Recombinant human glaze matrix protein preparation method based on double salting-out and surfactant denaturation and renaturation

Abstract

The invention relates to a recombinant human glaze matrix protein preparation method based on double salting-out and surfactant denaturation and renaturation, and belongs to the technical field of bioengineering. A first salting-out step: adding a first inorganic salt into the lysate supernatant to carry out first salting-out, and centrifuging after salting-out to obtain a first salting-out centrifugal precipitate; a surfactant modification and renaturation step; a second salting-out step: adding a second inorganic salt into the renaturation solution for second salting-out, and centrifuging after salting-out to obtain a second salting-out centrifugal precipitate; and drying the second salting-out centrifugal precipitate to obtain the recombinant human glaze matrix protein. The method is simple in process and low in cost, and the prepared recombinant human glaze matrix protein has an excellent anti-inflammatory effect and is particularly suitable for development of anti-periodontitis drugs, oral care products and skin care products.

Description

Technical Field

[0001] This invention relates to a method for preparing recombinant human ameliorative matrix protein based on double salting out and surfactant refolding, belonging to the field of bioengineering technology. Background Technology

[0002] Amelium matrix proteins, as key signaling molecules in periodontal tissue regeneration, have shown that their core component, amelogenin, can effectively promote the migration, proliferation, adhesion, and differentiation of various cell types, including osteoblasts and periodontal ligament stem cells, demonstrating significant clinical application potential in the treatment of periodontitis and alveolar bone regeneration. However, how to economically and efficiently obtain high-purity recombinant amelium matrix proteins while maintaining their natural biological activity has always been a bottleneck restricting their clinical translation and commercial application. Existing technical routes each have significant drawbacks: First, traditional animal-derived extraction methods (such as isolation from pig teeth) are not only limited in source and low in yield, but also carry the risks of zoonotic pathogen transmission, immune rejection reactions caused by foreign proteins, and uncontrollable batch-to-batch quality variations. Secondly, mainstream gene recombination strategies generally rely on the "tag-enzyme digestion" purification pathway. This involves constructing fusion tags such as GST or SUMO for initial affinity chromatography purification, followed by tag excision using expensive thrombin or SUMO proteases. This process not only significantly increases production costs but also introduces complex process steps, strict control of enzyme digestion conditions, and the crucial challenges of enzyme residue removal and detection, making this pathway neither economically nor safely advantageous for large-scale production. Furthermore, another common strategy utilizes prokaryotic expression systems. However, the target protein often exists as inactive inclusion bodies, requiring cumbersome, time-consuming, and highly unstable renaturation operations. These operations include dissolution with high-concentration denaturing agents and painstaking attempts to restore its correct three-dimensional structure and activity through fine gradient dilution or dialysis. The entire process is inefficient and has poor reproducibility. Most related technologies follow the tag-enzyme digestion route described above. Some employ GST tags combined with thrombin digestion, which can yield the target protein, but the inherent drawbacks of high cost and process complexity make them unsuitable for industrial production. In summary, developing a novel preparation method that can completely avoid animal-derived risks, does not rely on affinity tags and protease cleavage, avoids inefficient inclusion body refolding processes, and is simple and cost-effective has become an urgent and important technological need in this field. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention provides a method for preparing recombinant human ameliorative matrix protein based on double salting out and surfactant refolding, comprising the following steps: (a) Cell lysis: Cells expressing recombinant human ameliomatic protein were lysed, and centrifuged after lysis to obtain the supernatant of the lysate; (b) First salting out: Add a first inorganic salt to the supernatant of the lysis solution to perform the first salting out. After the salting out is completed, centrifuge to obtain the first salting out precipitate. (c) Surfactant denaturation and refolding: The first salting-out centrifuged precipitate is resuspended in water to obtain a resuspension; a surfactant is added to the resuspension for denaturation treatment, and centrifugation is performed after denaturation to obtain a denatured centrifugation supernatant; then phosphate buffer is added to the denatured centrifugation supernatant for dilution to reduce the surfactant concentration and achieve refolding to obtain a refolded solution. (d) Second salting out: A second inorganic salt is added to the refolding solution to perform a second salting out. After the salting out is completed, centrifugation is performed to obtain the second salting out precipitate. (e) The recombinant human ameliorative matrix protein is obtained by taking the second salting-out precipitate, centrifuging, and drying.

[0004] Preferably, the specific method for lysis in step (a) is as follows: take bacterial cells that are induced to express recombinant human ameliorative matrix protein, add phosphate buffer for resuspending, and then perform high-pressure homogenization.

