Method for purifying highly bioactive and stable recombinant collagen type xvii

Abstract

The present patent application discloses a method for purifying high-bioactivity and high-stability recombinant collagen XVII. 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 end according to the amino acid sequence of natural collagen XVII. The recombinant collagen XVII has high proliferation activity for hair follicle stem cells (HSFC) and high stability. The method for purifying the recombinant collagen XVII of the present application is particularly suitable for large-scale purification production of the above-mentioned recombinant collagen XVII, and can obtain purified recombinant collagen XVII with a purity of up to 99% and meeting the requirements of raw materials for injection products.

Description

Technical Field

[0001] This invention relates to the field of biomanufacturing technology. Specifically, this invention relates to a highly bioactive and stable recombinant type XVII collagen and its efficient purification method. 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.

[0004] On the other hand, hair loss prevention / hair growth promotion products based on recombinant type XVII collagen are usually formulated for topical application to the scalp, such as shampoos and conditioners. These topical products are convenient to use, but their effectiveness is limited by the transdermal absorption of collagen. Injectable products, such as microneedles, can effectively solve the technical problem of the poor transdermal absorption of recombinant type XVII collagen, but the preparation of injectable products requires obtaining high-purity recombinant type XVII collagen. Summary of the Invention

[0005] 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, and a purification method specifically for said recombinant type XVII collagen.

[0006] 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.

[0007] 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.

[0008] 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.

[0009] In addition, the inventors have developed a purification method specifically for the recombinant type XVII collagen of this invention. Using this purification method, high-purity recombinant type XVII collagen can be obtained, which meets the quality requirements (purity of 98% or higher) for recombinant type XVII collagen raw materials in injectable anti-hair loss / hair growth products.

[0010] That is, the present invention includes: 1. A polypeptide, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0011] 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.

[0012] 3. The recombinant type XVII collagen according to claim 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.

[0013] 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.

[0014] 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.

[0015] 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.

[0016] SEQ ID NO: 5 MGPQGPKGQKGSVGDPGMEGPMGQRGREGP MGPQGPKGQKGSVGDPGMEGPMGQRGREGP MGPQGPKGQKGSVGDPGMEGPMGQRGREGP MGPQGPKGQKGSVGDPGMEGPMGQRGREGP MGPQGPKGQKGSVGDPGMEGPMGQRGREGPM 7. Nucleic acid encoding the above-mentioned polypeptide or recombinant type XVII collagen.

[0017] 8. An expression vector comprising the nucleic acid of claim 7.

[0018] 9. A host cell in which the expression vector of claim 8 has been introduced. The host cell is preferably *Escherichia coli*.

[0019] 10. A method for producing the above-described polypeptide or recombinant type XVII collagen, comprising culturing the host cells of claim 9 to express the polypeptide or recombinant type XVII collagen, and collecting the culture containing the polypeptide or recombinant type XVII collagen.

[0020] 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.

[0021] 12. The purification method for the above-mentioned recombinant type XVII collagen includes: (1). Take a liquid containing the recombinant type XVII collagen (e.g., fermentation broth) and adjust its pH to 7.0 ± 0.2; (2) Keep the liquid at 60~70℃ (e.g. 65℃) for 1~2 hours (e.g. 1.5 hours); (3). After the above solution has cooled to room temperature, adjust the pH to 3.5±0.2, and then keep it at 50~60℃ (e.g. 55℃) for 1~2 hours (e.g. 1.5 hours); (4) After the above solution has cooled to room temperature, adjust the pH to 7.0±0.2 and set aside. (5) Add ammonium sulfate slowly to the above-mentioned solution and allow it to dissolve completely, according to a weight ratio of solution to ammonium sulfate of 1:0.2~0.5 (e.g., 1:0.3). (6) Centrifuge the above liquid mixture, discard the supernatant, and collect the precipitate; (7) Suspend the above precipitate in 50 mM, pH 7.0 PB buffer and incubate it at 40-50°C (e.g., 45°C) for 0.1-1 hour (e.g., 0.5 hours); (8) After the above solution has cooled to room temperature, centrifuge, discard the precipitate, and collect the supernatant; and (9) The supernatant above is concentrated by ultrafiltration through a membrane with a molecular weight of 5kD and desalted until the conductivity is less than 2ms / cm. The volume is then adjusted to 200mL per 1kg of the solution described in step (1).

