Efficient preparation method of recombinant human glaze matrix protein by combining sequence optimization with affinity chromatography

Abstract

The invention discloses a sequence optimization and affinity chromatography combined recombinant human glaze matrix protein efficient preparation method, and belongs to the technical field of bioengineering.The preparation method comprises the following steps that an expression strain is constructed, specifically, a human glaze matrix protein gene subjected to signal peptide removal and codon optimization is connected with an expression plasmid, a recombinant plasmid is constructed, and the expression strain is obtained; transforming to an expression host bacterium to obtain a recombinant strain; inducing expression; carrying out affinity chromatography purification; performing enzyme digestion to remove a label: cutting the primarily purified fusion protein by using specific protease to remove the label; and finely purifying. Through innovative combination of sequence optimization and a purification process, the problems that the recombinant glaze matrix protein is low in expression quantity, complex in purification process and high in cost are solved, and the prepared protein is high in purity and yield, has remarkable anti-periodontitis activity and is suitable for development of drugs, oral care products and skin care products.

Description

TECHNICAL FIELD

[0001] The present application relates to a high-efficiency preparation method of recombinant human amelogenin by sequence optimization and affinity chromatography, and belongs to the field of bioengineering technology. BACKGROUND

[0002] Amelogenin, especially amelogenin which plays a core role in tooth enamel formation and periodontal tissue regeneration, has great application potential in the fields of biological medicine and oral care due to its ability to effectively promote cell migration, proliferation, adhesion and differentiation. However, its large-scale preparation faces severe challenges. There are mainly three paths in the prior art: firstly, direct extraction from animal tissues such as pig teeth, which has inherent defects such as pathogen risk, high immunogenicity and large batch difference; secondly, fusion expression and affinity purification using GST or SUMO tags, and then using expensive tools such as thrombin or SUMO protease to remove the tags, which not only has high cost and complicated steps, but also introduces the safety risk of enzyme residues, making it difficult to be applied to industrial production; thirdly, in vitro renaturation after inclusion body expression, which has a complicated process, low efficiency and unstable activity recovery rate. The prior art has disclosed a technical solution of GST tag combined with thrombin cutting, which is restricted in large-scale application due to cost and complexity. Therefore, developing a new method for preparing recombinant human amelogenin which can avoid the above defects and has high yield, high purity, high activity and controllable cost has become a technical bottleneck that needs to be broken through in the field. SUMMARY

[0003] In view of the defects in the prior art, the present application provides a high-efficiency preparation method of recombinant human amelogenin by sequence optimization and affinity chromatography, comprising the following steps:

[0004] (1) Constructing an expression strain: link the human amelogenin gene without signal peptide and optimized by codon with an expression plasmid to construct a recombinant plasmid, and transform it into an expression host strain to obtain a recombinant strain;

[0005] (2) Inducing expression: culture the recombinant strain and induce expression of the fusion protein with the tag;

[0006] (3) Affinity chromatography purification: after lysing the bacterial cells, use an affinity chromatography medium corresponding to the tag to capture the fusion protein, and obtain a preliminarily purified fusion protein through elution;

[0007] (4) Enzymatic removal of the tag: use a specific protease to cut the preliminarily purified fusion protein to remove the tag;

[0008] (5) Fine purification: separate and remove the tag and the specific protease through a salting-out step to obtain high-purity recombinant human amelogenin.

[0009] Preferably, in step (1), the amino acid sequence of the human amelogenin is: .

[0010] Preferably, in step (1), the expression plasmid is pET-32a (+), the tag is 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, 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.

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

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

[0014] Preferably, the Ni-NTA affinity chromatography medium is a large particle medium, which does not need to be packed and can be directly combined by stirring incubation in the lysate.

[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 TEV protease.

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

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

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

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

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

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

[0024] Preferably, the recombinant human amelogenin prepared by the method is used for preparing a medicament for treating periodontitis.