[0005] Preferably, the weight ratio of the bacterial cells inducing recombinant human ameliorative matrix protein expression to phosphate buffer is 1:5~10; more preferably 1:9; the phosphate buffer is 15~25mM phosphate buffer with pH 6.0~8.0; more preferably 20mM phosphate buffer with pH 7.0.

[0006] Preferably, in step (b), the content of the first inorganic salt in the supernatant of the lysis solution after its addition is 50~60 g / L; more preferably 56 g / L; the first inorganic salt is ammonium sulfate.

[0007] Preferably, in step (b), the specific method for the first salting out is as follows: add the first inorganic salt to the supernatant of the lysis solution, stir to dissolve, and let stand for 1-2 hours.

[0008] Preferably, in step (c), the denaturation method is as follows: a surfactant is added to the resuspended solution, stirred and dissolved, and then allowed to stand at 2-10°C for 6-12 hours to denature.

[0009] Preferably, in step (c), the mass concentration of the surfactant in the resuspension after addition is 5-6%; the surfactant is sodium lauroyl sarcosinate; the mass concentration of the surfactant in the denaturing centrifugation supernatant is diluted to 1% by adding phosphate buffer; the weight ratio of the first salting-out centrifugation precipitate to water is 1:5-10; preferably 1:9.

[0010] Preferably, in step (d), the content of the second inorganic salt in the refolding solution after addition is 100~120g / L; more preferably 112g / L; the second inorganic salt is ammonium sulfate; the specific method of the second salting out is: add the second inorganic salt to the refolding solution, stir to dissolve, and let stand for 1~2h.

[0011] Preferably, the method further includes a Q-column chromatography purification step after step (d) and before step (e); the eluent is collected, concentrated, and dried to obtain the recombinant human ameliorative matrix protein.

[0012] Preferably, the specific method of the Q-column chromatography purification step is as follows: the second salting-out centrifuged precipitate is resuspended in 20 mM phosphate buffer at pH 7.0 and loaded onto a Q-column; impurities are first removed by elution with 2-4 column volumes of 0.2 M sodium chloride aqueous solution; then, elution is performed with 5-10 column volumes of 0.5 M sodium chloride aqueous solution, and the eluent obtained from the 0.5 M sodium chloride aqueous solution is collected, concentrated, and dried to obtain recombinant human ameliorative matrix protein; in the Q-column chromatography purification step, impurities are first removed by elution with 3 column volumes of 0.2 M sodium chloride aqueous solution; then, elution is performed with 7 column volumes of 0.5 M sodium chloride aqueous solution.

[0013] The recombinant human enamel matrix protein prepared by the above method can be used to prepare products with anti-inflammatory effects; the anti-inflammatory effect includes anti-periodontitis; the products include drugs, oral care products or skin care products.

[0014] The beneficial effects of this invention are: Compared with existing technologies, the technical route based on double salting out and surfactant refolding provided by this invention has significant advantages: Uniqueness of the technical approach: This invention does not rely on affinity tags at all, thus avoiding the enzymatic digestion process and all its associated costs and risks from the source. Furthermore, this invention does not form inclusion bodies, employing a unique surfactant-mediated renaturation strategy instead of the traditional inclusion body renaturation pathway. This differs from existing technical approaches such as animal extraction, tag-enzyme digestion, and inclusion body renaturation, representing a completely new, simpler, and more efficient technical approach.

[0015] Low cost and easy to scale up: The core purification steps are based on salting out and refolding, with low raw material costs and simple operation, making it very suitable for large-scale industrial production.

[0016] High product yield and good activity: As shown in the following examples, the method of the present invention consistently achieves a high yield of protein per unit cell. Through optimized renaturation parameters and purification conditions, the prepared protein exhibits excellent anti-inflammatory activity.

[0017] Product quality is stable and controllable: the purity of the prepared protein can reach over 90%, and the electrophoretic characterization is consistent with literature reports. It should be noted that there is a difference between the theoretical molecular weight of the enamel matrix protein and its apparent molecular weight in SDS-PAGE; this is a common phenomenon in this protein family and does not affect the implementation and effectiveness of this invention. Attached Figure Description

[0018] Figure 1 This is an electrophoresis diagram of the recombinant human ameliorative matrix protein of the present invention; Figure 2 Figure 1 shows the results of HE staining experiments on periodontal tissues. In the figure, A represents HE staining in the model group mice, B represents HE staining in the normal control group mice, C represents HE staining in experimental group 1 mice, and D represents HE staining in experimental group 2 mice. A: Normal control group; B: Model group; C: Model group using 0.315 ppm of enamel matrix protein prepared in Example 1; D: Model group using 3.15 ppm of enamel matrix protein prepared in Example 1. Detailed Implementation

[0019] 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.

