Bionic recombinant mussel myoglobin of type III and preparation method and application thereof

Abstract

The application discloses a kind of bionic recombination III type Mytilus edulis mucin and its preparation method and application.The recombination III type Mytilus edulis mucin is fused by Mfp-3 of natural Mytilus edulis and the protein that constitutes Mytilus edulis silk fiber skeleton.In the preparation process, fusion gene is co-expressed with tyrosinase gene, and the tyrosine residue in recombination Mytilus edulis mucin is modified with dopa using expressed tyrosinase, to obtain the recombination III type Mytilus edulis mucin rich in dopa group, which can be dissolved and stable at neutral pH, and has high yield, high purity, low endotoxin content, fully meet the application needs in the field of drug, medical device and cosmetic.

Description

A biomimetic recombinant type III mussel adhesive protein, its preparation method and application Technical Field

[0001] This invention belongs to the field of genetic engineering and relates to recombinant mussel adhesive protein formed by the fusion expression of Mfp-3 and byssal fiber skeletal protein fragments of natural mussels, and the modification of tyrosine residues in the recombinant mussel adhesive protein with dopa by co-expression of tyrosinase. Background Technology

[0002] Mussel adhesive protein (MAP), also known as mussel byssal 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 fiber 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 within 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 the formation of strong adhesion in mussels. Waite et al. believe that the adhesive properties of natural mussel adhesive proteins are positively correlated with the content of their DOPA groups.

[0003] Mfp-3 has a relatively small molecular weight, ranging from 5 to 7 kDa, with a dopa group content of 20 mol% to 28 mol%, and also contains a large number of glycine and aspartic acid residues. Mfp-3 possesses structural characteristics such as carrying a high positive charge, dopa groups capable of oxidizing and forming films, and good hydrophobicity. It can form a nanoscale mesh-like microscaffold with antioxidant capabilities, thereby inhibiting inflammation and promoting the adhesion and migration of various cells, making it an ideal medical adhesive and skin repair material. Analysis of the adhesion force of Mfp-3 on mica by Lin et al. showed that Mfp-3 has a very strong binding force with mica, and the adhesion of Mfp-3 is much greater than that of Mfp-1.

[0004] Currently, mussel adhesive protein products are mainly obtained by direct extraction from mussel byssal threads. The commercially available mussel adhesive, Cell Tak... TMAs 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] Because the secretion of natural mussel adhesive protein is very low—approximately 10,000 mussels are needed to extract 1 mg of mussel adhesive protein—direct extraction is inefficient and costly, resulting in expensive products. Furthermore, in vitro culture of mussel foot gland cells is challenging. In the production of mussel adhesive protein using genetically engineered yeast, the recombinant protein obtained directly through fermentation does not contain DOPA groups. Therefore, after in vitro extraction of the recombinant protein, tyrosinase is added, followed by a lengthy modification reaction. For example, Chinese patent CN101948519A describes a recombinant byssal adhesive protein prepared based on the cDNA sequence of the thick-shelled mussel Mfp-3. Furthermore, the study found that, similar to the pH of the solvent used to extract the recombinant protein, mussel adhesive protein with its natural sequence needs to be dissolved in an acidic solution with a pH of around 4.0. Once the pH exceeds this range, the corresponding protein is prone to precipitation when compounded with excipients in topical formulations, such as carbomer, xanthan gum, carboxymethyl cellulose, sodium hyaluronate, and sodium polyglutamate, resulting in precipitation in the compounded system. This seriously affects the application of mussel adhesive protein in topical formulations. Summary of the Invention

[0006] The purpose of this invention is to provide a biomimetic recombinant type III 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 type III mussel adhesive protein, comprising a protein constituting the cytoskeleton of mussels and an Mfp-3 protein expressed in fusion with the protein, wherein some or all of the tyrosine residues of the Mfp-3 protein are modified into dopa groups by co-expressed tyrosinase.

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

[0010] Preferably, the amino acid sequence of the Mfp-3 protein is any one of the different variants of the natural mussel Mfp-3.

[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-3 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-3 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 III mussel adhesive protein.

