Method for producing recombinant I-type mussel mucin rich in dopa

Abstract

The invention discloses a method for producing recombinant I-type mussel mucin rich in dopa, and belongs to the technical field of genetic engineering. According to the invention, Ala-Lys-Pro-Ser-Tyr-Pro-Pro-Thr-Tyr-Lys decapeptide of a natural type I mussel mucin sequence is taken as a repetitive unit, a gene expression vector for tandem expression of peptide fragments with different repetitive numbers is constructed, and pichia pastoris host bacteria are transformed, so that type I mussel mucin precursors with different molecular weights are obtained; in vitro, a precursor is subjected to dopa modification by high-activity tyrosinase obtained through screening in a mixed system containing ascorbic acid ethyl ether, copper sulfate and the like, and the recombinant I-type mussel mucin rich in dopa groups is obtained after purification. The recombinant I-type mussel mucin rich in dopa obtained by the invention has the advantages of high purity and high dopa modification rate, and has wide application prospects in the fields of biomedicine, surface chemistry and the like.

Description

Technical Field

[0001] This invention relates to a method for producing recombinant type I mussel adhesive protein rich in dopamine, belonging to the field of genetic engineering technology. Background Technology

[0002] Mussel adhesive protein (MAP) is a protein complex secreted by the foot glands of mussels. It possesses advantages such as high strength, high toughness, water resistance, and superior adhesion in wet environments, earning it the nickname "marine soft gold." Furthermore, mussel adhesive protein promotes cell adhesion and crawling, accelerates wound healing, inhibits itching, provides broad-spectrum adhesion, and forms a water-resistant protective film, attracting widespread attention in recent years. Currently, the identified mussel adhesive proteins mainly include six subspecies: mfp-1, mfp-2, mfp-3, mfp-4, mfp-5, and mfp-6. Among them, mfp-1 is the most abundant component, primarily covering the surface of the byssal threads and forming a protective layer that prevents other adhesive proteins within the byssal threads from being dissolved by seawater and degraded by marine microorganisms. mfp-2 to mfp-6 are mainly located in the byssal disc of mussels and are rich in L-3,4-dihydroxyphenylalanine (i.e., the dopa functional group), serving as the main protein components for strong adhesion in mussels. Currently, mussel adhesive protein products are mainly obtained by extracting from mussel byssal threads. The commercially available mussel adhesive CellTak™, as a medical cell and tissue adhesive, uses extracted natural mussel Mfp-1 and Mfp-3 / 5 as its main components. However, the traditional extraction process is extremely costly, requiring 10,000 mussels to extract 1 mg of mussel adhesive protein.

[0003] Therefore, the fermentation production of recombinant mussel adhesive protein using biotechnology has become an important direction for technological breakthroughs. Currently, recombinant mussel adhesive protein is mainly expressed through the *E. coli* expression system. Its drawback is that the target protein exists in the form of inclusion bodies, requiring subsequent acid extraction (acetic acid or citric acid). The product's pH cannot exceed 5.0, and precipitation occurs within the physiological pH range, greatly limiting its application. In contrast, the *Pichia pastoris* expression system offers advantages such as high-density fermentation, low culture costs, and high protein expression levels, making it suitable for large-scale production. Currently, there are no reports on the recombinant expression of type I mussel adhesive protein using *Pichia pastoris*.

[0004] Tyrosinase is a copper-containing multifunctional oxidoreductase, also known as polyphenol oxidase, which is a type of phenol oxidase and belongs to monooxygenases. Tyrosinase utilizes molecular oxygen to catalyze two different enzymatic reactions: (1) catalyzing the hydroxylation of tyrosine to dopa (monophenolase activity) and (2) catalyzing the oxidation of dopa to dopaquinone (bisphenolase activity). Molecular oxygen acts as an electron acceptor and is reduced to water in both reactions, while active quinone compounds can spontaneously polymerize to generate melanin. When using tyrosinase to oxidize and modify substrates in vitro, for small molecule substrates like tyrosine, it is easy to enter the active site of tyrosinase and obtain oxygen atoms, thus converting monohydroxy tyrosine into orthohydroxy DOPA; however, for protein substrates containing tyrosine residues, due to the existence of helical, folded, and tertiary conformations of the protein secondary structure, the probability of limited collisions between the active site of tyrosinase and tyrosine residues in the substrate protein is reduced. This steric hindrance makes it difficult to achieve the ideal level of tyrosine residue oxidation modification when attempting to use tyrosinase to modify recombinant mussel adhesive protein expressed by the host in vitro with dopa. Therefore, screening for tyrosinases that can be used to produce dopa-rich recombinant mussel adhesive protein is of great significance. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a method for preparing dopamine-rich recombinant type I mussel adhesive protein. This invention uses the Ala-Lys-Pro-Ser-Tyr-Pro-Pro-Thr-Tyr-Lys decapeptide of the natural type I mussel adhesive protein sequence as a repeating unit, constructs gene expression vectors expressing peptides with different numbers of repeating peptides in tandem, and introduces them into Pichia pastoris host cells to obtain recombinant type I mussel adhesive protein precursors of different molecular weights. These precursors are then subjected to dopamine oxidation modification in vitro using a screened highly active tyrosinase, and after purification, finally yield dopamine-rich recombinant type I mussel adhesive protein. The specific technical solution of this invention is as follows: In one aspect, the present invention provides an engineered bacterium that secretes and expresses a type I mussel adhesive protein precursor, characterized in that the engineered bacterium expresses a type I mussel adhesive protein composed of a repeating tandem decapeptide repeat sequence; Wherein, the decapeptide repeat sequence is AKPSYPPTYK, and the number of tandem repeats of the decapeptide repeat sequence is m, 5≤m≤20; preferably, m is selected from at least 5, 10 or 20. The starting strain of the engineered bacteria is Pichia pastoris. Preferably, the Pichia pastoris includes Pichia pastoris GS115, Pichia pastoris KM71, Pichia pastoris SMD1168 or Pichia pastoris X33. More preferably, the Pichia pastoris is selected from Pichia pastoris GS115.