[0020] 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.

[0021] 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.

[0022] Preparation example: (1) Recombinant plasmid: The DNA fragment optimized according to the amino acid sequence of human amelioma matrix protein was ligated to pET-28a(+) plasmid to obtain the recombinant plasmid; (2) Recombinant strain: The recombinant plasmid was transformed into BL21(DE3) competent cells, single colonies were screened, expanded and preserved; the recombinant strain was obtained. (3) Induction of expression: The recombinant strain was cultured in a large scale and then induced to express by IPTG. The bacterial cells were harvested by centrifugation to obtain the strain that induced the expression of recombinant human amelena matrix protein.

[0023] The amino acid sequence of the human amber matrix protein in step 1) is:

[0024] The human amelioma matrix protein DNA sequence in step 1) is:

[0025] The plasmid transformation method in step 2) is as follows: ① Remove BL21(DE3) competent cells from -80℃ and quickly place them on ice. After 5 minutes, wait for the bacterial block to thaw, add the target plasmid, and gently mix by tapping the bottom of the EP tube. Let it stand on ice for 25 minutes; ② Heat shock in a 42℃ water bath for 45 seconds, quickly return to ice and let it stand for 2 minutes. Shaking will reduce the transformation efficiency; ③ Add 700 μl of antibiotic-free sterile LB medium to the centrifuge tube, mix well, and revive at 37℃, 200 rpm for 60 minutes; ④ Centrifuge at 5000 rpm for one minute to collect the bacteria, keep about 100 μl of supernatant, gently pipette to resuspend the bacterial block and spread it on LB medium containing kanamycin. Invert the plate and incubate overnight at 37℃ (pET-28a(+) plasmid is kanamycin resistant).

[0026] The method for screening single colonies, expanding culture, and preserving bacteria in step 2) is as follows: ① Prepare 20 ml of liquid LB medium containing kanamycin in a breathable Erlenmeyer flask, and inoculate a fresh single colony containing the target plasmid; ② Incubate the bacteria overnight at 37℃ and 200 rpm for about 10 hours; ③ Take 10 ml of liquid culture, add 10 ml of sterile 50% glycerol, mix well, dispense into centrifuge tubes, and preserve the bacteria at -80℃.

[0027] The method for expanding the bacterial strain and inducing expression in step 3) is as follows: ① Inoculate the bacterial solution preserved in step 2) at a ratio of 1% into 50ml of liquid LB medium containing kanamycin. To increase dissolved oxygen, use a 500ml Erlenmeyer flask; ② Shake at 37℃ and 150rpm until the OD600 value is 0.5-0.8; ③ Add IPTG to the Erlenmeyer flask to a final concentration of 0.4mM, and continue to shake at 37℃ and 120rpm for 3h; ④ Remove the Erlenmeyer flask from the shaker, bury it in ice for 10 minutes, centrifuge at 4℃, 5000×g for 10 minutes, discard the supernatant, and store the precipitate at -20℃.

[0028] Example 1: Preparation of recombinant human ameliorative matrix protein by double salting out and refolding process Bacterial cell lysis 100.00 g of wet *E. coli* cells expressing recombinant human ameliorative matrix protein were removed from an ultra-low temperature freezer at -80°C and placed in a 2-liter beaker. 900 mL of pre-chilled 20 mM PBS (pH 7.0) buffer was added to the beaker. The beaker was placed in an ice-water bath. A high-speed homogenizer was turned on, and the homogenization probe was immersed below the surface of the bacterial suspension. Homogenization was performed at 10,000 rpm for 3 minutes, or until the suspension became homogeneous and free of visible bacterial clumps. The homogenized suspension was then transferred to the feed tank of a high-pressure homogenizer. Homogenization was performed twice at a pressure of 1000 bar under a cooling environment of 5°C. This process aims to thoroughly disrupt the bacterial cell walls.

[0029] Pour the homogenized pyrolysis liquid into the feed tank of a tubular centrifuge. Start the tubular centrifuge, set the centrifuge chamber cooling temperature to 5°C, the rotor speed to 14000 rpm, and the feed flow rate to 1 L / min. Begin centrifugation, collecting the clear pyrolysis supernatant flowing from the centrifuge outlet. Discard the precipitate. Record the volume of the obtained supernatant.

