Recombinant mussel-like mucin as well as coding gene and application thereof

Abstract

The invention relates to a recombinant mussel-like mucoprotein as well as a coding gene and application thereof, and belongs to the technical field of gene engineering and protein engineering. According to the invention, partial functional sequences of different types of mussel mucin are screened, assembled and spliced to obtain a section of brand new mussel mucin-like sequence, and the sequence is optimized by escherichia coli codon preference to construct a high-expression recombinant strain. According to the recombinant strain, the soluble expression quantity of the target protein is remarkably improved by optimizing culture conditions. The recombinant protein does not carry any exogenous tag, and the provided purification method is low in cost, high in yield and suitable for large-scale production. The prepared recombinant mussel-like protein has good adhesiveness and oxidation resistance, and the modified recombinant mussel-like protein is further provided, and the biological performance of the modified recombinant mussel-like protein is better than that of unmodified recombinant mussel-like protein.

Description

Technical Field

[0001] The invention relates to a recombinant mussel-like mucin and a coding gene and application thereof, belonging to the technical field of genetic engineering and protein engineering. Background Art

[0002] Marine mussels, such as the blue mussel, Mediterranean mussel, thick-shelled mussel, and green mussel, secrete byssal threads from their glands that allow them to adhere to a variety of substrates, allowing them to remain firmly attached despite the impact of heavy waves. This adhesive ability is primarily attributed to a series of specialized amino acids contained in mussel adhesive proteins (MAPs), such as DOPA (derived from the hydroxylation of tyrosine), which contains catechol groups. To date, at least 13 mussel adhesive proteins have been studied and identified.

[0003] As a novel biomaterial extracted from marine mussels, mussel mucin exhibits high biocompatibility and can form nanoscale meshwork scaffolds with antioxidant properties, thereby inhibiting inflammation. MAP has been successfully demonstrated as a potential bioadhesive in experiments involving cell or tissue adhesion, wound closure, and cell-to-cell toxicity, making it an important raw material for applications in the daily chemical industry and medical devices. However, its application is significantly limited by the difficulty in obtaining sufficient quantities of mussel raw materials. Recombinant mussel mucin (rMAP) is produced through genetic engineering techniques using microbial hosts through fermentation and purification. It possesses the same adhesive properties, biocompatibility, and biodegradability as natural mussel mucin, and its production is not limited by raw material sources, allowing for large-scale production. It has become a promising bioadhesive material for medical and tissue engineering applications. Therefore, the use of genetically engineered mussel mucin for its production is an inevitable future development trend.

[0004] Currently, rMAP production is low, and most contain fusion tags, resulting in high purification costs and low yields. The adhesion properties of recombinant proteins are far inferior to those of native mussel mucins, limiting their in-depth research and practical applications. Therefore, targeted design is being conducted based on existing mussel mucin sequences to specifically modify the coding sequence of the protein molecule, thereby improving its expression and adhesion properties. Summary of the Invention

[0005] In response to the deficiencies of the prior art, the present invention provides a recombinant mussel-like mucin, its encoding gene, and its application. The present invention screens, assembles, and splices partial functional sequences of different types of mussel mucins to obtain a completely new mussel-like mucin sequence. The soluble expression level of the recombinant protein in Escherichia coli is significantly improved by optimizing the culture conditions. The recombinant protein of the present invention does not carry any exogenous tags, and the purification method provided is low-cost and high-yield, making it suitable for large-scale production. The recombinant protein of the present invention has good adhesion and antioxidant properties, and the biological properties of the recombinant mussel-like mucin modified by tyrosinase are better.

[0006] In order to achieve the above technical objectives, the technical solutions of the present invention are as follows: A recombinant mussel-like mucin, the amino acid sequence of the recombinant mussel-like mucin is shown in SEQ ID NO.1.

[0007] A gene encoding a recombinant mussel-like mucin, which encodes the recombinant mussel-like mucin.

[0008] According to the present invention, preferably, the nucleotide sequence of the coding gene is shown as SEQ ID NO.2.

