A biomimetic recombinant V-type mussel adhesive protein, its preparation method and application

Abstract

This invention discloses a biomimetic recombinant V-type mussel adhesive protein, its preparation method, and its applications. The recombinant V-type mussel adhesive protein is composed of the Mfp-5 protein from natural mussels and proteins that constitute the fibrous skeleton of mussel byssal fibers. During preparation, the fusion gene and a tyrosinase gene are co-expressed, and the expressed tyrosinase is used to modify the tyrosine residues in the recombinant mussel adhesive protein with dopa, thereby obtaining a bioactive recombinant V-type mussel adhesive protein with high yield, high purity, and low endotoxin content, fully meeting the application requirements of pharmaceuticals, medical devices, and cosmetics.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and relates to the structural design and application of biomimetic mussel byssal protein, specifically involving the fusion expression and dopa modification of Mfp-5 and byssal fiber skeletal protein from natural mussels. Background Technology

[0002] Mussel adhesive protein (MAP), also known as mussel byssal silk protein, is produced and stored in the glands of the mussel's foot. It possesses high strength, high toughness, water resistance, and excellent adhesiveness. A key characteristic of mussel adhesive protein is the presence of a large number of dopa (3,4-dihydroxyphenylalanine, Dopa) groups, and this abundance of dopa groups is a major factor contributing to its adhesive properties. At least 13 mussel adhesive proteins have been studied and identified, including 8 adhesive proteins (Mfp-1, Mfp-2, Mfp-3F, Mfp-3S, Mfp-4, Mfp-5, Mfp-6, and Mfp-7) and 5 byssal silk fibrous skeletal proteins (preCOL-D, preCOL-P, preCOL-NG, PTMP-1, and TMP-1). Among them, preCOL-D, preCOL-P, and preCOL-NG mainly control the core skeleton of mussel byssal fibers and their extension; Mfp-1 covers the surface of the byssal fibers and forms a protective layer, which can prevent other MAPs in the byssal fibers from being dissolved by seawater and degraded by marine microorganisms; Mfp-2 to Mfp-6 are mainly located in the byssal disc of mussels and are the main protein components for mussels to form strong adhesion.

[0003] Mfp-5 has a molecular weight of approximately 9 kDa and contains 20 mol%–30 mol% of dopa groups, making it the mussel adhesive protein with the highest dopa group content discovered to date. It is also rich in serine, glycine, and lysine. Mfp-5 is present in relatively small amounts in the byssal disc, but it has been reported that Mfp-5 may be a byssal protein that directly performs adhesive functions.

[0004] Currently, mussel adhesive protein products are mainly obtained by direct extraction from mussel byssal threads. The commercially available mussel adhesive, Cell Tak... TM As a medical cell and tissue adhesive, it is mainly composed of Mfp-1, Mfp-2 and Mfp-3 extracted from natural mussels. Based on these protein components, it plays a cell adhesion role and is mainly used to adhere cells or tissue slices to the surface of various types of substrates such as plastics, glass, metals, FEP polymers, and biomaterials.

[0005] Compared to extracted mussel adhesive protein, recombinant mussel adhesive protein obtained through genetic engineering exhibits relatively weaker adhesion. To address this, Chinese patent CN105936916A employs a plant expression system, aiming to increase adhesion and achieve large-scale production by leveraging eukaryotic expression to achieve higher levels of glycosylation and phosphorylation in the translated recombinant protein. However, the plant expression system in this patent has a complex construction process and lacks experiments on the fermentation, purification, and dopa modification of the recombinant protein; therefore, the actual expression effect has not been verified. Although there are reports of dopa modification of tyrosine residues in the host cell during prokaryotic expression of mussel adhesive protein, such co-expression modification has not yet been achieved in eukaryotic expression hosts such as yeast. Therefore, after obtaining unmodified recombinant mussel adhesive protein through fermentation, the recombinant protein needs to be extracted in vitro and subjected to a prolonged modification reaction using tyrosinase. Chinese patent CN107084954A discloses a method for detecting tyrosinase, which uses reagents to effectively activate tyrosinase in the detection system, specifically sodium ascorbate and copper sulfate, enabling a detection signal response to be generated within tens of minutes. However, detection systems typically do not include cellular structures. There are currently no reports on the fermentation and dopa modification of recombinant Mfp-5. Furthermore, studies have found that mussel adhesive proteins with natural sequences are prone to precipitation and sedimentation when dissolved at pH levels exceeding approximately 4.0, severely impacting their application in topical formulations. Summary of the Invention

[0006] The purpose of this invention is to provide a biomimetic recombinant V-type mussel adhesive protein, its preparation method, and its application.