[0030] First salting out Place the collected lysate supernatant in a large magnetically stirred flask, with a clean stir bar at the bottom. Place the flask on the magnetic stirrer in an ice-water bath and start stirring slowly. Weigh the calculated amount of ammonium sulfate powder using an electronic balance. The calculation formula is: required ammonium sulfate mass (g) = V1 (mL) × 0.056. For example, if V1 is 800 mL, then weigh 800 × 0.056 = 44.80 g. Add the weighed ammonium sulfate powder very slowly and in multiple additions to the stirred supernatant, ensuring that each addition is completely dissolved before adding the next. After all the ammonium sulfate has been added and completely dissolved, stop stirring. Incubate the flask at 4°C for 1 hour to allow the protein to precipitate. After 1 hour, centrifuge again using a tube centrifuge at 5°C, 14000 rpm, and 1 L / min to collect the precipitate. After this centrifugation, carefully discard the supernatant and retain the first salting-out precipitate (a pale yellow solid) at the bottom of the tube or in the collector.

[0031] Surfactant refolding Denaturation: Transfer the first salting-out centrifuged precipitate to a clean beaker. Add 9 times the weight of the precipitate in pre-cooled deionized water. Gently resuspend the precipitate using a glass rod or pipette. Then, weigh out sodium lauroyl sarcosinate powder and add it to the resuspension to achieve a final concentration of 5%. Calculation formula: Required sodium lauroyl sarcosinate mass (g) = Resuspension volume (mL) × 0.05. After stirring to dissolve, place the beaker in a 5°C refrigerator and allow it to denature for 12 hours.

[0032] Centrifuge and collect the supernatant: After denaturation, aliquot the solution into 50mL centrifuge tubes and centrifuge at 4°C and 10,000rpm for 10 minutes using a low-temperature high-speed centrifuge. Carefully combine the supernatants from all tubes; this is the denaturation centrifugation supernatant. Discard the precipitate.

[0033] Renaturation: Measure the volume of the denatured centrifuged supernatant. Add pre-cooled 20mM PBS (pH 7.0) buffer to dilute the sodium lauroyl sarcosinate concentration to 1%. Calculation formula: Required PBS volume (mL) = V² × (5% / 1% - 1) = V² × 4. Mix slowly until homogeneous; this mixture is the renaturation solution.

[0034] Second salting out Place the refolding solution on a magnetic stirrer and stir slowly in an ice-water bath. Weigh out ammonium sulfate powder using the formula: required ammonium sulfate mass (g) = refolding solution volume (mL) × 0.112. Slowly add the powder to the refolding solution, following the same procedure as the first salting-out. After addition, let it stand at 4°C for 1 hour. Centrifuge again using a tubular centrifuge at 5°C, 14000 rpm, and 1 L / min, collect the precipitate, and discard the supernatant. The precipitate obtained this time is the second salting-out centrifugal precipitate.

[0035] freeze-dried The second salting-out centrifugation precipitate was transferred to a pre-weighed lyophilization bottle. The lyophilization bottle was rapidly placed in liquid nitrogen for 5 minutes, then transferred to a freeze dryer. The freeze dryer was turned on, and the sample was freeze-dried until completely dry and appeared as a white flocculent solid. The total weight of the lyophilization bottle was weighed, and the weight of the bottle was subtracted to obtain the yield of recombinant human ameliorative matrix protein.

[0036] Example 2: Preparation of recombinant human ameliorative matrix protein by double salting out and refolding process (Q column purification) Bacterial cell lysis 100.00 g of wet *E. coli* cells expressing recombinant human ameliorative matrix protein were removed from an ultra-low temperature freezer at -80°C and placed in a 2-liter beaker. 900 mL of pre-chilled 20 mM PBS (pH 7.0) buffer was added to the beaker. The beaker was placed in an ice-water bath. A high-speed homogenizer was turned on, and the homogenization probe was immersed below the surface of the bacterial suspension. Homogenization was performed at 10,000 rpm for 3 minutes, or until the suspension became homogeneous and free of visible bacterial clumps. The homogenized suspension was then transferred to the feed tank of a high-pressure homogenizer. Homogenization was performed twice at a pressure of 1000 bar under a cooling environment of 5°C. This process aims to thoroughly disrupt the bacterial cell walls.