[0006] The type I mussel adhesive protein has a histidine tag fused to its N-terminus; preferably, the histidine tag sequence is HHHHHH.

[0007] Preferably, the amino acid sequence corresponding to the decapeptide repeat sequence includes SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3.

[0008] More preferably, the nucleotide sequence corresponding to the decapeptide repeat sequence includes any of the nucleotide sequences shown in SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6, or a nucleotide sequence having 98% homology with any of the nucleotide sequences in SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6.

[0009] The recombinant yeast strain recombinant vector includes pPIC9K, or Preferably, pPIC9K is used as the expression vector.

[0010] The application of the above-mentioned recombinant yeast strain or microbial preparations containing the above-mentioned recombinant yeast strain in the production of type I mussel adhesive protein precursor.

[0011] Another aspect of the present invention provides a method for preparing a type I mussel adhesive protein precursor, characterized in that the method includes the steps of fermenting and induced expression of the above-mentioned recombinant yeast strain.

[0012] Specifically, the fermentation medium is BMMY or BSM, and methanol is used to induce the secretion and expression of type I mussel adhesive protein precursor.

[0013] In one specific embodiment, the method includes using BMMY medium and methanol-induced fermentation to produce a fermentation broth containing a recombinant type I mussel adhesive protein precursor.

[0014] In one specific embodiment, Pichia pastoris is inoculated into a seed culture medium to obtain a seed liquid; the seed liquid is then inoculated into a BSM fermentation medium for fermentation to obtain a fermentation broth containing recombinant type I mussel adhesive protein precursor, wherein the fermentation process includes four stages in sequence: initial fermentation, fed-batch culture, starvation culture, and methanol induction.

[0015] The initial fermentation conditions are: fermentation temperature of 25~30℃, pH of 4.0~7.0, with the preferred fermentation temperature being 30℃ and pH of 5.0.

[0016] The starvation culture time is 2-3 hours.

[0017] The temperature for the methanol induction stage is 25~30℃.

[0018] The fermentation time is 80-120 hours.

[0019] Another aspect of the present invention provides a biomaterial, characterized in that the biomaterial comprises any one of the following: A1) A nucleotide molecule comprising any of the nucleotide sequences shown in SEQ ID NO. 4, SEQ ID NO. 5, or SEQ ID NO. 6, or a nucleotide sequence having 98% homology with any of the nucleotide sequences in SEQ ID NO. 4, SEQ ID NO. 5, or SEQ ID NO. 6; A2) Expression cassette containing the nucleic acid molecule described in A1); A3) A recombinant vector containing the nucleic acid molecule described in A1) and / or containing the expression cassette described in A2); A4) Recombinant microorganisms containing the nucleic acid molecule described in A1), the expression cassette described in A2), and / or the recombinant vector described in A3); A5) Recombinant cells containing the nucleic acid molecule described in A1), the expression cassette described in A2), and / or the recombinant vector described in A3); A6) A whole-cell catalyst containing the nucleic acid molecule described in A1), the expression cassette described in A2), the recombinant vector described in A3), the recombinant microorganism described in A4), and / or the recombinant cell described in A5).

[0020] In another aspect of the present invention, a method for producing dopa-rich recombinant type I mussel adhesive protein is provided, wherein a purified type I mussel adhesive protein precursor is obtained using the above-mentioned recombinant bacteria, the purified product is modified with dopa in vitro by tyrosinase, and the dopa-rich recombinant type I mussel adhesive protein is obtained by separation and purification. The tyrosinase gene sequence mentioned above is derived from Wiedmannii bacillus or Bacillus wiedmannii bv. Thuringiensis Preferred Wiedmannii bacillus source.

[0021] Specifically, the aforementioned Wiedmannii bacillus The source is tyrosinase, and its amino acid sequence is shown in SEQ ID NO. 9.

[0022] Specifically, the aforementioned Wiedmannii bacillus The source is tyrosinase, whose nucleotide sequence is shown in SEQ ID NO. 10.

[0023] Specifically, the aforementioned wiedmannii bv. Thuringiensis The source is tyrosinase, and its amino acid sequence is shown in SEQ ID NO.11.

[0024] Specifically, the aforementioned wiedmannii bv. Thuringiensis The source is tyrosinase, and its nucleotide sequence is shown in SEQ ID NO.12; The in vitro modification mixture includes: 20-30 mM borate, 0.1-0.5 mM sodium dihydrogen phosphate, 10-20 mM ascorbic acid ethyl ether, 3-5 mM glutamate, 0.01-0.05 mM copper sulfate, 0.5-10 g / L recombinant mussel adhesive protein precursor, and 50-200 U / mL tyrosinase.

[0025] The separation and purification method is selected from one or more of affinity chromatography, chromatographic chromatography, salting out, acid-base precipitation and membrane separation, preferably a combination of membrane separation and chromatographic chromatography.

[0026] Another aspect of the present invention provides a dopa-rich recombinant type I mussel adhesive protein, wherein the recombinant type I mussel adhesive protein is a type I mussel adhesive protein composed of a repeating decapeptide repeating sequence, wherein the decapeptide repeating sequence is AKPSYPPTYK; the number of repeating sequences in the tandem is m, 5≤m≤20; preferably, m is selected from at least 5, 10 or 20. The recombinant type I mussel adhesive protein has a dopa content of 0.4-1.5% and a purity of 90-97%.

[0027] Optionally, the dopa content of the recombinant type I mussel adhesive protein is not less than 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5% and any value between them.

[0028] Another aspect of the present invention provides a dopa-rich recombinant type I mussel adhesive protein, wherein it is prepared by the above-described preparation method.