[0009] A recombinant expression vector contains the coding gene of the recombinant mussel-like mucin.

[0010] Preferably according to the present invention, the plasmid vector of the recombinant expression vector is pET28a.

[0011] A recombinant expression strain contains the coding gene of the recombinant mussel-like mucin or the recombinant expression vector.

[0012] According to the present invention, preferably, the host bacteria of the recombinant expression strain is Escherichia coli.

[0013] More preferably, the Escherichia coli is Escherichia coli BL21 (DE3).

[0014] The application of the recombinant expression strain in the preparation of recombinant mussel-like mucin.

[0015] A method for preparing recombinant mussel-like mucin by fermenting the above-mentioned recombinant expression strain comprises the steps of: inoculating the recombinant expression strain into LB liquid culture medium to obtain seed liquid, inoculating the seed liquid into TB culture medium to induce expression to obtain the recombinant mussel-like mucin.

[0016] A method for preparing recombinant mussel-like mucin by fermenting the above-mentioned recombinant expression strain comprises the steps of: inoculating the recombinant expression strain into LB liquid culture medium to obtain seed liquid, inoculating the seed liquid into M9 culture medium to induce expression to obtain the recombinant mussel-like mucin.

[0017] A method for purifying recombinant mussel-like mucin comprises the following steps: subjecting the fermentation liquid after induction expression of the recombinant expression strain to centrifugation to obtain bacterial sludge; and subjecting the bacterial liquid after resuspending the bacterial sludge to homogenization, protein removal, centrifugation, clarification, ion exchange chromatography, ultrafiltration desalination and freeze-drying to obtain a purified sample of the recombinant mussel-like mucin.

[0018] Preferably, according to the present invention, the impurity protein is removed by acid precipitation, and the pH of the homogenized bacterial solution is adjusted to 2.9-3.1.

[0019] According to the preferred embodiment of the present invention, the pH of the centrifugal supernatant is adjusted to 7.4-7.6 after the centrifugation.

[0020] According to the preferred embodiment of the present invention, the clarification is performed by filtering through a 0.45µm PES membrane to sterilize and remove macromolecular impurities.

[0021] According to the present invention, preferably, the ion exchange chromatography is cation exchange chromatography.

[0022] Preferably according to the present invention, the molecular weight cut-off of the ultrafiltration is 3 kDa.

[0023] The invention relates to a modified recombinant mussel-like mucin, which is obtained by treating the recombinant mussel-like mucin with tyrosinase to modify the tyrosine residue into DOPA.

[0024] Application of the above recombinant mussel-like mucin or the above modified recombinant mussel-like mucin in the preparation of food, medicine, cosmetics, health care products or device products.

[0025] Beneficial effects: The present invention screens, assembles and splices partial functional sequences of different types of mussel mucins to obtain a completely new mussel-like mucin sequence. The sequence is optimized by the codon preference of Escherichia coli to construct a high-expression recombinant strain. The recombinant strain significantly improves the soluble expression level of the target protein by optimizing the culture conditions. The recombinant protein of the present invention does not carry any exogenous tags, and the purification method provided is low in cost and high in yield, making it suitable for large-scale production. The recombinant mussel-like protein prepared by the present invention has good adhesion and antioxidant properties, and the present invention also provides a modified recombinant mussel-like protein whose biological properties are better than those before modification. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Recombinant bacteria E. coil SDS-PAGE protein electrophoresis after BL21 / pET28a-1MAP induction expression; Lane M represents a standard protein with a molecular weight of 180 kDa; Lane 1 represents a blank control bacterium E. coilBL21 / pET28a cell lysis supernatant; Lane 2 represents the blank control bacteria E. coil BL21 / pET28a cell lysis precipitation; Lane 3 represents the recombinant bacteria E. coil BL21 / pET28a-1MAP cell lysis supernatant; Lane 4 represents the recombinant bacteria E. coil BL21 / pET28a-1MAP cell lysis and precipitation.

