Preparation method of recombinant mussel mucoprotein

Abstract

The invention discloses a preparation method of recombinant mussel mucoprotein, which comprises the following steps: S1) extracting total RNA (Ribonucleic Acid) of mussels, and synthesizing cDNA (Complementary Deoxyribose Nucleic Acid) through reverse transcription; s2) designing a primer according to a mussel mucoprotein gene sequence, and amplifying a target gene segment from cDNA by adopting a PCR (Polymerase Chain Reaction) method; s3) connecting the target gene segment obtained by amplification to an expression vector to construct a recombinant expression vector; s4) transforming the recombinant expression vector into a host cell, screening positive clones, and inducing expression of recombinant mussel mucoprotein; s5) crushing the positive cloned host cells, and separating and purifying the recombinant mussel mucoprotein to obtain a high-purity product; and S6) performing dialysis and freeze-drying treatment on the purified recombinant mussel mucoprotein to prepare freeze-dried powder. According to the method, the expression level of the recombinant mussel mucoprotein is remarkably improved, and the yield of the recombinant protein can reach 50-200 mg / L of fermentation liquor by adopting the optimized expression vector and induction conditions and is several times that of a traditional method.

Description

Technical Field

[0001] This invention relates to the field of mussel adhesive protein preparation technology, specifically a method for preparing recombinant mussel adhesive protein. Background Technology

[0002] Mussels are an important type of marine bivalve mollusks, widely distributed in seas around the world. Mussels contain a special adhesive protein called mussel mucilage, which possesses unique adhesive properties and underwater solidification capabilities, showing broad application prospects in biomedical engineering, underwater coatings, and ship antifouling. Currently, methods for extracting mucilage from natural mussels have been reported, mainly including acid extraction, organic solvent extraction, and enzymatic hydrolysis. Among these, acid extraction is the most commonly used method, which involves breaking down the mussel's adhesive glands or byssal threads under weakly acidic conditions, followed by centrifugation and dialysis to extract and purify the mucilage. However, natural mussel resources are limited, the extraction process is time-consuming and labor-intensive, and the yield is low, making it difficult to meet the growing industrial demand.

[0003] With the development of genetic engineering technology, recombinant expression has become a promising alternative method for preparing mussel adhesive proteins. Researchers have successfully cloned various mussel adhesive protein genes and achieved recombinant expression in heterologous expression systems such as E. coli, yeast, and insect cells. However, existing recombinant expression methods still have some shortcomings: low expression levels of exogenous genes, difficulty in purifying recombinant proteins, unstable activity, and differences in physicochemical properties compared to natural adhesive proteins. Furthermore, optimizing the expression conditions of recombinant proteins to improve yield and quality while reducing production costs remains a critical issue that urgently needs to be addressed.

[0004] Given the limitations of existing technologies, there is an urgent need to develop an efficient, economical, and scalable method for preparing recombinant mussel adhesive protein. This method should significantly improve the expression level of exogenous genes, simplify the separation and purification process of recombinant proteins, and ensure that the obtained product meets or exceeds the levels of natural mussel adhesive protein in terms of yield, purity, activity, and stability. Furthermore, it should possess advantages such as simple processing, low cost, and suitability for industrial production. This will lay a solid material foundation for the widespread application of mussel adhesive protein and promote technological progress and industrial development in related fields. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing recombinant mussel adhesive protein, so as to solve the problems existing in the above-mentioned background art methods for preparing recombinant mussel adhesive protein.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing recombinant mussel adhesive protein, comprising the following steps: S1) Total RNA was extracted from mussels and cDNA was synthesized by reverse transcription; the mussels were bay scallops, comb scallops and noble comb scallops.

[0007] S2) Primers were designed based on the mussel adhesive protein gene sequence, and the target gene fragment was amplified from cDNA using PCR. S3) The amplified target gene fragment is ligated into an expression vector to construct a recombinant expression vector; S4) The recombinant expression vector was transformed into host cells, positive clones were screened, and recombinant mussel adhesive protein was induced to be expressed; S5) The host cells of the positive clones were broken, and the recombinant mussel adhesive protein was isolated and purified to obtain a high-purity product; S6) The purified recombinant mussel adhesive protein was dialyzed and freeze-dried to prepare freeze-dried powder.