[0029] The beneficial effects of this application include, but are not limited to, the following: This invention utilizes the Ala-Lys-Pro-Ser-Tyr-Pro-Pro-Thr-Tyr-Lys decapeptide sequence of natural type I mussel adhesive protein as a repeating unit to construct gene expression vectors for tandem expression of peptide segments with different numbers of repeats. These vectors are then transformed into Pichia pastoris host cells to obtain recombinant type I mussel adhesive protein precursors of different molecular weights. These precursors are then subjected to dopa oxidation modification in vitro using a highly active tyrosinase obtained through screening. After separation and purification, dopa-rich recombinant type I mussel adhesive protein is finally obtained. The recombinant type I mussel adhesive protein of this application has the advantages of high purity and high dopa modification rate, and has broad application prospects in biomedicine, surface chemistry, and other fields. Attached Figure Description

[0030] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings: Figure 1 The images show SDS-PAGE and Western blotting data of the GS115 / pPIC9k-Mfp-1 recombinant yeast strain during shake-flask horizontal fermentation. Figure 1 A is an SDS-PAGE image, with lanes 1, 2, 3, and 4 representing recombinant bacteria. P. pastoris GS115 / pPIC9K-Mfp-1-RE5 P. pastoris GS115 / pPIC9K-Mfp-1-RE10 P. pastoris GS115 / pPIC9K-Mfp-1-RE20 and P. pastoris GS115 / pPIC9K-Mfp-1-RE40 fermentation supernatant; Figure 1 B is a Western blotting image, with lanes 5, 6, 7, and 8 representing recombinant bacteria. P. pastoris GS115 / pPIC9K-Mfp-1-RE5 P. pastoris GS115 / pPIC9K-Mfp-1-RE10 P. pastoris GS115 / pPIC9K-Mfp-1-RE20 and P. pastoris GS115 / pPIC9K-Mfp-1-RE40 fermentation supernatant.

[0031] Figure 2 SDS-PAGE analysis of the fermentation supernatant of the GS115 / pPIC9k-Mfp-1 recombinant yeast strain in a 5 L fermenter over 94 h. Figure 2 A is a recombinant bacterium. P. pastoris SDS-PAGE of fermentation supernatant in a 5 L fermenter (GS115 / pPIC9K-Mfp-1-RE5) within 94 h. Lanes 1-8 represent fermentation supernatant after 17 h, 29 h, 41 h, 53 h, 65 h, 77 h, 89 h, and 94 h of fermentation, respectively. Figure 2 B is a recombinant bacterium. P. pastoris SDS-PAGE of fermentation supernatant in a 5 L fermenter (GS115 / pPIC9K-Mfp-1-RE10) within 94 h. Lanes 9-16 represent fermentation supernatant after 17 h, 29 h, 41 h, 53 h, 65 h, 77 h, 89 h, and 94 h of fermentation, respectively. Figure 2 C represents recombinant bacteria. P. pastoris SDS-PAGE of fermentation supernatant in a 5 L fermenter (GS115 / pPIC9K-Mfp-1-RE20) over 94 h. Lanes 17-24 represent fermentation supernatant after 17 h, 29 h, 41 h, 53 h, 65 h, 77 h, 89 h, and 94 h of fermentation, respectively.

[0032] Figure 3 SDS-PAGE protein electrophoresis (A) and Western Blot results (B) of recombinant *E. coli* BL21 strains expressing ten different tyrosinase genes were obtained. Lane M represents a standard protein with a molecular weight of 180 kDa; BL21 / pET28a is the negative control; BL21 / pET28a-TYR-BWT represents the expression of... Bacillus wiedmannii bv. Thuringiensis Source: Recombinant tyrosinase strain; BL21 / pET28a-TYR-SR-1 represents the expression... Stenotrophomonas rhizophila (Oligotrophomonas rhizophila) tyrosinase-derived recombinant bacteria; BL21 / pET28a-TYR-BW represents expression. Wiedmannii bacillus (Bacillus videmannii)-derived recombinant tyrosinase strain; BL21 / pET28a-TYR-PY represents expression Pseudomonas yamanorum Source: Recombinant tyrosinase strain; BL21 / pET28a-TYR-SR-2 represents expression Streptomyces rimosus Source: Recombinant tyrosinase strain; BL21 / pET28a-TYR-AB represents expression Actinobacteria bacterium OV320-derived recombinant tyrosinase strain; BL21 / pET28a-TYR-TS represents expression. Thalassospira sp. KO164 (Indian Ocean Cyclostome)-derived recombinant tyrosinase strain; BL21 / pET28a-TYR-PM represents the expression... Priestia megaterium (Bacillus megaterium)-derived recombinant tyrosinase strain; BL21 / pET28a-TYR-BJ represents expression Bradyrhizobium japonicum (Rhizobium japonicum) source tyrosinase recombinant strain; BL21 / pET28a-TYR-PS represents expression Pseudomonas synxantha Source: Recombinant tyrosinase bacteria; Figure 4Figure 1 shows the identification of DOPA modification of recombinant mussel adhesive protein precursor by crude tyrosinase enzyme solutions from different sources. Figure A shows the identification of DOPA modification of recombinant mussel adhesive protein precursor by crude tyrosinase enzyme solutions from prokaryotic sources. Samples 1-10 represent the catalytic systems of crude tyrosinase enzyme solutions numbered BWT, PS, SR-2, AB, TS, SR-1, PY, BW, BJ, and PM, respectively. Sample 11 is the negative control, i.e., the catalytic system of cell lysis supernatant without tyrosinase gene expression. Sample 12 is the mussel adhesive protein precursor without DOPA modification. Sample 13 is the catalytic system without mussel adhesive protein and any crude tyrosinase enzyme solution (to verify the effect of substances such as copper sulfate, ascorbic acid ethyl ether, and glutamic acid in the catalytic system on the DOPA identification reaction). Samples 14 and 15 are 1 g / L and 0.5 g / L commercially available recombinant mussel adhesive protein containing DOPA modification, respectively. Figure B shows the identification of the modification of recombinant mussel adhesive protein precursor dopa by crude eukaryotic tyrosinase enzyme solution. Sample 1 is the negative control, i.e., the fermentation supernatant catalytic system of recombinant Pichia pastoris strain that does not express the tyrosinase gene; Samples 2-11 represent the fermentation supernatant catalytic systems of recombinant tyrosinase strains numbered TS, NC, LB, AL, PC170, H, DC, RS, PC, and TV, respectively.

