A recombinant mussel adhesive protein, its preparation method and application

Abstract

This invention discloses a recombinant mussel adhesive protein, its preparation method, and its applications. The recombinant mussel adhesive protein includes a mussel adhesive protein tag, the amino acid sequence of which is shown in SEQ ID NO.2. The recombinant mussel adhesive protein is obtained by recombinantly fusing the aforementioned mussel adhesive protein tag with mussel adhesive protein, followed by in vitro hydroxylation modification. This recombinant mussel adhesive protein exhibits advantages such as high expression level, high purity, high Dopa content, high adhesion, and promotion of various cell growth mechanisms. It can be used in the preparation of adhesives or in the preparation of formulations that promote cell growth.

Description

Technical Field

[0001] This invention relates to the fields of genetic engineering and recombinant proteins, specifically to a bifunctional mussel adhesive protein tag, the construction, expression, purification, and application of recombinant mussel adhesive protein. Background Technology

[0002] Mussel foot protein (MFP), also known as mussel adhesive protein (MAP), is a class of proteins secreted by the byssal threads of natural marine mussels, containing at least 13 different proteins. Marine mussels possess exceptionally strong adhesiveness in wet environments, which research indicates is closely related to the proteins secreted by their byssal threads. Among these, the proteins responsible for adhesion to contact surfaces are primarily type III (mefp-3, mcofp-3) and type V (mefp-5, mcofp-5). Type VI protein mfp-6 mainly provides reducing power to maintain the correct oxidative state of Dopa. Other proteins such as mfp-1, mfp-2, mfp-4, mfp-7, preCOL-D / P / NG, PTMP-1, and TMP-1 primarily play a supporting and protective role. Among mussel adhesive proteins, mfp-5 has the highest Tyr residue content (~30%) and, after hydroxylation modification, exhibits the highest Dopa content, thus demonstrating the strongest adhesive ability. Meanwhile, the mfp-5 protein sequence also contains a high content of positively charged amino acids such as lysine and arginine, which gives it a high positive charge characteristic. This provides an advantage for the cross-linking of mussel adhesive proteins and their binding with negatively charged substances, broadening its applications beyond adhesion, such as promoting cell proliferation and anti-inflammation. It has the potential to become a raw material for medical devices and cosmetics.

[0003] Extracting 1 mg of mussel adhesive protein from natural marine mussels requires approximately 10,000 mussels. Due to the large molecular weight of type I / II mussel adhesive protein, the mussel adhesive protein obtained by natural extraction mainly consists of mfp-1 and mfp-2, which provide support and protection, rather than mfp-3 and mfp-5. This method does not achieve optimal biological properties such as adhesion and growth promotion, and is also expensive. Mussel adhesive protein can also be obtained through recombinant expression, which is less costly than natural extraction. The mussel adhesive protein gene is expressed exogenously in host bacteria such as *Escherichia coli*, *Pichia pastoris*, and *Bacillus subtilis*, and purified using methods such as column chromatography, ion exchange column chromatography, salting out, and acid extraction. However, in the process of recombinant expression of mussel adhesive protein, additional purification tags such as 6×His and Flags are inevitably added to improve the efficiency of protein purification. This results in the recombinant protein not being a complete mussel protein sequence, and the tags need to be removed during subsequent purification. Currently, there are several novel mussel adhesive protein fusion strategies. For example, Chinese invention patent publication number CN115894655A fuses mussel adhesive protein with mussel byssal skeletal protein, using Pichia pastoris as the host strain to achieve protein expression. However, even after large-scale, high-density fermentation, the yield is only 1.1 g / L. Furthermore, although the aforementioned patent does not use exogenous purification tags in the protein purification process, it requires multiple purification steps; this method significantly increases the complexity of the purification process and is not conducive to efficient protein extraction. Based on literature reports, the expression level of monotype mussel adhesive protein in E. coli BL21(DE3) remains low, failing to reach the gram level, which hinders the widespread application of mussel adhesive protein. Summary of the Invention

[0004] This invention aims to address the problems of low expression levels of recombinant monotype mussel adhesive protein and the presence of non-mussel-derived sequences in the protein. It provides a method for preparing recombinant type V mussel adhesive protein and its applications, which not only increases the yield of mussel adhesive protein but also greatly simplifies the protein purification process. Through molecular design, a peptide from type IV mussel adhesive protein is used as a bifunctional tag for both expression and purification, fused with type V mussel adhesive protein mcofp-5 for expression, thereby increasing protein yield. The recombinant mussel adhesive protein exhibits excellent properties in mechanical and cellular applications.