[0008] In step S1), the reverse transcription primer is 5'-TTTTTTTTTTTTTTTTTT-3', as shown in SEQ ID NO.1; In step S2), the primer sequences for the Gulf scallop are as follows: Forward primer sequence: 5'-CCGAATTCTATAAACCTAAGAAAAACTTATCC-3', SEQ ID NO.2; Reverse primer sequence: 5'-ATGTCGACTTACGTTGGTGGATAGCGTATC-3, SEQ ID NO.3; The primer sequences for the scallop *Ctenopharynx* are as follows: Forward primer: 5'-CATATGATGGCTGGTGGTGGTGGTGGTGGTGGTG-3', SEQ ID NO.4; Reverse primer: 5'-AAGCTTTTATTTGTAGTTCTTTCTTGTTG-3', SEQ ID NO.5; The primer sequences for *Scallop luxuriantis* are as follows: Forward primer sequence: 5'-GCNGARAAYGCNTTYGGN-3', SEQ ID NO.6; Reverse primer: 5'-CCRTTNGCRTTRTCRTC-3', SEQ ID NO.7.

[0009] Preferably, the expression vector in step S3 is one of pET-22b(+), pET-28a(+), pET-30a(+), pGEX-4T-1, pGEX-6P-1, or pMAL-c5X.

[0010] Preferably, the host cell in step S4 is one of Escherichia coli BL21 (DE3), Escherichia coli Rosetta (DE3), or Escherichia coli Origami B (DE3).

[0011] Preferably, step S4 includes the following specific steps: S4.1) The recombinant expression vector was transformed into host cells, plated on LB agar plates containing antibiotics, and incubated overnight at 37°C; S4.2) Select single colonies and incubate them in LB liquid medium at 37°C with shaking until the OD600 reaches 0.6-0.8; S4.3) Add IPTG to a final concentration of 0.1-1.0 mM, cool to 16-30℃ to induce expression for 12-24 h.

[0012] Preferably, step S5 includes the following specific steps: S5.1) Collect the induced bacterial cells by centrifugation, resuspend them in lysis buffer, and sonicate to disrupt them; S5.2) Centrifuge the supernatant, load it onto a Ni ion affinity chromatography column, elute and collect the target protein peak; S5.3) The collected target protein was further purified by cation exchange chromatography and molecular sieve chromatography.

[0013] Preferably, in step S6, the dialysis treatment uses a dialysis bag with a molecular weight cutoff of 3-30 kDa, and the dialysis solution is a 10-50 mM acetic acid solution with a pH of 4.0-6.0.

[0014] Preferably, the freeze-drying process in step S6 is carried out under a vacuum of 1-100 Pa and a condensation temperature of -40°C to -80°C, with a drying time of 12-48 h.

[0015] Preferably, the yield of the recombinant mussel adhesive protein obtained is 50-200 mg / L of fermentation broth.

[0016] Compared with the prior art, the present invention has the following beneficial effects: 1) This application significantly improves the expression level of recombinant mussel adhesive protein. By using optimized expression vectors and induction conditions, the yield of recombinant protein can reach 50-200 mg / L of fermentation broth, which is several times that of traditional methods. 2) This application simplifies the separation and purification process of recombinant mussel adhesive protein. By designing affinity tags and combining them with high performance liquid chromatography, a one-step separation and purification of recombinant protein can be achieved, which greatly shortens the production cycle and reduces production costs. 3) The recombinant mussel adhesive protein obtained in this application has high purity, strong activity, and good stability. The freeze-dried product prepared can reach a purity of over 95% and a moisture content of less than 5%. It can be stored for a long time at room temperature. The physicochemical properties and biological functions of the recombinant protein are comparable to those of natural mussel adhesive protein, such as molecular weight, subunit composition, and adhesion activity. 4) The raw materials for this application are widely available and have good prospects for industrialization. It uses common marine shellfish as the gene source, and the expression host is an industrialized strain of Escherichia coli. The fermentation process can be scaled up on a large scale, and the production process is mature and reliable, making it highly promising for industrial application. Attached Figure Description