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

[0008] A biomimetic recombinant V-type mussel adhesive protein, comprising a protein constituting the fibrous skeleton of mussel byssal fibers and an Mfp-5 protein expressed fused with the protein, wherein some or all of the tyrosine residues of the Mfp-5 protein are modified into dopa groups by co-expressed tyrosinase.

[0009] Preferably, the mass percentage of dopa groups in the recombinant V-type mussel adhesive protein is >1%, meeting the industry standard requirement (≥0.3%).

[0010] Preferably, the amino acid sequence of the Mfp-5 protein is any one of the different variants of the natural mussel Mfp-5.

[0011] Preferably, the protein constituting the byssal fiber skeleton of the mussel is any one of the following: preCOL-D, preCOL-P, preCOL-NG, PTMP-1, and TMP-1, either full-length or truncated.

[0012] Preferably, the protein constituting the byssal fiber skeleton of the mussel is selected from some or all of the Gly-XY structural region peptides contained in natural mussel preCOL-D, preCOL-P, preCOL-NG, PTMP-1 or TMP-1. The repeated Gly-XY in the peptide segment makes the peptide segment satisfy: (1) having a structure and properties similar to collagen, such as X and Y being mainly amino acid residues other than Gly (and not Tyr), and low immunogenicity; (2) since it does not contain Tyr residues, the length of the peptide segment is controlled so that the recombinant mussel adhesive protein obtained by the above fusion expression has or exceeds the dopamine content required by the industry standard after being acted upon by tyrosinase.

[0013] Preferably, the tyrosinase co-expressed with the Mfp-5 protein and the proteins constituting the byssal fiber skeleton of mussels is derived from humans, fungi (e.g., mushrooms) or bacteria.

[0014] Preferably, the tyrosinase co-expressed with the Mfp-5 protein and the proteins constituting the byssal fiber skeleton of mussels is derived from Bacillus megaterium. The amino acid sequence of the tyrosinase from this source is shown in SEQ.ID.NO.1, and the corresponding nucleotide sequence is contained in SEQ.ID.NO.2.

[0015] An expression system for mussel adhesive protein, comprising a host bacterium and a nucleic acid molecule located within the host bacterium for expressing the aforementioned biomimetic recombinant type V mussel adhesive protein.

[0016] Preferably, the nucleic acid molecule specifically includes the fusion gene sequence of the Mfp-5 protein and the protein constituting the byssal fiber skeleton of mussels, and the gene sequence of the tyrosinase. These two gene sequences are co-expressed in the host bacteria, thereby achieving the modification of tyrosine residues in the recombinant mussel adhesive protein into dopa groups.

[0017] Preferably, the nucleic acid molecule further includes a secretory-inducible expression vector backbone for introducing the fusion gene sequence and the tyrosinase gene sequence into the host bacterium, respectively, and the vector backbone includes elements for forming an expression cassette with the corresponding gene sequences.

[0018] Preferably, the host bacteria is selected from any one of yeast, Escherichia coli, and Bacillus subtilis.

[0019] Preferably, the host bacteria is Pichia pastoris or Saccharomyces cerevisiae.

[0020] Preferably, the expression system further includes a tyrosinase modification reaction system comprising 50–150 μM copper sulfate and 5–20 μM ascorbic acid, wherein the reaction system is situated in the fermentation substrate of the host bacteria.

[0021] A method for preparing biomimetic recombinant V-type mussel adhesive protein, the method comprising the following steps:

[0022] The fusion gene sequence of the Mfp-5 protein, the protein constituting the byssal fiber skeleton of mussels, and the gene sequence of tyrosinase were co-expressed in the host bacteria.