[0033] Figure 5 SDS-PAGE electrophoresis analysis of purified recombinant type I mussel adhesive proteins. Lanes 1, 2, and 3 represent purified recombinant type I mussel adhesive proteins Mfp-1-RE5, Mfp-1-RE10, and Mfp-1-RE20, respectively.

[0034] Figure 6 RP-HPLC purity analysis of lyophilized recombinant type I mussel adhesive protein. Figure 6 A and C are RP-HPLC purity analysis chromatograms of freeze-dried recombinant type I mussel adhesive proteins Mfp-1-RE5, Mfp-1-RE10, and Mfp-1-RE20. Detailed Implementation

[0035] The invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the following embodiments are only for illustrating this application and are not intended to limit the scope of this application.

[0036] In the embodiments of this invention, unless otherwise described in detail, conventional molecular biology experimental methods were used. The processes involved in the embodiments, such as enzyme digestion, DNA purification and recovery, and preparation of Pichia pastoris competent cells, are all understandable and easily implemented by those skilled in the art based on product instructions or basic knowledge in the field, and therefore will not be described in detail. Unless otherwise stated, all instruments, reagents, and materials used in this invention are commercially available products.

[0037] Example 1: Type I mussel adhesive protein precursor 1) Sequence design of type I mussel adhesive protein This invention utilizes the NCBI (https: / / www.ncbi.nlm.nih.gov / ) and Uniprot (https: / / www.uniprot.org / ) websites to obtain the amino acid sequences of four natural type I mussel adhesive proteins Mefp-1, Mcfp-1, Mcofp-1, and Mgfp-1 from different mussel sources (reference sequences Genebank accession number: AAY29131.1; Uniprot accession numbers Q25460.1, Q27409.1, and Q25434.1 sequences). The decapeptide repeat units (Ala-Lys-Pro-Ser-Tyr-Pro-Pro-Thr-Tyr-Lys) are extracted and compared to design three tandemly expressed type I mussel adhesive protein sequences Mfp-1-RE5, Mfp-1-RE10, and Mfp-1-RE20, forming the amino acid sequences shown in SEQ ID NO. 1, SEQ ID NO. 2, and SEQ ID NO. 3. To facilitate the detection and purification of protein expression, this invention fuses a histidine tag (HHHHHH) to the N-terminus of the designed three amino acid sequences of type I mussel adhesive protein.

[0038] 2) Gene synthesis of type I mussel adhesive protein Based on the codon preference of *Pichia pastoris* in expressing heterologous proteins, the coding nucleotide sequences corresponding to the amino acid sequences in step 1 were codon-optimized. To facilitate the construction of the expression vector, restriction enzyme sites were added to the 5' end of the coding sequence, and stop codon and restriction enzyme sites were added to the 3' end. The optimized sequences were subcloned into the pPIC9K plasmid; the specific sequences are detailed in SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6 in the sequence listing. The whole gene synthesis was commissioned to Nanjing Genscript Biotech Co., Ltd., and the recombinant plasmid vectors were named pPIC9K-Mfp-1-RE5, pPIC9K-Mfp-1-RE10, and pPIC9K-Mfp-1-RE20.

[0039] 3) Construction and screening of recombinant yeast strains The recombinant expression plasmids pPIC9K-Mfp-1-RE5, pPIC9K-Mfp-1-RE10, and pPIC9K-Mfp-1-RE20 from step 2 were linearized using SalI restriction enzyme (purchased from Thermo Scientific, and the specific operation was performed according to the instructions). The linearized plasmids were then recovered using a DNA purification and recovery kit (purchased from Tiangen Biotech (Beijing) Co., Ltd.).

[0040] The SalI-restricted plasmids pPIC9K-Mfp-1-RE5, pPIC9K-Mfp-1-RE10, and pPIC9K-Mfp-1-RE20 were linearized with 100 μL of each plasmid. P. pastoris GS115 competent cells were mixed thoroughly and transferred to ice-cold electroporation cuvettes with an electrode spacing of 0.1 cm for electroporation transformation. The electroporation parameters were set as follows: capacitance 25 μF, resistance 200 ohms, and voltage 1 kV. After electroporation, 1 mL of ice-cold 1 M sorbitol solution was added to mix the cells thoroughly. The mixture was then spread on MD (Minimal Dextrase; YNB 3.4 g / L, biotin 0.2 g / L, glucose 20 g / L, agar powder 20 g / L) agar plates and incubated upside down at 30°C. Recombinant Pichia pastoris, a high-copy-count precursor of type I mussel adhesive protein, was obtained through screening with genistein G418. P .pastoris GS115 / pPIC9K-Mfp-1-RE5 P. pastoris GS115 / pPIC9K-Mfp-1-RE10 P .pastoris GS115 / pPIC9K-Mfp-1-RE20.

[0041] Example 2: Shake-flask fermentation of recombinant type I mussel adhesive protein precursor The recombinant engineered bacteria obtained in Example 1 were screened. P. pastoris GS115 / pPIC9K-Mfp-1-RE5 P .pastoris GS115 / pPIC9K-Mfp-1-RE10 P. pastoris GS115 / pPIC9K-Mfp-1-RE20 were cultured using shake flask fermentation. The shake flask fermentation steps were as follows: The constructed strain was streaked on YPD solid medium (yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L, agar powder 20 g / L) for 2-3 days; single colonies were picked and inoculated into 40 mL of YPD liquid medium and cultured at 30℃ and 220 rpm for 24 h; 10% inoculum was transferred to 40 mL of initial expression medium BMGY (yeast extract 10 g / L, peptone 20 g / L, K2HPO4 3 g / L, KH2PO4 11.8 g / L, YNB 3.4 g / L, (NH4)2SO4 10 g / L, biotin 4×10⁻⁶). -4In a medium containing 10 mL / L yeast extract and 10 mL / L glycerol, the cells were cultured at 30°C and 220 rpm for 24 h. The cells were collected by low-speed centrifugation, washed twice with physiological saline to remove glycerol, and then resuspended in 40 mL of fresh BMMY induction expression medium (10 g / L yeast extract, 20 g / L peptone, 3 g / L K₂HPO₄, 11.8 g / L KH₂PO₄, 3.4 g / L YNB, 10 g / L (NH₄)₂SO₄, 4 × 10⁻⁶ biotin). -4 The culture was carried out in a solution of 10 mL / L g / L methanol (10 mL / L) at 30 °C and 220 rpm. Methanol was added every 24 h to a final concentration of 1.0% (v / v) to continue induction, for a total induction period of 96 h. The culture was then centrifuged at 8000 rpm for 10 min at 4 °C, and the fermentation supernatant was collected for SDS-PAGE electrophoresis and Western blotting analysis. The results are as follows: Figure 1 As shown.