[0017] Figure 1 This is a flowchart of the preparation method of this application; Figure 2 This is a detailed step diagram of step S4 in this application; Figure 3 This is a detailed step diagram of step S5 in this application; Figure 4 In Example 1 of this application, the recombinant protein was purified using nickel column affinity, and the protein concentration was determined by the BCA method. A bar chart was then calculated to obtain the yield of the target protein for different recombinant strains. Figure 5 For the nickel column affinity purification of recombinant protein in Comparative Example 1 of this application, the protein concentration was determined by BCA method, and bar charts were calculated to obtain the target protein yield of different recombinant strains. Figure 6 For the nickel column affinity purification of recombinant protein in Comparative Example 2 of this application, the protein concentration was determined by BCA method, and the bar charts of the yield of the target protein under different induction conditions were calculated. Figure 7 In Example 3 of this application, the lyophilized product was reconstituted, and the protein concentration was determined by the BCA method. Line graphs of lyophilized protein yield and moisture content under different conditions were calculated. Figure 8 This is a bar chart showing the yield of the target protein in each embodiment and comparative example of this application. Detailed Implementation

[0018] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0019] In the description of this invention, it should be noted that the execution order of the steps is not limited by the sequence number. The possible changes in the order of some steps, the synchronous execution of steps, and the split execution of steps are all within the protection scope of this application.

[0020] Please see Figure 1-8 This invention provides a technical solution: a method for preparing recombinant mussel adhesive protein, comprising the following steps: S1) Total RNA was extracted from mussels and cDNA was synthesized by reverse transcription; the mussels were bay scallops, comb scallops and noble comb scallops.

[0021] S2) Primers were designed based on the mussel adhesive protein gene sequence, and the target gene fragment was amplified from cDNA using PCR. S3) The amplified target gene fragment is ligated into an expression vector to construct a recombinant expression vector; S4) The recombinant expression vector was transformed into host cells, positive clones were screened, and recombinant mussel adhesive protein was induced to be expressed; S5) The host cells of the positive clones were broken, and the recombinant mussel adhesive protein was isolated and purified to obtain a high-purity product; S6) The purified recombinant mussel adhesive protein was dialyzed and freeze-dried to prepare freeze-dried powder.

[0022] Specifically, steps S1 and S2 utilize molecular biology techniques to extract total RNA from mussels, synthesize cDNA through reverse transcription, and then use the cDNA as a template to amplify the target gene fragment using PCR.

[0023] In step S1), the reverse transcription primer is 5'-TTTTTTTTTTTTTTTTTT-3', as shown in SEQ ID NO.1; In step S2), the primer sequences for the Gulf scallop are as follows: Forward primer sequence: 5'-CCGAATTCTATAAACCTAAGAAAAACTTATCC-3', SEQ ID NO.2; Reverse primer sequence: 5'-ATGTCGACTTACGTTGGTGGATAGCGTATC-3, SEQ ID NO.3; The primer sequences for the scallop *Ctenopharynx* are as follows: Forward primer: 5'-CATATGATGGCTGGTGGTGGTGGTGGTGGTGGTG-3', SEQ ID NO.4; Reverse primer: 5'-AAGCTTTTATTTGTAGTTCTTTCTTGTTG-3', SEQ ID NO.5; The primer sequences for *Scallop luxuriantis* are as follows: Forward primer sequence: 5'-GCNGARAAYGCNTTYGGN-3', SEQ ID NO.6; Reverse primer: 5'-CCRTTNGCRTTRTCRTC-3', SEQ ID NO.7.

[0024] This process allows for the rapid and accurate acquisition of the gene sequence encoding mussel adhesive protein, laying the foundation for subsequent heterologous expression. Steps S3 and S4 employ genetic engineering techniques to clone the target gene into an expression vector to construct a recombinant plasmid, which is then transformed into host cells to induce expression. By optimizing the expression vector and host bacteria, the expression efficiency of the exogenous gene and the yield of the recombinant protein can be significantly improved. Step S5 uses biochemical separation technology to prepare high-purity recombinant mussel adhesive protein by disrupting host cells and purifying the protein. Step S6 uses freeze-drying technology to prepare freeze-dried mussel adhesive protein powder, which extends the product's shelf life and facilitates transportation and storage.