[0023] Preferably, the preparation method specifically includes the following steps:

[0024] 1) Construct expression vectors corresponding to recombinant mussel adhesive protein and tyrosinase; wherein the expression vector of recombinant mussel adhesive protein includes the fusion gene sequence of Mfp-5 protein and the protein constituting the skeleton of mussel byssal fibers;

[0025] 2) Transform the host bacteria with the expression vector of recombinant mussel adhesive protein and the expression vector of tyrosinase, and then screen to obtain co-expressing recombinant strains;

[0026] 3) The co-expressed recombinant strain was fermented and cultured. During the fermentation process, the co-expressed tyrosinase and the above-mentioned tyrosinase modification reaction system were used to modify the tyrosine residues in the recombinant mussel adhesive protein into dopa groups, thereby generating recombinant V-type mussel adhesive protein in the fermentation culture system.

[0027] 4) Isolate and purify recombinant V-type mussel adhesive protein.

[0028] Preferably, in step 3, the above-mentioned tyrosinase modification reaction system is added to the fermentation substrate at the beginning of the induction expression.

[0029] Preferably, step 4 specifically includes the following steps: centrifuging the fermentation broth obtained from the fermentation culture, using an ultrafiltration system to remove the fermentation substrate components and pigments from the centrifuged fermentation supernatant, and then sequentially performing cation exchange chromatography, anion exchange chromatography, and freeze drying to obtain pure recombinant V-type mussel adhesive protein.

[0030] Preferably, the elution reagent used in the cation exchange chromatography is a 0.1% acetic acid solution containing 0.1–1.0 M NaCl.

[0031] The above-mentioned biomimetic recombinant V-type mussel adhesive protein is used in the preparation of drugs, medical devices or cosmetics with healing-promoting effects.

[0032] Preferably, the recombinant V-type mussel adhesive protein, as a biomaterial, can be specifically applied to products for treating dermatitis, eczema, acne, skin trauma, and psoriasis, and can also be applied to post-medical aesthetic procedures, scalp care products, scar repair products, stretch mark repair products, etc.

[0033] Preferably, the recombinant V-type mussel adhesive protein, after being dissolved at different pH levels (such as neutral pH), forms a stable compound system with excipients such as carbomer, xanthan gum, carboxymethyl cellulose, hydroxyethyl cellulose, sodium hyaluronate, and sodium polyglutamate.

[0034] The beneficial effects of this invention are reflected in:

[0035] The recombinant V-type mussel adhesive protein of this invention adopts a biomimetic mussel byssal structure (the byssal fiber skeleton of mussels gives the byssal fibers toughness and elasticity, while Mfp-5 is positively charged at physiological pH and rich in dopa groups, exhibiting excellent adhesion). Experimental results show that the recombinant V-type mussel adhesive protein, by fusing natural mussel Mfp-5 with the protein constituting the byssal fiber skeleton of mussels, has excellent cell proliferation-promoting activity and can be used as a healing-promoting active ingredient.

[0036] The method for preparing recombinant V-type mussel adhesive protein of the present invention utilizes the principle of co-expression, enabling the simultaneous expression of recombinant mussel adhesive protein (fusion protein) and dopa modification of its tyrosine residues, thereby obtaining dopa-rich recombinant V-type mussel adhesive protein. The process is simple, easy to implement, and yields high quantities, meeting the needs for applications in pharmaceuticals, medical devices, and cosmetics.

[0037] Furthermore, the proteins constituting the byssal fiber skeleton of mussels in this invention not only increase the toughness and elasticity of Mfp-5, but are also rich in collagen structures (collagen is composed of repeated tandem peptide segments with a Gly-XY structure), which is beneficial for expression in the host bacteria, thereby guiding Mfp-5 to achieve high expression. In addition, the solubility of recombinant V-type mussel adhesive protein is significantly improved, and it can exist stably under different pH conditions (including at neutral pH), with stable dissolution and low precipitation. It can also be stably compounded with external preparation excipients such as carbomer, xanthan gum, carboxymethyl cellulose, sodium hyaluronate, and sodium polyglutamate.

