Recombinant mytilus edulis mucin polypeptides, methods of making and uses thereof

Abstract

This application discloses a recombinant mussel adhesive protein polypeptide, its preparation method, and its application, relating to the field of genetic engineering technology. The amino acid sequence of the recombinant mussel adhesive protein polypeptide is as shown in SEQ ID NO.1; or a sequence variant obtained by substitution, deletion, or addition of one or more amino acids based on SEQ ID NO.1, exhibiting melanin production inhibitory activity. The recombinant mussel adhesive protein polypeptide proposed in this application possesses melanin production inhibitory activity. Compared to long-chain proteins, it has a smaller molecular weight, better solubility, and is less prone to aggregation, enabling effective compounding with various pharmaceutical or materials science excipients, thus overcoming the bottleneck of difficult compounding of long-chain proteins.

Description

Technical Field

[0001] This application relates to the field of genetic engineering technology, and in particular to a recombinant mussel adhesive protein polypeptide, its preparation method, and its application. Background Technology

[0002] Mussel adhesive proteins have broad application prospects in fields such as biomedical materials due to their excellent biocompatibility and underwater adhesion properties. However, the yield from natural extraction is extremely low and the cost is high. Therefore, recombinant expression using genetic engineering techniques has become a major research direction.

[0003] In existing technologies, plasmid vectors are commonly used to express recombinant mussel adhesive proteins or their truncated variants in hosts such as *E. coli*. However, this method has significant drawbacks: First, due to the unique nature of the mussel adhesive protein's natural sequence (e.g., high GC content, complex secondary structure), its expression level is generally low, making it difficult to meet the yield requirements of industrial production. Second, the expressed products are mostly long-chain proteins with large molecular weights and strong hydrophobicity, resulting in poor compatibility with various excipients such as thickeners, surfactants, and preservatives in practical applications. This leads to aggregation and precipitation, making compounding difficult and product performance unstable. Furthermore, plasmid vector-dependent expression systems inherently suffer from poor genetic stability. Plasmids are easily lost without antibiotic selection pressure, and expression levels decrease with each generation during passaging. Continuous antibiotic addition introduces cost and safety risks.

[0004] Therefore, existing technologies still need improvement. Summary of the Invention

[0005] The main purpose of this application is to propose a recombinant mussel adhesive protein polypeptide, its preparation method and application, aiming to solve or at least partially alleviate the problems of low expression level of recombinant mussel adhesive protein, difficulty in compounding long-chain proteins with excipients and poor genetic stability of plasmid expression systems in the prior art.

[0006] To achieve the above objectives, in a first aspect, this application proposes a recombinant mussel adhesive protein polypeptide, the amino acid sequence of which is as follows: The sequence shown in SEQ ID NO.1; or a sequence variant obtained by substitution, deletion or addition of one or more amino acids based on SEQ ID NO.1, and having melanin production inhibitory activity.

[0007] In some embodiments, the amino acid sequence of the sequence variant is selected from SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4.

[0008] Secondly, this application also proposes a nucleic acid molecule comprising a nucleotide sequence encoding a tandem repeat protein, which, upon cleavage by a protease, releases the recombinant mussel adhesive protein polypeptide proposed in the first aspect of this application, wherein the nucleotide sequence comprises, from the 5' end to the 3' end: At least two copies of the coding region, each coding region encoding a polypeptide with an amino acid sequence as shown in any one of SEQ ID NO. 1-4; and A linker coding region located between any two adjacent coding regions, the linker coding region encoding a protease recognition site.

[0009] In some implementations, the number of copies of the coding region is 2-25; Preferably, the number of copies of the encoding region is 6-16; More preferably, the number of copies of the coding region is 8 or 12.

[0010] Thirdly, this application also proposes a recombinant expression vector, which includes the nucleic acid molecule proposed in the second aspect of this application.

[0011] Fourthly, this application also proposes a recombinant engineered bacterium, the genome of which integrates the nucleic acid molecules proposed in the second aspect of this application.

[0012] In some embodiments, the recombinant engineered bacteria is Escherichia coli; the nucleic acid molecule is integrated into one or more sites in the genome of the recombinant engineered bacteria, the sites being selected from any one or more of the codA, ptsG, ydgA, yeiI, yfhL, yhaK, yiiM and IS186 sites.

[0013] Fifthly, this application also proposes a method for preparing recombinant mussel adhesive protein polypeptide, characterized by comprising the following steps: Step S1: Construct the recombinant engineered bacteria proposed in the fourth aspect of this application; Step S2: Cultivate the recombinant engineered bacteria and induce expression to obtain a culture; Step S3: The tandem protein is recovered from the culture. Step S4: The tandem protein is subjected to enzymatic digestion and purification to obtain the recombinant mussel adhesive protein polypeptide.

[0014] In some embodiments, in step S1, the nucleic acid molecule is integrated into the genome of the recombinant engineered bacteria via homologous recombination or CRISPR / Cas9 gene editing technology.

[0015] In some embodiments, step S4 includes enzymatic digestion of the tandem protein using trypsin.

[0016] In a sixth aspect, this application also proposes a composition comprising the recombinant mussel adhesive protein polypeptide proposed in the first aspect of this application, and pharmaceutically or materials-acceptable excipients.

[0017] In some embodiments, the pharmaceutically or materials-acceptable excipients include one or more of thickeners, surfactants, preservatives, crosslinking agents, and buffer salts.

[0018] Seventhly, this application also proposes the use of the recombinant mussel adhesive protein polypeptide proposed in the first aspect of this application in the preparation of products for inhibiting melanin production.

[0019] In some embodiments, the product for inhibiting melanin production includes skin whitening products, medications for treating pigmentation-related diseases, medical dressings, or tissue-engineered materials.