[0025] The mussels mentioned in step S1 are *Argopecten irradians*, *Chlamys farreri*, and *Chlamys nobilis*. Specifically, the genes used in this application to prepare recombinant mussel adhesive proteins are derived from *Argopecten irradians*, *Chlamys farreri*, and *Chlamys nobilis*. These shellfish resources are abundant, easy to cultivate and collect, providing a sufficient and stable source of raw materials for gene extraction and recombinant protein expression. Furthermore, the adhesive protein genes from different shellfish exhibit certain differences in sequence and structure, which can endow the recombinant products with unique physicochemical properties and biological functions. Taking *Argopecten irradians* as an example, its adhesive protein is renowned for its excellent adhesion strength and underwater solidification speed, showing broad application prospects in underwater engineering, tissue adhesion, and other fields. In conclusion, selecting suitable mussel species as gene sources can not only broaden the application areas of recombinant mussel adhesive proteins but also contribute to the sustainable utilization of resources and the diversified development of the industry.

[0026] Example 1, S1) Total RNA was extracted from scallops, scallops and noble scallops, respectively, and cDNA was synthesized by reverse transcription, and the mucin gene was amplified; wherein, the reverse transcription primer was 5'-TTTTTTTTTTTTTTTTTT-3', as shown in SEQ ID NO.1; The primer sequences for Gulf scallops are as follows: Forward primer sequence: 5'-CCGAATTCTATAAACCTAAGAAAAACTTATCC-3', SEQ ID NO.2; Reverse primer sequence: 5'-ATGTCGACTTACGTTGGTGGATAGCGTATC-3, SEQ ID NO.3; The primer sequences for the scallop *Ctenopharynx* are as follows: Forward primer: 5'-CATATGATGGCTGGTGGTGGTGGTGGTGGTGGTG-3', SEQ ID NO.4; Reverse primer: 5'-AAGCTTTTATTTGTAGTTCTTTCTTGTTG-3', SEQ ID NO.5; The primer sequences for *Scallop luxuriantis* are as follows: Forward primer sequence: 5'-GCNGARAAYGCNTTYGGN-3', SEQ ID NO.6; Reverse primer: 5'-CCRTTNGCRTTRTCRTC-3', SEQ ID NO.7; S2) The amplified fragment was cloned into the pET-22b(+) vector to construct the recombinant plasmids pET-AIMP, pET-CFMP, and pET-CNMP; S3) was transformed into Escherichia coli BL21(DE3), and induced to express the expression in accordance with the method described in this application, with strains transformed with empty vector pET-22b(+) as negative controls; S4) Bacterial cells were disrupted by sonication, and the supernatant was analyzed by SDS-PAGE electrophoresis. The results are as follows: Lane 1: Protein Marker; Lane 2: Lysis supernatant of pET-22b(+) empty vector strain; Lane 3: The target band is visible at approximately 55 kDa in the lysate supernatant of the pET-AIMP recombinant strain. Lane 4: The target band is visible at approximately 57 kDa in the lysate supernatant of the pET-CFMP recombinant strain. Lane 5: The target band is visible at approximately 58 kDa in the lysate supernatant of the pET-CNMP recombinant strain. S5) The target bands were extracted from the gel and identified by mass spectrometry. The results showed that they were all target mussel adhesive proteins. S6) Recombinant protein was purified using nickel column affinity purification, and protein concentration was determined by the BCA method. The yields of the target protein for different recombinant strains were calculated as follows: pET-AIMP: 85 mg / L; pET-CFMP: 78 mg / L; pET-CNMP: 74 mg / L.

[0027] Reference manual attached Figure 4 Therefore, the results of Example 1 show that mucin genes from different mussel species can be expressed in Escherichia coli, but the expression level is the highest in Gulf scallops.

[0028] The expression vector described in step S3 is pET-22b(+). The pET series is a common plasmid vector used to construct recombinant expression vectors. These commercially available plasmid vectors have characteristics such as high copy number, strong promoter, multiple cloning site, and inducible expression, which can significantly improve the transcription and translation efficiency of foreign genes, thereby increasing the yield of recombinant proteins. This vector contains a T7 promoter and a lacI regulatory gene, which can achieve controlled expression of recombinant proteins through IPTG induction; the vector also introduces the pelB signal peptide sequence, which can guide the target protein to the intracellular transmembrane pathway, facilitating the secretory expression and purification of recombinant proteins.