[0016] Preferably, the nucleic acid molecule specifically includes the fusion gene sequence of the Mfp-3 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 type III mussel adhesive protein, the method comprising the following steps:

[0022] The fusion gene sequence of the Mfp-3 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-3 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. 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 type III mussel adhesive protein in the fermentation culture system.

[0027] 4) Isolate and purify recombinant type III 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 type III 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 type III mussel adhesive protein is used in the preparation of pharmaceuticals, medical devices or cosmetics with healing-promoting effects.

[0032] Preferably, the recombinant type III 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 type III 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 type III 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-3 is positively charged at physiological pH and rich in dopa groups, exhibiting excellent adhesion). Experimental results show that the recombinant type III mussel adhesive protein, by fusing natural mussel Mfp-3 with the protein constituting the byssal fiber skeleton of mussels, has excellent cell migration-promoting activity, is easily dissolved and stable, and can be used as a healing-promoting active ingredient.

[0036] The method for preparing recombinant type III mussel adhesive protein of the present invention utilizes the principle of co-expression, enabling simultaneous expression of recombinant mussel adhesive protein (fusion protein) and dopa modification of its tyrosine residues, thereby obtaining dopa-rich recombinant type III mussel adhesive protein. The process is simple and easy to implement. Furthermore, compared with the expression of small-molecule mussel adhesive protein alone (such as natural mussel Mfp-3), the yield is higher, which can meet the application requirements in the fields of 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-3, 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 high expression of Mfp-3. In addition, the solubility of recombinant type III mussel adhesive protein is significantly improved, and it can remain stable at different pH levels (including at neutral pH) without easily precipitating. 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 type III mussel adhesive protein with high purity and low endotoxin content, which is less costly than extracting natural mussel adhesive protein. Attached Figure Description

[0039] Figure 1 shows the pPIC9K-Mfp3P plasmid map.

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

[0041] Figure 3 shows the electrophoresis diagram of tyrosinase (TYR) and recombinant mussel adhesive protein (Mfp3P) obtained by co-expression shake-flask fermentation.

[0042] Figure 4 shows the electrophoresis diagram of the purified product from the fermentation broth of recombinant type III mussel adhesive protein.

[0043] Figure 5 shows the colorimetric pattern for identifying recombinant type III mussel adhesive protein with dopa.

[0044] Figure 6 shows the migration detection analysis of recombinant type III mussel adhesive protein on cells.

[0045] Figure 7 shows a comparison of the solubility and stability of recombinant type III mussel adhesive protein and commercially available mussel adhesive protein (DOPA-modified recombinant Mfp-3) (after standing at 37°C for 24 hours at pH 7.0). In this figure, bottle A contains commercially available mussel adhesive protein, and bottle B contains recombinant type III mussel adhesive protein.

[0046] Figure 8 shows a comparison of the compatibility of recombinant type III mussel adhesive protein, commercially available mussel adhesive protein (DOPA-modified recombinant Mfp-3), and sodium hyaluronate (room temperature). In this figure, bottle A is a combination of commercially available mussel adhesive protein and sodium hyaluronate, and bottle B is a combination of recombinant type III mussel adhesive protein and 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 type III mussel adhesive protein, which is composed of the fusion of Mfp-3 from natural mussels and proteins constituting the fibrous skeleton of mussel byssal fibers (i.e., adopting a biomimetic mussel byssal structure). To obtain this recombinant type III mussel adhesive protein, the recombinant mussel adhesive protein was expressed using Pichia pastoris host strain, while Bacillus megaterium tyrosinase was co-expressed within the Pichia pastoris host strain to achieve modification by converting tyrosine residues in the recombinant mussel adhesive protein expressed by the Pichia pastoris host strain 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 Mfp3P) consists of the amino acid sequence of the thick-shelled mussel Mfp-3 (GenBank:ACT66140.1, SEQ.ID.NO.3) and a partial peptide segment selected from the purple mussel preCol-P (GenBank:AAB80719.1) (SEQ.ID.NO.4). For the specific amino acid sequence of Mfp3P, please refer to 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 Mfp3P, and following the codon preference of Pichia pastoris, a corresponding nucleotide sequence (i.e., the coding sequence of Mfp3P) was designed. For subsequent molecular manipulation, 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-Mfp3P. The plasmid map is shown in Figure 1. As shown in Figure 1, the recombinant mussel adhesive protein expression vector contains an expression cassette containing the fusion gene sequence (i.e., the coding sequence of Mfp3P). This expression cassette specifically includes the AOX1 promoter, α-factor, KEX2 restriction site, coding sequence of Mfp3P, stop codon, and TT.