[0042] from Figure 1 As can be seen from the SDS-PAGE protein electrophoresis analysis of the fermentation supernatant, the recombinant engineered bacteria... P .pastoris GS115 / pPIC9K-Mfp-1-RE5 P. pastoris GS115 / pPIC9K-Mfp-1-RE20 exhibits relatively distinct bands at higher molecular weights (14000 Da and 40000 Da) (indicated by arrows), indicating the presence of recombinant engineered bacteria. P. pastoris GS115 / pPIC9K-Mfp-1-RE10 showed a weak band. Western blotting further confirmed protein expression, and the results indicated that the recombinant engineered bacteria... P. pastoris GS115 / pPIC9K-Mfp-1-RE5 P .pastoris GS115 / pPIC9K-Mfp-1-RE10 P. pastoris GS115 / pPIC9K-Mfp-1-RE20 showed clear and distinct bands at approximately 14,000 Da, 25,000 Da, and 40,000 Da, respectively, indicating that the recombinant engineered bacteria could successfully express the secretory type I mussel adhesive protein precursor. The protein secretion expression levels were Mfp-1-RE20 > Mfp-1-RE5 > Mfp-1-RE10, which was consistent with the SDS-PAGE results.

[0043] Example 3: High-density fermentation of recombinant type I mussel adhesive protein precursor in a 5-L tank The recombinant engineered bacteria obtained in Example 3 were screened. P. pastoris GS115 / pPIC9K-Mfp-1-RE5 P.pastoris GS115 / pPIC9K-Mfp-1-RE10 P. pastoris GS115 / pPIC9K-Mfp-1-RE20 were cultured at high density in 5L fermenters. The recombinant engineered bacteria were activated by streak plating on YPD plates; after single colonies grew, they were picked using a sterile inoculation loop under a clean bench and transferred to 50 mL YPD shake flasks. The culture was incubated at 30℃ and 220 rpm for 24 h to obtain the primary seed culture. A 10% inoculum was then transferred to 250 mL BMGY medium and incubated at 30℃ and 220 rpm for 24 h to obtain the secondary seed culture. The secondary seed culture was inoculated into a 5 L fermenter containing 2 L of BSM fermentation medium (glycerol 40 g / L, K2SO4 18 g / L, MgSO4·7H2O 14.9 g / L, KOH 4.13 g / L, 85% H3PO4 26.7 mL / L, CaSO4·2H2O 0.93 g / L, PTM1 4.4 mL / L; PTM1 formulation: CuSO4·5H2O 6 g / L, KI 0.09 g / L, MnSO4·H2O 3 g / L, H3BO3 0.02 g / L, MoNa2O4·2H2O 0.2 g / L, CoCl2·6H2O 0.92 g / L, ZnCl2 20 g / L, FeSO4·7H2O 65 g / L, biotin 0.2 g / L, H2SO4 5.0 mL / L). Initial fermentation parameters were set as follows: temperature 30°C, pH 5.0, aeration rate 2.0 vvm, and turbine speed 500 rpm. During fermentation, ammonia was automatically added to maintain the pH at 5.0. Turbine speed and dissolved oxygen were coupled, with a maximum turbine speed of 800 rpm. Once dissolved oxygen rapidly rebounded, indicating glycerol depletion in the BSM medium, the fed-batch stage began. 50% (v / v) glycerol (containing 12 mL / L PTM1) was added exponentially. The feeding rate should be adjusted appropriately based on a dissolved oxygen level of 30%. Oddi concentration (OD) was measured during the process. 600 Once the concentration reaches 200 or higher, stop adding glycerol and starve the culture for 2-3 hours until the residual glycerol is depleted, then proceed to the methanol induction stage. Adjust the temperature to 25℃ and begin adding methanol (containing 12 mL / L PTMI). The initial addition rate is 5 mL / h, gradually adjusting the rate to maintain dissolved oxygen at around 30%. Induce fermentation for 94 hours. Electrophoresis results of the fermentation broth supernatant are shown below. Figure 2 Lane M is a protein marker. After 94 h of methanol induction, the recombinant engineered bacteria... P .pastoris GS115 / pPIC9K-Mfp-1-RE5 P. pastoris GS115 / pPIC9K-Mfp-1-RE10 P.pastoris The fermentation supernatant of GS115 / pPIC9K-Mfp-1-RE20 showed clear bands of recombinant type I mussel adhesive protein precursors at the corresponding locations. The protein expression levels obtained through high-density fermentation were significantly higher than those obtained through shake-flask fermentation, with expression levels of 1.0 g / L, 35 mg / L, and 1.2 g / L, respectively. P. pastoris The yields of GS115 / pPIC9K-Mfp-1-RE5 and P. pastoris GS115 / pPIC9K Mfp-1-RE20 are far higher than the highest reported yield of E. coli expression systems to date (300 mg / L), making industrial production possible.