[0029] Comparative Example 1, S1) The scallop mucin gene was cloned into the pET-22b(+), pET-28a(+), pGEX-4T-1, and pMAL-c5X vectors, respectively, to construct recombinant plasmids; S2) was transformed into Escherichia coli BL21(DE3) and induced to express according to the method of this application; S3) The bacterial cells were sonicated and the supernatant was analyzed by SDS-PAGE electrophoresis. The expression levels of the target proteins of each vector varied greatly. Among them, pET-22b(+) and pET-28a(+) had the highest expression levels, followed by pMAL-c5X, and pGEX-4T-1 had the lowest expression levels. S4) Recombinant protein was purified by nickel column affinity chromatography, and protein concentration was determined by BCA method. The yields of the target protein for different recombinant strains were calculated as follows: pET-22b(+): 85 mg / L; pET-28a(+): 81 mg / L; pMAL-c5X: 65 mg / L; pGEX-4T-1: 45mg / L.

[0030] Reference manual attached Figure 5 Therefore, the results of Comparative Example 1 show that the pET series vectors, especially pET-22b(+), are more conducive to the efficient expression of recombinant mussel adhesive protein, while pGEX-4T-1 and pMAL-c5X may have affected the expression and activity of recombinant protein due to the excessively large fusion tags.

[0031] The host cell mentioned in step S4 is one of *Escherichia coli* BL21(DE3), *Escherichia coli* Rosetta(DE3), or *Escherichia coli* Origami B(DE3). Specifically, BL21(DE3), Rosetta(DE3), and Origami B(DE3) are commonly used engineered *E. coli* strains used as expression hosts for recombinant mussel adhesive protein. These host strains have been genetically engineered to remove endogenous protease genes and introduce T7 RNA polymerase genes, enabling efficient expression of exogenous genes carrying the T7 promoter under IPTG induction. Furthermore, different engineered strains have their own advantages: for example, Rosetta(DE3) contains additional tRNA genes, which can improve the translation efficiency of rare codons; Origami B(DE3) has mutated reductase genes trxB and gor, which is beneficial for disulfide bond formation and correct folding of recombinant proteins.

[0032] Specifically, *Escherichia coli*, as a prokaryotic expression system, has advantages such as rapid growth, low fermentation cost, high expression level, and simple genetic manipulation, making it the most widely used and mature heterologous protein expression host currently. Using *E. coli* as an expression host for mussel adhesive proteins can not only significantly improve the yield and quality of recombinant proteins, but also facilitate large-scale production and industrial application, thus possessing significant technological and economic value.

[0033] Example 2, S1) The pET-AIMP plasmid was transformed into Escherichia coli BL21(DE3), Rosetta(DE3), and Origami B(DE3), respectively, and expression was induced according to the method of this application; S2) Bacterial cells were disrupted by sonication, and the supernatant was analyzed by SDS-PAGE electrophoresis. The expression levels of the target protein among different host strains showed little difference. S3) Recombinant protein was purified by nickel column affinity chromatography, and protein concentration was determined by BCA method. The yields of the target protein for different recombinant strains were calculated as follows: BL21(DE3): 85 mg / L; Rosetta (DE3): 89 mg / L; OrigamiB(DE3): 82 mg / L; The results of Example 2 showed that the expression levels of recombinant mussel adhesive proteins were slightly increased by Rosetta (DE3) and Origami B (DE3) compared to BL21 (DE3), but the difference was not significant. Considering that BL21 (DE3) has a faster growth rate and is easier to genetically manipulate, BL21 (DE3) was preferred as the expression host.

[0034] Step S4 includes the following specific steps: S4.1) The recombinant expression vector was transformed into host cells, plated on LB agar plates containing antibiotics, and incubated overnight at 37°C; S4.2) Select single colonies and incubate them in LB liquid medium at 37°C with shaking until the OD600 reaches 0.6-0.8; S4.3) Add IPTG to a final concentration of 0.1-1.0 mM, cool to 16-30℃ to induce expression for 12-24 h.

[0035] Specifically, step S4.1 employs conventional transformation and screening methods to transform the recombinant plasmid into host cells, and then screens for positive clones on a selective medium containing antibiotics. This step ensures that only host cells carrying the recombinant plasmid can grow, thus avoiding contamination and interference from wild-type cells. Steps S4.2 and S4.3 involve the induction expression conditions of the recombinant protein, including cell concentration, inducer concentration, induction temperature, and induction time. By optimizing these key parameters, efficient expression of the exogenous gene can be induced at the optimal time point, while inhibiting excessive cell growth and degradation of the recombinant protein. For example, lowering the induction temperature to 16-30℃ can improve the solubility and stability of the recombinant protein; extending the induction time to 12-24 h can further increase the accumulation of the target protein.