[0056] (3) Construction of tyrosinase expression vector

[0057] Based on the amino acid sequence (SEQ.ID.NO.1) of the tyrosinase (TYR) of Bacillus megaterium, 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 of this nucleotide sequence, 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, resulting in the tyrosinase expression vector, named pPICZαA-TYR. The plasmid map is shown in Figure 2. As shown in Figure 2, 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-Mfp3P 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 type III mussel adhesive protein genetic engineering strain. The SDS-PAGE electrophoresis results of the expression product of this engineered strain are shown in Figure 3. The theoretical molecular weight of the recombinant mussel adhesive protein is 34.98 kDa, and the theoretical molecular weight of the tyrosinase is 34.42 kDa. As can be seen from Figure 3, the target protein band is located at Marker 35 kDa, which is consistent with the theoretical expectation, and the tyrosinase band is below Marker 35 kDa, which is also consistent with the theoretical expectation.

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

[0064] The recombinant type III 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, 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 to dopa groups. The yield of recombinant type III mussel adhesive protein was approximately 1.4 g / L.

[0065] 4. Purification and endotoxin removal of recombinant type III 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 type III 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 (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 III mussel adhesive protein was collected. The 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 endotoxin (the retentate was directly loaded onto the column, the endotoxin bound to the column, and the protein flowed through). The percolation solution was collected and freeze-dried to obtain recombinant type III mussel adhesive protein with a purity ≥95%, endotoxin <0.5 EU / mg, and rich in dopa groups. The electrophoretic detection results of the purified recombinant type III mussel adhesive protein are shown in Figure 4.

[0067] 5. Identification and content determination of recombinant type III 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 type III 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] As shown in Figure 5, the recombinant type III mussel adhesive protein obtained by fermentation with co-expressed recombinant strains (referred to as co-expression or modification) has purple spots on the NC membrane, while the recombinant mussel adhesive protein that has not been co-expressed (obtained by fermentation with recombinant mussel adhesive protein expression strains, referred to as non-co-expression or unmodified) 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 type III mussel adhesive protein was tested and found to contain 1.05 wt% dopa groups, which meets the industry standard requirements.

[0083] 6. Cell migration assay (scratch assay)

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

[0085] Reagents: DMEM medium, PBS, 75% ethanol.

[0086] Materials: Cell culture flasks, 6-well plates, pipettes, centrifuge tubes, alcohol lamp, 1mL syringe, 0.22μL filter membrane, steel ruler, marker pen.

[0087] Cells and test samples: mouse fibroblasts (L929), recombinant mussel adhesive protein (unmodified with dopa), recombinant type III mussel adhesive protein (modified with dopa), and dopa-modified recombinant Mfp-3 (commercially available mussel adhesive protein, representing natural mussel Mfp-3).

[0088] Operating procedures:

[0089] ① Laying boards: Place 2×10 6 A number of L929 cells were inoculated into 6-well plates, cultured for 24 hours, streaked with a pipette tip, and washed three times with PBS.

[0090] ② Subsequently, serum-free culture medium containing different concentration gradients of the test samples were added, with the test samples being 50 μg / mL, 100 μg / mL, 200 μg / mL, and 500 μg / mL, respectively. A blank control was set up with only serum-free culture medium added. Images were taken under a microscope after 24, 48, and 72 hours of incubation.

[0091] ③ Perform data analysis on the photos using ImageJ and Prism 8.

[0092] The results are shown in Figure 6. At 48 and 72 hours, the cell migration rate of recombinant type III mussel adhesive protein gradually increased with increasing concentration, and the migration rate at each concentration was significantly higher than that of the blank control. The difference at 200 μg / mL was extremely significant at both 48 and 72 hours. A significant increase in migration rate was observed at 72 hours with recombinant mussel adhesive protein. In contrast, the migration rate of commercially available mussel adhesive protein reached its peak at 50 μg / mL, and then decreased with increasing concentration. At 48 and 72 hours, the migration rate of recombinant type III mussel adhesive protein at all concentrations was significantly higher than that of commercially available mussel adhesive protein at the same concentration. Furthermore, at 48 and 72 hours, the migration rates of recombinant type III mussel adhesive protein at concentrations of 100 μg / mL, 200 μg / mL, and 500 μg / mL were significantly higher than those of commercially available mussel adhesive protein. These results suggest that recombinant type III mussel adhesive protein has a superior wound healing effect compared to natural mussel adhesive protein.

