A method for efficiently preparing recombinant human amelogenin by optimizing sequence and affinity chromatography

Abstract

The application discloses a kind of sequence optimization combined with high-efficiency preparation method of recombinant human enamel matrix protein of affinity chromatography, belong to the field of bioengineering technology, and preparation method includes the following steps: construction expression strain: remove signal peptide and the human enamel matrix protein gene of codon optimization is connected with expression plasmid, constructs recombinant plasmid, and is transformed into expression host bacteria, obtains recombinant strain;Induction expression;Affinity chromatography purification;Tag is removed by enzyme cutting: the fusion protein of preliminary purification is cut using specific protease to remove the label;Refine pure.The application solves the problems of low expression level of recombinant enamel matrix protein, complex purification process and high cost by the innovation of sequence optimization and purification process, the prepared protein is high in purity, high in yield, and has significant anti-periodontitis activity, and is suitable for the development of drugs, oral care products and skin care products.

Description

Technical Field

[0001] This invention relates to a highly efficient method for preparing recombinant human ameliorative matrix proteins using sequence optimization combined with affinity chromatography, belonging to the field of bioengineering technology. Background Technology

[0002] Ameliomatrix proteins, particularly amelogenin which plays a central role in enamel formation and periodontal tissue regeneration, have shown great application potential in the biomedical and oral care fields due to their ability to effectively promote cell migration, proliferation, adhesion, and differentiation. However, their large-scale preparation faces severe challenges. Existing technologies mainly involve three pathways: First, direct extraction from animal tissues such as pig teeth carries inherent drawbacks such as pathogen risk, high immunogenicity, and large batch-to-batch variability. Second, after fusion expression and affinity purification using tags such as GST or SUMO, expensive enzymes such as thrombin or SUMO protease are required to remove the tags. This process is not only costly and cumbersome but also introduces the safety risk of enzyme residues, making it unsuitable for industrial production. Third, in vitro refolding after expression in inclusion body form is a complex, inefficient process with extremely unstable activity recovery. Existing technologies include GST tag-based thrombin digestion solutions, but their large-scale application is limited by cost and complexity. Therefore, developing a new method for preparing recombinant human ameliorative matrix proteins that can circumvent the above-mentioned defects and has the advantages of high yield, high purity, high activity, and controllable cost has become a technical bottleneck that urgently needs to be overcome in this field. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention provides a method for the efficient preparation of recombinant human ameliorative matrix proteins through sequence optimization combined with affinity chromatography, comprising the following steps:

[0004] (1) Construction of expression strain: The human amelioma matrix protein gene with the signal peptide removed and codon optimized was linked to the expression plasmid to construct a recombinant plasmid, which was then transformed into the expression host bacteria to obtain a recombinant strain;

[0005] (2) Induced expression: The recombinant strain was cultured and the expression of the fusion protein with the tag was induced;

[0006] (3) Affinity chromatography purification: After lysing the bacterial cells, the fusion protein was captured using the affinity chromatography medium corresponding to the tag, and the fusion protein was obtained by elution.

[0007] (4) Tag removal by enzyme digestion: The initially purified fusion protein is digested with a specific protease to remove the tag;

[0008] (5) Purification: The tag and the specific protease are separated and removed by salting out to obtain high-purity recombinant human ameliorative matrix protein.

[0009] Preferably, in step (1), the amino acid sequence of the human ameliorative matrix protein is as follows: .

[0010] Preferably, in step (1), the expression plasmid is pET-32a(+), the tag is the Trx-His tag carried by the plasmid, and the expression host bacterium is Escherichia coli BL21(DE3).

[0011] Preferably, in step (2), IPTG is used for induction, with a final IPTG concentration of 0.2-0.6 mM, an induction temperature of 30-34°C, and an induction time of 2-4 hours.

[0012] Preferably, the final concentration of IPTG is 0.4 mM and the induction temperature is 34°C.

[0013] Preferably, in step (3), the affinity chromatography medium is a Ni-NTA affinity chromatography medium.

[0014] Preferably, the Ni-NTA affinity chromatography medium is a large-particle medium, which does not require column packing and can be directly bound in the lysis solution by stirring and incubation.