[0022] 13. The above purification method further includes: (10) Mix the two solutions according to the volume ratio of the concentrated solution after volume adjustment in step (9) above: solution A = 1:3~5 (e.g. 1:2) to obtain the loading solution; the solution A is 20mM PB buffer with pH 7.0; (11) Load the sample solution onto a chromatography column that has been equilibrated with 10 column volumes of solution A, and perform column chromatography at a flow rate of 0.5 to 1 (e.g., 0.6) column volumes / min; the packing material of the chromatography column is SP Seplife XL (Suzhou Lanxiao Biotechnology Co., Ltd.); (12) Wash with a mixture of solution A and solution B in a volume ratio of 85:15 for 5 column volumes at a flow rate of 0.5 to 1 (e.g., 0.6) column volumes / min; solution B is a 20 mM PB buffer containing 1 M NaCl, pH 7.0; and (13) Elute with a mixture of solution A and solution B in a volume ratio of 70:30 for 12 column volumes at a flow rate of 0.5 to 1 (e.g., 0.6 times) column volumes / min and collect the target eluent.

[0023] 14. The above purification method further includes: (14) The collected target eluent is concentrated and desalted by ultrafiltration using a membrane with a molecular weight of 5 kD, and the transmembrane pressure (TMP) is controlled to be 0.06~0.1 MPa (e.g. 0.08 MPa).

[0024] The target eluent obtained from each 1 kg feed solution described in step (1) can be concentrated and desalted to 100 mL, then the membrane pack can be rinsed with water and finally brought to a final volume of 200 mL.

[0025] 15. The above purification method further includes: (15) The concentrated and desalted protein solution obtained in step (14) is dispensed and freeze-dried.

[0026] 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.

[0027] 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.

[0028] 3. The purification method of the recombinant type XVII collagen of the present invention is particularly suitable for the large-scale purification and production of the recombinant type XVII collagen of the present invention, and can obtain purified recombinant type XVII collagen with a purity of up to 99% that meets the requirements of raw materials for injectable products. Attached Figure Description

[0029] 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).

[0030] Figure 2 SDS-PAGE protein electrophoresis image of purified recombinant type XVII collagen with sequences 4 and 5.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] Figure 8 This is a statistical graph showing the effects of sequences 4 and 5 on the proliferative activity of hair follicle dermal papilla.

[0037] 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).

[0038] Figure 10SDS-PAGE protein electrophoresis images of target protein samples at each stage of crude purification.

[0039] Figure 11 SDS-PAGE protein electrophoresis images of target protein samples at each stage of purification.

[0040] Figure 12 The results are from the liquid chromatography analysis of the sample after crude purification.

[0041] Figure 13 The results are from the liquid chromatography analysis of the purified sample. Detailed Implementation

[0042] 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.

[0043] 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).

[0044] 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.

[0045] (2) The disrupted 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.

[0046] In this embodiment, the following were prepared and purified respectively: (1) Recombinant type XVII collagen of sequence 2 (SEQ ID NO: 2).

[0047] (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).

[0048] 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).

[0049] 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 .

[0050] 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.

[0051] 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 ).

[0052] 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.

[0053] 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).

[0054] Sequence 3 (SEQ ID NO: 8) MGPQGPKGQKGSVGDPGMEGPMGQRGREGP 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 .

[0055] 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).

[0056] 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).

[0057] 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 .

[0058] 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).

[0059] Example 5: Large-scale production of high-purity, high-bioactivity, and high-stability recombinant type XVII collagen 1. Induced expression of target protein (1) Pick a single colony of Escherichia coli BL21(DE3) strain that expresses recombinant type XVII collagen with sequence 5 (SEQ ID NO: 5) prepared in Example 4, add it to 5 mL of LB liquid medium (0.5% yeast extract, 1% peptone and 1% sodium chloride), and incubate at 37°C and 200 rpm for 12 h for activation; (2) Transfer 1% of the inoculum to a 500ml Erlenmeyer flask (containing 200ml of LB culture medium), and incubate at 37℃ and 200rpm for 10h as the seed for the upper tank; (3) Prepare 3L of TB culture medium and add it to a 5L fermenter. Sterilize at 121℃ for 20min. After cooling to 37℃, adjust the pH to 7.0. Add the seed prepared in (2) to the fermenter in the form of flame inoculation for fermentation culture. (4) When the OD600 reaches 30, add 50% glycerol until the OD600 reaches about 60, then add 0.5mM IPTG to induce expression; (5) During the induction process, the dissolved oxygen should be controlled to be no less than 30%, the pH should be 7.0, the induction temperature should be 25℃, and the induction should be carried out for 8-10 hours before the fermentation is stopped.