[0020] The recombinant mussel adhesive protein polypeptide proposed in this application is a decapeptide composed of 10 amino acids. Compared with long-chain proteins, it has a small molecular weight, good solubility, and is not prone to aggregation. Therefore, it can be effectively compounded with a variety of pharmaceutical or materials science excipients, overcoming the bottleneck of difficult compounding of long-chain proteins. More importantly, this application clarifies for the first time that the provided recombinant mussel adhesive protein polypeptide has melanin production inhibitory activity, while its longer protein sequences (including natural full-length proteins or tandem proteins) lack this activity. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0022] Figure 1 The plasmid pDonor-8 provided in this application; Figure 2 The plasmid map of plasmid pQCasTns(Ptr)-IS186 provided in this application; Figure 3 The plasmid map of plasmid pQCasTns(Ptr)-array8 provided in this application; Figure 4 The plasmid map of the plasmid pCutamp provided in this application; Figure 5 This application provides SDS-PAGE gel images of repeating tandem unit sequences of varying lengths. Figure 6This application provides another set of SDS-PAGE gel images of repeating tandem unit sequences of different lengths. Figure 7 SDS-PAGE gel images of eight tandem repeat decapeptide units assembled into different sites of the Escherichia coli genome, as provided in this application; Figure 8 SDS-PAGE gel images of tandemly 12 repeating decapeptide units assembled into different sites of the Escherichia coli genome, as provided in this application; Figure 9 SDS-PAGE gel images of 14 tandem repeat decapeptide units assembled into different sites of the Escherichia coli genome, as provided in this application; Figure 10 SDS-PAGE gel images of the stability test of the tandem eight repeating decapeptide units assembled into the E. coli genome from passages 1 to 32 provided in this application: A shows the stability results of plasmid expression of a protein with tandemly repeated decapeptide units after 32 passages; B shows the stability results of integrating the tandemly repeated decapeptide units into the E. coli genome after 32 passages. Figure 11 SDS-PAGE gel images were used to verify the enzymatic digestion effect of 8 repeating units in series. Figure 12 Images showing the effect of removing melanin from mussel adhesive protein with different tandem lengths.

[0023] The realization of the purpose, functional features and advantages of this application will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0024] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0025] It should be noted that if the embodiments of this application involve directional indicators (such as up, down, left, right, front, back, etc.), the directional indicators are only used to explain the relative positional relationship and movement of the components in a specific posture. If the specific posture changes, the directional indicators will also change accordingly.

[0026] Furthermore, if the embodiments of this application involve descriptions such as "first" or "second," these descriptions are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include at least one of those features. Additionally, the use of "and / or" or "and / or" throughout the text includes three parallel solutions. For example, "A and / or B" includes solution A, solution B, or a solution that simultaneously satisfies A and B. Furthermore, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or impossible to implement, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed in this application.

[0027] Mussel adhesive protein (MAP) is a natural adhesive protein extracted from the byssal threads of mussels, exhibiting excellent underwater adhesion properties, good biocompatibility, and biodegradability. Compared to traditional chemical adhesives, MAP maintains strong adhesion even in humid environments, is non-cytotoxic, and does not induce significant immune rejection. Based on these characteristics, MAP shows broad application prospects in biomedical adhesives, tissue engineering scaffolds, wound dressings, drug delivery carriers, surgical suture substitutes, and marine antifouling coatings. However, the natural extraction of MAP faces several bottlenecks. The yield of mussel byssal threads is extremely limited, with only micrograms to milligrams of MAP extracted from each mussel. Furthermore, the extraction process involves complex dissolution and purification steps, resulting in a high risk of organic solvent residue, poor batch-to-batch consistency, and extremely high production costs. Therefore, utilizing genetic engineering technology to construct recombinant expression systems to achieve efficient expression of recombinant MAP in heterologous hosts has become a major technological pathway to overcome the raw material bottleneck.

[0028] In existing technologies, the expression levels of natural mussel adhesive proteins in heterologous hosts are generally low, making it difficult to meet the needs of industrial production. Natural mussel adhesive proteins and their recombinant long-chain proteins have large molecular weights, making them prone to aggregation and precipitation in formulation applications. They also have poor compatibility with various excipients such as thickeners, surfactants, preservatives, and cross-linking agents, limiting their practical applications. In existing plasmid vector expression systems, plasmids are easily lost without selective pressure, and the expression level decreases with each generation during passage. Furthermore, continuous addition of antibiotics is required, posing safety risks and cost burdens.

[0029] In view of the above problems, firstly, this application provides a recombinant mussel adhesive protein polypeptide, the amino acid sequence of which is as follows: The sequence shown in SEQ ID NO.1; or A sequence variant of SEQ ID NO.1 obtained by substitution, deletion or addition of one or more amino acids, and having melanin production inhibitory activity.

[0030] The recombinant mussel adhesive protein polypeptide proposed in this application is a decapeptide composed of 10 amino acids and possesses melanin production inhibitory activity, while longer-sequence proteins of the recombinant mussel adhesive protein polypeptide (including natural full-length proteins, tandem polymers containing two or more decapeptide units, and various truncated polymers) lack this activity. Furthermore, compared to long-chain proteins, it has a smaller molecular weight, better solubility, and is less prone to aggregation, thus enabling effective compounding with various pharmaceutical or materials science excipients, overcoming the bottleneck of difficult compounding of long-chain proteins.

[0031] In some embodiments, the amino acid sequence of the sequence variant is selected from SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.4.

[0032] Systematic screening revealed that, based on SEQ ID NO.1, replacing serine (Ser) at specific positions in the sequence with threonine (Thr), or making a conserved substitution of specific tyrosine (Tyr), resulted in variants that, despite differences in amino acid sequences, still maintained melanin production inhibition activity similar to SEQ ID NO.1.

[0033] This application also proposes a nucleic acid molecule comprising a nucleotide sequence encoding a tandem repeat protein, which, upon cleavage by a protease, releases the aforementioned recombinant mussel adhesive protein polypeptide. The nucleotide sequence, from the 5' end to the 3' end, includes: At least two copies of the coding region, each coding region encoding a polypeptide with an amino acid sequence as shown in any one of SEQ ID NO. 1-4; and The adapter coding region is located between any two adjacent coding regions and encodes the protease recognition site.

[0034] The nucleic acid molecule provided in this embodiment can generate a large-molecule tandem repeat protein after transcription and translation. This "large to achieve small" design strategy, on the one hand, effectively avoids recognition and degradation by the host cell's proteolytic system due to its large molecular volume and complex spatial structure, allowing it to accumulate more stably within the cell; on the other hand, by doubling the gene copy number, it significantly enhances the transcription level of the target gene, synergistically achieving an increase in protein expression. Simultaneously, the linker coding regions located between the coding regions encode specific protease recognition sites, enabling the purified tandem protein to be precisely cleaved at predetermined sites through enzymatic digestion, thereby efficiently releasing the recombinant mussel adhesive protein polypeptide.

[0035] In some implementations, the number of copies of the coding region is 2-25. For example, the number of copies of the coding region can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, etc.

[0036] The copy number of the coding region is directly related to the expression level of the tandem protein and the total yield of recombinant mussel adhesive protein polypeptide after enzymatic digestion. A copy number that is too low results in the direct expression of a single decapeptide (recombinant mussel adhesive protein polypeptide), leading to extremely low yield. A copy number that is too high, while increasing the expression level of the tandem protein, may cause premature translation termination, protein misfolding, or aggregation and precipitation due to the large molecular weight, resulting in a decrease in effective expression levels. When the copy number is between 2 and 25, the host can maintain a high expression level while ensuring the expression quality of the tandem protein and the completeness of subsequent enzymatic digestion. In some preferred embodiments, the copy number of the coding region is 6-16. In some more preferred embodiments, the copy number of the coding region is 8 or 12.