[0044] Example 4 Construction of engineered bacteria for tyrosinase gene expression 1. Collection, assembly, and expression construction of gene sequences This invention collected ten prokaryotic tyrosinase gene sequences from the NCBI database (specific sequence information is shown in Table 1). Based on the encoded amino acid sequences SEQ ID NO. 9, SEQ ID NO. 11, and SEQ ID NO. 13~SEQ ID NO. 20, codon optimization was performed with reference to the codon preference of *E. coli*, resulting in DNA sequences SEQ ID NO. 10, SEQ ID NO. 12, and SEQ ID NO. 21~SEQ ID NO. 28. The codon-optimized tyrosinase DNA sequences were synthesized in their entirety by Suzhou Hongxun Biotechnology Co., Ltd., and cloned into the EcoRI and NotI restriction sites of the *E. coli* expression vector pET28a, respectively, to obtain a series of recombinant expression vectors pET28a-TYR. The recombinant expression plasmids were transformed into *E. coli* BL21(DE3) host cells, and after resistance selection, ten recombinant strains expressing tyrosinase BL21(DE3) from different sources were obtained.

[0045] 2. Shake-flask fermentation of recombinant bacteria and preparation of crude enzyme solution Successfully transformed tyrosinase gene-expressing engineered bacteria were selected for shake-flask fermentation. The recombinant bacteria were then activated and amplified to obtain a seed culture. This seed culture was inoculated into TB fermentation medium at a 1% inoculum size. The culture was allowed to ferment until the OD (digesterone oxide) of the bacterial culture was reached. 600When the tyrosinase content reaches 0.6-0.8, add an inducer to a final concentration of 0.5 mM and incubate overnight at 25°C. After fermentation, centrifuge at 10,000 rpm for 10 min using a refrigerated centrifuge, collect the cells, wash 1-2 times with 0.9% NaCl solution, and centrifuge again to collect the cells. Resuspend the cells in 5 ml of extraction buffer (20 mM Tris-HCl, pH=8.5). Disrupt the cells using an ultrasonic disruptor under the following conditions: power 40 W, working time 2 s, interval 4 s, disruption time 25 min. After ultrasonication, centrifuge and collect the supernatant to obtain crude enzyme solutions of ten tyrosinases.

[0046] Table 1 Information on prokaryotic tyrosinase genes screened in this invention

[0047] Example 5: Detection of Tyrosinase Protein Expression and Determination of Enzyme Activity 1. Detection of expression of ten tyrosinases The supernatant from the cell lysis of the recombinant engineered bacteria and the supernatant from the cell lysis of the control bacteria were analyzed by SDS-PAGE protein electrophoresis and Western blotting. The results are as follows: Figure 3 As shown in A and B. The Western Blot results show that, except for... Stenotrophomonas rhizophila (SR-1) Streptomyces rimosus (SR-2) and Priestia megaterium Tyrosinase derived from (PM) was not expressed, while the other seven tyrosinases were successfully expressed.

[0048] Example 6: In vitro dopa modification of recombinant type I mussel adhesive protein The mussel agaric precursor prepared in Example 1 was prepared into a 2 g / L liquid. The pH was adjusted to 3.5 with 30% citric acid solution and allowed to stand at room temperature for 20 min. Then, 25 mM boric acid, 0.2 mM sodium dihydrogen phosphate, 15 mM ascorbic acid ethyl ether, 4 mM glutamic acid and 0.02 mM copper sulfate were added to a final concentration. The pH was adjusted to 5.8 with NaOH solution. Finally, 100 U / mL crude tyrosine enzyme solution was added. The catalytic system was placed in a shaker at 22℃ and 200 rpm for 3 h. After the reaction, 5% glacial acetic acid was added to inactivate the enzyme. The mixture was centrifuged at 10,000 rpm for 10 min, the precipitate was discarded and the supernatant was retained.

[0049] Example 7: Identification and content determination of dopamine-rich recombinant type I mussel adhesive protein. 1. Identification of dopa groups Basis: YY / T 1293.6~2020 Contact Wound Dressings, Part 6 Mussel Adhesive Dressings, Method A. The identification principle is as follows: Under alkaline conditions and in the presence of glycine as a reducing agent, the 1,2-benzenediol of the DOPA residues within the protein molecule can be oxidized to quinone compounds. Upon addition of nitrotetrazole blue chloride (NBT), the NBT reacts with the quinone compounds to generate insoluble blue-purple crystalline formazan. The specific experimental steps are as follows: 1) Take 2 μL of sample from the catalytic reaction system obtained in Example 6 and place it on a 0.2 μm nitrocellulose membrane (NC membrane) of 5 cm × 5 cm, and mark the sample position.

[0050] 2) After the sample is absorbed by the NC membrane, place the NC membrane containing the sample in a 500 mL beaker, add 300 mL of pure water, and sonicate for 10 min.

[0051] 3) Remove the NC membrane and place it in a culture dish. Add NBT staining solution and stain in the dark for 45 min.

[0052] 4) Take out the NC membrane and rinse it twice with boric acid solution. Store it in sodium borate solution overnight, then rinse it three times with purified water. Observe the formation of blue-purple spots at the sample markings.

[0053] like Figure 4 As shown, samples 11, 12, and 13 showed no color reaction, proving that the recombinant mussel protein precursor does not contain a dopa group. This also verifies that substances such as copper sulfate, ascorbic acid ethyl ether, glutamic acid, and the supernatant from the engineered bacteria that does not express the tyrosinase gene have no effect on the dopa identification reaction. Samples 1, 2, 6, 8, and 9 all showed purple spots on the NC membrane, with sample 8 being the darkest, followed by sample 1. This indicates that among the 10 different tyrosinase sources, ... Bacillus wiedmannii (BW) and wiedmannii bv. Thuringiensis Tyrosinase from (BWT) can modify recombinant type I mussel adhesive protein precursors with dopa, and the recombinant type I mussel adhesive protein modified with crude enzyme solution of BW-derived tyrosinase has a higher dopa oxidation rate.

[0054] 2. Determination of dopa group content Basis: YY / T 1293.6~2020 Contact Wound Dressings, Part 6 Mussel Adhesive Dressings, Appendix B. The experimental principle is as follows: Substances containing the 3,4-dihydroxyphenylalanine (DOPA) structure are yellow under acidic conditions, turning deep orange-red when excess alkali is added. The specific experimental steps are as follows: Hydrochloric acid solution (0.012 mol / L): Take 0.2 mL of hydrochloric acid and dilute it with pure water to a final volume of 200 mL.