[0015] Preferably, in step (3), the Ni equilibration solution used is: 20 mM Tris, 200 mM NaCl, 5 mM imidazole, pH 8.0; the Ni washing solution is: 20 mM Tris, 200 mM NaCl, 50-100 mM imidazole, pH 8.0; and the Ni elution solution is: 20 mM Tris, 200 mM NaCl, 200-300 mM imidazole, pH 8.0.

[0016] Preferably, the imidazole concentration of the Ni washing solution is 50 mM, 75 mM, or 100 mM; and the imidazole concentration of the Ni elution solution is 200 mM, 250 mM, or 300 mM.

[0017] Preferably, in step (4), the specific protease is a TEV protease.

[0018] Preferably, the amount of TEV protease added is 1 KU enzyme activity unit per 1g of fusion protein.

[0019] Preferably, the enzyme digestion reaction temperature is 30°C and the enzyme digestion time is 2 hours.

[0020] Preferably, in step (5), the salt used for salting out is ammonium sulfate; the final concentration of ammonium sulfate in the solution after enzymatic digestion is 50-60 g / L.

[0021] Preferably, the final concentration of the ammonium sulfate is 56 g / L.

[0022] Preferably, in step (5), after salting out, a tubular centrifuge is used to collect the precipitate. The centrifugation conditions are: cooling temperature 2-10℃, rotation speed 14000rpm, and feed flow rate 1L / min.

[0023] Preferably, the purity of the recombinant human ameliorative matrix protein finally obtained by the method is not less than 90%.

[0024] Preferably, the recombinant human enamel matrix protein prepared by the above method can be used to prepare drugs for the treatment of periodontitis.

[0025] Preferably, the recombinant human ameliorative matrix protein prepared by the above method can be used to prepare oral care products.

[0026] Preferably, the recombinant human ameliorative matrix protein prepared by the above method can be used to prepare skin care products.

[0027] The beneficial effects of this invention are:

[0028] Significantly improved yield: By specifically removing the hydrophobic signal peptide and fusing it with a soluble Trx tag, the soluble expression of the target protein in E. coli was greatly improved, with a protein yield of up to 15-20 g / kg per bacterial cell, which is much higher than the 6-8 g / kg of the traditional method.

[0029] Excellent purity and quality: Utilizing an optimized Trx-His dual-tag system for high-efficiency affinity chromatography, combined with TEV enzymatic digestion and targeted salting out for purification, the final product purity is consistently above 90%, with a significant reduction in dimers and degradation bands, resulting in a significant improvement in product quality.

[0030] The process is simplified and the cost is low: the use of a new type of large-particle Ni-NTA medium eliminates the cumbersome step of high-speed centrifugation of the lysis buffer; the use of self-produced low-cost TEV protease to replace expensive commercial enzymes significantly reduces the overall production cost.

[0031] Clear Activity and Efficacy: The recombinant human enamel matrix protein prepared by this invention has demonstrated excellent anti-periodontitis activity and the mechanism of action by inhibiting the NF-κB signaling pathway in both animal models and at the cellular level, laying a solid foundation for its clinical application.

[0032] Suitable for industrial scale-up: The process route is simple, the number of centrifugations is small, and the operating conditions are mild and easy to control, making it very suitable for large-scale industrial production. Attached Figure Description

[0033] Figure 1 SDS-PAGE electrophoresis image of the purified TEV digestion results.

[0034] Figure 2Immunohistochemical staining results of TNF-α in periodontal tissues of SD rats. A: Normal control group; B: Negative control group; C: Low concentration recombinant amelogenin (Example 1, 0.315 mg / kg) treatment group; D: High concentration recombinant amelogenin (Example 1, 3.15 mg / kg) treatment group.

[0035] Figure 3 Results of qPCR detection of inflammatory factor mRNA expression levels. A: IL-1β mRNA expression level detection results; B: IL-6 mRNA expression level detection results (n=3, *P<0.05 vs. TSH group; **P<0.01 vs. TSH group; ***P<0.001 vs. TSH group; ****P<0.0001 vs. TSH group). Detailed Implementation

[0036] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0038] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the experimental materials used in the following examples are all purchased from commercial channels.