[0037] This application also proposes a recombinant expression vector comprising the nucleic acid molecules described above. This recombinant expression vector can serve as a convenient tool for introducing the coding sequence of a tandem repeat protein into a host cell. The recombinant expression vector can be a plasmid vector commonly used in prokaryotic expression systems, such as the pET series or pCold series.

[0038] This application also proposes a recombinant engineered bacterium whose genome integrates the nucleic acid molecules described above.

[0039] In some embodiments, the recombinant engineered bacteria is Escherichia coli; the nucleic acid molecule described above is integrated into one or more sites in the genome of the recombinant engineered bacteria (Escherichia coli), the sites being selected from any one or more of the codA, ptsG, ydgA, yeiI, yfhL, yhaK, yiiM and IS186 sites.

[0040] This application's embodiments target and integrate a series of non-essential gene loci or high-frequency insertion sequence loci on the *E. coli* chromosome. Among these, codA (cytosine deaminase), ptsG (glucose transporter), ydgA, yeiI, yfhL, yhaK, and yiiM are genes that are not essential for *E. coli* growth under conventional culture conditions; their insertion and inactivation do not affect the normal growth and metabolism of the strain in rich media such as LB. IS186, on the other hand, is a multi-copy insertion sequence present in the genome. By inserting expression cassettes (as described above) into these loci, the multi-copy characteristic can be utilized to rapidly increase the expression level of the target gene.

[0041] This application also proposes a method for preparing recombinant mussel adhesive protein polypeptide, comprising the following steps: S1. Construct the recombinant engineered bacteria as described above; S2. Cultivate the recombinant engineered bacteria and induce expression to obtain a culture; S3. Tandem protein was recovered from the culture; S4. The tandem protein was subjected to enzymatic digestion and purification to obtain recombinant mussel adhesive protein polypeptide.

[0042] Specifically, in step S1, nucleic acid molecules are integrated into the genome of the recombinant engineered bacteria via homologous recombination or CRISPR / Cas9 gene editing technology. Homologous recombination technology relies on constructing a donor plasmid with hundreds of base pairs of homologous arms upstream and downstream of the selected integration site at both ends. After being introduced into the host cell, the exogenous expression cassette is precisely replaced or inserted into the target chromosomal location under the action of the host itself or an exogenously provided recombinase (such as Red / ET recombinase). CRISPR / Cas9 technology, on the other hand, can guide the Cas9 endonuclease protein to create double-strand breaks at a specified location in the genome by designing site-specific guide RNA. Subsequently, the cell initiates a homologous targeted repair mechanism, and the target nucleic acid molecule can be integrated in situ using the introduced repair template plasmid.

[0043] In step S2, when Escherichia coli is selected as the recombinant engineered bacterium, it can be cultured using conditions known to those skilled in the art that are suitable for E. coli culture. Induction is achieved by adding inducers such as IPTG in the late logarithmic growth phase, which initiates efficient transcription and translation of the exogenous gene, allowing the bacteria to accumulate a large amount of the target tandem repeat protein.

[0044] Step S3 involves cell collection, cell disruption (e.g., high-pressure homogenization), and preliminary purification of the released tandem protein (e.g., using His-tagged affinity chromatography or ion-exchange chromatography targeting the protein's physicochemical properties) to obtain a relatively pure tandem protein.

[0045] In step S4, the tandem protein is cleaved into individual decapeptide units using pre-designed protease recognition sites. Then, through fine purification, the target peptides released by enzymatic digestion are separated from the incompletely cleaved tandem protein, protease, and other impurities, ultimately obtaining high-purity recombinant mussel adhesive protein polypeptide monomers with melanin-inhibiting activity. In some embodiments, the enzymatic digestion process includes using trypsin to digest the tandem protein. Trypsin has the advantages of high activity, low cost, well-defined recognition sites, and simple operation, making it suitable for enzymatic digestion processes in large-scale industrial production.

[0046] This application also proposes a composition comprising the recombinant mussel adhesive peptide as described above, and pharmaceutically or materials-acceptable excipients.

[0047] In some embodiments, pharmaceutically or materials-acceptable excipients include one or more of thickeners, surfactants, preservatives, crosslinking agents, and buffer salts.

[0048] This application also proposes the application of the recombinant mussel adhesive protein polypeptide as described above in the preparation of products for inhibiting melanin production.

[0049] In some implementations, products used to inhibit melanin production include skin whitening products, medications for treating pigmentation-related diseases, medical dressings, or tissue-engineered materials.

[0050] Based on the foregoing, the recombinant mussel adhesive protein polypeptide or its sequence variants provided in this application have biological activity that inhibits melanin synthesis. Using it as a core ingredient, products with whitening and anti-pigmentation effects can be manufactured. Whitening products may include face creams, serums, masks, etc.; drugs for treating pigmentation-related diseases can be used to improve and / or treat melasma, freckles, post-inflammatory hyperpigmentation, and other skin pigmentation-related diseases. Preparing the recombinant mussel adhesive protein polypeptide provided in this application into medical dressings or tissue engineering materials can promote wound healing while inhibiting abnormal melanin deposition during the healing process, achieving a dual function of repair and whitening.

[0051] The following specific examples provide further details.

[0052] First, the materials and methods used in the embodiments will be described.

[0053] 1. Strains and plasmids Expression strain: E. coli BL21(DE3) (carrying the chromosome-integrated T7 RNA polymerase gene, regulated by the lacUV5 promoter).

[0054] Expression plasmid: pET-22b(+)-target (containing kanamycin resistance gene, N-terminal His tag, T7 / lac promoter).

[0055] Control plasmid: pET-22b(+) empty vector.

[0056] 2. Culture medium and reagents LB liquid medium: 1% tryptone, 0.5% yeast extract, 1% NaCl, pH 7.0.

[0057] LB solid medium: LB liquid medium, 1.5% agar powder.

[0058] Kanamycin: 50g / mL aqueous solution, filtered for sterilization.

[0059] IPTG: 0.5M aqueous solution, filtered for sterilization.

[0060] Lysis buffer: 10M urea, 20mM PB, pH 6-7.

[0061] SDS-PAGE reagents: 30% acrylamide, 1.5M Tris-HCl (pH 8.8), 1.0M Tris-HCl (pH 6.8).

