which the recombinant expression vector as described in claim 14 is introduced, characterized in that... The host cells are Escherichia coli, Lactobacillus, Bacillus subtilis, Saccharomyces cerevisiae, Candida albicans, Pichia pastoris, and tobacco cells.

16. A scalp care or hair repair composition comprising the recombinant fusion protein of claim 1, and cosmetically acceptable excipients.

17. The scalp care or hair repair composition according to claim 16, characterized in that, The cosmetic excipients acceptable to the product include thickeners, stabilizers, preservatives, pH adjusters, and polyols.

18. The use of the recombinant fusion protein according to any one of claims 1-11 in the preparation of cosmetics or skin care products for scalp care and / or hair repair.

Images