Recombinant membrane-targeted collagen type 17 and uses thereof

Abstract

The application discloses a recombinant membrane-targeting type 17 collagen protein and application thereof. The application realizes gene fusion of a COL17A1 key function domain and an independent membrane-targeting structure domain for the first time, constructs a novel "activity-targeting" integrated recombinant membrane-targeting type 17 collagen protein (M-C17a1), effectively integrates function activity and cell targeting property through ingenious structure design, and exhibits excellent performance in resisting skin aging, tissue repair and promoting hair growth. The application also proposes a complete preparation method based on a Pichia pastoris expression system. The expression system has high-density fermentation capacity and secretory expression characteristics, significantly simplifies a purification process, and has relatively low production cost, so that the expression system is suitable for large-scale production of the recombinant protein and has good industrial application prospect.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, and more specifically, to a recombinant membrane-targeting type 17 collagen and its applications. Background Technology

[0002] As the largest organ in the human body, the skin's structural integrity and homeostasis depend on the delicate interface between the epidermis and dermis—the dermal-epidermal junction (DEJ). This region is mainly composed of the basement membrane zone (BMZ), which consists of core molecules such as laminin, type IV collagen, and type XVII collagen (COL17A1). It is a key structural basis for maintaining the skin's mechanical stability, cell polarity, and signal transduction.

[0003] Collagen, the most abundant structural protein in animals, possesses excellent tensile strength and elasticity due to its typical triple-helix conformation. COL17A1, a unique transmembrane collagen, is located in the extracellular region of hemidesmosomes in epidermal basal cells, serving as a key "anchoring" molecule connecting intracellular keratin intermediate filaments to extracellular basement membrane components (such as laminin-332). Recent studies have shown that the function of COL17A1 extends far beyond its structural support role. This protein is a core factor in the regulation of epidermal stem cell (EpSC) fate, playing a decisive role in maintaining skin homeostasis, promoting damage repair, and regulating the hair follicle cycle by stabilizing basement membrane adhesion and mediating competitive proliferation among stem cells. Related studies have also found that COL17A1 expression levels decline significantly with age and are closely related to skin aging, tissue atrophy, and impaired hair regrowth. Therefore, targeted activation or supplementation of COL17A1 function has become a highly promising therapeutic strategy in the fields of anti-aging, skin repair, and hair regrowth.

[0004] However, the development of active ingredients based on COL17A1 faces numerous challenges. Firstly, as a transmembrane protein, COL17A1 has a large molecular weight and highly complex structure, making it difficult to extract directly from natural tissues. Simultaneously, its complete recombinant expression suffers from secretion barriers, easy degradation, and misfolding, resulting in low yields and high costs of the active product. To address these bottlenecks, existing research mainly focuses on the recombinant expression of its extracellular functional domains. For example, some patented technologies aim to achieve efficient and soluble expression of this active fragment in heterologous expression systems by selecting specific amino acid sequences, tandem repeat structures, or optimizing codons. More advanced design strategies employ a "membrane-targeted biomimetic" approach, which uses engineering to enable recombinant fragments to simultaneously and specifically bind to targets on both the cell membrane and basement membrane, mimicking the high-affinity "bridging" function of natural hemidesmosomes, thereby hoping to achieve more robust tissue repair effects. Despite some progress made by these methods, a key limitation of existing technologies remains: the vast majority of recombinant COL17A1 fragments lack effective and programmable cell membrane targeting capabilities. These fragments typically exist in the cellular microenvironment as soluble factors, making it difficult to effectively enrich and anchor them on the surface of target cells (such as basal stem cells and hair follicle cells). Consequently, they cannot accurately mimic the transmembrane localization and local signal regulation function of endogenous COL17A1, thus limiting their bioavailability and functional potency.