[0036] As an additional note, different recombinant proteins may have different optimal induction conditions. Optimizing induction expression conditions is crucial for improving the yield and quality of recombinant proteins and is also a key factor affecting subsequent separation, purification, and industrial production.

[0037] Comparative Example 2, S1): pET-AIMP / BL21(DE3) strain was induced to express the protein under different conditions as shown in the table below:

[0038] S2) Bacterial cells were disrupted by sonication, and the supernatant was analyzed by SDS-PAGE electrophoresis. The expression level of recombinant protein increased significantly with decreasing induction temperature and increasing induction time, with the highest expression level observed after induction at 16℃ and 0.5 mM IPTG for 18 h. S3) Nickel column affinity purification of recombinant protein, BCA method for protein concentration determination, and calculation of the yield of target protein under different induction conditions:

[0039] Reference manual attached Figure 6Therefore, the results of Comparative Example 2 show that low temperature and prolonged induction time are beneficial to increasing the expression level of recombinant mussel adhesive protein. The highest yield was obtained by induction at 16℃ and 0.5 mM IPTG for 18 h, while the yield was lower by conventional induction at 37℃ and high IPTG. This is because the bacterial cells grew too fast and the synthesis of recombinant protein was inhibited.

[0040] Step S5 includes the following specific steps: S5.1) Collect the induced bacterial cells by centrifugation, resuspend them in lysis buffer, and sonicate to disrupt them; S5.2) Centrifuge the supernatant, load it onto a Ni ion affinity chromatography column, elute and collect the target protein peak; S5.3) The collected target protein was further purified by cation exchange chromatography and molecular sieve chromatography.

[0041] Specifically, firstly, bacterial cells are collected by centrifugation and then sonicated to release recombinant proteins into the supernatant. This step utilizes the mechanical vibration and cavitation effect of ultrasound to efficiently disrupt the cell wall and cell membrane, releasing intracellular proteins without causing denaturation or inactivation. Next, Ni ion affinity chromatography is used to separate and purify the recombinant proteins in the supernatant. This method, based on the specific chelation between the 6×His tag fused to the N-terminus or C-terminus of the recombinant protein and Ni ions, enables one-step separation and purification of the recombinant protein, offering simple operation and high purification efficiency. Further cation exchange chromatography and molecular sieve chromatography are employed to remove residual contaminating proteins, improving the purity and homogeneity of the target protein. Cation exchange chromatography utilizes the electrostatic interaction between the recombinant protein and the positively charged groups on the surface of the chromatography medium, combined with the principle of pH and ionic strength gradient elution, to separate proteins with different isoelectric points. Molecular sieve chromatography utilizes the differences in protein molecule size and shape, achieving separation under the sieving effect of the gel matrix, removing aggregates and degradation fragments, and improving the homogeneity of the recombinant protein. This allows for the effective isolation of high-purity recombinant mussel adhesive protein from complex bacterial proteins, providing high-quality material for subsequent research and applications.

[0042] Example 3, S1) Following the method of this application, the bacterial cells of pET-AIMP / BL21(DE3) strain induced to express were broken down and purified; S2) The supernatant from the fragmentation, Ni column eluent, cation exchange eluent, and gel filtration eluent were analyzed by SDS-PAGE electrophoresis. The results showed: Lane 1: Protein Marker; Lane 2: The supernatant was fragmented and contained a large amount of extraneous proteins, making the target band unclear; Lane 3: Ni column elution buffer, significantly reduced contaminating proteins, target band clearly visible; Lane 4: Cation exchange elution buffer further removes impurities and significantly improves the purity of the target protein; Lane 5: Gel filtration eluent, single band on electrophoresis, target protein purity greater than 95%; The protein concentration in the gel filtration eluent was determined by the BCA method (S3), and the total purified yield was calculated to be 108 mg / L, with a recovery rate of 78.5%.

[0043] Therefore, the results of Example 3 show that a stepwise purification strategy using Ni affinity chromatography, cation exchange chromatography, and molecular sieve chromatography can efficiently separate and purify electrophoretic grade recombinant mussel adhesive protein from bacterial cell lysates, with a purity greater than 95% and high yield and recovery rate. This separation and purification method is feasible and easy to operate.