[0038] Furthermore, this invention achieves high expression and dopa modification of recombinant mussel adhesive protein during the secretion process of host bacteria, and combines this with separation and purification processes to obtain recombinant V-type mussel adhesive protein with high purity and low endotoxin content at a low cost. Attached Figure Description

[0039] Figure 1 This is the pPIC9K-Mfp5P plasmid map.

[0040] Figure 2 This is the pPICZαA-TYR plasmid map.

[0041] Figure 3 Electrophoresis diagram of tyrosinase (TYR) co-expressed by shake-flask fermentation and recombinant mussel adhesive protein (Mfp5P).

[0042] Figure 4 Electrophoresis diagram of the purified product from the fermentation broth of recombinant V-type mussel adhesive protein.

[0043] Figure 5 Colorimetric pattern for identifying recombinant V-type mussel adhesive protein with dopa.

[0044] Figure 6 This is a diagram illustrating the effect of recombinant V-type mussel adhesive protein on cell proliferation.

[0045] Figure 7 The figure shows a comparison of the solubility and stability of recombinant V-type mussel adhesive protein and self-made dopamine-modified recombinant Mfp-5 (after standing at 37℃ for 24 hours at pH 7.0). In this figure, bottle A contains dopamine-modified recombinant Mfp-5 and bottle B contains recombinant V-type mussel adhesive protein.

[0046] Figure 8 The image shows a comparison of the compatibility of recombinant V-type mussel adhesive protein, self-made dopa-modified recombinant Mfp-5, and sodium hyaluronate (at room temperature). Bottle A shows the compatibility of dopa-modified recombinant Mfp-5 with sodium hyaluronate, and bottle B shows the compatibility of recombinant V-type mussel adhesive protein with sodium hyaluronate. Detailed Implementation

[0047] The invention will be further described in detail below with reference to the accompanying drawings and embodiments. The embodiments are only used to explain the invention and are not intended to limit the scope of protection of the invention.

[0048] Example 1

[0049] This embodiment provides a biomimetic recombinant V-type mussel adhesive protein, which is composed of the Mfp-5 protein of natural mussels and proteins that constitute the fibrous skeleton of mussel byssal fibers (i.e., it adopts a biomimetic mussel byssal structure). To obtain this recombinant V-type mussel adhesive protein, the recombinant mussel adhesive protein was expressed by fusion with Pichia pastoris host strain, and Bacillus megaterium tyrosinase was co-expressed in the Pichia pastoris host strain to achieve the modification of the recombinant mussel adhesive protein expressed by the Pichia pastoris host strain by converting tyrosine residues into dopa groups (i.e., dopa modification).

[0050] 1. Construction of expression vector

[0051] (1) Sequence structure

[0052] The amino acid sequence of the recombinant mussel adhesive protein (named Mfp5P) consists of the amino acid sequence of the mussel Mfp-5 (GenBank: AAS00463.1, SEQ.ID.NO.3) and a partial peptide segment selected from the mussel preCol-P (GenBank: AAB80719.1) (SEQ.ID.NO.4). The specific amino acid sequence of Mfp5P can be found in SEQ.ID.NO.5.

[0053] The amino acid sequence of the tyrosinase of Bacillus megaterium can be found in SEQ.ID.NO.1.

[0054] (2) Construction of recombinant mussel adhesive protein expression vector

[0055] Based on the amino acid sequence of Mfp5P, and following the codon preference of Pichia pastoris, a corresponding nucleotide sequence (i.e., the coding sequence of Mfp5P) was designed. For subsequent molecular operations, an Xho I restriction endonuclease site CTCGAG and a KEX2 restriction endonuclease site AAAAGA were added to the 5' end of this nucleotide sequence (this site is cleaved when the target protein is secreted into the extracellular space of Pichia pastoris to remove the N-terminal α-factor signal peptide), and a stop codon and a Not I restriction endonuclease site GCGGCCGC were added to the 3' end. The designed nucleotide sequence (SEQ.ID.NO.6) was then synthesized as a whole gene. This synthesized sequence was ligated into the Pichia pastoris vector pPIC9K (Invitrogen, V17520) after double digestion with Xho I and Not I to obtain the recombinant mussel adhesive protein expression vector, named pPIC9K-Mfp5P. The plasmid map is shown below. Figure 1 .according to Figure 1 As shown, the recombinant mussel adhesive protein expression vector contains an expression cassette containing the fusion gene sequence (i.e., the coding sequence of Mfp5P). This expression cassette specifically includes the AOX1 promoter, α-factor, KEX2 restriction site, coding sequence of Mfp5P, stop codon, and TT.