[0039] Example 1

[0040] 1. Bacterial culture and induced expression

[0041] The recombinant BL21(DE3) strain containing the pET-32a(+)-optimized human ameliorative matrix protein gene, preserved at -80℃, was inoculated at a 1% inoculum into 50 mL of LB liquid medium containing 100 μg / mL ampicillin. The culture was incubated at 37℃ and 200 rpm for approximately 3 hours until the OD600 reached 0.6. IPTG was added to the culture to a final concentration of 0.4 mM, and the shaker temperature was adjusted to 34℃ and the shaking speed to 120 rpm. Expression was induced for another 3 hours. After induction, the bacterial culture was centrifuged at 4℃ and 5000 × g for 10 minutes, and the bacterial cells were collected, weighed to approximately 0.5 g (wet weight), and stored at -20℃ for later use.

[0042] 2. Affinity chromatography purification

[0043] 0.5 g of bacterial cells were suspended in 4.5 mL of pre-chilled Ni equilibration buffer (20 mM Tris, 200 mM NaCl, 5 mM imidazole, pH 8.0) and sonicated under ice bath conditions. The sonication power was 300 W, with a 2-second interval and a 3-second rest, for a total of 10 minutes. The lysate was then mixed with 1 mL of Ni-NTA medium (ChuanBio) in a centrifuge tube and incubated at 4°C for 30 minutes. After incubation, the mixture was centrifuged at 500 × g for 2 minutes, and the supernatant was carefully discarded. 5 mL of Ni equilibration buffer was added to the medium, and the cells were gently resuspended and washed. The mixture was centrifuged at 500 × g for 2 minutes, and the supernatant was discarded. 5 mL of Ni washing buffer (20 mM Tris, 200 mM NaCl, 50 mM imidazole, pH 8.0) was added to the medium, and the cells were gently resuspended and washed. The mixture was centrifuged at 500 × g for 2 minutes, and the supernatant was discarded. Add 2 mL of Ni elution buffer (20 mM Tris, 200 mM NaCl, 250 mM imidazole, pH 8.0) to the medium, gently resuspend, and incubate at 4 °C for 20 minutes by rotation. After incubation, centrifuge at 500 × g for 2 minutes and carefully collect the supernatant, which is the preliminarily purified Trx-His-enamel matrix protein fusion protein solution.

[0044] 3. Removal of the tag by enzyme digestion

[0045] Determine the protein concentration of the elution buffer. Take a solution containing approximately 10 mg of fusion protein, add 1×TEV enzyme buffer to make up the volume, then add 10 KU of self-produced TEV protease and mix well. Place the reaction system in a 30°C water bath for 2 hours for enzymatic digestion.

[0046] 4. Salting out for purification

[0047] Solid ammonium sulfate was slowly added to the enzyme-digested solution while stirring until a final concentration of 56 g / L was reached. The solution was incubated on ice for 1 hour. The solution was then centrifuged at 4°C and 14000 rpm for 20 minutes, and the supernatant was carefully discarded. The precipitate was dissolved in a small amount of deionized water and then freeze-dried to obtain the final recombinant human ameliorative matrix protein product. The yield and purity were calculated.

[0048] Example 2

[0049] 1. Bacterial culture and induced expression

[0050] A glycerol tube containing the pET-32a(+)-optimized human ameliorative matrix protein gene, preserved from a -80°C ultra-low temperature freezer, was taken out. On a sterile work surface, a small amount of bacterial culture was inoculated onto an LB agar plate containing 100 μg / mL ampicillin and streaked. The plate was then inverted and incubated overnight (approximately 16 hours) at 37°C. The next day, a single, plump white colony was picked and inoculated into a 500 mL Erlenmeyer flask containing 50 mL of LB liquid medium containing 100 μg / mL ampicillin. The flask was placed in a shaker at 37°C and incubated at 200 rpm for approximately 3 hours, until the OD600 value reached between 0.6 and 0.8. IPTG stock solution was added to the flask to a final concentration of 0.4 mM. The shaker temperature was adjusted to 34°C and the shaking speed to 120 rpm, and expression was induced for another 3 hours. After induction, remove the Erlenmeyer flask and immediately immerse it in an ice-water mixture to cool for 10 minutes. Then centrifuge at 4°C and 5000×g for 10 minutes, carefully discarding the supernatant. Weigh the wet bacterial cells precipitated at the bottom of the centrifuge tube using an electronic balance; the weight should be approximately 0.5 grams. The bacterial cells can be stored for a short period at -20°C.