[0062] 3. Plasmids for genome integration pDonor: A donor plasmid containing a complete expression cassette of the target gene (including promoter-target tandem sequence-terminator), ampicillin resistance, for example, when containing 8 repeating tandem decapeptide units, the plasmid map is as follows. Figure 1 As shown (pDonor-8); pQCasTns(Ptr)-IS186: A helper plasmid carrying a transposase system, used to integrate the target gene into five identical IS186 sites on the genome, for kanamycin resistance (e.g., Figure 2 (as shown) pQCasTns(Ptr)-array8: A helper plasmid carrying a Tn7-like transposase system, used to integrate the target gene into eight specific sites on the genome (codA, ptsG, ydgA, yeiI, yfhL, yhaK, yiiM, and an IS186 site), kanamycin resistance (e.g., Figure 3 (as shown) pCutamp: Carries the SpCas9 expression cassette, rhamnose-induced sgRNA, SacB expression cassette, and abramycin resistance gene, used to eliminate pDonor and helper plasmids (such as...). Figure 4 (As shown).

[0063] 4. Experimental Procedure 1) Plasmid transformation of competent cells (heat shock method) (1) Take 100 μL of BL21(DE3) competent cells thawed on an ice bath, add 1 μL (about 50 ng) of recombinant plasmid, gently mix, and incubate on ice for 30 min.

[0064] (2) Heat shock in a 42℃ water bath for 45 seconds, followed immediately by an ice bath for 2 minutes.

[0065] (3) Add 500 μL of antibiotic-free LB medium and incubate at 37°C with shaking for 45 min (to restore growth).

[0066] (4) Spread 100 μL of bacterial culture onto an LB plate containing kanamycin (50 μg / mL) and incubate upside down at 37°C for 12-16 h.

[0067] 2) Single colony screening and seed culture (1) Pick a single colony (3-5) and inoculate it into 5 mL of LB medium (containing 50 μg / mL kanamycin), shake at 37℃ and 220 rpm overnight (12-16 h).

[0068] (2) The next day, transfer the culture to 50 mL of fresh LB (containing kanamycin) at a ratio of 1:100 and incubate at 37°C until OD. 600 = 0.4-0.6 (approximately 2–3 h).

[0069] 3) Induced expression (1) Take 1 mL of pre-induction bacterial culture as an uninduced control, centrifuge to collect the bacterial cells, and store at -20℃.

[0070] (2) Add IPTG to the remaining bacterial culture to a final concentration of 0.5 mM.

[0071] (3) Transfer the shake flask to 37°C and continue induction culture at 220 rpm for 6 h.

[0072] (4) After induction, take 1 mL of bacterial solution as a whole bacterial control after induction.

[0073] 4) Bacterial cell collection and lysis (1) Transfer the culture to a 50 mL centrifuge tube, centrifuge at 4 °C and 5000 × g for 10 min, and discard the supernatant.

[0074] (2) Resuspend the bacterial pellet in 10 mL of pre-cooled lysis buffer.

[0075] (3) Ultrasonic breakage (ice bath): power 200 W, working for 3 seconds, interval 5 seconds, total effective time 10 min (repeat 2-3 times to prevent overheating).

[0076] (4) Centrifuge the lysate at 4°C and 12000×g for 20 min, and collect the supernatant (soluble components) and precipitate (inclusion body components).

[0077] 5) SDS-PAGE detection of expression (1) Take 20 μL of each of the following samples and add 5 μL of 5× SDS loading buffer: whole bacteria before and after induction (OD 600 Adjust to consistency), lysate supernatant, lysate precipitate (resuspended with an equal volume of lysate), boil for 10 min, and briefly centrifuge.

[0078] (2) Prepare 12% separating gel (pH 8.8) and 5% stacking gel (pH 6.8), and load 10-15 μL of each.

[0079] (3) Electrophoresis at 120 V for about 90 minutes (until bromophenol blue reaches the bottom of the gel).

[0080] (4) Stain with Coomassie Brilliant Blue R-250 for 30 min, decolorize with decolorizing solution (10% acetic acid, 40% methanol) until the background is transparent, and take a picture to record.

[0081] 6) 5L tank fermentation with microbial culture Following the mussel protein fermentation process, the bacterial strain was activated on plates. After primary and secondary seed culture, the culture conditions were 37℃, 220 rpm, and 8-14 hours. Fermentation was then carried out in a 5 L tank at 37℃, with dissolved oxygen (DO) > 30%. 14% ammonia was added to maintain the pH at 7.0 ± 0.2. The fermentation speed and dissolved oxygen were linked to maintain DO above 35%. Once the aeration rate reached its maximum of 1-2 vvm, the maximum speed and aeration rate were maintained during fermentation. Samples were taken during fermentation to measure the OD of the fermentation broth. 600 Value, fermentation culture for about 4-5 hours (OD) 600 When the dissolved oxygen level rapidly rises to above 45%, fed-batch culture is initiated, maintaining the dissolved oxygen level above 10%, and the culture continues for approximately 1-1.5 hours (OD). 600 IPTG induction was performed at a final concentration of 0.5 mM / L for 28-32 hours. The induction culture temperature was 37±1℃, pH 6.5±0.2, and the feed flow rate was controlled at 0.7 mL / min for carbon source and 1.4 mL / min for nitrogen source. Fermentation was continued for 4-6 hours. After fermentation, samples were taken to detect OD. 600 The bacterial cells were collected by centrifugation in a container and the protein yield was then measured.

[0082] 7) Method for determining the yield of 5L fermentation culture (1) Reagents and consumables Urea, disodium hydrogen phosphate, sodium dihydrogen phosphate, tris(hydroxymethyl)aminomethane, sodium chloride, sodium hydroxide, purified water.

[0083] (2) Instruments Ultra-high pressure crusher, chromatography system, ultra-micro spectrophotometer, electronic balance.

[0084] (3) Solution preparation (1L) ① Homogenized bacterial breaking solution (10M urea, 10mM disodium hydrogen phosphate): Weigh 600.06g urea, 3.58g disodium hydrogen phosphate dodecahydrate and 530.02g pure water, and dissolve and mix well.

[0085] ② Diluent (10mM disodium hydrogen phosphate): Weigh 3.58g of disodium hydrogen phosphate dodecahydrate and 997.62g of pure water, and dissolve and mix well.

[0086] ③ Chromatography solution: 20 mmol / L phosphate (pH 7); 6 mol / L industrial grade urea + 10 mmol / L phosphate (pH 7); 1 mol / L sodium chloride + 10 mmol / L tris(hydroxymethyl)aminomethane (pH 7); 6 mol / L industrial grade urea + 0.3 mol / L sodium chloride + 10 mmol / L tris(hydroxymethyl)aminomethane (pH 7); 6 mol / L industrial grade urea + 1 mol / L sodium chloride + 10 mmol / L tris(hydroxymethyl)aminomethane (pH 7); 0.5 mol / L sodium hydroxide.