[0056] (3) Construction of tyrosinase expression vector

[0057] Based on the amino acid sequence (SEQ.ID.NO.1) of the Bacillus megaterium tyrosinase (TYR), a corresponding nucleotide sequence (i.e., the coding sequence of TYR) was designed according to the codon preference of Pichia pastoris. To facilitate subsequent molecular manipulation, an Xho I restriction endonuclease site CTCGAG and a KEX2 restriction endonuclease site AAAAGA were added to the 5' end, and a stop codon and a Not I restriction endonuclease site GCGGCCGC were added to the 3' end. The designed nucleotide sequence (SEQ.ID.NO.2) was then synthesized as a whole gene. This synthesized sequence was ligated into the Pichia pastoris vector pPICZαA (Invitrogen, V19020) after double digestion with Xho I and Not I, yielding the tyrosinase expression vector, named pPICZαA-TYR. The plasmid map is shown below. Figure 2 .according to Figure 2 As shown, the tyrosinase expression vector contains a tyrosinase gene sequence expression cassette, which specifically includes the AOX1 promoter, α-factor, KEX2 restriction site, TYR coding sequence, stop codon, and TT.

[0058] 2. Construction of genetically engineered bacteria

[0059] (1) Construction of recombinant mussel adhesive protein expression strain

[0060] The expression vector pPIC9K-Mfp5P was linearized using Sal I restriction enzyme and then electroporated into Pichia pastoris host GS115 competent cells. Positive strains that were correctly transformed were screened using G418 antibiotic, and the expression of positive strains was identified by shaking flasks. The optimal strain was selected for later use.

[0061] (2) Transformation of tyrosinase expression vector

[0062] The recombinant mussel adhesive protein expression strain obtained in the previous step was used to prepare competent cells according to the Pichia pastoris competent cell preparation method ("Pichia pastoris Expression Operation Manual"). The expression vector pPICZαA-TYR was linearized with Sac I restriction enzyme and then electroporated into the competent cells. Zeocin was used to screen for positive transformation strains (i.e., co-expressing recombinant strains). The expression status was identified by shaking flasks, and the optimal co-expressing recombinant strain was selected as the recombinant V-type mussel adhesive protein genetic engineering strain. The SDS-PAGE electrophoresis results of the expression product of this engineered strain are shown in the figure. Figure 3 The theoretical molecular weight of recombinant mussel adhesive protein is 37.24 kDa, and the theoretical molecular weight of tyrosinase is 34.42 kDa. Figure 3 As can be seen, the target protein band is located between Marker 35 kDa and 45 kDa, which is in line with theoretical expectations, and the tyrosinase band is located below Marker 35 kDa, which is also in line with theoretical expectations.

[0063] 3. Fermentation and modification of recombinant mussel adhesive protein

[0064] The recombinant V-type mussel adhesive protein genetically engineered bacteria obtained through screening were fermented using inorganic salt BSM medium as the substrate. The pH was controlled at 5.0, the temperature at 29.0℃, and the dissolved oxygen at 30%. Methanol induction was initiated when the wet weight of the bacterial cells in the 100L fermenter reached 180–200 mg / mL. Simultaneously, 100 μM copper sulfate and 10 μM ascorbic acid were added. The copper ions served as the substrate for tyrosinase reaction, while the addition of ascorbic acid inhibited the conversion of L-DOPA to dopaquinone. After 40–50 hours of fermentation induction, the fermentation broth was transferred to another fermentation tank, completing the fermentation production and the modification of the recombinant mussel adhesive protein by tyrosinase to convert tyrosine residues into dopa groups. The yield of recombinant V-type mussel adhesive protein was approximately 1.1 g / L.