[0051] 2. Affinity chromatography purification (high-rigor washing)

[0052] 0.5 g of wet bacterial cells were suspended in 4.5 mL of pre-chilled Ni equilibration buffer (20 mM Tris, 200 mM NaCl, 5 mM imidazole, pH 8.0). The suspension was vortexed thoroughly until completely resuspended with no visible bacterial clumps. The suspension was transferred to a 15 mL centrifuge tube and placed in an ice-water bath. An ultrasonic cell disruptor was used with a 3 mm probe, set to the following parameters: 300 W power, 2 seconds of sonication followed by a 3-second interval, for a total duration of 10 minutes. The sample was kept in an ice bath during the process to prevent overheating. After sonication, the lysis buffer was centrifuged at 4 °C and 12000 × g for 20 minutes. The supernatant was carefully transferred to a new 15 mL centrifuge tube. The precipitate was discarded. 1 mL of Ni-NTA affinity chromatography medium (ChuanBio) pre-equilibrated with Ni equilibration buffer was added to the supernatant. Secure the centrifuge tubes to a rotary mixer and incubate slowly at 4°C for 30 minutes to allow the His-tagged fusion protein to fully bind to the medium. After incubation, centrifuge the tubes at 500×g for 2 minutes to allow the medium to settle. Carefully aspirate the supernatant using a pipette.

[0053] Washing: Add 5 mL of pre-chilled Ni washing buffer (20 mM Tris, 200 mM NaCl, 100 mM imidazole, pH 8.0) to the sedimented medium. Gently resuspend the medium by pipetting, then return it to the rotary mixer and wash at 4°C for 15 minutes. Afterward, centrifuge at 500×g for 2 minutes and discard the supernatant. This step uses a higher concentration of 100 mM imidazole to more effectively wash away non-specifically bound proteins. Add 2 mL of pre-chilled Ni elution buffer (20 mM Tris, 200 mM NaCl, 250 mM imidazole, pH 8.0) to the medium. Gently resuspend the medium and incubate at 4°C for 20 minutes to dissociate the target protein from the medium. After incubation, centrifuge at 500×g for 2 minutes. Carefully collect the supernatant containing the target protein, i.e., the eluent, into a new centrifuge tube. This is the preliminarily purified Trx-His-enamel matrix protein fusion protein solution.

[0054] 3. Removal of the tag by enzyme digestion

[0055] Take 50 μL of eluent and determine its protein concentration using a BCA protein quantification kit. Calculate the total protein content in the eluent based on the measured concentration. Transfer the remaining eluent to a 5 mL dialysis bag. Based on the total protein content, add the calculated volume of self-produced TEV enzyme stock solution at a ratio of 1 KUTEV enzyme / 1 g fusion protein. Add 1×TEV enzyme buffer to the appropriate total volume. Incubate the reaction system in a 30°C water bath for 2 hours to perform the enzymatic digestion reaction.

[0056] 4. Salting out for purification

[0057] After the enzymatic digestion reaction is complete, transfer the reaction solution to a 15 mL centrifuge tube. Place the tube on a magnetic stirrer in an ice-water bath and stir slowly. Accurately weigh the required amount of ammonium sulfate solid powder using an electronic balance. Calculation formula: Required ammonium sulfate mass (g) = Enzyme digestion solution volume (mL) × 0.056. Slowly and in portions, add the ammonium sulfate powder to the stirred enzyme digestion solution, ensuring each addition is completely dissolved before adding the next. After all ammonium sulfate has been added, stop stirring. Seal the centrifuge tube tightly and incubate at 4°C for 1 hour to allow the target protein to precipitate fully. After incubation, centrifuge the tube at 4°C and 14000 rpm for 20 minutes. At this point, recombinant human ameliorative matrix protein will precipitate at the bottom of the tube, while the excised Trx-His tag and TEV enzyme will mainly remain in the supernatant. Thoroughly discard the supernatant using a pipette. Retain the white precipitate at the bottom of the tube. Add 1 mL of pre-cooled deionized water to the precipitate and gently pipette to dissolve it completely.