[0025] Preferably, the recombinant human amelogenin prepared by the method is used for preparing an oral care product.

[0026] Preferably, the recombinant human amelogenin prepared by the method is used for preparing a skin care product.

[0027] Advantages of the present application:

[0028] Significant increase in yield: By specifically removing the hydrophobic signal peptide and fusing the soluble Trx tag, the solubility expression of the target protein in Escherichia coli is greatly improved, and the unit bacterial protein yield is as high as 15-20 g / kg, which is much higher than the traditional method of 6-8 g / kg.

[0029] Excellent purity and quality: The optimized Trx-His double-tag system is used for high-efficiency affinity chromatography, and combined with TEV enzyme cutting and targeted salting-out, the purity of the final product is stable at more than 90%, and the dimer and degradation band are significantly reduced, and the product quality is significantly improved.

[0030] Simplified process and low cost: The new type of large particle Ni-NTA medium is used, which saves the complicated step of high-speed centrifugation of lysis solution; the self-produced low-cost TEV protease is used instead of expensive commercial enzymes, which greatly reduces the overall production cost.

[0031] Clear activity and efficacy: The recombinant human amelogenin prepared by the present application has been proved to have excellent anti-periodontitis activity and mechanism of action by inhibiting the NF-κB signaling pathway in animal models and cell levels, which lays a solid foundation for its clinical application.

[0032] Suitable for industrial amplification: The process route is simple, the centrifugation times are few, the operation conditions are mild and easy to control, and it is very suitable for large-scale industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 SDS-PAGE electrophoretogram of the purification result after TEV enzyme cutting.

[0034] Figure 2SD rat periodontal tissue immunohistochemical staining TNF-α results. A: normal control group; B: negative control group; C: low concentration of recombinant amelogenin (Example 1, 0.315 mg / kg) treatment group; D: high concentration of recombinant amelogenin (Example 1, 3.15 mg / kg) treatment group.

[0035] Figure 3 qPCR detection of inflammatory factor mRNA expression level results. 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 DESCRIPTION

[0036] The technical solutions in the embodiments of the present application will be clearly and completely described below in combination with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments, and all other embodiments obtained by those of ordinary skill in the art without creative work based on the embodiments in the present application shall fall within the scope of the present application.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art. In addition, any method and material similar or equivalent to those described herein can be used in the present application. The preferred methods and materials described herein are only for demonstration, but cannot limit the content of the present application.

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

[0039] Example 1

[0040] 1. Bacterial culture and induction of expression

[0041] The recombinant BL21(DE3) strain containing the pET-32a(+)-optimized human amelogenin gene preserved at -80℃ was inoculated into 50 mL of LB liquid medium containing 100 μg / mL ampicillin at a 1% inoculation amount, and cultured at 37℃ with 200 rpm shaking for about 3 hours until the OD600 was 0.6. IPTG was added to the culture to a final concentration of 0.4 mM, and the shaking bed temperature was adjusted to 34℃ and the rotation speed was adjusted to 120 rpm, and the induction of expression was continued for 3 hours. After the induction was completed, the bacterial solution was centrifuged at 4℃ and 5000 x g for 10 minutes, the bacterial cells were collected, weighed about 0.5 grams (wet weight), and stored at -20℃ for standby use.