[0005] To achieve the above objectives, the present invention is carried out according to the following scheme:

[0006] A histidine-rich peptide segment (HXHX structure) was selected from type IV mussel adhesive protein derived from the California mussel (Mytilus californianus) as a bifunctional mussel adhesive protein tag that can replace non-target proteins in the CDS region of the expression vector, enabling both expression promotion and purification. The amino acid sequence of the mussel adhesive protein tag is shown in SEQ ID NO.2.

[0007] The mussel adhesive protein tag can be used to increase the expression level of mussel adhesive protein and / or to purify mussel adhesive protein.

[0008] A recombinant type V mussel adhesive protein is composed of mcofp-5 from the natural marine mussel *Mytilus coruscus* and a mussel adhesive protein tag. By using the mussel adhesive protein tag to recombinantly fuse with the mussel adhesive protein, the expression level of the mussel adhesive protein can be significantly increased, and high-purity mussel adhesive protein can be successfully purified. In the preparation process, an in vitro hydroxylation modification strategy was employed to obtain mussel adhesive protein with high Dopa content.

[0009] The method for preparing the recombinant mussel adhesive protein includes the following steps:

[0010] (1) The gene sequence of the mussel adhesive protein tag and the fusion gene sequence of the mussel adhesive protein are linked to the CDS region on the vector to obtain a plasmid, and the plasmid is recombinantly expressed by Escherichia coli to obtain recombinant mussel adhesive protein; the gene sequence of the mussel adhesive protein tag is a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.2;

[0011] (2) The mussel adhesive protein expressed in step (1) was modified by hydroxylation using tyrosinase to obtain recombinant mussel adhesive protein.

[0012] Preferably, the tyrosinase in step (2) is derived from the tyrosinase AbPPO4 of Agaricus bisporus, and its amino acid sequence is as shown in SEQ ID NO.10.

[0013] Preferably, the tyrosinase in step (2) is obtained by ligating the gene sequence of the tyrosinase into a vector to obtain a plasmid, and then co-transforming the molecular chaperone Tf16 with the plasmid into competent Escherichia coli cells to obtain recombinant tyrosinase.

[0014] Preferably, the gene sequence of the mussel adhesive protein tag in step (1) is shown in SEQ ID NO.1, and the gene sequence of the tyrosinase is shown in SEQ ID NO.9; the Escherichia coli in steps (1) and (2) is Escherichia coli BL21(DE3); and the mussel adhesive protein is type III or type V.

[0015] Preferably, the hydroxylation modification reaction system in step (2) contains Cu at a concentration of 125-1500 μM. 2+ The pH was 4-10, the reaction temperature was 4-37℃, and the reaction time was 30-120 min; the amount of recombinant tyrosinase added was 0.5-5.0 wt.% of the recombinant mussel adhesive protein.

[0016] Preferably, Cu in the reaction system described in step (2) 2+ The concentration was 1000±200μM, pH=6±1, reaction temperature was 25±5℃, and reaction time was 60±20min; the amount of recombinant tyrosinase added was 1.0±0.5wt. of the recombinant mussel adhesive protein.

[0017] Preferably, the hydroxylation modification reaction system in step (2) further contains 5-50 mM ascorbic acid and 5-50 mM sodium borate; the fusion gene sequence of the mussel adhesive protein in step (1) is shown in SEQ ID NO.3, 5 or 7.

[0018] Characterization of mussel adhesive protein: Morphological characterization of mussel adhesive protein was performed using scanning electron microscopy; hydroxylation degree and underwater adhesion of mussel adhesive protein were characterized by NBT and Coomassie brilliant blue staining; mechanical properties of mussel adhesive protein were characterized using a universal testing machine.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] (1) Excellent purification tag performance: By fusing the peptide tag (Mfp-4) in type IV mussel adhesive protein with type V mussel adhesive protein, a recombinant protein of completely mussel origin is achieved. Furthermore, the tag (Mfp-4) can completely replace the traditional purification tag, achieving high purity, high yield and efficient purification of the protein.