[0055] DOPA standard solution (200 μg / mL): Weigh 20 mg of DOPA standard and dilute to 100 mL with hydrochloric acid solution. Prepare fresh before use.

[0056] Acidic reagent (0.516 mol / L): Take 4.3 mL of hydrochloric acid and dilute it with pure water to a final volume of 100 mL.

[0057] Alkaline reagent (1 mol / L): Weigh 4 g of sodium hydroxide (NaOH) and dilute with pure water to a final volume of 100 mL.

[0058] Nitrous acid reagent: Weigh 10 g of sodium molybdate dihydrate and 10 g of sodium nitrite, dissolve them in pure water and bring the volume to 100 mL.

[0059] Preparation of DOPA standard solutions: Take 0 mL, 0.1 mL, 0.5 mL, 1.0 mL, 1.5 mL, 2.0 mL, 2.5 mL, and 3 mL of DOPA standard solutions respectively, dilute with hydrochloric acid solution to a final volume of 10 mL, and shake well. Take 1 mL of each of the above standard solutions, add 0.5 mL of acidic reagent to each test tube, then add 1.5 mL of nitrite reagent and 2 mL of alkaline reagent to each test tube sequentially (the alkaline reagent should be added within 5 minutes after the nitrite reagent is added), and shake well. Take 1 mL of the sample to be tested in a 10 mL test tube and perform the above procedure, using tube 0 as a blank. Measure the absorbance of the prepared standard solutions and sample solutions at a wavelength of 500 nm using a 1 cm cuvette. Plot a standard curve with DOPA concentration on the x-axis and absorbance on the y-axis (Table 2), and then calculate the DOPA content of the sample based on the measured absorbance of the sample solution.

[0060] The results are shown in Table 3. Screening of ten different tyrosinases for the dopa modification rate of recombinant type I mussel adhesive protein precursor revealed that... Wiedmannii bacillus (BW) and wiedmannii bv. Thuringiensis The recombinant mussel adhesive protein modified with tyrosinase from (BWT) had a high dopa content, while the modification rates of the other eight enzymes were negligible. Wiedmannii bacillus The dopa modification rate of the derived tyrosinase (1.239%) was superior to that of the other sources. Bacillus wiedmannii bv. Thuringiensis The content of both tyrosinases (0.459%) exceeded the standard for recombinant mussel adhesive protein dopa content (0.3%) for medical device grade. The amino acid sequences of the two tyrosinases were compared with those of the Bacillus megaterium-derived tyrosinases disclosed in patents CN111004785A and CN116947996A. Bacillus wiedmannii The source tyrosinase has a sequence similarity of 27.61%. Bacillus wiedmannii bv.ThuringiensisThe source tyrosinase has a sequence similarity of 43.65%. The screening and identification of two dopa-modifiable tyrosinases provides a possibility for the subsequent application of recombinant type I mussel adhesive protein in the field of medical devices.

[0061] Table 2 Standard Curve of DOPA Concentration and Absorbance

[0062] Table 3. Modification rate of dopa on recombinant type I mussel adhesive protein precursor by tyrosinases from ten different sources.

[0063] Example 8 Chromatographic separation and purification of recombinant type I mussel adhesive protein The catalytic system solution was pressure filtered to remove suspended solids that could not be removed by centrifugation. A filter plate with a pore size of 0.1–0.2 μm was selected. Before pressure filtration, 20 g of diatomaceous earth was pre-coated onto the plate to clarify the solution. The filtrate was treated with a 2000 Da molecular weight cutoff organic ultrafiltration membrane to remove salts and small molecule impurities and to concentrate the solution. The concentrated solution was then subjected to chromatographic chromatography. The column was equilibrated with 5–10 column volumes of equilibration buffer (0.2 M NaAc-HAc buffer, pH=4). After loading the sample, impurities were washed away with the equilibration buffer, followed by gradient elution of the target protein using eluent (0.2 M NaAc-HAc buffer + 2 M NaCl, pH=4). The eluent was treated with a 300 Da molecular weight cutoff organic nanofiltration membrane for further desalting and concentration. The concentrated solution obtained from nanofiltration was decolorized with activated carbon (1% activated carbon added) and stirred at room temperature for 1 h. A filter with a pore size of 0.1–0.2 μm was then selected. The solution was filtered through a 0.22 μm filter plate, and the filtered solution was further filtered through a 0.22 μm filter membrane to remove carbon. The protein content of the solution after activated carbon treatment was verified by SDS-PAGE, and the results are as follows. Figure 5 As shown, recombinant type I mussel adhesive proteins Mfp-1-RE5, Mfp-1-RE10 and Mfp-1-RE20 were obtained, and the protein bands were single and pure.

[0064] Example 9: Lyophilization and Purity Analysis of Recombinant Type I Mussel Adhesive Protein The clarified liquid obtained in Example 8 was pre-frozen in an ultra-low temperature freezer at -80°C, and then freeze-dried in a freeze dryer to obtain recombinant type I mussel protein lyophilized powder. After dissolving the lyophilized powder in deionized water, the purity of the recombinant type I mussel adhesive protein was analyzed using a C8 reversed-phase bonded column. The results are as follows: Figure 6 As shown, the purities of the recombinant type I mussel adhesive proteins Mfp-1-RE5, Mfp-1-RE10, and Mfp-1-RE20 obtained in this embodiment are 96.26%, 88.05%, and 97.15%, respectively.

[0065] Comparative Example 1 The amino acid sequence (SEQ ID NO. 7) of type I mussel adhesive protein Mfp-1-RE40, which tandemly expresses 40 repeating peptides, was designed according to the method described in Example 1 or 2. The codon-optimized sequence (SEQ ID NO. 8) was subcloned into the pPIC9K plasmid. The whole genome was synthesized by Nanjing Genscript Biotech Co., Ltd., and the recombinant plasmid vector was named pPIC9K-Mfp-1-RE40. The recombinant pPIC9K was transformed into Pichia pastoris GS115, and high-copy-count recombinant Pichia pastoris containing type I mussel adhesive protein was obtained through screening. P. pastoris GS115 / pPIC9K-Mfp-1-RE40. Shake-flask fermentation was performed according to the method described in Example 4. The collected fermentation supernatant was analyzed by SDS-PAGE electrophoresis and Western blotting. The results are as follows: Figure 1 As shown.