[0042] 2. Affinity chromatography purification

[0043] The 0.5 g of bacteria was suspended in 4.5 mL of pre-cooled Ni equilibrium solution (20 mM Tris, 200 mM NaCl, 5 mM imidazole, pH 8.0), and was broken by ultrasonic in ice bath, with 300 W ultrasonic power, 2 seconds of work and 3 seconds of interval, for a total of 10 minutes. The broken lysate was mixed with 1 mL of Ni-NTA medium (ChuanBio) in a centrifuge tube, and was incubated at 4°C for 30 minutes. After incubation, it was centrifuged at 500 x g for 2 minutes, and the supernatant was carefully discarded. 5 mL of Ni equilibrium solution was added to the medium, and was gently resuspended and washed, centrifuged at 500 x g for 2 minutes, and the supernatant was discarded. 5 mL of Ni wash solution (20 mM Tris, 200 mM NaCl, 50 mM imidazole, pH 8.0) was added to the medium, and was gently resuspended and washed, centrifuged at 500 x g for 2 minutes, and the supernatant was discarded. 2 mL of Ni elution solution (20 mM Tris, 200 mM NaCl, 250 mM imidazole, pH 8.0) was added to the medium, and was gently resuspended and incubated at 4°C for 20 minutes. After incubation, it was centrifuged at 500 x g for 2 minutes, and the supernatant was carefully collected, which was the primary purified Trx-His-Enamel Matrix Protein fusion protein solution.

[0044] 3. Enzymatic removal of tag

[0045] The protein concentration of the above eluate was determined. About 10 mg of fusion protein was taken, 1 x TEV enzyme buffer was added to make up the system, and then 10 KU of self-produced TEV protease was added and mixed. The reaction system was placed in a 30°C water bath for 2 hours of enzymatic digestion.

[0046] 4. Salting-out purification

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

[0048] Example 2

[0049] 1. Bacterial culture and induction of expression

[0050] A vial of preserved gylcerol stock of recombinant BL21(DE3) strain containing pET-32a(+)-optimized human amelogenin gene was taken out from -80°C ultra-low temperature freezer. A small amount of bacterial liquid was taken with a sterile inoculating loop and streaked on a LB solid plate containing 100 μg / mL ampicillin. The plate was inverted and incubated in a 37°C incubator overnight (about 16 hours). The next day, a single, white, and well-grown colony was picked and inoculated into a 500 mL vented flask containing 50 mL of LB liquid medium with 100 μg / mL ampicillin. The flask was placed in a 37°C shaker at 200 rpm for about 3 hours until the OD600 value of the bacterial liquid reached 0.6-0.8. 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 speed to 120 rpm. The induction was continued for 3 hours. After the induction, the flask was taken out and immediately inserted into an ice-water mixture for 10 minutes. Then the flask was centrifuged at 4°C and 5000 x g for 10 minutes. The supernatant was carefully discarded. The wet bacterial pellet at the bottom of the centrifuge tube was weighed with an electronic balance. The weight was about 0.5 gram. The bacterial pellet could be stored in a -20°C freezer for a short period of time.

[0051] 2. Affinity chromatography purification (high stringency wash)

[0052] The 0.5 gram wet bacterial pellet was suspended in 4.5 mL pre-cooled Ni equilibration buffer (20 mM Tris, 200 mM NaCl, 5 mM imidazole, pH 8.0). The bacterial pellet was fully resuspended by vortexing. The bacterial suspension was transferred to a 15 mL centrifuge tube and placed in an ice-water bath. An ultrasonic cell disruptor with a 3 mm probe was used. The working parameters were set as follows: power 300 W, sonication 2 seconds, interval 3 seconds, total time 10 minutes. The sample was kept in an ice bath during the process to prevent overheating. After the ultrasonic treatment, the lysate was centrifuged at 4°C and 12000 x g for 20 minutes. The lysate supernatant was carefully transferred to a new 15 mL centrifuge tube. The pellet was discarded. 1 mL of Ni-NTA affinity chromatography medium (ChuanBio) pre-equilibrated with Ni equilibration buffer was added to the lysate supernatant. The centrifuge tube was fixed on a rotary mixer and incubated in a 4°C refrigerator for 30 minutes to allow the His-tagged fusion protein to bind to the medium. After the incubation, the centrifuge tube was centrifuged at 500 x g for 2 minutes to allow the medium to settle. The supernatant was carefully removed with a pipette.