[0058] 5. Freeze-drying

[0059] Aliquot the dissolved protein solution into 1.5mL centrifuge tubes. Immediately place the aliquoted samples into a -80°C ultra-low temperature freezer for 2 hours. Quickly transfer the completely frozen samples to a freeze dryer, start the main program, and freeze-dry until the samples completely transform into white flocculent solids. After drying, weigh the total weight of the freeze-dried product and subtract the tare weight of the container; this is the final yield of recombinant human ameliorative matrix protein.

[0060] Example 3

[0061] 1. Bacterial culture and induced expression

[0062] The steps are exactly the same as in Example 1.

[0063] 2. Affinity chromatography purification (high concentration elution)

[0064] The steps of cell lysis, centrifugation to collect the supernatant, incubation with Ni-NTA medium, and washing with equilibration solution were exactly the same as in Example 1.

[0065] Elution: Add 2 mL of pre-chilled Ni elution buffer (20 mM Tris, 200 mM NaCl, 300 mM imidazole, pH 8.0) to the bound and washed medium. Gently resuspend the medium and incubate at 4°C with rotation for 20 minutes. After incubation, centrifuge at 500 × g for 2 minutes. Carefully collect the supernatant, which is the elution buffer. This step uses a high concentration of 300 mM imidazole to more thoroughly elute all fusion proteins bound to the medium.

[0066] 3. Removal of the tag by enzyme digestion

[0067] The steps are exactly the same as in Example 1.

[0068] 4. Salting out for purification

[0069] The steps are exactly the same as in Example 1.

[0070] 5. Freeze-drying

[0071] The steps are exactly the same as in Example 1.

[0072] Comparative Example 1 (signal peptide not removed)

[0073] 1. Bacterial culture and induced expression

[0074] The strain used in this comparative example was a recombinant BL21(DE3) strain containing the full-length human enamel matrix protein gene with pET-32a(+)- signal peptide. Its amino acid sequence begins with "MGTWILFACLLGAAFA...". The steps for picking single colonies, scaling up culture, and IPTG-induced expression were exactly the same as in Example 1. After induction, the bacterial cells were collected by centrifugation. It was observed that the amount of bacterial precipitate was less than in Example 1. Approximately 0.5 grams of wet bacterial cells were weighed.

[0075] 2. Affinity chromatography purification

[0076] The pyrolysis steps are the same as in Example 1.

[0077] After centrifugation, the supernatant was observed to be very clear and in small quantity, while a large amount of white precipitate was found at the bottom of the tube. A small amount of the supernatant was incubated with Ni-NTA medium, and subsequent washing and elution steps were the same as in Example 1. After the elution step, the collected eluent was almost colorless and transparent. A small amount was subjected to SDS-PAGE electrophoresis, and only weak bands were observed at very high molecular weight positions, which were likely undissolved aggregates. No clear main band was observed at the target molecular weight positions.

[0078] 3. Removal of the tag by enzyme digestion

[0079] This step could not be performed effectively because a sufficient amount of the initially purified fusion protein could not be obtained.

[0080] Ultimately, it was not possible to obtain a sufficient quantity of recombinant human ameliorative matrix protein lyophilized powder for testing.

[0081] Comparative Example 2 (using GST tag and thrombin)

[0082] 1. Bacterial culture and induced expression

[0083] The strain used in this comparative example was the recombinant BL21(DE3) strain containing the pGEX-4T-1-signal peptide-free human ameliomatic protein gene. LB medium containing 100 μg / mL ampicillin was used. Single colonies were picked, and the expansion culture procedure was the same as in Example 1. When the OD600 reached 0.6, IPTG was added to a final concentration of 0.4 mM, and expression was induced at 37°C for 4 hours. The bacterial cells were collected by centrifugation, and approximately 0.5 g of wet bacterial cells were weighed.