[0037] Pour the homogenized pyrolysis liquid into the feed tank of a tubular centrifuge. Start the tubular centrifuge, set the centrifuge chamber cooling temperature to 5°C, the rotor speed to 14000 rpm, and the feed flow rate to 1 L / min. Begin centrifugation, collecting the clear pyrolysis supernatant flowing from the centrifuge outlet. Discard the precipitate. Record the volume of the obtained supernatant.

[0038] First salting out Place the collected lysate supernatant in a large magnetically stirred flask, with a clean stir bar at the bottom. Place the flask on the magnetic stirrer in an ice-water bath and start stirring slowly. Weigh the calculated amount of ammonium sulfate powder using an electronic balance. The calculation formula is: required ammonium sulfate mass (g) = V1 (mL) × 0.056. For example, if V1 is 800 mL, then weigh 800 × 0.056 = 44.80 g. Add the weighed ammonium sulfate powder very slowly and in multiple additions to the stirred supernatant, ensuring that each addition is completely dissolved before adding the next. After all the ammonium sulfate has been added and completely dissolved, stop stirring. Incubate the flask at 4°C for 1 hour to allow the protein to precipitate. After 1 hour, centrifuge again using a tube centrifuge at 5°C, 14000 rpm, and 1 L / min to collect the precipitate. After this centrifugation, carefully discard the supernatant and retain the first salting-out precipitate (a pale yellow solid) at the bottom of the tube or in the collector.

[0039] Surfactant refolding Denaturation: Transfer the first salting-out centrifuged precipitate to a clean beaker. Add 9 times the weight of the precipitate in pre-cooled deionized water. Gently resuspend the precipitate using a glass rod or pipette. Then, weigh out sodium lauroyl sarcosinate powder and add it to the resuspension to achieve a final concentration of 5%. Calculation formula: Required sodium lauroyl sarcosinate mass (g) = Resuspension volume (mL) × 0.05. After stirring to dissolve, place the beaker in a 5°C refrigerator and allow it to denature for 12 hours.

[0040] Centrifuge and collect the supernatant: After denaturation, aliquot the solution into 50mL centrifuge tubes and centrifuge at 4°C and 10,000rpm for 10 minutes using a low-temperature high-speed centrifuge. Carefully combine the supernatants from all tubes; this is the denaturation centrifugation supernatant. Discard the precipitate.

[0041] Renaturation: Measure the volume of the denatured centrifuged supernatant. Add pre-cooled 20mM PBS (pH 7.0) buffer to dilute the sodium lauroyl sarcosinate concentration to 1%. Calculation formula: Required PBS volume (mL) = V² × (5% / 1% - 1) = V² × 4. Mix slowly until homogeneous; this mixture is the renaturation solution.

[0042] Second salting out Place the refolding solution on a magnetic stirrer and stir slowly in an ice-water bath. Weigh out ammonium sulfate powder using the formula: required ammonium sulfate mass (g) = refolding solution volume (mL) × 0.112. Slowly add the powder to the refolding solution, following the same procedure as the first salting-out. After addition, let it stand at 4°C for 1 hour. Centrifuge again using a tubular centrifuge at 5°C, 14000 rpm, and 1 L / min, collect the precipitate, and discard the supernatant. The precipitate obtained this time is the second salting-out centrifugal precipitate.

[0043] Q-column chromatography purification Sample preparation: The centrifuged precipitate obtained after the second salting-out was resuspended and dissolved in 10 times its weight of Q column equilibration buffer (20 mM PBS, pH 7.0). The solution was filtered through a 0.22 μm filter membrane to remove particulate matter that might clog the chromatography column (Q column equilibration buffer: 20 mM PBS, pH 7.0; Q column wash buffer: 20 mM PBS, pH 7.0, containing 0.2 M NaCl; Q column eluent: 20 mM PBS, pH 7.0, containing 0.5 M NaCl).

[0044] Column equilibration: Connect the Q column to the chromatography system. Wash the column with at least 5 column volumes (CV) of Q column equilibration buffer at a flow rate of 2 mL / min until the UV absorption baseline is stable.

[0045] Sample loading: Load the filtered sample solution onto the equilibrated Q column at a flow rate of 1 mL / min.

[0046] Washing: After sample loading, wash the column with 3 column volumes (CV) of Q column wash buffer (containing 0.2M NaCl) at a flow rate of 2 mL / min. This step is used to wash away unbound or weakly bound contaminating proteins. Monitor the absorbance using UV until it returns to baseline.