[0027] Figure 2 Recombinant bacteria E. coil SDS-PAGE protein electrophoresis of BL21 / pET28a-lMAP after induction expression under different fermentation conditions; Lane M represents a standard protein with a molecular weight of 180 kDa; Lane 1 represents the recombinant bacteria E. coil BL21 / pET28a-1MAP cell lysis supernatant; Lane 2 represents the recombinant bacteria E. coil BL21 / pET28a-1MAP cell lysis precipitation; Lane 3 represents the recombinant bacteria E. coil BL21 / pET28a-1MAP cell lysis supernatant after fermentation conditions were optimized; Lane 4 represents the recombinant bacteria E. coil Cell lysis and precipitation after optimization of BL21 / pET28a-lMAP fermentation conditions.

[0028] Figure 3 The figure shows the adhesion results of the glass lap test of different samples; among them, the blank control represents BSA, lMAP represents the unmodified recombinant mussel-like mucin, and lMAP-T represents the modified recombinant mussel-like mucin.

[0029] Figure 4 The figure shows the DPPH radical scavenging rate results of different samples; among them, the blank control represents water, lMAP represents unmodified recombinant mussel-like mucin, and lMAP-T represents modified recombinant mussel-like mucin. DETAILED DESCRIPTION

[0030] The technical solutions of the present invention are further described in detail below with reference to the examples and accompanying drawings, but the scope of protection of the present invention is not limited thereto. The drugs and reagents involved in the examples, unless otherwise specified, are all common commercially available products; the experimental procedures involved in the examples, unless otherwise specified, are all routine operations in the art. The percentages involved in the examples, unless otherwise specified, are all percentages by mass.

[0031] Example 1: Design of recombinant mussel-like mucin sequences In order to make the expressed recombinant mussel-like mucin have better biological function, the present invention selected partial functional sequences of natural type I blue mussel (GenBank: AAX23968.1), type III thick-shelled mussel (GenBank: ACT66140.1) and natural type V green mussel (GenBank: KF318695.1) for assembly and splicing to obtain a new mussel-like mucin sequence. The sequence has a total of 134 amino acids, and the specific amino acid sequence is shown in SEQ ID NO.1. The sequence contains the main biological active sites of different types of mussel mucins, ensuring the realization of the biological function of the recombinant mussel-like mucin.

[0032] Example 2: Recombinant bacteria E. coil Construction of BL21 / pET28a-1MAP The coding gene of the recombinant mussel-like mucin shown in SEQ ID NO.1 in Example 1 was codon-optimized according to the codon preference of E. coli. The optimized coding gene nucleotide sequence is shown in SEQ ID NO.2 and is named 1MAP. The nucleotide sequence of 1MAP was synthesized by Nanjing GenScript Biotech Co., Ltd. and cloned into the E. coli expression vector pET28a and transformed into E. coil Among the TOP10, the recombinant expression vector pET28a-lMAP was obtained while ensuring that the reading frame was not shifted. After DNA sequencing and comparison, the recombinant expression vector pET28a-lMAP was successfully constructed.

[0033] The successfully verified recombinant expression vector pET28a-lMAP was transformed into Escherichia coli BL21 (DE3) according to the Escherichia coli operation manual, and the recombinant expression transformants were screened using kanamycin antibiotics. After PCR verification, the recombinant bacteria expressing recombinant mussel-like mucin were E. coil BL21 / pET28a-1MAP was constructed successfully.