[0084] 2. Affinity chromatography purification (GST tag)

[0085] The bacterial resuspended in 4.5 mL of pre-chilled PBS buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.4). Sonication was performed under the same conditions as in Example 1. After centrifugation, the supernatant was incubated with 1 mL of pre-equilibrated GST affinity chromatography medium at 4°C for 30 minutes. Centrifugation was performed at 500 × g for 2 minutes, and the supernatant was discarded. The medium was washed twice with 5 mL of PBS buffer. 2 mL of freshly prepared reduced glutathione elution buffer (50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0) was added to the medium, and the medium was incubated at 4°C for 20 minutes. Centrifugation was performed at 500 × g for 2 minutes, and the supernatant was collected; this was the GST-enamel matrix protein fusion protein.

[0086] 3. Removal of the tag (thrombin) by enzyme digestion

[0087] Determine the protein concentration in the eluent. Add commercial thrombin at a ratio of 10 units of thrombin / 1g fusion protein, according to the supplier's instructions. Incubate overnight at room temperature for 16 hours.

[0088] 4. Pure

[0089] After enzymatic digestion, attempts were made to dialyze the reaction solution to remove glutathione and thrombin. However, due to the lack of a specific separation step as effective as salting out, thrombin and the cleaved GST tag could not be effectively removed.

[0090] 5. Freeze-drying

[0091] The dialysis solution was freeze-dried.

[0092] This comparative example yielded approximately 4.0 mg of lyophilized powder from 0.5 g of wet bacterial cells. However, testing revealed the presence of GST labeling and thrombin residue.

[0093] Comparative Example 3 (without salting out purification step)

[0094] 1. Bacterial culture and induced expression

[0095] The steps are exactly the same as in Example 1.

[0096] 2. Affinity chromatography purification

[0097] The steps are exactly the same as in Example 1.

[0098] 3. Removal of the tag by enzyme digestion

[0099] The steps are exactly the same as in Example 1.

[0100] 4. Purity (critical deviation)

[0101] After the enzymatic digestion reaction, ammonium sulfate precipitation is not performed. All the digested protein is transferred into a dialysis bag with a molecular weight cutoff of 10 kDa. The dialysis bag is placed in 2 liters of 20 mM Tris-HCl buffer (pH 8.0) at 4°C and slowly stirred on a magnetic stirrer. The dialysis buffer is changed every 4-6 hours, for a total of 3 dialysis cycles, with a total duration of approximately 20 hours. This step aims to remove small molecules and salts through diffusion, but it cannot effectively separate the Trx-His tag and TEV enzyme, which have similar molecular weights to the target protein.

[0102] 5. Freeze-drying

[0103] The solution in the dialysis bag was transferred to a centrifuge tube and freeze-dried.

[0104] This comparative example yielded approximately 6.9 mg of lyophilized powder from 0.5 g of wet bacterial cells. However, this product is a mixture of the target protein, the Trx-His tag, and the TEV enzyme.

[0105] Detection method:

[0106] Protein concentration determination: The BCA protein quantification kit was used, and the procedure was strictly followed according to the instructions.

[0107] Protein purity analysis: SDS-PAGE was used. After Coomassie brilliant blue staining, the gel was scanned using a gel imaging system, and the percentage of the target band in the total protein was calculated using software.

[0108] Anti-inflammatory activity assay: A periodontitis model was established in SD rats (ligature method). After grouping, protein samples (0.315 mg / kg) prepared in each example and comparative example were injected locally into the periodontal tissue. After 4 weeks of treatment, periodontal tissue was taken for RNA extraction, and the relative mRNA expression levels of inflammatory factors IL-1β and IL-6 were detected by qPCR.

[0109] The above methods were used to test all embodiments and comparative examples, and the results are shown in Table 1.

[0110] Table 1. Preparation and activity data of recombinant human ameliomatic proteins

[0111]

[0112] Regarding sequence optimization: In Comparative Example 1, the absence of signal peptide removal resulted in severe protein aggregation and inclusion bodies, making it almost impossible to obtain active protein via soluble pathways. This contrasts sharply with Example 1, which achieved highly efficient soluble expression by removing the signal peptide. This difference demonstrates the importance of the signal peptide removal sequence optimization step, which is key to solving the problem of soluble expression of matrix proteins in prokaryotic expression systems, and brings a revolutionary increase in yield to this invention.