[0021] (2) Significantly increased protein expression: After fusion with peptide Tag (Mfp-4), the expression of various mussel adhesive proteins was significantly increased, reaching 2 g / L after being scaled up in a 7L fermenter, which is beneficial for the large-scale preparation and application of mussel adhesive proteins.

[0022] (3) Excellent mechanical properties of the protein: The protein produced by fusing with Tag (Mfp-4) peptide has a high Dopa content and exhibits excellent mechanical properties in tensile tests, which are superior to the adhesion properties of commercial adhesives Cell-Tak and Boyin MAP-Reno, and can be used to prepare adhesives.

[0023] (4) It has the effect of promoting the growth of multiple cells: This recombinant protein also has the effect of promoting the growth of multiple cells. It can be used as a biomedical material and cosmetic raw material, and is suitable for applications in tissue engineering, wound repair, anti-aging and other fields. Attached Figure Description

[0024] Figure 1Image A shows the PCR results of a single colony containing the recombinant plasmid pET28a-Tag(Mfp-4)-mcofp5. The marker is the DL2000 DNA marker, and lanes 2-17 show the PCR results of a single colony containing the recombinant plasmid pET28a-Tag(Mfp-4)-mcofp5.

[0025] Figure 1 B is a schematic diagram of constructing the vector pET28a-Tag(Mfp-4)-mcofp5 by replacing the redundant sequence on the pET28a vector with the type IV mussel adhesive protein source tag Tag(Mfp-4).

[0026] Figure 2 This is a diagram showing the results of the fusion expression of the tag (Mfp-4) with type III / V proteins. Among them, Figure 2 A is a comparison of the expression of the unmodified protein mcofp5 and the modified protein Tag(Mfp-4)-mcofp5; Figure 2 B is a comparison of the expression of the unmodified protein mefp3 / mgfp3B and the modified protein Tag(Mfp-4)-mefp3 / mgfp3B; Figure 2 C-2E are statistical graphs showing the expression of protein Tag(Mfp-4)-mefp3 / mgfp3B / mcofp5.

[0027] Figure 3 This is a diagram showing the results of expression and purification of recombinant mussel adhesive protein Tag(Mfp-4)-mcofp5. Among them, Figure 3 A is a Western blotting image comparing the expression of unmodified mcofp5 protein and modified Tag(Mfp-4)-mcofp5 protein; Figure 3 Figure B shows the purification results of recombinant mussel adhesive protein Tag(Mfp-4)-mcofp5. Figure 3 C is the graph showing the purification yield of protein Tag(Mfp-4)-mcofp5; Figure 3 Statistical chart of the soluble expression percentages of unmodified mcofp5 protein and modified Tag(Mfp-4)-mcofp5 protein; Figure 3 E is a statistical graph comparing the expression of unmodified mcofp5 protein and modified Tag(Mfp-4)-mcofp5 protein.

[0028] Figure 4 This is a diagram showing the results of the hydroxylation system optimization. Among them, Figure 4 A is the graph showing the expression results of tyrosinase AbPPO4; Figure 4 B is the NBT staining diagram corresponding to each parameter in the hydroxylation system optimization; Figure 4C-4F correspond to the optimized results of reaction pH, reaction time, reaction temperature, and copper ion concentration in the hydroxylation system, respectively.

[0029] Figure 5 This is a graph characterizing the hydroxylation efficiency of mussel adhesive proteins. Among them, Figure 5 A is a comparison of the NBT staining results of four groups: mussel adhesive protein, Cell-Tak, MAP-Reno, and BSA. Figure 5 B shows the Coomassie Brilliant Blue staining results of mussel adhesive protein, Cell-Tak, and BSA on tin foil and glass surfaces, respectively. Figure 5 C represents the results of analysis of hydroxylated mussel adhesive protein using an amino acid analyzer.

[0030] Figure 6 These are scanning electron microscopy results of mussel adhesive proteins. Among them, Figure 6 A is an electron micrograph of the unhydroxylated mussel adhesive protein Tag(Mfp-4)-mcofp5; Figure 6 B is an electron micrograph of mussel adhesive protein Tag(Mfp-4)-mcofp5 in the successfully hydroxylated state.