[0034] Example 3: Recombinant bacteria E. coil Inducible expression of BL21 / pET28a-1MAP Picking recombinant bacteria E. coil A single BL21 / pET28a-lMAP colony was inoculated into 5 mL of LB liquid medium (1% NaCl, 0.5% yeast extract, 1% peptone) containing 50 μg / mL kanamycin and cultured overnight at 37°C and 200 rpm. Then, a 2% inoculum size was inoculated into TB medium (1.18% peptone, 2.36% yeast extract, 0.94% K2HPO4, 0.22% KH2PO4, 0.4% glycerol) and cultured at 37°C and 200 rpm until the OD 600The expression of the recombinant mussel-like mucin was induced by adding IPTG (isopropyl-β-D-thiogalactopyranoside) at a final concentration of 0.5 mM, culturing at 25 ° C for 16 h, collecting the bacteria by centrifugation, resuspending them in Tris buffer (20 mM Tris, pH 7.5), and then ultrasonically disrupting them. The supernatant and precipitate of the cells were subjected to SDS-PAGE detection. The theoretical molecular weight of the recombinant mussel-like mucin was 15.03 kDa. The SDS-PAGE results are as follows Figure 1 As shown, the recombinant bacteria E. coil BL21 / pET28a-lMAP cell lysis supernatant (lane 3) and blank control bacteria E. coil Compared with BL21 / pET28a (lane 1), there is an obvious protein band (indicated by the arrow) at the theoretical molecular weight of the target protein. E. coil The BL21 / pET28a-1MAP cell lysis pellet (lane 4) showed a higher concentration of the target protein than the cell lysis supernatant (lane 3). These results demonstrate that the recombinant mussel-like mucin of the present invention can be successfully induced for expression, but under the culture conditions of this example, insoluble expression of the target protein was greater than soluble expression.

[0035] Example 4: Recombinant bacteria E. coil Optimization of BL21 / pET28a-1MAP culture conditions In order to improve the soluble expression of recombinant mussel-like mucin, the recombinant bacteria E. coil The culture conditions of BL21 / pET28a-1MAP were optimized.

[0036] Picking recombinant bacteria E. coil A single BL21 / pET28a-lMAP colony was inoculated into 5 mL of LB liquid medium (1% NaCl, 0.5% yeast extract, 1% peptone) containing 50 μg / mL kanamycin and cultured overnight at 37°C and 200 rpm. Then, a 3% inoculum size was inoculated into M9 medium (M9 mother liquor: 64 g disodium hydrogen phosphate, 15 g potassium dihydrogen phosphate, 2.5 g sodium chloride, 5.0 g ammonium chloride, dilute to 1 L and then sterilize; 200 mL of M9 mother liquor was added to 700 mL of sterile water, 2 mL of sterile 1 mol / L magnesium sulfate solution, and 100 μL of sterile 1 mol / L calcium chloride solution) and cultured at 37°C and 200 rpm until the OD 600The expression of the recombinant mussel-like mucin was induced by adding IPTG (isopropyl-β-D-thiogalactopyranoside) at a final concentration of 0.25 mM, culturing at 25 ° C for 16 h, collecting the bacteria by centrifugation, resuspending them in Tris buffer (20 mM Tris, pH 7.5), and then ultrasonically disrupting them. The supernatant and precipitate of the cells were subjected to SDS-PAGE detection. The theoretical molecular weight of the recombinant mussel-like mucin was 15.03 kDa. The SDS-PAGE results are as follows Figure 2 As shown, the recombinant bacteria E. coil The supernatant of BL21 / pET28a-1MAP cell lysis (lane 3) and the recombinant bacteria in Example 3 E. coil Compared to the supernatant of BL21 / pET28a-1MAP (lane 1), the expression of the soluble target protein increased by 5.3-fold (indicated by the arrow), while the insoluble expression of the recombinant mussel-like mucin 1MAP (lane 4) was significantly reduced. The increased soluble protein expression is hypothesized to be due to the relatively richer nutrients in the TB medium used in Example 3 compared to the M9 medium used in Example 4. This leads to faster bacterial growth and metabolism during fermentation, and consequently, faster protein production. This hinders subsequent protein folding, and improperly folded proteins precipitate as inclusion bodies. The M9 medium used in Example 4 is relatively nutrient-poor, primarily consisting of inorganic salts, which slows bacterial growth and metabolism, resulting in slower protein production and more time for the cells to complete proper protein folding.