[0044] In step S6, dialysis is performed using a dialysis bag with a molecular weight cutoff of 3-30 kDa and a 10-50 mM acetic acid solution with a pH of 4.0-6.0. Specifically, dialysis is a commonly used desalting and buffer replacement technique. Utilizing the diffusion effect caused by the concentration difference of molecules across a semi-permeable membrane, it removes small molecule impurities from protein samples, such as salt ions, denaturants, and reducing agents, while simultaneously replacing the protein sample with a suitable buffer system. Choosing appropriate dialysis conditions is crucial for maintaining the native conformation and biological activity of recombinant mussel adhesive protein. Using a dialysis bag with a molecular weight cutoff of 3-30 kDa removes small molecule impurities while avoiding the loss of the target protein. Using an acetic acid solution with a pH in the range of 4.0-6.0 as the dialysis solution simulates the natural environment of mussel adhesive protein, maintaining its stability and adhesive activity. Furthermore, controlling the dialysis temperature at around 4°C minimizes protein denaturation and degradation.

[0045] In step S6, the freeze-drying process is carried out under a vacuum of 1-100 Pa and a condensation temperature of -40℃ to -80℃ for 12-48 hours. Specifically, freeze-drying is a commonly used end-processing technology for biological products. Through steps such as sample pre-freezing, sublimation, and desorption, moisture in the sample can be removed to prepare a loose, porous dry powder product. Compared with traditional spray drying and vacuum drying, freeze-drying involves lower sample processing temperatures, higher vacuum levels, and more thorough water removal, thus maximizing the preservation of the protein's natural structure and bioactivity. Freeze-drying the purified recombinant mussel adhesive protein can significantly extend its shelf life, facilitating transportation and storage, and also providing a high-quality raw material for subsequent formulation development.

[0046] As an additional note, to obtain high-quality freeze-dried products, it is necessary to strictly control the freeze-drying process parameters: for example, controlling the vacuum degree at 1-100 Pa can accelerate the sublimation and precipitation of water; controlling the condensation temperature at -40℃ to -80℃ can improve the water capture efficiency; and controlling the drying time at 12-48 h can ensure that the sample is fully dried. Optimizing freeze-drying conditions can not only improve the yield and quality of recombinant mussel adhesive protein freeze-dried powder, but also significantly reduce production energy consumption and shorten the drying cycle, resulting in good economic and environmental benefits.

[0047] Comparative Example 3, S1) The recombinant mussel adhesive protein purified in Example 3 was subjected to dialysis and freeze-drying under different conditions as shown in the table below:

[0048] S2) Reconstitute the lyophilized product, determine the protein concentration using the BCA method, and calculate the lyophilized protein yield and moisture content under different conditions:

[0049] S3) The lyophilized protein was prepared with a certain concentration using a 10 mM acetic acid solution at pH 5.5. After being stored at 4℃ for 4 weeks, the changes in its purity and concentration were examined by Coomassie brilliant blue staining and BCA method. The results showed that samples No. 1 and No. 3 had the best stability, maintaining a purity of over 95% and no significant change in concentration. Other samples showed a certain degree of degradation and precipitation.

[0050] Reference manual attached Figure 7 Therefore, the results of Comparative Example 3 show that dialyzing recombinant mussel adhesive protein with 10 mM acetic acid solution for 24 h, followed by freeze-drying at 10 Pa vacuum and -50℃ for 24 h, can yield freeze-dried products with low moisture content and good stability, and a protein yield of more than 90%. This is the optimal process for obtaining high-quality recombinant mussel adhesive protein freeze-dried powder.

[0051] Reference manual attached Figure 8 Set up an unloaded comparison, attached Figure 8 The yields of the target proteins in Examples 1-3 and Comparative Examples 1-3 are visually demonstrated. It can be seen that the empty control showed no expression of the target protein, while the yield of recombinant mussel adhesive protein obtained under the optimal conditions of this invention (Example 1: Gulf scallop; Comparative Example 1: pET-22b(+); Example 2: BL21(DE3); Examples 1 and Comparative Example 2: 16℃ induction for 18h with 0.5mM IPTG) was significantly higher than that under other comparative conditions.

[0052] The recombinant mussel adhesive protein obtained in this application has a yield of 50-200 mg / L fermentation broth. Specifically, 50-200 mg of the target protein can be obtained per liter of fermentation broth. Compared with traditional methods for extracting natural mussel adhesive protein, this invention uses genetic engineering technology to significantly improve the yield and quality of recombinant mussel adhesive protein and achieve large-scale preparation by optimizing gene expression elements, host cells, induction conditions, and separation and purification methods.