[0031] Figure 7 These are images of the results of a universal testing machine tensile test on mussel adhesive protein (modified and unmodified), Cell-Tak, and BSA. Detailed Implementation

[0032] The present invention will now be described in more detail with reference to the embodiments and accompanying drawings. Unless otherwise specified, the methods described below are conventional methods.

[0033] Experimental materials: Escherichia coli DH5α competent cells were purchased from Beijing Qingke Biotechnology Co., Ltd., and Escherichia coli BL21(DE3) competent cells were purchased from Beijing Qingke Biotechnology Co., Ltd.

[0034] Main reagents: DL2000 / 5000 DNA Marker purchased from Sangon Biotech (Shanghai) Co., Ltd., KOD one TM PCR Master Mix was purchased from TOYOBO; 2×ES Taq MasterMix (Dye) was purchased from Kangwei Reagent Biotechnology Co., Ltd.; chloramphenicol and L-arabinose were purchased from BBI Life Sciences Co., Ltd.; kanamycin sulfate and sodium glycine were purchased from Shanghai Maclean's; Nitro blue tetrazolium chloride (NBT) was purchased from Sangon Biotech (Shanghai) Co., Ltd.; plasmid extraction kit and DNA purification and recovery kit were purchased from Nanjing Novizan Biotechnology Co., Ltd.; Price TMThe BCA protein quantification kit was purchased from ThermoFisher; the CCK8 cytotoxicity-proliferation kit was purchased from Biosharp.

[0035] Example 1: Construction, transformation and identification of recombinant mussel adhesive protein expression plasmid

[0036] This embodiment uses the construction process of Tag(Mfp-4)-mcofp5 as an example; the construction methods for other fusion proteins are completely consistent with this method. Using the existing plasmid pET28a as a template, upstream and downstream primers were designed. A 102bp redundant sequence, including 6×His, thrombin site, and T7 Tag, was deleted from the plasmid, and the remaining portion was linearized. Using pET28a-mfp5 as a template, upstream and downstream primers were designed to amplify the fusion fragment of the Tag and mussel adhesive protein. The primer sequences are as follows:

[0037] Table 1. Primers used for constructing the recombinant mussel adhesive protein expression plasmid

[0038]

[0039] Using plasmid pET28a as a template, the plasmid template was linearized after removing redundant sequences using pET28-Tag-F / R primers. The Tag-mfp5 target gene fragment was amplified using plasmid pET28a-mcofp5 as a template, with Mcofp5-Tag-F / R primers. The PCR system and procedure are as follows:

[0040] Table 2. PCR reaction system and procedure for constructing recombinant mussel adhesive protein plasmids

[0041]

[0042] After purifying the linearized vector and the target gene fragment, seamless cloning ligation was performed using a seamless cloning ligase. The system and procedure used are as follows:

[0043] Table 3. Seamless clonal linking system and reaction conditions

[0044]

[0045]

[0046] The product from the seamless cloning was transformed into *E. coli* DH5α. After incubation at 37°C for 40-60 min, the resulting bacterial suspension was plated onto LB agar plates containing 50 μg / mL kanamycin and incubated overnight at 37°C inverted. Several single colonies were selected from the plates and amplified using T7 / T7-TER primers for verification. The PCR system and procedure are as follows:

[0047] Table 4. PCR reaction system and procedure for positive clone verification

[0048]

[0049] The PCR amplification products were subjected to agarose gel electrophoresis, and the results are as follows: Figure 1 As shown, the target gene was successfully amplified, and the positive colonies were sent to Guangzhou Tianyi Huiyuan Company for sequencing. The sequencing results were compared with the recombinant plasmid template, confirming successful construction of the recombinant plasmid, named pET28a-Tag(Mfp-4)-mcofp5. The recombinant plasmid was transformed into E. coli BL21(DE3) competent cells using the heat shock method. The positive recombinant strain E. coli BL21(DE3)(pET28a-Tag(Mfp-4)-mcofp5) was obtained through kanamycin resistance screening and PCR verification.