[0047] Elution: Elute with 7 column volumes (CV) of Q column eluent (containing 0.5 M NaCl) at a flow rate of 2 mL / min. Collect the eluent from this stage using a fraction collector; the fraction corresponding to the UV absorption peak is the component rich in recombinant human ameliorative matrix protein.

[0048] Concentration and freeze drying The collected eluents were combined and concentrated by centrifugation at 4°C and 4000 rpm using ultrafiltration centrifuge tubes (molecular weight cutoff 10 kD) to reduce the volume to less than 10 mL. The concentrated protein solution was then freeze-dried, following the same procedure as in Example 1.

[0049] Example 3: Preparation of recombinant human ameliorative matrix protein using double salting-out and refolding processes. Step 1: Cell lysis. Same as Example 1.

[0050] Step 2: First salting out. The procedure is the same as in Example 1, but the final concentration of ammonium sulfate is adjusted to 60 g / L.

[0051] Step 3: Surfactant refolding.

[0052] Denaturation: The procedure is the same as in Example 1, but the final concentration of sodium lauroyl sarcosinate is adjusted to 6%.

[0053] Refolding: The procedure is the same as in Example 1, except that the concentration of sodium lauroyl sarcosinate in the denatured centrifugation supernatant is diluted to 1%.

[0054] Step 4: Second salting out. The procedure is the same as in Example 1, but the final concentration of ammonium sulfate is adjusted to 100 g / L.

[0055] Step 5: Q-column chromatography purification. The procedure is the same as in Example 2, but the washing buffer is 2 column volumes of 0.2M NaCl, and the elution buffer is 5 column volumes of 0.5M NaCl.

[0056] Step 6: Concentration and freeze-drying. Same as Example 2.

[0057] The difference between Comparative Example 1 and Example 1 is that the reversibility parameter deviates (at high concentration). Step 1: Cell lysis is the same as in Example 1.

[0058] Step 2: The first salting out is the same as in Example 1.

[0059] Step 3: Surfactant refolding (parameter deviation) Denaturation: The procedure is the same as in Example 1, but the final concentration of sodium lauroyl sarcosinate is adjusted to 10%.

[0060] Refolding: The procedure is the same as in Example 1, but the concentration of sodium lauroyl sarcosinate in the denatured centrifugation supernatant is diluted to 2%.

[0061] Step 4: The second salting out is the same as in Example 1.

[0062] Step 5: Freeze-dry as in Example 1.

[0063] The difference between Comparative Example 2 and Example 1 is that the refoldability parameter deviates (at medium concentration). Step 1: Cell lysis is the same as in Example 1.

[0064] Step 2: The first salting out is the same as in Example 1.

[0065] Step 3: Surfactant refolding (parameter deviation) Denaturation: The procedure is the same as in Example 1, but the final concentration of sodium lauroyl sarcosinate is adjusted to 8%.

[0066] Refolding: The procedure is the same as in Example 1, but the concentration of sodium lauroyl sarcosinate in the denatured centrifugation supernatant is diluted to 3%.

[0067] Step 4: The second salting out is the same as in Example 1.

[0068] Step 5: Freeze-dry as in Example 1.

[0069] The difference between Comparative Example 3 and Example 2 is that the Q-column washing conditions deviate (low salt). Steps 1 to 4: The lysis of bacterial cells to the second salting out is exactly the same as in Example 2.

[0070] Step 5: Q-column chromatography (washing conditions deviated) The operating procedure is the same as in Example 2, but in the washing step, 0.1M NaCl with 3 column volumes is used for washing. The elution step is the same as in Example 2.

[0071] Step 6: Concentration and freeze-drying are the same as in Example 2.

[0072] The difference between Comparative Example 4 and Example 2 is that the Q-column elution conditions deviated (high salt). Steps 1 to 4: The lysis of bacterial cells to the second salting out is exactly the same as in Example 2.

[0073] Step 5: Q-column chromatography (elution condition deviation) The operation process is the same as in Example 2, and the washing steps are the same as in Example 2.

[0074] However, in the elution step, 7 column volumes of 2.0 M NaCl were used for elution.

[0075] Step 6: Concentration and freeze-drying are the same as in Example 2.

[0076] The following methods were used to test all embodiments and comparative examples, and the results are shown in Table 1.