[0065] 4. Purification and endotoxin removal of recombinant V-type mussel adhesive protein

[0066] The fermentation broth was centrifuged, and the supernatant was collected. The supernatant was then concentrated by ultrafiltration using a hollow fiber ultrafiltration system with a molecular weight cutoff of 3.0 kDa. The retentate was collected, and the recombinant V-type mussel adhesive protein in the retentate was purified by gradient elution using an SP cation exchange chromatography column. The loading buffer was a 0.1% aqueous acetic acid solution (0.1% acetic acid, pH 3.5), and the mobile phase was 0.1% acetic acid + 1M... NaCl concentration was automatically adjusted in the gradient elution range of 0.1–1.0 M by the instrument. The eluent containing recombinant type V mussel adhesive protein was collected. This eluent was then desalted and concentrated using a hollow fiber ultrafiltration system with a molecular weight cutoff of 3.0 KD. The retentate was collected and then subjected to DEAE anion exchange chromatography to remove endotoxins (the retentate was directly loaded onto the column, the endotoxins bound to the column, and the protein flowed through). The percolate was collected and freeze-dried to obtain recombinant type V mussel adhesive protein with a purity ≥95%, endotoxin <0.5 EU / mg, and rich in dopa groups. Electrophoretic analysis results of the purified recombinant type V mussel adhesive protein are shown below. Figure 4 .

[0067] 5. Identification and content determination of recombinant V-type mussel adhesive protein dopa

[0068] 5.1 Identification of dopa groups

[0069] 5.1.1 Basis: YY / T 1293.6-2020 Contact Wound Dressings, Part 6 Mussel Adhesive Dressings, Appendix A.

[0070] 5.1.2 Principle of identification experiment: Under alkaline conditions and in the presence of glycine as a reducing agent, the 1,2-benzenediol of DOPA residues in protein molecules can be oxidized into quinone compounds. After the addition of nitrotetrazole blue chloride (NBT), insoluble blue-purple crystal formazan is generated by the reaction of NBT with quinone compounds.

[0071] 5.1.3 Experimental Procedure

[0072] (1) Prepare a 1 mg / mL liquid by purifying the above-purified recombinant V-type mussel adhesive protein with purified water. Take 2 μL of the sample and place it on a 0.2 μm nitrocellulose membrane (NC membrane) of 5 cm × 5 cm and mark the sample position.

[0073] (2) After the sample is absorbed by the NC membrane, place the NC membrane containing the sample in a 500mL beaker, add 300mL of pure water and sonicate for 10min.

[0074] (3) Take out the NC membrane and place it in a culture dish. Add NBT staining solution and stain for 45 min in the dark.

[0075] (4) After taking out the NC membrane, rinse it twice with boric acid solution, store it in sodium borate solution overnight, rinse it three times with purified water, and observe whether blue-purple spots are generated at the sample marking area.

[0076] 5.1.4 Experimental Results

[0077] from Figure 5 As can be seen, the recombinant V-type mussel adhesive protein obtained by fermentation through co-expression of recombinant strains (referred to as co-expression or modification) has purple spots generated on the NC membrane, while the recombinant mussel adhesive protein that has not been co-expressed (obtained by fermentation through recombinant mussel adhesive protein expression strains, referred to as non-co-expression or non-modification) has no obvious purple spots.

[0078] 5.2 Determination of Dopa Group Content

[0079] 5.2.1 Basis: YY / T 1293.6-2020 Contact Wound Dressings, Part 6 Mussel Adhesive Dressings, Appendix B.

[0080] 5.2.2 Principle: Substances containing the 3,4-dihydroxyphenylalanine (DOPA) structure are yellow under acidic conditions, and turn deep orange-red when an excess of alkali is added.

[0081] 5.2.3 Results

[0082] The obtained recombinant V-type mussel adhesive protein was tested and found to contain 1.18 wt% dopa groups, which meets the industry standard requirements.

[0083] 6. Cell proliferation experiment

[0084] Instruments: Clean bench, cell culture incubator, inverted microscope, centrifuge, enzyme-linked immunosorbent assay (ELISA) reader.

[0085] Reagents: DMEM medium (containing 10% fetal bovine serum), PBS, 75% alcohol.