[0060] 2. Crude purification of the target protein (1). Take 1 kg of fermentation liquid after fermentation in a tank and place it in a 2 L glass bottle, and adjust the pH to 7.0 ± 0.2; (2). Place the 2L glass bottle containing the fermentation liquid in a water bath, set the temperature to 65℃, and start timing after the liquid temperature reaches the set temperature. Remove the bottle after 1.5 hours. (3). After the above solution has cooled to room temperature, adjust the pH to 3.5±0.2, then place it in a water bath and set the temperature to 55℃. Once the solution temperature reaches the set temperature, start timing and remove it after 1 hour. (4) After the above solution has cooled to room temperature, adjust the pH to 7.0±0.2 and set aside. (5). According to the ratio of fermentation liquid (g): ammonium sulfate (g) = 1:0.3, slowly add 300g of ammonium sulfate to the above liquid while stirring to ensure that the ammonium sulfate is completely dissolved, and let stand for 10 minutes; (6). Centrifuge the above liquid at 6000 RPM for 30 min, discard the supernatant, and collect the precipitate into a 2L beaker; (7). Add 1 kg of 50 mM, pH 7.0 PB to the precipitate obtained in the above steps, stir well, place it in a water bath, set the temperature to 45°C, start timing after the temperature of the liquid reaches the set temperature, and take it out after 0.5 h. (8) After the above liquid has cooled to room temperature, centrifuge at 6000 RPM and 30℃ for 30 min, discard the precipitate, and collect the supernatant into a 2L beaker for later use.

[0061] (9) The supernatant was concentrated by ultrafiltration through a membrane with a molecular weight of about 5kD. When the concentration was about 200ml, desalting was started. When the conductivity was less than 2ms / cm, desalting was stopped and the volume was adjusted to 200ml for later use.

[0062] 3. Purification of the target protein 100 ml of SP Seplife XL (Suzhou Lanxiao Biotechnology Co., Ltd.) was used for column chromatography. The purification steps are as follows: (1) Buffer preparation: 1) Solution A: Prepare 5L of 20mM PB solution, adjust the pH to approximately 7.0, filter, and set aside; 2) Solution B: Prepare 5L of 20mM PB + 1M NaCl, adjust the pH to around 7.0, filter, and set aside; 3) Regeneration solution: Prepare 1L of 1M NaOH + 1M NaCl, filter, and set aside; 4) Preservative solution: Prepare 1L of 0.02M sodium hydroxide solution, filter and use for later use.

[0063] (2) Preparation of sample loading solution: Take 20 mL of the concentrated fermentation broth, add 80 mL of solution A, shake well, and adjust the pH to about 7.0. Set aside for later use. (3) Column purification: 1) Equilibration: Equilibrate solution A for 10 column volumes, flow rate 60 mL / min, column pressure difference ≤ 0.3 MPa; 2) Sample loading: Load the sample solution prepared above at a flow rate of 60 mL / min and a column pressure difference of ≤0.3 MPa; 3) Washing: Washing was performed using a mixture of solution A and solution B in a volume ratio of 85:15 for 5 column volumes at a flow rate of 60 mL / min and a column pressure difference ≤0.3 MPa. 4) Elution: Elution was performed using a mixture of solution A and solution B in a volume ratio of 70:30 for 12 column volumes. The target eluent was collected at a flow rate of 60 mL / min and a column pressure difference of ≤0.3 MPa. 5) Regeneration: Wash with 3 column volumes of regeneration solution at a flow rate of 40 mL / min and a column pressure difference ≤ 0.3 MPa.

[0064] 6) Purified water flushing: 6 column volumes were flushed with purified water at a flow rate of 40 mL / min and a column pressure difference of ≤0.3 MPa.

[0065] 7) Preservation: Four column volumes were flushed with 0.02M preservation solution for preservation at a flow rate of 40 kg / h and a column pressure difference of ≤0.3 MPa.

[0066] (4) 5kD membrane-encapsulated ultrafiltration concentration and desalination: 1) Membrane pretreatment: Wash the ultrafiltration membrane with purified water until the permeate and reflux are neutral, and set aside.

[0067] 2) Ultrafiltration concentration: The collected protein solution is concentrated and desalted by ultrafiltration using a treated ultrafiltration system. During ultrafiltration, the transmembrane pressure (TMP) is controlled at 0.08 MPa. When the concentration and desalting reaches 100 mL, the membrane is rinsed with water and finally brought to a final volume of 200 mL.

[0068] 3) Membrane pack post-treatment: First, rinse the membrane pack with 1L of purified water, then rinse it with 0.5mol / L sodium hydroxide solution and cycle 2-3 times. Finally, store the membrane pack in 0.1mol / L sodium hydroxide solution.

[0069] (5) Freeze-drying: The desalted solution was filtered, dispensed, freeze-dried, and the samples were collected.

[0070] 4. Sample Testing (1) Take 1 ml of the target protein sample from each of the above stages and perform SDS-PAGE protein electrophoresis. The results are as follows: Figure 10 and Figure 11 As shown.

[0071] Figure 10 In the table, 1 is the fermentation broth after being placed in the tank; 2 is the sample after the crude purification step (4) of the target protein; 3 is the precipitate and supernatant of the sample after the crude purification step (6) of the target protein; 4 is the precipitate and supernatant of the sample after the crude purification step (8) of the target protein; and 5 is the sample after the ultrafiltration concentration step (9) of the target protein.