[0077] Data detection methods: Yield Assay (BCA Method): Standard Curve Preparation: Dilute BSA standards with deionized water to prepare a series of concentrations of 0, 125, 250, 500, 750, 1000, and 1500 μg / mL. Add 25 μL of each concentration of standard and the sample to be tested (appropriately diluted with PBS) to a 96-well plate, performing three replicates for each concentration. Reaction: Add 200 μL of BCA working solution (prepared according to the kit instructions) to each well and vortex for 30 seconds. Incubation and Measurement: Cover the 96-well plate with the sealing film and incubate at 37°C for 30 minutes. Then remove the plate and measure the absorbance (OD value) at 562 nm using a microplate reader. Calculation: Plot a standard curve with BSA concentration on the x-axis and the average OD562 value on the y-axis. Calculate the protein concentration from the standard curve based on the sample's OD value, multiply by the dilution factor and volume to obtain the total protein content, and finally calculate the yield per 100 g of wet cells.

[0078] Anti-inflammatory efficacy (mouse periodontitis model and ELISA): Modeling and grouping: Balb / C male mice weighing 30g±3g were anesthetized with isoflurane inhalation. 10μL of 2mg / mL lipopolysaccharide (LPS) solution was injected into the buccal and palatal gingival sulci between the first and second molars on the right maxilla using a microsyringe. Injections were given every 2 days for 4 weeks. After modeling, 80 mice were randomly divided into 8 groups (model group, experimental groups 1-7 correspond to the examples and comparative examples, respectively). Drug administration: The experimental groups were administered the corresponding protein sample suspended in physiological saline by gavage once daily at a dose of 0.315mg protein / kg body weight. The model group and a separate normal control group (10 non-modeled mice) were administered the same volume of physiological saline by gavage daily for 4 weeks. Serum collection: After the last administration, mice were fasted but allowed free access to water for 12 hours. Blood was collected by enucleation into 1.5mL centrifuge tubes and allowed to stand at room temperature for 2 hours. Then, centrifuge at 3000 rpm for 15 minutes at 4°C, carefully aspirate the pale yellow serum from the top layer using a pipette, aliquot, and store at -80°C. ELISA assay: Use a mouse IL-6 and TNF-α ELISA kit. Thaw the frozen serum on ice. In a 96-well plate coated with antibody, add 50 μL of standard, 50 μL of sample, and immediately add 50 μL of biotinylated antibody to each well. Cover with a plate patch and incubate at 37°C for 60 minutes. Wash the plate 5 times. Add 100 μL of horseradish peroxidase-labeled streptavidin to each well and incubate at 37°C for 30 minutes. Wash the plate 5 times. Add 90 μL of chromogenic substrate TMB to each well and incubate at 37°C in the dark for 15 minutes. Add 50 μL of stop solution to each well. Immediately measure the OD value at 450 nm using a microplate reader. Calculate the concentrations of IL-6 and TNF-α in the serum based on the standard curve.

[0079] The above methods were used to test all embodiments and comparative examples, and the results are shown in Table 1.

[0080] Table 1. Yield and anti-inflammatory efficacy data of recombinant human ameliomatic protein.

[0081] As can be seen, the yields of all three examples remained consistently high, demonstrating the stability and reliability of the core process of double salting out and surfactant refolding. The yields of Comparative Examples 1 and 2 were not significantly different from those of Example 1, indicating that deviations in the refolding parameters had little impact on the final protein quantity. Comparative Example 4 had the lowest yield, suggesting that elution under harsh conditions with 2.0M NaCl might cause some target proteins to bind too tightly to the chromatography medium, preventing effective elution, or to aggregate and precipitate under high salt conditions, resulting in loss. This conversely demonstrates the optimization of the 0.5M NaCl elution conditions. The IL-6 and TNF-α levels in the model group were significantly higher than those in the normal control group, indicating the successful construction of the periodontitis model. The inflammatory factor levels in Example 1 were significantly lower than those in the model group, demonstrating that the protein prepared solely through the core double salting out and refolding process of this invention already possesses good anti-inflammatory activity. The inflammatory factor levels in Example 2 were reduced to near those of the normal control group, with no statistically significant difference. This indicates that adding a specific Q-column purification step to the core process can effectively enrich the protein components with the highest activity, which is one of the important conditions for obtaining the optimal product. The anti-inflammatory effects of Comparative Examples 1 and 2 were far inferior to those of Example 1, despite their similar yields. This strongly demonstrates that the specific concentration range of sodium lauroyl sarcosinate in the surfactant refolding step is a decisive factor in ensuring the high biological activity of the resulting protein; deviations from this range will severely impair protein function. While Comparative Examples 3 and 4 were better than Experimental Group 1, they were significantly worse than Example 2. This indicates that Q-column chromatography must be performed under the specific elution conditions of this invention to maximize anti-inflammatory activity. Too low a concentration of eluting salt may result in impurity residue, while too high a concentration may elute different components or cause protein denaturation. Therefore, the complete set of process parameters based on double salting-out and surfactant refolding provided by this invention, including salting-out concentration, surfactant refolding concentration, and Q-column purification conditions, forms an organic whole. They work together to ensure that the final product achieves optimal yield and biological activity; none of them can be omitted. Deviations from any key parameter will lead to a significant decrease in yield or activity.