[0086] Materials: Cell culture flasks, 96-well plates, pipettes, centrifuge tubes, alcohol lamp, 10mL syringe, 0.22μL filter membrane, steel ruler, marker pen.

[0087] Cells and test samples: 3T3 mouse fibroblasts, recombinant mussel adhesive protein (unmodified with dopa), recombinant V-type mussel adhesive protein (modified with dopa), and dopa-modified recombinant Mfp-5 (self-made, representing natural mussel Mfp-5).

[0088] Operating procedures:

[0089] (1) 30 3 3 T3 cells per well were inoculated into 96-well plates and cultured for 24 hours. Different concentration gradients (50 μg / mL, 200 μg / mL, 500 μg / mL) of the test sample, namely recombinant mussel adhesive protein (unmodified), recombinant V-type mussel adhesive protein (modified), or dopamine-modified recombinant Mfp-5, were added to the medium diluted with 10% serum. The blank control group was only added to the medium containing 10% serum.

[0090] (2) After culturing for 72 hours, the cell state was recorded using an inverted imaging microscope. Then the culture medium was removed, and 50 μL of MTT solution was added to each well. The cells were incubated at 37°C and 5% CO2 for 2 hours.

[0091] (3) Then remove the MTT solution, add 100 μL of isopropanol to each well, and incubate for 30 min. Measure the absorbance of each well at 570 nm using an ELISA reader.

[0092] (4) Record the data and use Prism 8 for data analysis.

[0093] See results Figure 6Compared with the control, recombinant V-type mussel adhesive protein at concentrations of 50 μg / mL and 500 μg / mL significantly increased cell proliferation rate (p<0.01), and at a concentration of 200 μg / mL, it significantly increased cell proliferation rate (p<0.001). At a concentration of 50 μg / mL, the cell proliferation rate was significantly increased compared with the control (p<0.05), but other concentrations did not show a significant increase. This indicates that dopa modification promotes cell proliferation. Furthermore, dopa-modified recombinant Mfp-5 had no significant effect on promoting cell proliferation, and cell fragmentation occurred with increasing concentration. These results suggest that recombinant V-type mussel adhesive protein has a superior wound healing effect compared to natural mussel adhesive protein.

[0094] 7. Recombinant V-type mussel adhesive protein dissolution and compounding test

[0095] This experiment mainly examines the solubility and stability of the recombinant V-type mussel adhesive protein and the dopamine-modified recombinant Mfp-5 under different pH conditions and their compatibility with different excipients.

[0096] 7.1 Solubility stability

[0097] Using buffer solutions of different pH values ​​as solvents, the recombinant V-type mussel adhesive protein of this invention and the dopamine-modified recombinant Mfp-5 were respectively prepared into 0.1% (w / w) solutions. After dissolution, the samples were incubated at 37°C for 24 hours, and the solution state was observed. The results are shown in Table 1. The results show that the recombinant V-type mussel adhesive protein of this invention is stable in dissolution at pH 3.0–9.0 and does not easily precipitate. While the dopamine-modified recombinant Mfp-5 is stable in dissolution under acidic conditions at pH 3.0 and 4.0, precipitation occurs upon standing as the pH increases (see Table 1). Figure 7 ).

[0098] Table 1. Observation results of the test solution after standing at 37℃ for 24 hours

[0099]

[0100] 7.2 Compatibility

[0101] At room temperature, solutions of 0.1% (w / w) each of carbomer 940, xanthan gum, carboxymethyl cellulose, hydroxyethyl cellulose, sodium hyaluronate, and sodium polyglutamate were prepared. Then, 0.1 wt% of either the recombinant V-type mussel adhesive protein of this invention or DOPA-modified recombinant Mfp-5 was added, and the solution states were observed. The results are shown in Table 2. The results indicate that the recombinant V-type mussel adhesive protein of this invention can be compounded with carbomer 940, xanthan gum, carboxymethyl cellulose, hydroxyethyl cellulose, sodium hyaluronate, and sodium polyglutamate without precipitation and the solution is clear. However, DOPA-modified recombinant Mfp-5 only showed no precipitation reaction when compounded with hydroxyethyl cellulose; when compounded with other excipients, flocculent precipitates appeared (see Table 2). Figure 8 ).