[0050] Example 2: Fusion expression of tag (Mfp-4) with various mussel adhesive proteins

[0051] To verify the expression-promoting function of the type IV mussel adhesive protein source tag, multiple type III / V mussel adhesive proteins were fused with the tag (Mfp-4) and expressed in E. coli BL21(DE3). Strains containing the recombinant plasmid pET28a-Tag(Mfp-4)-mfps were removed from glycerol tubes and streaked onto plates. Single colonies were selected and inoculated into TB liquid medium containing 50 μg / mL kanamycin as a seed culture and incubated overnight at 37°C and 220 rpm. The seed culture was then transferred to TB liquid medium containing 50 μg / mL kanamycin at a 1% inoculation rate until bacterial OD... 600 When the expression level reached 0.6–0.8, 1 mM IPTG was added to induce protein expression for 6–8 hours. Protein samples were collected at 500°C and validated by SDS-PAGE. It was found that the expression levels of several recombinant mussel adhesive proteins fused with the tag (Mfp-4) were increased to varying degrees compared to monotype mussel adhesive proteins without the fused tag. Results are as follows: Figure 2 As shown.

[0052] Example 3: Purification of recombinant mussel adhesive protein

[0053] After fermentation, the bacterial cells were collected by centrifugation at 6000 rpm for 10 min using a refrigerated centrifuge. The cells were resuspended in Buffer A (4 M Urea, 0.5 M NaCl, 0.02 M Tris, pH 7.0) at a ratio of 10 OD / mL, and then the cells were disrupted using a high-pressure homogenizer. The disruption pressure was adjusted according to the actual situation until the bacterial solution was clear. The completely disrupted solution was centrifuged at 8000 g for 30 min, and the supernatant was collected and filtered through a 0.22 μm filter membrane to obtain the unpurified sample.

[0054] Remove the nickel column from the container at 4°C and allow the preservative solution to slowly drain under gravity. Then add 3-5 column volumes of ddH2O. Equilibrate the column with 3-5 column volumes of Buffer A (4M Urea, 0.5M NaCl, 0.02M Tris, pH=7.0). Load the filtered purified sample at a rate of 5 mL / time, allowing it to flow out under gravity. Collect the eluent during this process; after loading, rinse the nickel column with 3-5 column volumes of Buffer A (4M Urea, 0.5M NaCl, 0.02M Tris, pH=7.0) and collect the effluent; this is the equilibration solution. Subsequently, perform gradient elution with different imidazole concentrations (10%, 20%, 30%, 50%, 100%) of Buffer B (4M Urea, 0.5M NaCl, 0.02M Tris, 0.5M Imidazole, pH=7.0), collecting the eluent for each gradient. SDS-PAGE analysis confirmed that the target protein began to elute at a 30% gradient. Purification using nickel column affinity chromatography achieved a yield of 81.1% and a protein purity exceeding 95%, as shown in the results. Figure 3 As shown in the figure, this demonstrates that fusion with the peptide tag (Mfp-4) not only promotes protein expression but also enables high-purity, high-yield, and efficient purification of the protein.

[0055] The eluent containing a single target protein band was collected and dialyzed in 5% acetic acid using a dialysis bag with a molecular cutoff of 3.5 kD. The medium was changed every 8-12 hours. After dialysis, the protein was pre-frozen at -80°C and then lyophilized in a low-temperature vacuum freeze dryer. The protein was stored as a lyophilized powder.

[0056] Example 4: Optimization of the mussel adhesive protein hydroxylation system

[0057] Mussel adhesive protein was hydroxylated using the recombinant tyrosinase AbPPO4. The tyrosinase concentration was 1% (v / v), and the hydroxylation system solution was 1×PBS (20 mM ascorbic acid, 25 mM sodium borate). To achieve the best hydroxylation effect, single-factor optimization was used to control the hydroxylation temperature, hydroxylation time, pH of the hydroxylation system, and copper ion concentration. The hydroxylation reaction was terminated by adding acid after the reaction. Since components such as sodium borate in the reaction system may interfere with the detection results, the hydroxylated mussel adhesive protein was dialyzed into a 1×PBS system.