[0082] 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.

[0083] 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 preparing recombinant human ameliorative matrix protein based on double salting out and surfactant refolding, characterized in that, Includes the following steps: (a) Cell lysis: Cells expressing recombinant human ameliomatic protein were lysed, and centrifuged after lysis to obtain the supernatant of the lysate; (b) First salting out: Add a first inorganic salt to the supernatant of the lysis solution to perform the first salting out. After the salting out is completed, centrifuge to obtain the first salting out precipitate. (c) Surfactant denaturation and refolding: The first salting-out centrifuged precipitate is resuspended in water to obtain a resuspension; a surfactant is added to the resuspension for denaturation treatment, and centrifugation is performed after denaturation to obtain a denatured centrifugation supernatant; then phosphate buffer is added to the denatured centrifugation supernatant for dilution to reduce the surfactant concentration and achieve refolding to obtain a refolded solution. (d) Second salting out: A second inorganic salt is added to the refolding solution to perform a second salting out. After the salting out is completed, centrifugation is performed to obtain the second salting out precipitate. (e) The recombinant human ameliorative matrix protein is obtained by taking the second salting-out precipitate, centrifuging, and drying.

2. The method according to claim 1, characterized in that, The specific method for lysis in step (a) is as follows: take bacterial cells that are induced to express recombinant human ameliorative matrix protein, add phosphate buffer for resuspending, and then perform high-pressure homogenization.

3. The method according to claim 2, characterized in that, The weight ratio of the bacterial cells inducing recombinant human ameliorative matrix protein expression to phosphate buffer is 1:5~10; the phosphate buffer is a 15~25mM phosphate buffer with a pH of 6.0~8.

0.

4. The method according to claim 1, characterized in that, In step (b), the content of the first inorganic salt in the supernatant of the lysis solution after its addition is 50~60 g / L; the first inorganic salt is ammonium sulfate.

5. The method according to claim 1, characterized in that, In step (b), the specific method for the first salting out is as follows: add the first inorganic salt to the supernatant of the lysis solution, stir to dissolve, and let stand for 1 to 2 hours.

6. The method according to claim 1, characterized in that, In step (c), the specific method of denaturation is as follows: a surfactant is added to the resuspended solution, stirred and dissolved, and then allowed to stand at 2-10°C for 6-12 hours to denature.

7. The method according to claim 1, characterized in that, In step (c), the surfactant has a mass concentration of 5-6% in the resuspension after addition; the surfactant is sodium lauroyl sarcosinate; the mass concentration of the surfactant in the denaturing centrifugation supernatant is diluted to 1% by adding phosphate buffer; the weight ratio of the first salting-out centrifugation precipitate to water is 1:5-10.

8. The method according to claim 1, characterized in that, In step (d), the content of the second inorganic salt in the refolding solution after addition is 100~120g / L; the second inorganic salt is ammonium sulfate; the specific method of the second salting out is: add the second inorganic salt to the refolding solution, stir to dissolve, and let stand for 1~2h.

9. The method according to claim 1, characterized in that, The method further includes a Q-column chromatography purification step after step (d) and before step (e); the eluent is collected, concentrated, and dried to obtain the recombinant human ameliorative matrix protein.

10. The method according to claim 9, characterized in that, The specific method of the Q-column chromatography purification step is as follows: the second salting-out centrifuged precipitate is resuspended in 20 mM phosphate buffer (pH 7.0) and loaded onto a Q-column; impurities are removed by elution with 2-4 column volumes of 0.2 M sodium chloride aqueous solution; then, elution is performed with 5-10 column volumes of 0.5 M sodium chloride aqueous solution. The eluent obtained from the 0.5 M sodium chloride aqueous solution is collected, concentrated, and dried to obtain recombinant human ameliorative matrix protein; in the Q-column chromatography purification step, impurities are removed by elution with 3 column volumes of 0.2 M sodium chloride aqueous solution; then, elution is performed with 7 column volumes of 0.5 M sodium chloride aqueous solution.

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