Highly bioactive and stable recombinant collagen xvii and uses thereof

Abstract

The present patent application discloses a high-bioactivity and high-stability recombinant collagen XVII and its application. The recombinant collagen XVII is constructed by splicing two segments of amino acid sequences derived from natural collagen XVII, repeating multiple times, and adding amino acids at the C-terminal according to the amino acid sequence of natural collagen XVII. The recombinant collagen XVII has high proliferation activity on hair follicle stem cells (HSFC) and high stability, and can be used as an active ingredient of anti-hair loss and / or hair growth products, such as shampoos, hair conditioners, transdermal patches, and injection products such as micro-needle, skin-breaking introduction products, and roller needle introduction products.

Description

Technical Field

[0001] This invention relates to the field of synthetic biology. Specifically, this invention relates to a highly bioactive and stable recombinant type XVII collagen and its application in promoting hair growth. Background Technology

[0002] Hair loss is a common physical and mental health problem that affects quality of life. Hair follicles are microscopic organs that regenerate cyclically, and their homeostasis depends on hair follicle stem cells and their microenvironment (nest). Type XVII collagen (COL17A1, BP180) is a key transmembrane component of hemidesmosomes and plays an indispensable role in maintaining hair follicle stem cell function, anchoring the basement membrane, and regulating the hair follicle cycle.

[0003] Type XVII collagen has a large molecular weight, making recombinant expression of the complete type XVII collagen extremely difficult. Researchers are considering constructing various short proteins derived from type XVII collagen using techniques such as truncation, repetition, and splicing of the amino acid sequence to facilitate their production in recombinant expression systems. Bioactivity and stability are important indicators for evaluating the performance of the obtained short proteins; however, a major technical challenge is that good bioactivity and good stability are not correlated, making it difficult to obtain recombinant type XVII collagen with both good bioactivity and stability. Summary of the Invention

[0004] In view of the above-mentioned technical problems existing in the prior art, the object of the present invention is to provide a recombinant type XVII collagen with good biological activity and good stability.

[0005] The inventors first obtained a highly bioactive amino acid sequence (SEQ ID NO: 1) derived from natural type XVII collagen through screening. They then constructed a highly bioactive recombinant type XVII collagen (SEQ ID NO: 2) by repeatedly repeating this sequence. Studies showed that this highly bioactive recombinant type XVII collagen could be efficiently expressed in an *E. coli* expression system and exhibited superior bioactivity compared to full-length type XVII collagen; however, its stability was poor.

[0006] To address the technical problem of poor stability in the highly bioactive recombinant type XVII collagen, the inventors spliced ​​the highly bioactive amino acid sequence shown in SEQ ID NO: 1 with another amino acid sequence, MGP, also derived from natural type XVII collagen, to obtain a new amino acid sequence (SEQ ID NO: 3). This new amino acid sequence was then repeated five times to obtain a new recombinant type XVII collagen (SEQ ID NO: 4). Studies have shown that this new recombinant type XVII collagen maintains the high bioactivity of the original recombinant type XVII collagen (SEQ ID NO: 2) and exhibits significantly improved stability.

[0007] Moreover, the inventors have demonstrated that by adding a methionine residue (as is done in natural type XVII collagen) to the C-terminus of the aforementioned new recombinant type XVII collagen (SEQ ID NO: 4), the bioactivity of the recombinant type XVII collagen shown in SEQ ID NO: 5 is further enhanced, while maintaining high stability.

[0008] That is, the present invention includes: 1. A polypeptide, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0009] SEQ ID NO: 1 QGPKGQKGSVGDPGMEGPMGQRGREGP 2. Recombinant type XVII collagen, which is composed of the amino acid sequence shown in SEQ ID NO: 1 repeated n times, where n is an integer greater than 1 and less than 50.

[0010] 3. The recombinant type XVII collagen according to item 2, wherein n is an integer greater than or equal to 2 and less than or equal to 10. For example, when n=5, the amino acid sequence of the recombinant type XVII collagen is as shown in SEQ ID NO: 2.

[0011] SEQ ID NO: 2 QGPKGQKGSVGDPGMEGPMGQRGREGP QGPKGQKGSVGDPGMEGPMGQRGREGPQGPKGQKGSVGDPGMEGPMGQRGREGP QGPKGQKGSVGDPGMEGPMGQRGREGPQGPKGQKGSVGDPGMEGPMGQRGREGP 4. Recombinant type XVII collagen, which is composed of the amino acid sequence shown in SEQ ID NO: 3 repeated n times, where n is an integer greater than 1 and less than 50.