[0037] Example 5: Purification and preparation of recombinant mussel-like mucin A fermentation broth was prepared according to the fermentation method of Example 4, and the broth was centrifuged at 8000 rpm to obtain bacterial sludge. The bacterial sludge was purified by the following steps to obtain a pure recombinant mussel-like mucin: (1) Protein removal: After the bacterial slurry is resuspended and homogenized, the pH of the bacterial solution is adjusted to 3.0 with a low concentration (1M) hydrochloric acid solution. The bacterial solution is stirred continuously during the acidification process to prevent local over-acidification during the acidification process, which will cause the recombinant mussel-like mucin to denature and precipitate; (2) Centrifugation: Remove the impurities precipitated by acid by centrifugation, and adjust the pH of the supernatant to 7.5 with a low concentration (1 M) sodium hydroxide solution; (3) Clarification: The centrifugal supernatant adjusted to pH 7.5 was filtered through a 0.45 µm PES membrane to sterilize and remove macromolecular impurities; (4) Ion exchange chromatography: using cation exchange chromatography, the recombinant mussel-like mucin is positively charged in a pH 7.5 buffer. During the loading process, the cationic filler binds to the target protein. After sufficient washing, it is eluted with 200 mM NaCl solution and the peaks are collected. The eluate obtained is the purified stock solution; (5) Ultrafiltration membrane desalination: Use a 3 kDa ultrafiltration membrane to desalinate the purified stock solution until the conductivity of the feed solution is less than 70 μs / cm; (7) Freeze-drying: The desalted liquid obtained above was filtered through a 0.45 μm microfiltration membrane and placed in a freeze-drying tray for freeze-drying.

[0038] The purity of the recombinant mussel-like mucin purified through the above steps is greater than 97%, and the yield is greater than 60%.

[0039] Example 6: In vitro modification, purification and DOPA content determination of recombinant mussel-like mucin The lyophilized recombinant mussel-like mucin prepared in Example 5 was weighed and dissolved in 5% acetic acid solution to a concentration of 1.5 mg / mL. Tyrosinase (Sigma, T3824) was added to a final concentration of 10 U / mL, and the mixture was reacted at 25°C for 2 hours to modify the tyrosine residues of the recombinant mussel-like mucin into DOPA, thereby obtaining the final product of the recombinant protein functional region modification, lMAP-T.

[0040] The DOPA content was detected using a DOPA decarboxylase ELISA kit (Zhongshuo Biotechnology, ZS16356S). Three samples were tested in parallel, and the test results showed an average DOPA content of 198 μmol / g.

[0041] Example 7: Adhesion performance test of recombinant mussel-like mucin - glass overlap experiment BSA (blank control), unmodified recombinant mussel-like mucin lMAP, and modified mussel-like mucin lMAP-T were dissolved in 150 mM sodium chloride saline to obtain different protein solutions at a protein concentration of 50 mg / mL. Approximately 10 μL of the protein solution was applied to the top of a bonded glass substrate (contact surface approximately 10 mm wide by 10 mm long) and cured at 37°C for 18 hours. Lap shear tests were performed using a universal testing machine, using a 100 N load cell and a shear speed of 10 mm / min. Shear strength (i.e., adhesion) was determined by dividing the maximum force by the bonded area.

[0042] The results are as follows Figure 3 As shown in the results, the modified recombinant mussel-like mucin lMAP-T and the unmodified recombinant mussel-like mucin lMAP had better adhesion properties than the blank control (BSA), and the modified recombinant mussel-like mucin lMAP-T had the best adhesion property.

[0043] Example 8: Antioxidant capacity detection of recombinant mussel-like mucin—DPPH free radical scavenging experiment Take 1 mL of unmodified recombinant mussel mucin lMAP solution and modified recombinant mussel mucin lMAP-T solution with a concentration of 3.5 g / L and place them in test tubes respectively. Add 2.5 mL of DPPH free radical solution (0.1 mmol / L) dissolved in 95% ethanol. Shake the mixture vigorously for 10 seconds and then place it at room temperature to react for 30 minutes. After the reaction is completed, measure the absorbance of the reaction mixture at 517 nm, and use distilled water instead of the added protein solution as a blank control.