[0058] Based on the reactivity of the Dopa group, the hydroxylation results were characterized using the acid-sodium borate method. The specific procedure is as follows: using a 96-well plate, 150 μL of 0.1M HCl / Na₂B₄O₇ was added to each well. Then, 50 μL of the protein solution to be tested was added to each well containing 0.1M HCl / Na₂B₄O₇. After mixing, the plate was immediately placed in a microplate reader, and the absorbance was measured at 293 nm. The difference in absorbance reflects the Dopa content in the protein sample.

[0059] The optimal reaction conditions were determined through the above-mentioned optimization experiments as follows: hydroxylation temperature 25℃, hydroxylation time 60 min, pH of the hydroxylation system 6.0, and copper ion concentration 1000 μM. The results are as follows: Figure 4 As shown.

[0060] Example 5 Characterization of mussel adhesive protein hydroxylation rate

[0061] The hydroxylation effect of NBT staining on mussel adhesive protein was compared with that of Cell-Tak and MAP-Reno. The protein concentration in each group was uniformly controlled at 0.1 mg / mL. 5 μL of protein solution was dropped onto a PVDF membrane, and then the PVDF membrane with the protein drop was immersed in 20 mL of 2M sodium glycinate solution (containing 0.6 mg / mL NBT, pH = 10.0) and incubated in a shaking, dark incubator for 45 min. The solution was discarded, and 10 mL of 0.16M sodium borate was added to wash the PVDF membrane, repeated twice. Then, 20 mL of 0.16M sodium borate solution was added and the membrane was shaken overnight. Finally, the membrane was developed and photographed. The NBT staining results clearly showed that the hydroxylated mussel adhesive protein was successfully hydroxylated and the staining degree was comparable to that of commercial reagents Cell-Tak and MAP-Reno. The results are as follows: Figure 5 As shown in Figure A.

[0062] The hydroxylation rate of mussel agarin was further analyzed using an amino acid analyzer. After hydroxylation of mussel agarin, 10 mL of 6M HCl and the protein sample were added to a protein hydrolysis tube. The tube was repeatedly evacuated and purged 2-3 times to maintain an anaerobic environment and avoid affecting the hydrolysis. The sealed tube was then placed in a 110℃ oven for 22-24 hours for hydrolysis. Since copper ions were added to the hydroxylation system, an equal mass of EDTA was added after hydrolysis to remove interference. The hydrolyzed solution was filtered through filter paper into a 25 mL volumetric flask, and ddH2O was added to bring the volume to 25 mL. 1 mL of the solution was transferred from the volumetric flask to a 10 mL beaker and deacidified in a 60℃ water bath under a fume hood until only solid residue remained at the bottom. 1 mL of sample buffer was added to dissolve the deacidified sample, which was then filtered through a 0.22 μm filter and placed into a sample vial for analysis. The amino acid analysis results are as follows: Figure 5As shown, calculations show that mussel adhesive protein was successfully hydroxylated, with a hydroxylation rate of up to 25%.

[0063] Example 6: Scanning electron microscopy (SEM) morphology characterization of mussel adhesive proteins

[0064] Cell slides were prepared, and 20 μL of both unhydroxylated and hydroxylated recombinant mussel adhesive protein (Tag-mcofp5) solution was dropped into the center of each slide. The slides were left at room temperature for 30–60 min to allow protein sedimentation. The slides were then embedded in filter paper and fixed with staples. They were subsequently immersed in 70%, 85%, and 95% ethanol for 15 min each, and finally in 100% ethanol for 15 min (this step was repeated three times) to ensure complete removal of moisture. The samples were then dried using a critical point desiccator. After drying, the slides were removed, and the samples were adhered to conductive adhesive for gold sputtering. After pretreatment, the samples were scanned using a field emission scanning electron microscope (FET). The scan results showed significant morphological differences between the unhydroxylated and hydroxylated mussel adhesive proteins. The unhydroxylated mussel adhesive protein exhibited a fibrous, sheet-like form, while the successfully hydroxylated mussel adhesive protein samples displayed large, fibrous protein patterns, and in some fields, also exhibited morphological characteristics consistent with the unhydroxylated mussel adhesive protein. The difference in morphological characteristics between the two protein samples is likely due to hydroxylation modifying tyrosine residues into dopa groups, altering the structure of mussel adhesive proteins. This difference also verifies the successful hydroxylation of mussel adhesive protein Tag(Mfp-4)-mcofp5. Scanning results are as follows... Figure 6 As shown.