[0012] SEQ ID NO: 3 MGPQGPKGQKGSVGDPGMEGPMGQRGREGP 5. The recombinant type XVII collagen according to claim 4, wherein n is an integer greater than or equal to 2 and less than or equal to 10. For example, when n=5, the amino acid sequence of the recombinant type XVII collagen is as shown in SEQ ID NO: 4.

[0013] SEQ ID NO: 4 MGPQGPKGQKGSVGDPGMEGPMGQRGREGP MGPQGPKGQKGSVGDPGMEGPMGQRGREGP MGPQGPKGQKGSVGDPGMEGPMGQRGREGP MGPQGPKGQKGSVGDPGMEGPMGQRGREGP MGPQGPKGQKGSVGDPGMEGPMGQRGREGP 6. A recombinant type XVII collagen, the amino acid sequence of which is shown in SEQ ID NO: 5.

[0014] SEQ ID NO: 5 MGPQGPKGQKGSVGDPGMEGPMGQRGREGP MGPQGPKGQKGSVGDPGMEGPMGQRGREGP MGPQGPKGQKGSVGDPGMEGPMGQRGREGP MGPQGPKGQKGSVGDPGMEGPMGQRGREGP MGPQGPKGQKGSVGDPGMEGPMGQRGREGPM 7. Nucleic acid encoding the above-mentioned polypeptide or recombinant type XVII collagen.

[0015] 8. An expression vector comprising the nucleic acid described in item 7.

[0016] 9. A host cell in which the expression vector described in item 8 has been introduced. The host cell is preferably *Escherichia coli*.

[0017] 10. A method for producing the above-mentioned polypeptide or recombinant type XVII collagen, comprising culturing the host cells described in item 9 to express the polypeptide or recombinant type XVII collagen, and collecting the culture containing the polypeptide or recombinant type XVII collagen.

[0018] 11. The use of the above-mentioned polypeptide or recombinant type XVII collagen in the preparation of products for preventing hair loss and / or promoting hair growth. Preferably, the product is formulated in a form suitable for application to the scalp, such as shampoo, conditioner, transdermal patch, and injectable products such as microneedles, dermal delivery products, and roller delivery products.

[0019] Beneficial effects of the invention 1. The recombinant type XVII collagen of the present invention is a humanized collagen whose amino acid sequence is entirely derived from natural human type XVII collagen without introducing amino acid sequences from other proteins.

[0020] 2. The recombinant type XVII collagen of the present invention, while ensuring that its amino acid sequence is completely derived from natural human type XVII collagen, achieves both high biological activity (e.g., HSFC proliferation activity) and high stability, making it particularly suitable as an active ingredient in anti-hair loss and / or hair growth promotion products, especially injectable products. Attached Figure Description

[0021] Figure 1 SDS-PAGE protein electrophoresis images of the purified recombinant type XVII collagen of sequence 2 (SEQ ID NO: 2), contrast sequence 2 (SEQ ID NO: 6), and contrast sequence 4 (SEQ ID NO: 7).

[0022] Figure 2 SDS-PAGE protein electrophoresis image of purified recombinant type XVII collagen with sequences 4 and 5.

[0023] Figure 3 SDS-PAGE protein electrophoresis images of recombinant type XVII collagen with sequence 2 (SEQ ID NO: 2), contrast sequence 2 (SEQ ID NO: 6), and contrast sequence 4 (SEQ ID NO: 7) after storage at 40°C for different times.

[0024] Figure 4 This is an SDS-PAGE protein electrophoresis image of recombinant type XVII collagen (Sequence 4) after storage at 40°C for different times.

[0025] Figure 5 This is an SDS-PAGE protein electrophoresis image of recombinant type XVII collagen (Sequence 5) after storage at 40°C for different times.

[0026] Figure 6 The graph shows the statistical effects of sequence 2 (SEQ ID NO: 2), contrast sequence 2 (SEQ ID NO: 6), and contrast sequence 4 (SEQ ID NO: 7) on the proliferative activity of hair follicle dermal papilla.

[0027] Figure 7 The graph shows the statistical effects of sequence 2 (SEQ ID NO: 2) and sequence 4 on the proliferative activity of hair follicle dermal papilla.

[0028] Figure 8 This is a statistical graph showing the effects of sequences 4 and 5 on the proliferative activity of hair follicle dermal papilla.

[0029] Figure 9 Electrophoresis diagram of purified protein from the full length of the triple helix region of natural type XVII collagen (amino acids 567-1482 of the a1 chain). Detailed Implementation

[0030] The present patent application is further illustrated below with reference to embodiments. It should be understood that the embodiments are only used to further illustrate and explain the present application and are not intended to limit the present invention.