[0044] DPPH free radical scavenging rate = (OD 空白 -OD 样品 ) / OD 空白 ×100%, Among them, OD 空白 is the absorbance value of the blank control group, OD 样品 is the absorbance value of the unmodified recombinant mussel-like mucin 1MAP or the modified recombinant mussel-like mucin 1MAP-T group.

[0045] The results are as follows Figure 4 As shown, the unmodified recombinant mussel-like mucin 1MAP and the modified recombinant mussel-like mucin 1MAP-T of the present invention have significant DPPH free radical scavenging ability compared to the blank control, and the modified recombinant mussel-like mucin 1MAP-T has better DPPH free radical scavenging ability than the unmodified recombinant mussel-like mucin 1MAP. In summary, the unmodified recombinant mussel-like mucin 1MAP and the modified recombinant mussel-like mucin 1MAP-T provided by the present invention both have significant antioxidant capacity, and the modified recombinant mussel-like mucin 1MAP-T has stronger antioxidant capacity than the unmodified mussel-like mucin 1MAP.

Claims

1. A recombinant mussel-like mucin, characterized in that: The amino acid sequence of the recombinant mussel-like mucin is shown in SEQ ID NO.

1.

2. A gene encoding a recombinant mussel-like mucin, characterized in that: It encodes the recombinant mussel-like mucin according to claim 1; Preferably, the nucleotide sequence of the encoding gene is shown as SEQ ID NO.

2.

3. A recombinant expression vector, characterized in that: Containing the gene encoding the recombinant mussel-like mucin according to claim 2; Preferably, the plasmid vector of the recombinant expression vector is pET28a.

4. A recombinant expression strain, characterized in that: Containing the coding gene of the recombinant mussel-like mucin according to claim 2 or the recombinant expression vector according to claim 3; Preferably, the host bacteria of the recombinant expression strain is Escherichia coli; More preferably, the Escherichia coli is Escherichia coli BL21 (DE3).

5. Use of the recombinant expression strain according to claim 4 in preparing recombinant mussel-like mucin.

6. A method for preparing recombinant mussel-like mucin by fermentation using the recombinant expression strain according to claim 4, characterized in that: The method comprises the following steps: inoculating the recombinant expression strain into LB liquid culture medium to obtain seed liquid, inoculating the seed liquid into TB culture medium to obtain recombinant mussel-like mucin by inducing expression.

7. A method for preparing recombinant mussel-like mucin by fermentation using the recombinant expression strain according to claim 4, characterized in that: The method comprises the following steps: inoculating the recombinant expression strain into LB liquid culture medium to obtain seed liquid, inoculating the seed liquid into M9 culture medium to obtain recombinant mussel-like mucin by inducing expression.

8. A method for purifying recombinant mussel-like mucin, characterized in that: The method comprises the following steps: subjecting the fermentation liquid after induction expression of the recombinant expression strain according to claim 6 or 7 to centrifugation to obtain bacterial sludge, and subjecting the bacterial liquid after resuspending the bacterial sludge to homogenization, protein removal, centrifugation, clarification, ion exchange chromatography, ultrafiltration desalination and freeze-drying to obtain a purified sample of recombinant mussel-like mucin; Preferably, the impurity protein is removed by acid precipitation, and the pH of the homogenized bacterial solution is adjusted to 2.9-3.1; Preferably, after the centrifugation, the pH of the supernatant is adjusted to 7.4-7.6; Preferably, the clarification is performed by filtration through a 0.45 μm PES membrane to sterilize and remove macromolecular impurities; Preferably, the ion exchange chromatography is cation exchange chromatography; Preferably, the molecular weight cut-off of the ultrafiltration is 3 kDa.

9. A modified recombinant mussel-like mucin, characterized in that: The recombinant mussel-like mucin according to claim 1 is treated with tyrosinase to modify the tyrosine residues into dopa.

10. Use of the recombinant mussel-like mucin according to claim 1 or the modified recombinant mussel-like mucin according to claim 9 in the preparation of food, medicine, cosmetics, health products or device products.

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