[0065] Example 7 Characterization of mussel adhesive protein adhesion ability

[0066] Remove the glass slides, immerse them in 100% ethanol, and then sonicate for 60 min. Next, immerse them in ddH2O and sonicate for another 60 min. After sonication, rinse with ddH2O and air dry. Five protein groups were prepared: BSA, Cell-Tak, MAP-Reno, and mussel adhesive protein Tag-mcofp5, with each protein concentration controlled at 0.2 mg / mL. 10 μL of protein solution was dropped onto the cleaned and dried glass slides. Two slides were overlapped and fixed at the protein addition site, with an overlap area of ​​25 mm × 26 mm. The fixed slides were placed at room temperature for 24–48 hours, followed by tensile testing using an Instron 5697 universal testing machine. The two ends of the slides were fixed to the clamps of the universal testing machine. The tensile speed was [value missing], and a 500 N sensor was used for testing. The tensile results are shown below. Figure 7As shown in the figure. The tensile test results of the universal testing machine show that the recombinant mussel adhesive protein Tag(Mfp-4)-mcofp5 designed and expressed in this invention has better adhesion performance than commercial adhesives Cell-Tak and Boyin MAP-Reno, and has a high expression level, which is expected to become a raw material for the mass production of adhesives.

[0067] The above embodiments are preferred experimental schemes of the present invention, but the implementation schemes of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A mussel agaric tag, characterized in that, The amino acid sequence of the mussel adhesive protein tag is shown in SEQ ID NO.

2.

2. The use of the mussel adhesive protein tag of claim 1 in increasing the expression level of mussel adhesive protein and / or in purifying mussel adhesive protein.

3. A method for preparing recombinant mussel adhesive protein, characterized in that, Includes the following steps: (1) The gene sequence of the mussel adhesive protein tag and the fusion gene sequence of the mussel adhesive protein are ligated to the CDS region on the vector to obtain a plasmid, and recombinant expression is performed using Escherichia coli to obtain recombinant mussel adhesive protein; the gene sequence of the mussel adhesive protein tag is a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.2; (2) The mussel adhesive protein expressed in step (1) was modified by hydroxylation using tyrosinase to obtain recombinant mussel adhesive protein.

4. The preparation method according to claim 3, characterized in that, The amino acid sequence of the tyrosinase in step (2) is as shown in SEQ ID NO.

10.

5. The preparation method according to claim 3, characterized in that, The tyrosinase described in step (2) is obtained by ligating the gene sequence of tyrosinase into a vector to obtain a plasmid, and then co-transforming the molecular chaperone Tf16 with the plasmid into competent E. coli cells to obtain recombinant tyrosinase.

6. The preparation method according to claim 5, characterized in that, The gene sequence of the mussel adhesive protein tag in step (1) is shown in SEQ ID NO.1, and the gene sequence of the tyrosinase is shown in SEQ ID NO.9; the Escherichia coli in steps (1) and (2) is Escherichia coli BL21(DE3); the mussel adhesive protein is type III or type V.

7. The preparation method according to any one of claims 3-6, characterized in that, The hydroxylation modification reaction system in step (2) contains Cu at a concentration of 125-1500 μM. 2+ The pH is 4-10, the reaction temperature is 4-37℃, and the reaction time is 30-120 min; the amount of tyrosinase added is 0.5-5.0 wt. of the recombinant mussel adhesive protein.

8. The preparation method according to claim 7, characterized in that, In the reaction system described in step (2), Cu 2+ The concentration of the enzyme was 1000±200 μM, pH=6±1, the reaction temperature was 25±5℃, and the reaction time was 60±20 min; the amount of tyrosinase added was 1.0±0.5 wt.% of the recombinant mussel adhesive protein. The hydroxylation modification reaction system in step (2) also contains 5-50 mM ascorbic acid and 5-50 mM sodium borate; the gene sequence of the mussel adhesive protein tag and the fusion gene sequence of the mussel adhesive protein in step (1) are shown in SEQ ID NO.3, 5 or 7.

9. Recombinant mussel adhesive protein prepared by the method according to any one of claims 3-8.

10. The use of the recombinant mussel adhesive protein of claim 9 in the preparation of adhesives.

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