[0113] Regarding the tagging and enzymatic digestion system: Comparative Example 2 uses a traditional GST / thrombin system, which not only has a yield only about half that of Example 1, but also carries the risk of tag and expensive protease residues, leading to product purity and safety issues, and is also costly. In contrast, the Trx-His / TEV enzyme system used in Example 1, combined with subsequent specific salting-out purification, successfully achieved high yield, high purity, and low-cost production. This demonstrates that the tagging, enzymatic digestion, and purification combination of this invention is a synergistic organic whole, and its overall technical effect is far superior to existing technical solutions.

[0114] Regarding the necessity of the purification step: Comparative Example 3 omitted the crucial salting-out step, resulting in a sharp drop in the purity of the final product to 65%, and the presence of a large amount of impurity proteins. Its anti-inflammatory effect was also significantly worse than that of Example 1. This demonstrates that the purification step of removing tags and proteases through salting-out at a specific concentration is essential for obtaining a high-purity, high-activity final product.

[0115] Regarding the optimization of process parameters: The three embodiments achieved fine-tuning of purity while maintaining high yield and high activity by adjusting the imidazole concentration in affinity chromatography. This demonstrates that the parameter range provided by this invention is effective, and the process possesses a certain degree of flexibility and robustness.

[0116] In summary, this invention successfully solves the long-standing industry problems of low yield, poor purity, high cost, and complex processes in the preparation of recombinant human ameliorative matrix proteins by combining sequence optimization (removing the signal peptide) with specific tags, enzymatic digestion, and purification processes.

[0117] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0118] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention. The actual application is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A method for the efficient preparation of recombinant human ameliorative matrix protein by sequence optimization combined with affinity chromatography, characterized in that, Includes the following steps: (1) Construction of expression strain: The human amelioma matrix protein gene with the signal peptide removed and codon optimized was linked to the expression plasmid to construct a recombinant plasmid carrying the Trx-His tag, and transformed into the expression host bacteria to obtain the recombinant strain; (2) Induced expression: The recombinant strain was cultured and the expression of the fusion protein with the tag was induced; (3) Affinity chromatography purification: After lysing the bacterial cells, the fusion protein was captured using the affinity chromatography medium corresponding to the tag, and the fusion protein was obtained by elution. (4) Tag removal by enzyme digestion: The initially purified fusion protein is digested with a specific protease to remove the tag; (5) Purification: The tag and the specific protease are separated and removed by salting out to obtain high-purity recombinant human ameliorative matrix protein; In step (1), the amino acid sequence of the human ameliorative matrix protein with the signal peptide removed is: MGTWILFACLLGAAFAMPVLTPLRWYQSIRPPYPSYGYDPMGGWLHHQIIPVLSQQHPPTHTLQPHKHIPVVPAQQPVIPQQPMMPVPGQHSMTPIQHHQPNLPPPAQQPYQPQPVQPQPHQPMQPQPPVHPMQPLPPQPPLPPMFPMQPLPPMLPDLTLDAWPSTDRTRRDDVD; In step (1), the expression plasmid is pET-32a(+); the expression host bacterium is Escherichia coli BL21(DE3); In step (2), IPTG is used for induction. The final concentration of IPTG is 0.2-0.6 mM, the induction temperature is 30-34℃, and the induction time is 2-4 hours. In step (3), the affinity chromatography medium is Ni-NTA affinity chromatography medium; In step (3), the Ni equilibration solution used is: 20 mM Tris, 200 mM NaCl, 5 mM imidazole, pH 8.0; the Ni washing solution is: 20 mM Tris, 200 mM NaCl, 50-100 mM imidazole, pH 8.0; and the Ni elution solution is: 20 mM Tris, 200 mM NaCl, 200-300 mM imidazole, pH 8.

0.

2. The method according to claim 1, characterized in that, In step (4), the specific protease is TEV protease.

3. The method according to claim 1, characterized in that, In step (5), the salt used for salting out is ammonium sulfate; the final concentration of ammonium sulfate in the solution after enzymatic digestion is 50-60 g / L.

4. The method according to claim 1, characterized in that, In step (5), after salting out, a tubular centrifuge is used to collect the precipitate. The centrifugation conditions are: cooling temperature 2-10℃, rotation speed 14000rpm, and feed flow rate 1L / min.

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