[0053] Washing: To the settled medium, 5 mL of pre-chilled Ni wash buffer (20 mM Tris, 200 mM NaCl, 100 mM imidazole, pH 8.0) was added. The medium was resuspended by gently pipetting and then placed back on the rotary mixer for 15 minutes at 4°C. After that, the medium was centrifuged at 500 x g for 2 minutes and the supernatant was discarded. The higher 100 mM imidazole concentration in this step was used to more effectively wash away the non-specifically bound contaminant proteins. To the medium, 2 mL of pre-chilled Ni elution buffer (20 mM Tris, 200 mM NaCl, 250 mM imidazole, pH 8.0) was added. The medium was resuspended by gentle pipetting and incubated at 4°C for 20 minutes to allow the target protein to be dissociated from the medium. After the incubation, the medium was centrifuged at 500 x g for 2 minutes. The supernatant containing the target protein, i.e., the eluate, was carefully collected into a new centrifuge tube. This was the solution of the Trx-His-Enamel Matrix Protein fusion protein after the initial purification.

[0054] 3. Enzymatic removal of the tag

[0055] Fifty microliters of the eluate was used to determine the protein concentration using the BCA protein quantification kit. Based on the measured concentration, the total protein amount in the entire eluate was calculated. The remaining eluate was transferred into a 5 mL dialysis bag. Based on the total protein amount, the calculated volume of the self-produced TEV enzyme stock solution was added at a ratio of 1 KTEV enzyme per 1 g of fusion protein. The total volume was adjusted by adding 1 x TEV enzyme buffer. The reaction system was placed in a 30°C constant temperature water bath and incubated for 2 hours for the enzymatic reaction.

[0056] 4. Salting-out purification

[0057] After the enzymatic reaction, the reaction solution was transferred into a 15 mL centrifuge tube. The tube was placed on a magnetic stirrer and stirred slowly in an ice water bath. The required amount of ammonium sulfate solid powder was accurately weighed using an electronic balance. The calculation formula was: required ammonium sulfate mass (g) = enzyme cutting liquid volume (mL) x 0.056. The ammonium sulfate powder was slowly and gradually added to the stirring enzyme cutting liquid, ensuring that each addition was completely dissolved before the next addition. After all the ammonium sulfate was added, the stirring was stopped. The centrifuge tube was tightly covered and placed in a 4°C refrigerator for 1 hour to allow the target protein to fully precipitate. After standing, the centrifuge tube was centrifuged at 14000 rpm for 20 minutes at 4°C. At this time, the recombinant human enamel matrix protein formed a precipitate at the bottom of the tube, while the cut Trx-His tag and TEV enzyme were mainly left in the supernatant. The supernatant was completely aspirated using a pipette. The white precipitate at the bottom of the tube was retained. One milliliter of pre-chilled deionized water was added to the precipitate, which was gently resuspended using a pipette to completely dissolve it.

[0058] 5. Lyophilization

[0059] The dissolved protein solution was aliquoted into 1.5 mL centrifuge tubes. The aliquoted samples were immediately placed into a -80°C ultra-low temperature freezer for pre-freezing for 2 hours. The completely frozen samples were immediately transferred into a freeze-dryer, and the main program was started for freeze-drying until the samples completely became white flocculent solids. After the drying was completed, the total weight of the freeze-dried product was weighed, and the container tare weight was subtracted to obtain the final yield of the recombinant human amelogenin.

[0060] Example 3

[0061] 1. Bacterial culture and induction of expression

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

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

[0064] The steps of bacterial lysis, centrifugal collection of supernatant, incubation and binding with Ni-NTA medium, and washing with equilibration buffer were exactly the same as in Example 1.

[0065] Elution: 2 mL of pre-cooled Ni elution buffer (20 mM Tris, 200 mM NaCl, 300 mM imidazole, pH 8.0) was added to the combined and washed medium. The medium was gently resuspended and incubated at 4°C for 20 minutes. After the incubation, the medium was centrifuged at 500 x g for 2 minutes. The supernatant, which was the eluate, was carefully collected. This step used a high concentration of 300 mM imidazole to more completely elute all of the fusion protein bound to the medium.