[0031] Example 1: Recombinant Type XVII Collagen Constructed by Repeating Amino Acid Sequences Derived from Natural Type XVII Collagen Gene synthesis and vector construction Based on the codon preference of *E. coli*, DNA sequences of sequence 2 (SEQ ID NO: 2), contrast sequence 2 (SEQ ID NO: 6), and contrast sequence 4 (SEQ ID NO: 7) were synthesized. These sequences were cloned into the pET-28a(+) vector to construct the corresponding expression plasmids, which were then transformed into *E. coli* BL21(DE3).

[0032] Expression and purification of recombinant proteins (1) Select single clones and inoculate them into LB medium, and incubate at 37°C until the OD600 is about 0.6-0.8. Add IPTG to a final concentration of 0.5 mM and induce expression at 25°C for 16 hours. Collect the bacterial cells by centrifugation and sonicate.

[0033] (2) The lysed bacterial cells were centrifuged and the supernatant was collected. The supernatant was then subjected to salting out and Q-column anion exchange chromatography to obtain high-purity recombinant type XVII collagen. Protein purity and molecular weight were determined by SDS-PAGE. (See attached image). Figure 1 The results showed that a single main band appeared at the expected molecular weight.

[0034] In this embodiment, the following were prepared and purified respectively: (1) Recombinant type XVII collagen of sequence 2 (SEQ ID NO: 2).

[0035] (2) The motif of sequence 1 (SEQ ID NO: 1) was moved forward by 13 amino acids in the natural type 17 collagen sequence to obtain the comparative sequence 1 (also 27 amino acids). The comparative sequence 1 was repeated 5 times to obtain the recombinant type XVII collagen of the comparative sequence 2 (SEQ ID NO: 6).

[0036] Compare sequence 2 (SEQ ID NO: 6): TPGIPGPLGHPGPQGPKGQKGSVGDPGTPGIPGPLGHPGPQGPKGQKGSVGDPGTPGIPGPLGHPGPQGPKGQKGSVGDPGTPGIPGPLGHPGPQGPKGQKGSVGDPGTPGIPGPLGHPGPQGPKGQKGSVGDPG (3) The motif of sequence 1 (SEQ ID NO: 1) was shifted 13 amino acids backward in the natural type 17 collagen sequence to obtain the comparative sequence 3 (also 27 amino acids). The comparative sequence 3 was repeated 5 times to obtain the recombinant type XVII collagen of the comparative sequence 4 (SEQ ID NO: 7).

[0037] Compare sequence 4 (SEQ ID NO: 7): GMEGPMGQRGREGPMGPRGEAGPPGSGGMEGPMGQRGREGPMGPRGEAGPPGSGGMEGPMGQRGREGPMGPRGEAGPPGSGGMEGPMGQRGREGPMGPRGEAGPPGSGGMEGPMGQRGREGPMGPRGEAGPPGSG Example 2: Stability and bioactivity tests of recombinant type XVII collagen 1. Stability test of recombinant type XVII collagen The three recombinant type XVII collagens purified in Example 1 were stored in a 40°C incubator. Samples were taken at the initial (0 months), 3 months, and 6 months, and equal amounts of protein samples were subjected to SDS-PAGE electrophoresis. The results are shown in the figure. Figure 3 .

[0038] 2. Proliferative Activity Test of Recombinant Type XVII Collagen The cell proliferation-promoting activities of several proteins were detected using third-generation hair follicle stem cells (HSFC). A negative control group (without collagen) and three experimental groups containing recombinant type XVII collagen (1 mg / mL) were included, with three replicates per group. HSFC were digested with trypsin and then processed at 5 × 10⁻⁶ ppm. 3Cells were plated at a density of 100 μL, and cell proliferation activity was measured using the CCK8 assay 48 h after collagen addition. Results are as follows: Figure 6 As shown.

[0039] In this embodiment, the bioactivity of sequence 2 (SEQ ID NO: 2), contrast sequence 2 (SEQ ID NO: 6), and contrast sequence 4 (SEQ ID NO: 7) was determined. The bioactivity of sequence 2 (SEQ ID NO: 2) was the best among the three sequences and superior to the full-length triple helix region of natural type XVII collagen (amino acids 567-1482 of the a1 chain of natural type XVII collagen, which was expressed and purified in-house; its protein electrophoresis diagram is shown below). Figure 9 ).

[0040] In this embodiment, the stability of sequence 2 (SEQ ID NO: 2), comparison sequence 2 (SEQ ID NO: 6), and comparison sequence 4 (SEQ ID NO: 7) was determined, and the stability of sequence 2 (SEQ ID NO: 2) was the worst among the three sequences.