[0005] Therefore, there is an urgent need in this field for an innovative recombinant protein design strategy that can not only achieve efficient expression of the active fragment but also integrate a highly efficient and specific cell membrane targeting domain. By constructing recombinant proteins that integrate a "functionally active domain" and a "membrane anchoring domain," these proteins can autonomously and precisely locate on the surface of target cell membranes, forming a high-concentration and persistent functional microenvironment locally, thereby more effectively mimicking and enhancing the biological functions of endogenous COL17A1. This strategy provides a novel core material basis for developing next-generation products that are highly effective in anti-aging, repairing, and promoting hair growth in the skin. Summary of the Invention

[0006] To address the technical challenges of existing recombinant type XVII collagen (COL17A1) fragments, such as insufficient membrane targeting ability, low bioavailability, and limited functional potency, this invention proposes an innovative recombinant membrane-targeting type XVII collagen. The core of this invention lies in constructing and expressing a fusion protein using genetic engineering technology. This fusion protein organically combines the key functional active domain of COL17A1 with a highly efficient cell membrane targeting domain, thereby endowing the recombinant product with the ability to autonomously anchor to the target cell membrane and significantly enhancing its biological efficacy.

[0007] To achieve the above objectives, the present invention proposes the following technical solution:

[0008] This invention first proposes a recombinant membrane-targeting type 17 collagen, abbreviated as M-C17a1. The amino acid sequence of this polypeptide includes an extracellular matrix-binding functional region (as the first region) and a cell membrane anchoring region (as the second region) from natural human type 17 collagen (COL17A1). Specifically, the first region is located at the N-terminus of the recombinant protein, and the second region is located at the C-terminus, with the two connected by a flexible linker peptide. This structural design mimics the transmembrane localization mechanism of natural COL17A1, enabling the functional region to accumulate on the cell surface via the membrane anchoring domain, thereby achieving its continuous regulatory effect within a short distance.

[0009] Specifically, the amino acid sequence of M-C17a1 is shown in SEQ ID NO:1. This invention also covers functional variants of the sequence shown in SEQ ID NO:1, which may undergo conserved amino acid substitutions, deletions, or insertions (e.g., 1 to 10 amino acids) in the first region (functional domain), provided that they retain the same or similar biological activities as the sequence of SEQ ID NO:1 in terms of anti-skin aging, promoting cell adhesion, and / or promoting hair growth. Preferably, such functional variants have at least 90% amino acid homology to the sequence of SEQ ID NO:1, more preferably at least 95%, 96%, 97%, 98%, 99%, or 100%. The second region (membrane-targeting domain) may be selected from, but is not limited to, a heparin-binding domain, an integrin-binding domain, or a positively charged membrane-binding peptide to achieve binding mediated by specific recognition of cell membrane components or charge interactions.

[0010] Secondly, the present invention provides a fusion polypeptide comprising the aforementioned M-C17a1. In a preferred embodiment, for ease of purification and detection, one or more (ranging from 1 to 10) histidine (His) tags are fused to the N-terminus and / or C-terminus of M-C17a1. The most preferred fusion polypeptide is fused with one histidine, and its amino acid sequence is shown in SEQ ID NO:2.

[0011] Thirdly, the present invention also provides a nucleic acid molecule encoding the above-mentioned fusion polypeptide, the nucleotide sequence of which is shown in SEQ ID NO:3.

[0012] Fourthly, the present invention provides a recombinant expression vector containing the above-mentioned nucleic acid molecules, specifically, for example, the pPICZ AM-C17a1 plasmid for Pichia pastoris expression constructed in the embodiments.

[0013] Fifthly, the present invention relates to the application of the aforementioned polypeptides, nucleic acid molecules, and carriers containing them. Specifically, these components can be used to prepare cosmetics (skincare products) that promote cell repair, or as functional additives in cell culture media; to prepare cosmetics (skincare products) that delay the natural aging of cells; and to prepare shampoos, hair-strengthening products, and hair-growth preparations that promote hair growth.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The present invention is the first to realize the gene fusion of the key functional domain of COL17A1 and the independent membrane targeting structural domain, and constructs a novel "active-targeting" integrated recombinant membrane-targeting type 17 collagen (M-C17a1). Through ingenious structural design, it effectively integrates functional activity and cell targeting, and exhibits excellent performance in anti-skin aging, tissue repair and hair growth promotion. (1) Highly efficient membrane targeting ability: as in Example 4 ( Figure 6 As shown in the figure, the M-C17a1 described in this invention can specifically and efficiently bind to and accumulate on the membrane surface of target cells (e.g., HaCat cells), while the C17a1 in the control group (lacking membrane targeting domains) is mainly distributed in the culture medium. This membrane localization characteristic forms the basis for its efficient function. (2) Significantly enhanced biological function: Effect of promoting cell adhesion: Example 5 (see Figure 7 and Figure 8 The results showed that M-C17a1 was superior to C17a1 in promoting keratinocyte adhesion, exhibiting a more significant promoting effect. (3) Anti-aging activity: Example 6 (see Figure 9 and Figure 10 The results showed that M-C17a1 could significantly reduce the activity of aging-related β-galactosidase, effectively delaying the aging process of skin fibroblasts, and its anti-aging effect was superior to that of C17a1. (4) Promote hair growth: Animal experiments (Example 7, Figure 11 The results showed that the introduction of M-C17a1 using microneedle technology significantly promoted the activation of hair follicles and hair growth in C57BL / 6 mice. (5) Scalable preparation: This invention proposes a complete preparation method based on the Pichia pastoris expression system (see Examples 1-3). This expression system has high-density fermentation capacity and secretory expression characteristics, significantly simplifies the purification process, and has relatively low production costs, thus making it suitable for the large-scale production of this recombinant protein and showing good prospects for industrial application. Attached Figure Description