[0087] ④ Crushing the mushroom sludge Dispersion: Weigh 1g of polypeptide-4 bacterial sludge, add it to the homogenized bacterial breaking solution to completely disperse the bacterial sludge, and then use the homogenized bacterial breaking solution to make up to 30mL.

[0088] Cleaning before homogenization: Set the chiller temperature to 10℃, drain the liquid from the homogenizer, circulate pure water under no pressure for 3 minutes, then drain; circulate pure water under 30MPa for 3 minutes, then drain; circulate 25% alcohol under 30MPa for 10-15 minutes, then drain; finally circulate pure water under 30MPa for 3 minutes, then drain.

[0089] Homogenization: Add the dispersed bacterial sludge solution to a cleaned homogenizer and homogenize at 90-100 MPa for approximately 15 minutes until the solution is clear and transparent. Then drain and collect the solution, using a small amount of lysis solution to push out any remaining liquid in the homogenizer and collect it. Finally, use the homogenization lysis solution to bring the collected solution to a final volume of 50 mL.

[0090] Wash after homogenization: Circulate with 25% ethanol at 30MPa for 10-15 minutes, then drain; then seal with 25% ethanol.

[0091] ⑤ Cation chromatography Treatment of homogenized collection liquid: Take 20 mL of homogenized collection liquid, add 4 mL of diluent, mix well, and then filter using a 0.2 μm filter.

[0092] Pass through a chromatography column.

[0093] ⑥ Detection and Calculation Detection: The collected solution was subjected to gel electrophoresis, and the absorbance of the eluted liquid was measured separately using A280 spectrophotometry.

[0094] If gel chromatography shows that the target protein band is clear and there are few impurities in the elution collection buffer, and the target protein band is absent or very faint in the other collection buffers, the A280 value of the elution collection buffer can be used to calculate the yield. .

[0095] Example 1: Construction of recombinant expression vector and acquisition of plasmid expression strain In this embodiment, a recombinant expression vector containing different tandem repeat units was constructed and transformed into an E. coli host, providing a basis for subsequent expression level screening and genome integration.

[0096] Based on the core sequence AKPSYPPTYK of the natural decapeptide repeat unit of mussel adhesive protein, repeat unit coding sequences with different tandem numbers were designed and synthesized. The tandem numbers were 1, 2, 3, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 18, 22, and 23 repeat units. In addition, 10 repeat unit tandem sequences based on decapeptide variant sequences (AKPTYPPTYK, AKPTYPSTYK, and AKPSYPSTYK) were synthesized. All of the above sequences were artificially synthesized and cloned into the pET-22b(+) plasmid to obtain a series of recombinant plasmids pET-22b-target, with the empty vector pET-22b(+) serving as a negative control.

[0097] The prepared series of recombinant plasmids and empty vectors were transformed into E. coli BL21(DE3) according to the above method. Positive clones were screened, induced expression was performed, and SDS-PAGE was used for verification. After confirming that the target protein expression band was correct, the positive clone strains were stored for later use.

[0098] Example 2: Screening of expression levels with different tandem lengths and different sequence backgrounds In this embodiment, protein expression levels of recombinant strains with different numbers of tandem repeat units and different sequence sources were compared by culturing in a 5L fermenter.

[0099] The positive clones obtained in Example 1 were fermented in a 5L tank according to the method described above, and the protein yield was measured after fermentation. The results are shown in Table 1.

[0100] Table 1. Sequence information and expression levels of decapeptides of different tandem lengths screened by plasmid expression.

[0101] As shown in Table 1, the protein expression level gradually increased as the tandem repeat number increased from 1 to 16, with the expression level of 16 tandem repeat units reaching a peak of 2.25 g / L; when the tandem repeat number continued to increase to 18, 22, and 23, the expression level showed a downward trend.

[0102] Simultaneously, the solution collected after fermentation was analyzed by SDS-PAGE, and the results are as follows: Figure 5 and Figure 6 As shown. FromFigure 5 and Figure 6 It can be seen that the expression levels of recombinant mussel adhesive proteins with different numbers of repeat units differ significantly under the same expression conditions. Among them, proteins containing 6-20 repeat units have higher expression levels, which are significantly better than proteins with other numbers of repeat units.

[0103] For recombinant mussel adhesive proteins from different sequence sources, high expression levels (1.29-1.59 g / L) were observed when the number of tandem repeat units was 10. This result indicates that the tandem arrangement of 10 repeat units is beneficial for efficient protein expression under different sequence backgrounds and has a certain degree of universality.

[0104] Example 3: Genome integration of tandem expression cassettes and screening of integration sites This embodiment describes a method for integrating tandem expression cassettes into specific sites in the Escherichia coli genome and a comparison of expression levels at different integration sites / copy numbers.

[0105] (1) Gene integration Genome integration was performed using tandem sequences of 8, 12, and 14 repeat units as examples. Complete expression cassettes containing the target gene and plasmid vectors were amplified using corresponding primers. After confirming the correct band size by agarose gel electrophoresis, the bands were excised and recovered. The recovered expression cassettes were ligated to the vectors using seamless cloning technology and transformed into *E. coli* Top10 competent cells. Transformants with correct sequencing were selected, expanded, and three recombinant plasmids were extracted and labeled pDonor (corresponding to tandem sequences of 8, 12, and 14 repeat units, respectively).

[0106] Three types of pDonor plasmids were mixed with pQCasTns(Ptr)-array8 plasmid at a 1:1 mass ratio and transformed into E. coli BL21(DE3) competent cells. The mixture was then plated on LB agar plates containing ampicillin (100 μg / mL) and kanamycin (100 μg / mL) and incubated at 37°C for 12–16 h. All colonies were scraped off using a disposable sterile inoculating loop (bent at the loop end) and transferred to a sterile 50 mL centrifuge tube. The cells were resuspended in 2 mL of LB liquid medium. A portion of the bacterial suspension was then plated on triple-antibody agar plates containing ampicillin (100 μg / mL), kanamycin (50 μg / mL), and adehydrotetracycline (100 ng / mL) and incubated at 37°C. Scrape off colonies and resuspend them. Spread a portion onto triple-antibody plates containing ampicillin (100 μg / mL), kanamycin (50 μg / mL), and dehydrotetracycline (1000 ng / mL), and incubate at 37°C. Repeat this step once to improve integration efficiency.

[0107] Single colonies were selected, and the original strain was used as a control. Colony PCR identification was performed using 8 pairs of specific primers targeting 8 pre-defined genomic integration sites (codA, ptsG, ydgA, yeiI, yfhL, yhaK, yiiM and IS186 sites). The primers for locus 1 are difsite1-F and difsite1-R; for locus 2, difsite2-F and difsite2-R; for locus 3, difsite3-F and difsite3-R; for locus 4, difsite4-F and difsite4-R; for locus 5, difsite5-F and difsite5-R; for locus 6, difsite6-F and difsite6-R; for locus 7, difsite7-F and difsite7-R; and for locus 8, difsite8-F and difsite8-R. Both forward and reverse primers were designed onto the *E. coli* genome. The primer sequences are shown in Table 2. If a band of approximately 3300 bp is amplified by PCR, the target gene is considered to have been successfully integrated at that locus.