[0066] from Figure 1 As can be seen from the SDS-PAGE protein electrophoresis analysis of the fermentation supernatant, the recombinant engineered bacteria... P .pastoris GS115 / pPIC9K-Mfp-1-RE20 showed no obvious bands. Figure 1 Lane A, 4). Western-blot analysis also failed to detect the target band. Figure 1 Lane B (8) further confirmed the recombinant engineered bacteria P. pastoris GS115 / pPIC9K-Mfp-1-RE40 failed to achieve successful expression and secretion of type I mussel adhesive protein Mfp-1-RE40.

[0067] Comparative Example 2 While screening prokaryotic tyrosinase genes as described in Example 5, this invention also identified 10 eukaryotic tyrosinase genes through screening the NCBI database. The specific sequence information is shown in Table 4. After codon optimization based on the codon preference of Pichia pastoris, the ten gene sequences were entrusted to Suzhou Hongxun Biotechnology Co., Ltd. for whole-gene synthesis and cloned into the Pichia pastoris expression vector pPIC9K. The six recombinant plasmids that were successfully constructed were linearized by SalI rapid digestion enzyme and then electroporated into P. pastoris GS115 expression host cells. The recombinant transformants were screened with genimycin G418 to obtain high-copy recombinant Pichia pastoris.

[0068] Ten recombinant Pichia pastoris strains were subjected to shake-flask fermentation. The fermentation supernatant was concentrated 5-fold via ultrafiltration and then subjected to in vitro dopa modification of the recombinant type I mussel protein precursor according to the method described in Example 8. The modified catalytic system was then subjected to dopa group identification according to the method described in Example 9. The results are as follows: Figure 4 As shown in B, sample point 2-11 and negative control sample point 1 (engineered bacteria) P .pastorisThe color development of GS115 / pPIC9K was consistent, with no purple spots produced, indicating that the corresponding ten eukaryotic tyrosinases had no dopa modification activity on the recombinant type I mussel protein precursor.

[0069] Table 4 Information on eukaryotic tyrosinase genes screened in this invention

[0070] The above description is merely an embodiment of this application, and the scope of protection of this application is not limited to these specific embodiments, but is determined by the claims of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principles of this application should be included within the scope of protection of this application.

Claims

1. A method of producing recombinant Mytilus galloprovincialis type I mucus glycoprotein enriched in dopamine, characterized in that, The method comprises the following steps: 1) obtaining a type I mussel-like myoglobin precursor by fermentation of a recombinant bacterium capable of expressing the type I mussel-like myoglobin precursor; 2) in vitro, the type I mussel-like myoglobin precursor in 1) is modified by tyrosinase to dopa; 3) the recombinant type I mussel-like myoglobin enriched in dopa is obtained by separation and purification; The tyrosinase is derived from at least one of Bacillus wiedmannii and / or Bacillus wiedmannii bv. Thuringiensis the tyrosinase is derived from one or more of the above.

2. The method of claim 1, wherein, The Bacillus wiedmannii The tyrosinase from the source comprises the amino acid sequence of SEQ ID NO. 9 or an amino acid sequence having more than 98% sequence homology with SEQ ID NO. 9; and / or, The Bacillus wiedmannii bv. Thuringiensis The tyrosinase from the source comprises the amino acid sequence of SEQ ID NO. 11 or an amino acid sequence having more than 98% sequence homology with SEQ ID NO.

11.

3. The method according to claim 1 or 2, characterized in that, The recombinant bacterium of 1) expresses a type I mussel-like myoglobin composed of repeated tandem decapeptide repeats. The decapeptide repeat comprises AKPSYPPTYK, and the number of repeated tandem decapeptide repeats is m, 5≤m≤20. The starting strain of the engineering bacterium is Pichia pastoris.

4. Use of an enzyme in the preparation of a type I Mytilus edulis mucin, characterised in that, The enzyme is derived from at least one of the following: Bacillus wiedmannii and / or Bacillus wiedmannii bv. Thuringiensis a tyrosinase derived from one of the above tyrosinases.

5. Use according to claim 4, characterized in that, The Bacillus wiedmannii The tyrosinase from the source comprises the amino acid sequence of SEQ ID NO. 9 or an amino acid sequence having more than 98% sequence homology with SEQ ID NO. 9; and / or, The Bacillus wiedmannii bv. Thuringiensis The tyrosinase from the source comprises the amino acid sequence of SEQ ID NO. 11 or an amino acid sequence having more than 98% sequence homology with SEQ ID NO.

11.

6. Use according to claim 4, characterized in that, The application comprises the step of in vitro modification of the type I mussel-like myoglobin precursor by the tyrosinase to dopa.

7. Use according to claim 6, characterized in that, The type I mussel-like myoglobin precursor comprises repeated tandem decapeptide repeats, the decapeptide repeat is AKPSYPPTYK, and the number of repeated tandem decapeptide repeats is m, 5≤m≤20.

8. A recombinant Mytilus galloprovincialis type I mucus glycoprotein, characterized in that, The recombinant type I mussel-like myoglobin is a type I mussel-like myoglobin composed of repeated tandem decapeptide repeats, the decapeptide repeat is AKPSYPPTYK, and the number of repeated tandem decapeptide repeats is m, 5≤m≤20. The dopa content of the recombinant type I mussel-like myoglobin is 0.4-1.5%, and the purity is 90-97%.

9. A recombinant bacterium secreting a Type I Mytilus edulis -mucin precursor, characterized in that, The engineering bacterium expresses a type I mussel-like myoglobin composed of repeated tandem decapeptide repeats. The decapeptide repeat is AKPSYPPTYK, and the number of repeated tandem decapeptide repeats is m, 5≤m≤20. The starting strain of the engineering bacterium is Pichia pastoris.

10. The recombinant bacterium of claim 9 is used for fermentation production of a type I mussel-like myoglobin precursor.

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