[0015] The present invention will be described in detail below with reference to the accompanying drawings.

[0016] Figure 1 This is a schematic diagram illustrating the construction of the Pichia pastoris recombinant expression plasmid pPICZ AM-C17a1 used in this embodiment of the invention.

[0017] Figure 2 The image shows the agarose gel electrophoresis results of the DNA amplification products of the target gene of the M-C17a1 screening strain in this invention; where "M" represents the DNA molecular weight standard marker, "1" is the negative control, "2" is the positive control, and "3~18" correspond to the electrophoretic bands of different monoclonal amplified fragments.

[0018] Figure 3 The screening results of the X33 / pPICZ AM-C17a1 expression strain were analyzed by polyacrylamide gel electrophoresis; where "M" represents the molecular weight standard marker of acrylamide gel electrophoresis, "1" represents the supernatant sample before induction, and "2~10" correspond to the supernatant samples after induction of different monoclonal strains 72 hours later.

[0019] Figure 4 The results are shown in the polyacrylamide gel electrophoresis (PAG) images of the M-C17a1 protein after purification by Ni affinity chromatography. "M" represents the molecular weight standard for acrylamide gel electrophoresis; "1" indicates the sample before loading onto the column, i.e., the supernatant from cell lysis 72 hours after fermentation induction; "2-7" represent the eluting components at different stages after loading; "8-9" represent the impurity peaks eluted with 50 mM imidazole; and "10-11" represent the target protein peaks eluted with 200 mM imidazole.

[0020] Figure 5 The results are from the immunoblotting experiment of M-C17a1 protein; where "M" represents the molecular weight standard marker used in acrylamide gel electrophoresis, and "1" indicates the sample of the target protein.

[0021] Figure 6 This illustrates the interaction between FITC-labeled M-C17a1 protein and the HaCat cell membrane.

[0022] Figure 7 Microscopic images showing the effect of M-C17a1 protein on the adhesion ability of HaCat cells, obtained by crystal violet staining.

[0023] Figure 8 The figure shows the effect of M-C17a1 protein on the adhesion ability of HaCat cells, and the statistical analysis results of cell adhesion rate are presented in the figure.

[0024] Figure 9 Microscopic images showing the anti-aging effect of M-C17a1 protein on human skin fibroblasts (HSF) in a galactose-induced aging model. Senescent cells were detected using β-galactosidase (SA β-Gal) staining.

[0025] Figure 10The figure shows the anti-aging effect of M-C17a1 protein on the galactose-induced aging model HSF, and the statistical analysis results of the β-galactosidase positivity rate in cells.

[0026] Figure 11 The effect of M-C17a1 micro-target on promoting hair growth in C57BL / 6 mice. Detailed Implementation

[0027] To more clearly illustrate the technical solutions of the embodiments of the present invention, the technical solutions of the present invention will be further described in detail below with reference to specific embodiments. However, the above description should not be regarded as any limitation on the present invention. Any changes or substitutions made based on the content disclosed in the present invention should be included within the protection scope of the present invention.