[0108] Table 2. Primer sequences for genomic integration sites

[0109] For IS186 site integration, pQCasTns(Ptr)-array8 was replaced with pQCasTns(Ptr)-IS186 plasmid for insertion into 5 identical IS186 sites on the genome, and the experimental method was the same as above.

[0110] (2) Plasmid elimination Positive strains identified as having multiple copies of integrated bacteria were prepared into competent bacteria according to the instructions of the Sangon Biotech Supercompetent Cell Preparation Kit (B529303-0200). The pCutamp plasmid was transformed into the competent bacteria and plated on LB agar plates containing abramycin sulfate (50 μg / mL) and 10 mM rhamnose, and cultured at 37°C for 12–16 h. The pCutamp plasmid carries the SpCas9 expression cassette, rhamnose-induced sgRNA, SacB expression cassette, and abramycin resistance gene. Rhamnose induces sgRNA transcription, which guides Cas9 to cleave the consistent sequence of the ampicillin resistance gene promoter on the pDonor series and pQCasTns(Ptr)-array8 plasmids, rendering it unable to be repaired and thus eliminating it.

[0111] Single colonies were selected and replicated, then spread onto three plates: an LB agar plate containing ampicillin (100 μg / mL), an LB agar plate containing kanamycin (50 μg / mL), and an LB agar plate containing 10% sucrose. If a colony did not grow on the ampicillin and kanamycin plates but grew on the sucrose plate, it indicated that pDonor and the helper plasmid had been eliminated. Colonies growing on the sucrose plate were then replicated and spread onto antibiotic-free LB agar plates and LB agar plates containing abramycin (50 μg / mL) to verify the elimination of the pCutamp plasmid. Colonies that did not grow on the abramycin plate but grew on the antibiotic-free plate were considered multi-copy integrated strains with all introduced plasmids eliminated. The sucrose plate was used to screen for colonies lacking the pCutamp plasmid, utilizing the characteristics of the SacB gene (which catalyzes the conversion of sucrose to toxic fructose).

[0112] The resulting multi-copy integrated strain was inoculated into LB liquid medium and cultured at 37°C with shaking at 220 rpm for strain preservation.

[0113] The prepared strains (containing tandem sequences of 8, 12, and 14 repeating units) were fermented using the aforementioned 5L tank fermentation method, and the yields were measured. The results are shown in Tables 3 to 5. The corresponding SDS-PAGE results are also shown below. Figures 7 to 9 As shown.

[0114] Table 3. Expression levels of the eight tandem repeat decapeptides assembled into different sites in the *E. coli* genome.

[0115] Table 4. Expression levels of the 12 tandem repeat decapeptides assembled at different sites in the *E. coli* genome.

[0116] Table 5. Expression levels of 14 tandem repeat decapeptides assembled into different sites in the *E. coli* genome.

[0117] Combine Tables 3 to 5 and Figures 7 to 9 It can be seen that: Compared with plasmid expression, integrating eight tandem repeat decapeptide units into different sites in the *E. coli* genome resulted in varying degrees of increased protein expression. Furthermore, significant differences in expression levels were observed at different integration sites and under different copy number conditions. After integration of the eight tandem repeat units, the highest yield was achieved with nine copies (sites: codA, ptsG, ydgA, yeiI, yfhL, yhaK, yiiM, IS186-1, IS186-5), reaching 1.75 g / L, which was superior to the 1.50 g / L of plasmid expression.

[0118] Compared with plasmid expression, integrating the tandemly repeated decapeptide unit sequence into different sites in the *E. coli* genome resulted in varying degrees of increased protein expression. Furthermore, significant differences in expression levels were observed at different integration sites and under different copy number conditions. After integration of the 12 tandemly repeated units, the highest yield (2.05 g / L) was achieved when all five IS186 sites were integrated, and the 3-copy combination (codA, ptsG, yiiM) also reached 2.05 g / L, both superior to plasmid expression (1.55 g / L).

[0119] Compared with plasmid expression, the integration of 14 tandemly repeated decapeptide units into different sites in the *E. coli* genome resulted in varying degrees of decreased protein expression. Expression levels were generally lower than plasmid expression (1.60 g / L), with a maximum of only 1.51 g / L, indicating that excessively high tandem numbers are actually detrimental to genome integration.

[0120] Based on the combined results, the strain that integrates 8 tandem repeat units into 9 sites (including the IS186 site) was selected as the production strain for subsequent process development.

[0121] Example 4: Validation of the passage stability of genome-integrated strains The genome-integrating strain (8 tandem repeat units, 9 copies integrated) and the corresponding plasmid expression strain obtained in Example 3 were inoculated into LB liquid medium (antibiotic-free) and cultured at 37°C with shaking at 220 rpm. Every 12 hours, the culture was transferred to fresh medium at a 1:100 ratio, and the culture was passaged for 32 generations. Samples were taken every 4 generations for shake-flask induction of expression and SDS-PAGE analysis as described above. The SDS-PAGE results are shown below. Figure 10 As shown. Figure 10 Figure A shows the stability results of a plasmid expressing a protein with a tandem repeat of eight decapeptide units after 32 passages. Figure 10 Figure B shows the stability results after 32 passages following integration of the sequence into the *E. coli* genome. The comparison reveals that after 32 passages, the target protein band in the plasmid-expressing strain significantly faded, indicating a decrease in expression level. In contrast, after 32 passages, the target protein band intensity remained essentially unchanged, and the expression level remained roughly the same in the genome-integrated strain. This demonstrates that the protein expression stability on the genome is significantly superior to that on the plasmid, with a lower loss rate during passages, making it more suitable for large-scale production applications.

[0122] Example 5 Preparation of recombinant mussel adhesive protein polypeptide (decapeptide) This embodiment uses the genome-integrating strain (8 tandem repeat units, 9 copies integrated) obtained in Example 3 as the subject to prepare recombinant mussel adhesive protein polypeptide. The specific preparation steps are as follows: 1. Bacterial resuscitation and lysis 1) Resuspension: Add industrial-grade urea, disodium hydrogen phosphate dodecahydrate, and pure water at a bacterial dry weight ratio of 1:6, stir until completely dissolved, and control the pH to 7.0-8.0.