[0093] 7. Recombinant Type III Mussel Adhesive Protein Dissolution and Compounding Test

[0094] This experiment mainly examines the solubility stability of the recombinant type III mussel adhesive protein obtained above and commercially available mussel adhesive protein under different pH conditions, as well as their compatibility with different excipients.

[0095] 7.1 Solubility stability

[0096] Using buffer solutions of different pH values ​​as solvents, the recombinant type III mussel adhesive protein of this invention and commercially available mussel adhesive protein were respectively prepared into 0.1% solutions by mass. After dissolution, the samples were placed in an incubator 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 type III mussel adhesive protein of this invention is stable in dissolution at pH 3.0–9.0 and does not easily precipitate. In contrast, commercially available mussel adhesive protein is stable in dissolution under acidic conditions at pH 3.0 and 4.0, but as the pH increases, it precipitates during standing (see Figure 7).

[0097] Table 1. Observation results of the test solution after standing at 37℃ for 24 hours

[0098]

[0099] 7.2 Compatibility

[0100] 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 type III mussel adhesive protein of this invention or commercially available mussel adhesive protein was added. The solution states were observed, and the results are shown in Table 2. The results indicate that the recombinant type III 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 remains clear. However, commercially available mussel adhesive protein showed no precipitation when compounded with hydroxyethyl cellulose, but flocculent precipitates appeared when compounded with other excipients (see Figure 8).

[0101] Table 2. Observation results after the test sample was compounded with excipients

[0102]

[0103] 8. Application of recombinant type III mussel adhesive protein

[0104] The above method for preparing recombinant type III 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-3 alone. Furthermore, the prepared recombinant type III mussel adhesive protein overcomes the problems of poor stability and difficulty in compounding natural mussel adhesive protein preparations. Therefore, the recombinant type III 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).

[0105] Example 2

[0106] This embodiment provides another biomimetic recombinant type III mussel adhesive protein. In the preparation of this recombinant type III mussel adhesive protein, the amino acid sequence of Mfp-3 is derived from other variants (GenBank: BAB16314.1). The prepared recombinant type III mussel adhesive protein also has the characteristics of high activity, high yield, stable solubility, and easy compounding with excipients.

Claims

1. A biomimetic recombinant type III mussel adhesive protein, characterized in that: This recombinant type III mussel adhesive protein consists of proteins constituting the fibrous backbone of mussel byssal fibers and Mfp-3 protein expressed fused with these proteins. The Mfp-3 protein and the proteins constituting the fibrous backbone are fused in direct tandem. Some or all tyrosine residues of the Mfp-3 protein are modified to dopa groups by co-expressed tyrosinase. The amino acid sequence of the Mfp-3 protein is shown in SEQ.ID.NO.

3. The proteins constituting the fibrous backbone of mussel byssal fibers are selected from natural mussels preCOL-D, preCOL-P, and... The preCOL-NG, PTMP-1, or TMP-1 contains some or all of the Gly-XY structural region peptides; 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 mass percentage of dopa groups in the recombinant mussel adhesive protein obtained by the above fusion expression is ≥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 type III mussel adhesive protein according to claim 1, characterized in that: The mass percentage of dopa groups in the recombinant type III mussel adhesive protein is >1%.

3. The biomimetic recombinant type III 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 type III mussel adhesive protein according to claim 1, characterized in that: The tyrosinase co-expressed with the Mfp-3 protein and the proteins that constitute the byssal fiber skeleton of mussels is derived from humans, fungi, or bacteria.

5. The biomimetic recombinant type III mussel adhesive protein according to claim 1, characterized in that: The recombinant type III 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 type III mussel adhesive protein as described in any one of claims 1 to 5.

7. A method for preparing biomimetic recombinant type III 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-3 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 type III 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.

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