[0028] The present invention will be further illustrated below through specific embodiments:

[0029] Example 1: Construction of pPICZ AM-C17a1 recombinant plasmid

[0030] Based on the polypeptide sequence M-C17a1 (amino acid sequence as shown in SEQ ID NO:1) and the fusion polypeptide containing an N-terminal His tag as shown in SEQ ID NO:2 according to the present invention, their corresponding optimized gene sequences (nucleotide sequences as shown in SEQ ID NO:3) were designed and synthesized. These gene sequences underwent codon optimization to adapt to the expression system of *Pichia pastoris*. The synthesized gene fragment was inserted into the *Pichia pastoris* expression vector pPICZA using restriction endonucleases Xho I and Xba I to obtain the vector pPICZ AM-C17a1.

[0031] Example 2: Expression of M-C17a1 protein

[0032] Recombinant plasmid pPICZ AM-C17a1 was extracted from the DH5α clone strain and linearized using Sac I enzyme digestion. 1 μg of the linearized plasmid was introduced into Pichia pastoris X33 competent cells via electroporation. After transformation, the cells were revived and cultured, and then plated on YPD selective medium containing bleomycin. Colony PCR was used to screen the obtained colonies to identify positive clones. Figure 2Positive clones were sequentially inoculated into YPD and YPG media containing bleomycin for amplification culture. When the OD600 of the culture medium reached 2.0 to 3.0, the bacterial cells were collected, and the cell suspension was adjusted to an OD600 of 1.0. Subsequently, the adjusted cells were transferred to YP media containing methanol and induced for expression for 72 hours. Cell samples were collected before and after induction, and after cell lysis, sonication, and centrifugation, the supernatant was used for SDS-PAGE analysis to detect protein expression (see...). Figure 3 Strains with obvious expression were selected for amplification culture, and the bacterial cells were collected and stored at -20℃ for subsequent purification.

[0033] Example 3: Purification and Identification of M-C17a1 Protein

[0034] The fermented cells were resuspended in 20 mM phosphate buffer (PB) at pH 7.8, and then homogenized at 1000 bar. The lysate was centrifuged at high speed, and the supernatant was collected. The supernatant was purified using a nickel affinity column (Ni Sepharose 6 Fast Flow, GE). During purification, the cells were washed with a 20 mM PB buffer containing 50 mM imidazole at pH 7.4 to remove non-specifically bound impurities; subsequently, the target protein was eluted using the same buffer containing 200 mM imidazole (see [link to purification instructions]). Figure 4 The elution products were finally desalted using a G25 molecular sieve column. The molecular weight and immunological validation of the recombinant protein were performed using SDS-PAGE and Western blotting (see [link to article]). Figure 5 ).

[0035] Example 4: Study on the targeting effect of M-C17a1 protein on HaCat cell membrane

[0036] The purified C17a1 and M-C17a1 protein solutions were lyophilized to prepare protein powder. The protein powder was then redissolved in a 0.1 mol / L Na₂CO₃-NaHCO₃ buffer solution (pH 9.0) to prepare a protein solution with a concentration of 2 mg / mL. 40 μL of a 1 mg / mL fluorescein isothiocyanate (FITC) solution was added to each mL of the protein solution, and the mixture was thoroughly mixed and stirred at 4°C in the dark for 12 hours to obtain FITC-labeled protein products. Subsequently, the FITC-labeled proteins were separated and purified using a Sephadex G-25 dextran gel column to finally obtain C17a1 and M-C17a1 proteins with green fluorescent labeling.

[0037] Suspensions of 1.5 × 10^5 to 2.0 × 10^5 HaCat cells (human immortalized keratinocytes, purchased from ATCC) were seeded into 24-well culture plates and incubated at 37°C with 5% CO2. 2 The cells were cultured under the specified conditions for 24 hours. Then, 0.25 μM of FITC-labeled C17a1 and FITC-labeled M-C17a1 were added, and incubation continued for another 4 hours. After the culture was completed, observation and imaging were performed using a fluorescence microscope, and the results are as follows: Figure 6 As shown.

[0038] Conclusion: M-C17a1 can specifically target the HaCat cell membrane and accumulate significantly at the cell membrane site; in contrast, C17a1 is mainly dispersed in the culture medium and does not show enrichment of the cell membrane.

[0039] Example 5: Experimental study on the adhesion ability of M-C17a1 protein to HaCat cells stained with crystal violet.