[0053] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing recombinant mussel adhesive protein, characterized in that, Includes the following steps: S1) Total RNA was extracted from mussels and cDNA was synthesized by reverse transcription; the mussels were bay scallops, comb scallops and noble comb scallops; S2) Primers were designed based on the mussel adhesive protein gene sequence, and the target gene fragment was amplified from cDNA using PCR. S3) The amplified target gene fragment is ligated into an expression vector to construct a recombinant expression vector; S4) The recombinant expression vector was transformed into host cells, positive clones were screened, and recombinant mussel adhesive protein was induced to be expressed; S5) The host cells of the positive clones were broken, and the recombinant mussel adhesive protein was isolated and purified to obtain a high-purity product; S6) The purified recombinant mussel adhesive protein was dialyzed and freeze-dried to prepare freeze-dried powder; In step S1), the reverse transcription primer is 5'-TTTTTTTTTTTTTTTTTT-3', as shown in SEQ ID NO.1; In step S2), the primer sequences for the Gulf scallop are as follows: Forward primer sequence: 5'-CCGAATTCTATAAACCTAAGAAAAACTTATCC-3', SEQ ID NO.2; Reverse primer sequence: 5'-ATGTCGACTTACGTTGGTGGATAGCGTATC-3, SEQ ID NO.3; The primer sequences for the scallop *Ctenopharynx* are as follows: Forward primer: 5'-CATATGATGGCTGGTGGTGGTGGTGGTGGTGGTG-3', SEQ ID NO.4; Reverse primer: 5'-AAGCTTTTATTTGTAGTTCTTTCTTGTTG-3', SEQ ID NO.5; The primer sequences for *Scallop luxuriantis* are as follows: Forward primer sequence: 5'-GCNGARAAYGCNTTYGGN-3', SEQ ID NO.6; Reverse primer: 5'-CCRTTNGCRTTRTCRTC-3', SEQ ID NO.

7.

2. The method for preparing recombinant mussel adhesive protein according to claim 1, characterized in that, The expression vector mentioned in step S3 is one of pET-22b(+), pET-28a(+), pET-30a(+), pGEX-4T-1, pGEX-6P-1, or pMAL-c5X.

3. The method for preparing recombinant mussel adhesive protein according to claim 1, characterized in that, The host cell mentioned in step S4 is one of Escherichia coli BL21 (DE3), Escherichia coli Rosetta (DE3), or Escherichia coli Origami B (DE3).

4. The method for preparing recombinant mussel adhesive protein according to claim 1, characterized in that, Step S4 includes the following specific steps: S4.1) The recombinant expression vector was transformed into host cells, plated on LB agar plates containing antibiotics, and incubated overnight at 37°C; S4.2) Select single colonies and incubate them in LB liquid medium at 37°C with shaking until the OD600 reaches 0.6-0.8; S4.3) Add IPTG to a final concentration of 0.1-1.0 mM, cool to 16-30℃ to induce expression for 12-24 h.

5. The method for preparing recombinant mussel adhesive protein according to claim 1, characterized in that, Step S5 includes the following specific steps: S5.1) Collect the induced bacterial cells by centrifugation, resuspend them in lysis buffer, and sonicate to disrupt them; S5.2) Centrifuge the supernatant, load it onto a Ni ion affinity chromatography column, elute and collect the target protein peak; S5.3) The collected target protein was further purified by cation exchange chromatography and molecular sieve chromatography.

6. The method for preparing recombinant mussel adhesive protein according to claim 1, characterized in that, In step S6, a dialysis bag with a molecular weight cutoff of 3-30 kDa is used for dialysis treatment, and the dialysate is a 10-50 mM acetic acid solution with a pH of 4.0-6.

0.

7. The method for preparing recombinant mussel adhesive protein according to claim 1, characterized in that, In step S6, the freeze-drying process is carried out under vacuum conditions of 1-100 Pa and condensation temperature of -40℃ to -80℃, and the drying time is 12-48 h.

8. The method for preparing recombinant mussel adhesive protein according to any one of claims 1-7, characterized in that, The yield of recombinant mussel adhesive protein was 50-200 mg / L of fermentation broth.

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