[0041] Example 3: Improving the stability of recombinant type XVII collagen shown in SEQ ID NO: 2 by amino acid sequence splicing. To improve the stability of the recombinant type XVII collagen shown in SEQ ID NO: 2, we spliced ​​the MGP tripeptide, which is also derived from natural type XVII collagen, with sequence 1 (SEQ ID NO: 1) to obtain a new motif—sequence 3 (SEQ ID NO: 8). We then repeated sequence 3 (SEQ ID NO: 8) 5 times to obtain a new recombinant type XVII collagen—sequence 4 (SEQ ID NO: 4).

[0042] Sequence 3 (SEQ ID NO: 8) MGPQGPKGQKGSVGDPGMEGPMGQRGREGP The expression and purification were performed as in Example 1 to obtain purified recombinant type XVII collagen of sequence 4 (SEQ ID NO: 4). Its SDS-PAGE electrophoresis image is shown below. Figure 2 .

[0043] The stability and bioactivity of recombinant type XVII collagen of sequence 4 (SEQ ID NO: 4) were determined as in Example 2, and the results are as follows: Figure 4 and Figure 7 As shown. Figure 4The experimental results showed that the recombinant type XVII collagen of sequence 4 (SEQ ID NO: 4) had significantly improved stability compared to the recombinant type XVII collagen of sequence 2 (SEQ ID NO: 2), and was superior to the control sequence 2 (SEQ ID NO: 6) and control sequence 4 (SEQ ID NO: 7). Figure 7 The experimental results showed that the recombinant type XVII collagen of sequence 4 (SEQ ID NO: 4) maintained the high biological activity of the recombinant type XVII collagen of sequence 2 (SEQ ID NO: 2).

[0044] Example 4: Preparation and performance testing of improved recombinant type XVII collagen with high bioactivity and high stability. To verify whether there is room for improvement in the recombinant type XVII collagen of sequence 4 (SEQ ID NO: 4), we added an amino acid M to the C-terminus of sequence 4 (SEQ ID NO: 4) (as is done in natural type 17 collagen) to construct the recombinant type XVII collagen of sequence 5 (SEQ ID NO: 5).

[0045] The expression and purification were performed as in Example 1 to obtain purified recombinant type XVII collagen with sequence 5 (SEQ ID NO: 5). Its SDS-PAGE electrophoresis image is shown below. Figure 2 .

[0046] The stability and bioactivity of recombinant type XVII collagen with sequence 5 (SEQ ID NO: 5) were determined as in Example 2, and the results are as follows: Figure 5 and Figure 8 As shown. The experimental results indicate that the recombinant type XVII collagen of sequence 5 (SEQ ID NO: 5) maintains the stability of the recombinant type XVII collagen of sequence 4 (SEQ ID NO: 4), and its biological activity is further improved compared with that of the recombinant type XVII collagen of sequence 4 (SEQ ID NO: 4).

Claims

1. A polypeptide, the amino acid sequence of which is shown in SEQ ID NO:

1.

2. Recombinant type XVII collagen, which is composed of the amino acid sequence shown in SEQ ID NO: 1 repeated n times, wherein, n is an integer greater than 1 and less than 50.

3. The recombinant type XVII collagen according to claim 2, wherein, The n is an integer greater than or equal to 2 and less than or equal to 10. For example, when n=5, the amino acid sequence of the recombinant type XVII collagen is shown in SEQ ID NO:

2.

4. Recombinant type XVII collagen, which is composed of the amino acid sequence shown in SEQ ID NO: 3 repeated n times, wherein, n is an integer greater than 1 and less than 50.

5. The recombinant type XVII collagen according to claim 4, wherein, The n is an integer greater than or equal to 2 and less than or equal to 10. For example, when n=5, the amino acid sequence of the recombinant type XVII collagen is shown in SEQ ID NO:

4.

6. A recombinant type XVII collagen, the amino acid sequence of which is shown in SEQ ID NO:

5.

7. A nucleic acid encoding the polypeptide of claim 1 or the recombinant type XVII collagen of any one of claims 2 to 6.

8. An expression vector comprising the nucleic acid of claim 7.

9. A host cell in which the expression vector of claim 8 has been introduced. The host cell is preferably *Escherichia coli*.

10. A method for producing the polypeptide of claim 1 or the recombinant type XVII collagen of any one of claims 2 to 6, comprising culturing the host cells of claim 9 to express the polypeptide or the recombinant type XVII collagen, and collecting the culture containing the polypeptide or the recombinant type XVII collagen.

11. Use of the polypeptide of claim 1 or the recombinant type XVII collagen of any one of claims 2 to 6 in the preparation of products for preventing hair loss and / or promoting hair growth. Preferably, the product is formulated in a form suitable for application to the scalp, such as shampoo, conditioner, transdermal patch, etc., as well as injectable products such as microneedles, transdermal delivery products, and microneedle roller delivery products.

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