[0072] Figure 11 In the table, "Sampling" refers to the prepared sample loading solution; "Flow-through" refers to the eluent of the sample loading solution after column chromatography; "15%" refers to the solution after washing with 15% solution B; "30%" refers to the target eluent after elution with 30% solution B; "Advanced" refers to the target eluent after elution with 30% solution B; "Post-advanced" refers to the sample solution after ultrafiltration and concentration of the collected target eluent; and "Ultra-permeable" refers to the permeate solution after ultrafiltration and concentration of the target eluent.

[0073] Depend on Figure 10 and Figure 11 It can be seen that the recombinant type XVII collagen of the present invention prepared by the above purification method achieved a high purity with only one band on electrophoresis.

[0074] (2) Take the crudely purified and finely purified samples respectively, filter them through a 0.22 μm membrane, and perform high-performance liquid chromatography (HPLC) for detection. Calculate the purity using the area normalization method. The chromatographic conditions are as follows: A hydrophilic spherical silica gel column (7.8 mm × 300 mm, 5 μm or equivalent) was used as the packing material. The mobile phase was 50 mmol / L phosphate buffer solution (adjusted to pH 7.0 ± 0.5). The flow rate was 0.8 mL / min, the column temperature was 25 °C, the detection wavelength was 220 nm, and the injection volume was 10 μL. The detection results are shown below. Figure 12 and Figure 13 .in, Figure 12 The liquid chromatography results of the crudely purified sample show that the target protein content is 94%. Figure 13 The liquid phase detection results of the purified sample show that the target protein content is over 99%.

Claims

1. Recombinant type XVII collagen, consisting 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.

2. The recombinant type XVII collagen according to claim 1, 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.

3. 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.

4. The recombinant type XVII collagen according to claim 3, 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.

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

5.

6. A method for purifying recombinant type XVII collagen according to any one of claims 1 to 5, comprising: (1). Take a liquid containing the recombinant type XVII collagen (e.g., fermentation broth) and adjust its pH to 7.0 ± 0.2; (2) Keep the liquid at 60~70℃ (e.g. 65℃) for 1~2 hours (e.g. 1.5 hours); (3). After the above solution has cooled to room temperature, adjust the pH to 3.5±0.2, and then keep it at 50~60℃ (e.g. 55℃) for 1~2 hours (e.g. 1.5 hours); (4) After the above solution has cooled to room temperature, adjust the pH to 7.0±0.2 and set aside. (5) Add ammonium sulfate slowly to the above-mentioned solution and allow it to dissolve completely, according to a weight ratio of solution to ammonium sulfate of 1:0.2~0.5 (e.g., 1:0.3). (6) Centrifuge the above liquid mixture, discard the supernatant, and collect the precipitate; (7) Suspend the above precipitate in 50 mM, pH 7.0 PB buffer and incubate it at 40-50°C (e.g., 45°C) for 0.1-1 hour (e.g., 0.5 hours); (8) After the above solution has cooled to room temperature, centrifuge, discard the precipitate, and collect the supernatant; and (9) The supernatant above is concentrated by ultrafiltration through a membrane with a molecular weight of 5kD and desalted until the conductivity is less than 2ms / cm. The volume is then adjusted to 200mL per 1kg of the solution described in step (1).

7. The purification method according to claim 6, further comprising: (10) Mix the two solutions according to the volume ratio of the concentrate after volume adjustment in step (9) above: solution A = 1:3~5 (e.g. 1:2) to obtain the loading solution; solution A is 20mM PB buffer with pH 7.

0. (11) Load the sample solution onto a chromatography column that has been equilibrated with 10 column volumes of solution A, and perform column chromatography at a flow rate of 0.5 to 1 (e.g., 0.6) column volumes / min; the packing material of the chromatography column is SP Seplife XL; (12) Wash with a mixture of solution A and solution B in a volume ratio of 85:15 for 5 column volumes at a flow rate of 0.5 to 1 (e.g., 0.6) column volumes / min; solution B is a 20 mM PB buffer containing 1 M NaCl, pH 7.0; and (13) Elute 12 column volumes of a mixture of solution A and solution B with a volume ratio of 70:30 at a flow rate of 0.5 to 1 (e.g., 0.6 times) column volume / min and collect the target eluent.

8. The purification method according to claim 7, further comprising: (14) The collected target eluent is concentrated and desalted by ultrafiltration using a membrane with a molecular weight of 5 kD, and the transmembrane pressure (TMP) is controlled to be 0.06~0.1 MPa (e.g. 0.08 MPa).

9. The purification method according to claim 8, further comprising: (15) The concentrated and desalted protein solution obtained in step (14) is dispensed and freeze-dried.

10. Use of the recombinant type XVII collagen prepared by the purification method according to any one of claims 6 to 9 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, transdermal delivery products, and microneedle roller delivery products.

Images