[0123] 2) Sterilization: The resuspended liquid was homogenized 4 times using a high-pressure homogenizer at a pressure of 900-1000 bar, with the temperature controlled at 20-30℃.

[0124] 2. Solid-liquid separation and clarification 1) pH adjustment: Slowly add glacial acetic acid to the homogenized solution to adjust the pH to 7.20-7.40.

[0125] 2) Plate and frame filtration: Use a plate and frame filter loaded with diatomaceous earth, pre-lay filter cake with 6 mol / L urea-10 mmol / L phosphate buffer (pH 7.00±0.10) and filter the liquid.

[0126] 3) Fine filtration: The plate and frame filter press is used to filter the filtrate again to obtain a clear liquid.

[0127] 3. Cation exchange chromatography 1) Chromatography procedure: Perform the following steps in sequence: equilibration, sample loading, rinsing, equilibration, impurity washing, equilibration, and elution.

[0128] 2) Sample testing and processing: Collect the eluent, measure A280, and calculate the protein concentration.

[0129] Add glacial acetic acid to a final concentration of approximately 1% (v / v) according to volume, stir well, and the chromatographic collection solution is obtained.

[0130] Samples from each step (loading, breakthrough, washing, elution, and regeneration) were analyzed by SDS-PAGE; if the target protein was not found in the breakthrough solution, it was discarded, otherwise it was retained.

[0131] 4. Step 1: Ultrafiltration Concentration: Using a spiral wound membrane, concentrate to a protein concentration of 20-40 mg / mL.

[0132] Collection: After changing the liquid, collect the ultrafiltrate.

[0133] 5. Enzyme-digested tags Add GT8 enzyme and stir at room temperature for ≥12 hours.

[0134] Take samples for SDS-PAGE to confirm complete enzyme digestion; if incomplete, add GT8 enzyme, adjust pH to 6.50±0.10, remove the precipitate, and collect the supernatant.

[0135] 6. Enzyme modification Based on the quality of the enzyme-modified solution, add the following components sequentially, maintaining the pH within the range of 4.50-5.50: ascorbic acid, copper sulfate, and SAT enzyme. When the required dopa concentration is reached, immediately add a terminator to terminate the reaction.

[0136] 7. Chromatographic filtration 1) Anion chromatography and cation II chromatography After the sample was purified by anion exchange chromatography and cation II chromatography in sequence, the cation II eluent was collected and proceeded to the next step of ultrafiltration.

[0137] 2) Second step: ultrafiltration Concentration: Use a spiral wound membrane to concentrate the protein to a concentration of 20-40 mg / mL.

[0138] Collection: After changing the liquid, collect the ultrafiltrate.

[0139] 8. Enzymatic hydrolysis Add trypsin to the collected solution and react with stirring at room temperature for at least 8 hours. Then, take a sample for SDS-PAGE to verify complete enzymatic digestion. If the solution is not completely dissolved, continue the reaction until complete dissolution. Afterward, add glacial acetic acid for fine filtration, and finally sterile filtration: filter the ultrafiltrate using a sterile 0.45 / 0.2 μm filter and collect it in a sterile clean container. SDS-PAGE analysis results are as follows: Figure 11 As shown. From Figure 11 It can be seen that the protein with 8 repeating unit sequences in tandem was digested by enzyme to obtain a single decapeptide. The large tandem protein band disappeared completely and was transformed into a single small peptide band, which confirms that the enzyme digestion was complete.

[0140] 9. Sample Preservation Protein samples should be stored at 2-8°C.

[0141] Example 6: Performance Test of Recombinant Mussel Adhesive Protein Peptide (Decapeptide) with Various Excipients This embodiment compares the compatibility of eight tandem repeat unit proteins (i.e., tandem large proteins and single decapeptides) with commonly used excipients before and after enzymatic hydrolysis.

[0142] The undigested tandem protein (8× decapeptide) and the single decapeptide obtained in Example 5 were each prepared into solutions of the same mass concentration. These solutions were then mixed with 22 commonly used excipients at recommended dosages, including: carbomer series (Carbopol 940, Carbopol 941, Carbopol 980, Carbopol 974, Carbopol 934, Carbopol ETD 2020), acrylate copolymers (U10, U20, U21, U30, TR-1, TR-2), sodium hyaluronate (HA 1.2-1.5 million, Hybloom HA-T), xanthan gum CG-T, Aristoflex AVC, ARISTOFLEX SILK, dipotassium glycyrrhizate, BLV, hydrolyzed sclerotium gum, Sepimax Zen, and β-glucan. The solution state was observed after mixing (clear and transparent was marked with "√", turbidity or precipitation was marked with "×"). The results are shown in Table 6.

[0143] Table 6. Comparison of compound states of different excipients

[0144] As shown in Table 6, the undigested tandem protein exhibited turbidity or precipitation when mixed with all tested excipients, resulting in formulation failure. In contrast, the enzymatically hydrolyzed single decapeptide remained clear and transparent without precipitation when mixed with all 22 excipients, demonstrating excellent formulation compatibility.

[0145] Example 7 Verification of the melanin production inhibitory activity of mussel adhesive proteins with different tandem lengths This embodiment uses a B16 mouse melanoma cell model to systematically evaluate the inhibitory activity of mussel adhesive proteins of different tandem lengths (all modified with DOPA) on melanin synthesis. The specific steps are as follows: 1. Experimental Materials Cell line: Mouse skin melanoma cells B16-F10 (Pronosei).

[0146] Main reagents: RPMI 1640 medium (Pronosai), fetal bovine serum FBS (Gibco), trypsin (Gibco), penicillin / strep antibiotics (Gibco), PBS (Sangon Biotech), α-MSH (MCE), arbutin (Sigma), DMSO (Solepro).

[0147] Melanin extract: an aqueous solution containing 10% DMSO and 1 M NaOH.

[0148] 2. Test Sample The test samples included: 10aa-1 (AKPSYPPTYK); 10aa-2 (AKPTYPPTYK); 10aa-3 (AKPTYPTYK); 10aa-4 (AKPSYPTYK); 2 repeating units in series (2×); 4 repeating units in series (4×); 8 repeating units in series (8×); 16 repeating units in series (16×); all samples were DOPA modified.

[0149] 3. Steps (1) Cell preparation One vial of cryopreserved B16-F10 cells was thawed and seeded into a 10cm culture dish, and passaged at least once before detection.

[0150] (2) Cell digestion Trypsin (EDTA) is used. The specific digestion concentration and dosage need to be determined according to the characteristics of the cells in the laboratory: the trypsin concentration should be 0.25%, the trypsin volume for a 10 cm culture dish is 1 mL, and the trypsin volume for a T75 culture flask is 2 mL.