[0040] Under aseptic conditions, 200 μL of C17a1 protein solution, M-C17a1 protein solution, and PBS control were added to 24-well plates and air-dried. The HaCat cell concentration was adjusted to prepare cell suspensions containing 2.0 × 10^5 to 2.5 × 10^5 cells per mL. 500 μL of cell suspension was added to each well and incubated at 37°C with 5% CO2 for 4 hours. Cells were then washed twice with PBS, fixed with 4% paraformaldehyde for 20 minutes, and stained with 1% crystal violet for 20 minutes. After staining, cells were washed with PBS. Randomly selected fields of view were photographed under a microscope, and the cell count was then performed and statistical analysis was conducted. The experimental results are as follows: Figure 7 As shown, the statistical analysis results are as follows: Figure 8 .

[0041] Conclusion: Figure 7 and Figure 8 As shown, under the same concentration conditions, M-C17a1 exhibits a superior ability to promote cell adhesion compared to C17a1.

[0042] Example 6: Age-related β-galactosidase (SA β-Gal) staining experiment

[0043] Human skin fibroblasts (HSF, purchased from ATCC) were seeded into 6-well plates, with 5 × 10^5 cells per well. The experiment was divided into the following groups: normal control group (Ctrl, cells cultured in standard medium); D-galactose-induced model group (Model); and C17a1 and M-C17a1 treatment groups, all with a galactose concentration of 50 mM. Twenty-four hours after seeding, the medium was replaced with 0.4% serum containing the corresponding components for each group, and the cells were cultured for another 24 hours at 37°C in a 5% CO2 incubator. Subsequently, the SA β-galactosidase (SA β-Gal) staining kit provided by Beyotime Biotechnology Research Institute was used to detect cell senescence according to the manufacturer's instructions. Cell images were acquired using an inverted microscope, and the total number of cells and the number of SAβ-Gal positive (blue) cells were counted. The proportion of senescent cells is expressed as the percentage of blue-positive cells to the total number of cells. For detailed results, please refer to [link to relevant information]. Figure 9 and Figure 10 .

[0044] Conclusion: Figure 9 and Figure 10 As shown, after co-culturing naturally senescent HSF cells with C17a1 and M-C17a1 for 24 hours, the results showed that both C17a1 and M-C17a1 significantly inhibited β-galactosidase activity associated with natural senescence and promoted the recovery of the viability of senescent cells. Among them, the inhibitory effect of M-C17a1 was more significant.

[0045] Example 7: Germ regrowth experiment in C57BL / C mice

[0046] Seven-week-old male C57BL / 6J mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital. After anesthesia, a 2 cm × 2 cm area of ​​hair was shaved along the spinal line on the back using a razor. Depilatory cream was then applied and left in place for 3 minutes, followed by thorough rinsing with 0.9% saline. The mice were randomly divided into 5 groups of 6 mice each. M-C17a1 protein solution was introduced into the shaved skin using a microneedling device. Photos were taken and observed on days 9, 12, and 15. Results are shown below. Figure 11 .

[0047] Conclusion: On day 9, the skin of mice in the Model group was pink, and hair follicles were still in the resting phase; while in some mice in the MN and M-C17a1 groups, hair follicles had been activated, melanocytes began to synthesize and secrete melanin, and melanin gradually deposited in the skin. On day 12, compared with the Model and MN groups, mice in the M-C17a1 group showed significant hair growth. By day 15, the melanin deposition coverage area in the skin of mice in the 200 μg / mL and 400 μg / mL M-C17a1 groups both exceeded 90%.

Claims

1. A recombinant membrane-targeting type 17 collagen, the amino acid sequence of which is shown in SEQ ID NO:

1.

2. A fusion polypeptide containing the recombinant membrane-targeting type 17 collagen as described in claim 1, characterized in that... The recombinant membrane targets the N-terminus or C-terminus of type 17 collagen by adding 1-10 histidine tags.

3. The fusion polypeptide of claim 2, wherein the amino acid sequence is shown in SEQ ID NO:

2.

4. A nucleic acid molecule encoding the fusion polypeptide of claim 3, the nucleotide sequence of which is shown in SEQ ID NO:

3.

5. A recombinant expression vector comprising the nucleic acid molecule of claim 4.

6. The use of the recombinant membrane-targeting type 17 collagen as described in claim 1 in the preparation of products for promoting cell repair.

7. The use of the recombinant membrane-targeting type 17 collagen as described in claim 1 in the preparation of products for anti-cellular aging.

8. The use of the recombinant membrane-targeting type 17 collagen as described in claim 1 in the preparation of products for promoting hair growth.

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