[0102] Table 2. Observation results after the test sample was compounded with excipients

[0103]

[0104] 8. Application of recombinant V-type mussel adhesive protein

[0105] The above method for preparing recombinant V-type mussel adhesive protein not only solves the problems of high cost and low purity in obtaining mussel adhesive protein products through extraction, but also increases the yield compared to expressing the relatively small molecular weight Mfp-5 alone. Furthermore, the prepared recombinant V-type mussel adhesive protein overcomes the problems of poor stability and difficulty in compounding natural mussel adhesive protein preparations. Therefore, the recombinant V-type mussel adhesive protein of this invention, as a highly active biomaterial, can be widely used in medical devices, cosmetics, and pharmaceuticals (such as products for treating dermatitis, eczema, acne, skin trauma, and psoriasis; post-medical aesthetic products; scalp care products; scar repair products; and stretch mark repair products).

[0106] Example 2

[0107] This embodiment provides another biomimetic recombinant V-type mussel adhesive protein. In the preparation of this recombinant V-type mussel adhesive protein, the amino acid sequence of Mfp-5 is derived from other variants (GenBank:ACT66141.2). The obtained recombinant V-type mussel adhesive protein also has the characteristics of high activity, high yield, stable solubility, and easy compatibility with excipients.

Claims

1. A biomimetic recombinant V-type mussel adhesive protein, characterized in that: The recombinant V-type mussel adhesive protein consists of a protein that forms the skeleton of mussel byssal fibers and an Mfp-5 protein expressed in fusion with the protein. Some or all of the tyrosine residues of the Mfp-5 protein are modified into dopa groups by co-expressed tyrosinase. The amino acid sequence of the Mfp-5 protein is shown in SEQ.ID.NO.3; The proteins constituting the byssal fiber skeleton of mussels are selected from some or all of the Gly-XY structural peptides contained in natural mussel preCOL-D, preCOL-P, preCOL-NG, PTMP-1, or TMP-1. The repeated Gly-XY in the peptides satisfy the following: (1) having a structure and properties similar to collagen, where X and Y are amino acid residues other than Gly and not Tyr; (2) the length of the peptides is controlled such that the recombinant mussel adhesive protein obtained by the above fusion expression has a dopa group mass percentage of ≥0.3% after being acted upon by tyrosinase. The amino acid sequence of the recombinant mussel adhesive protein is shown in SEQ.ID.NO.

5.

2. The biomimetic recombinant V-type mussel adhesive protein according to claim 1, characterized in that: The mass percentage of dopa groups in the recombinant V-type mussel adhesive protein is >1%.

3. The biomimetic recombinant V-type mussel adhesive protein according to claim 1, characterized in that: The amino acid sequence of the protein constituting the byssal fiber skeleton of mussels is shown in SEQ.ID.NO.

4.

4. The biomimetic recombinant V-type mussel adhesive protein according to claim 1, characterized in that: The tyrosinase co-expressed with the Mfp-5 protein and the proteins that constitute the byssal fiber skeleton of mussels is derived from humans, fungi, or bacteria.

5. The biomimetic recombinant V-type mussel adhesive protein according to claim 1, characterized in that: The recombinant V-type mussel adhesive protein is soluble and stable within the pH range of 3 to 9.

6. A mussel adhesive protein expression system, characterized in that: The expression system includes a host bacterium and a nucleic acid molecule located within the host bacterium for expressing the biomimetic recombinant V-type mussel adhesive protein as described in any one of claims 1 to 5.

7. A method for preparing biomimetic recombinant V-type mussel adhesive protein as described in any one of claims 1 to 5, characterized in that: Includes the following steps: The fusion gene sequences of Mfp-5 protein, proteins constituting the cytoskeleton of mussel byssal fibers, and tyrosinase gene sequences were co-expressed in the host bacteria.

8. The use of the biomimetic recombinant V-type mussel adhesive protein as described in any one of claims 1 to 5 in the preparation of pharmaceuticals, medical devices or cosmetics with healing-promoting effects.

Images