[0151] (3) Observe under a microscope until most cells become round and are in suspension. Add about 2-3 times the volume of trypsin in serum-containing DMEM medium to stop digestion and collect in a centrifuge tube. Centrifuge at 1000 r / min for 3 min. The speed and centrifugation time can be determined according to the characteristics of the cells in the laboratory.

[0152] (4) After centrifugation, discard the supernatant, add a certain volume of cell culture medium (about 4 mL) to the centrifuge tube, mix the cells with a 1 mL pipette, and count the cells using a cell counter or hemocytometer.

[0153] (5) Dilute the cells with cell culture medium to the seeding density, and seed them into 12-well plates, with 5 × 10⁶ cells per well. 4 1 cell per well, 1 mL of cell suspension per well.

[0154] (6) After overnight culture and cell attachment (approximately 16 hours), discard the culture medium and administer the drug: Add 1 mL of fresh 1640 complete culture medium (10% FBS) to the blank control group, add 1 mL of 1640 complete culture medium containing the corresponding drug and 0.2 μM α-MSH to the drug administration group; add 1 mL of 1640 complete culture medium containing 250 μM arbutin and 0.2 μM α-MSH to the positive control group; after drug administration, continue to culture the cells in a 5% CO2 incubator for 48 hours.

[0155] (7) 24 h after drug administration, wash the cells twice with 1×PBS, remove the PBS, add 200 μL of 0.25% trypsin to each well to digest the cells, and place them in a CO2 incubator for 2 min. Then add 1 mL of PBS to each well to agitate the cells, collect the cells in centrifuge tubes, and centrifuge at 4000 rpm for 5 min.

[0156] (8) Discard the supernatant, and use a pipette to remove as much PBS residue as possible from the centrifuge tubes. Add 200 μL of melanin extraction solution to each tube, shake well, and heat in an 80°C metal bath for 1 hour. After cooling, centrifuge to remove droplets from the tube wall, and transfer 150 μL of solution from each centrifuge tube into a 96-well plate. Measure the absorbance at 405 nm in each well using a microplate reader.

[0157] (9) Results calculation: The relative content of melanin or the melanin synthesis inhibition rate can be calculated.

[0158] Relative melanin content = ((T-C0) / (C-C0)) × 100% In the formula: T is the absorbance of the test sample well; C is the three-time average absorbance of the blank control group; C0 is the background absorbance of the melanin extract.

[0159] 4. Test Results Test results are as follows Figure 12 As shown, Figure 12 In the diagram, BC represents the blank control group, NC represents the model group (α-MSH only), and PC represents the positive control group. Figure 12 As can be seen, the blank control group had the lowest melanin content, while the model group showed a significant increase in melanin content, indicating successful modeling. The positive control group showed a significant decrease in melanin content, validating the reliability of the experimental system. The single decapeptide 10aa-1 and its variants 10aa-2, 10aa-3, and 10aa-4 all exhibited significant melanin-inhibiting activity, with the relative melanin content decreasing to levels close to the blank control group. However, the 2×, 4×, 8×, and 16× tandem sequences did not show melanin-inhibiting activity, and their relative melanin content was not significantly different from that of the model group. This demonstrates that only specific 10-amino acid sequences or their variant sequences possess significant melanin-inhibiting activity, while longer protein sequences completely lack this biological function.

[0160] The above description is merely an exemplary embodiment of this application and does not limit the patent scope of this application. Any equivalent structural transformations made based on the technical concept of this application and the contents of the specification and drawings of this application, or direct / indirect applications in other related technical fields, are included within the patent protection scope of this application.

Claims

1. A recombinant mussel adhesive protein polypeptide, characterized in that, Its amino acid sequence is as follows: The sequence shown in SEQ ID NO.1; or A sequence variant of SEQ ID NO.1 obtained by substitution, deletion or addition of one or more amino acids, and having melanin production inhibitory activity.

2. The recombinant mussel adhesive protein polypeptide as described in claim 1, characterized in that, The amino acid sequence of the sequence variant is selected from SEQ ID NO.2, SEQ ID NO.3 or SEQ ID NO.

4.

3. A nucleic acid molecule, characterized in that, It includes a nucleotide sequence encoding a tandem repeat protein, which, upon protease cleavage, releases the recombinant mussel adhesive protein polypeptide of claim 1 or 2, wherein the nucleotide sequence comprises, from the 5' end to the 3' end: At least two copies of the coding region, each coding region encoding a polypeptide with an amino acid sequence as shown in any one of SEQ ID NO. 1-4; and A linker coding region located between any two adjacent coding regions, the linker coding region encoding a protease recognition site.

4. The nucleic acid molecule as described in claim 3, characterized in that, The number of copies of the encoding region is 2-25.

5. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid molecule as described in claim 3 or 4.

6. A recombinant engineered bacterium, characterized in that, The genome of the recombinant engineered bacteria integrates the nucleic acid molecule described in claim 3 or 4.

7. The recombinant engineered bacteria as described in claim 6, characterized in that, The recombinant engineered bacteria is Escherichia coli; the nucleic acid molecule is integrated into one or more sites in the genome of the recombinant engineered bacteria, and the sites are selected from any one or more of the codA, ptsG, ydgA, yeiI, yfhL, yhaK, yiiM and IS186 sites.

8. A method for preparing recombinant mussel adhesive protein polypeptide, characterized in that, Includes the following steps: Step S1: Construct the recombinant engineered bacteria as described in claim 6 or 7; Step S2: Cultivate the recombinant engineered bacteria and induce expression to obtain a culture; Step S3: The tandem protein is recovered from the culture. Step S4: The tandem protein is subjected to enzymatic digestion and purification to obtain the recombinant mussel adhesive protein polypeptide.

9. The method for preparing the recombinant mussel adhesive protein polypeptide as described in claim 8, characterized in that, In step S1, the nucleic acid molecule is integrated into the genome of the recombinant engineered bacteria through homologous recombination or CRISPR / Cas9 gene editing technology.

10. The method for preparing the recombinant mussel adhesive protein polypeptide as described in claim 8, characterized in that, In step S4, the enzymatic digestion process includes enzymatic digestion of the tandem protein using trypsin.

11. A composition, characterized in that, It includes the recombinant mussel adhesive peptide as described in claim 1 or 2, and pharmaceutically or materials-acceptable excipients.

12. The composition according to claim 11, characterized in that, The pharmaceutically or materials-acceptable excipients include one or more of the following: thickeners, surfactants, preservatives, crosslinking agents, and buffer salts.

13. The use of the recombinant mussel adhesive peptide of claim 1 or 2 in the preparation of a product for inhibiting melanin production.

14. The application as described in claim 13, characterized in that, The products used to inhibit melanin production include skin whitening products, medications for treating pigmentation-related diseases, medical dressings, or tissue-engineered materials.

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