[0066] 3. Enzymatic removal of tag

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

[0068] 4. Salting-out purification

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

[0070] 5. Freeze-drying

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

[0072] Comparative Example 1 (without removal of signal peptide)

[0073] 1. Bacterial culture and induction of expression

[0074] The strain used in this comparative example was a recombinant BL21(DE3) strain containing the pET-32a(+) plasmid with the signal peptide-containing full-length human amelogenin gene. The amino acid sequence started with "MGTWILFACLLGAAFA...". The steps of picking a single colony, expanding the culture, and IPTG induction of expression were exactly the same as in Example 1. After the induction was completed, the bacterial cells were collected by centrifugation. It was observed that the bacterial cell pellet was less than in Example 1. About 0.5 grams of wet bacterial cells were weighed.

[0075] 2. Affinity chromatography purification

[0076] The lysis step was the same as in Example 1.

[0077] After centrifugation, the supernatant was observed to be very clear and in small volume, while there was a large amount of white precipitate at the bottom of the tube. A small amount of supernatant was incubated with Ni-NTA medium, and the subsequent washing and elution steps were the same as in Example 1. After the elution step, the collected eluate was almost colorless and transparent. A small amount was taken for SDS-PAGE electrophoresis, and only a faint band was observed at a very high molecular weight position, which was possibly an unsoluble aggregate, and there was no clear main band at the target molecular weight position.

[0078] 3. Enzymatic removal of tag

[0079] This step could not be effectively performed due to the failure to obtain sufficient amount of the primary purified fusion protein.

[0080] Finally, sufficient amount of the recombinant human amelogenin lyophilized powder for testing could not be obtained.

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

[0082] 1. Bacterial culture and induction of expression

[0083] The strain used in this comparative example was a recombinant BL21(DE3) strain containing the pGEX-4T-1 -signal peptide-free human amelogenin gene. LB medium containing 100 μg / mL ampicillin was used. The single colony was picked and the scale-up culture step 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 the expression was induced at 37°C for 4 hours. The bacterial cells were collected by centrifugation, and about 0.5 gram of wet bacterial cells was weighed.

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

[0085] The bacterial cells were resuspended in 4.5 mL of pre-cooled PBS buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4). The ultrasonic disruption conditions were the same as in Example 1. After centrifugation, the supernatant was incubated with 1 mL of GST affinity chromatography medium pre-equilibrated with PBS at 4°C for 30 minutes. Centrifugation was performed at 500 x 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, which was incubated at 4°C for 20 minutes. Centrifugation was performed at 500 x g for 2 minutes, and the supernatant was collected, which was the GST-amelogenin fusion protein.

[0086] 3. Enzymatic cleavage of tag (Thrombin)

[0087] The protein concentration of the eluate was determined. Commercial thrombin was added according to the supplier's instructions at a ratio of 10 units thrombin per 1 g of fusion protein. The enzyme cleavage was performed at room temperature overnight for 16 hours.

[0088] 4. Refinement

[0089] After the enzyme cleavage, the reaction was attempted to be dialyzed to remove glutathione and thrombin. However, due to the lack of a specific separation step as effective as ammonium sulfate precipitation, it was not possible to effectively remove thrombin and the cleaved GST tag.

[0090] 5. Lyophilization

[0091] The solution after dialysis was lyophilized.

[0092] The comparative example obtained about 4.0 mg of lyophilized powder from 0.5 g of wet cells. However, it was detected that it contained GST tag and thrombin residues.

[0093] Comparative Example 3 (without ammonium sulfate refinement step)

[0094] 1. Cell culture and induction of expression

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

[0096] 2. Affinity chromatography purification

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

[0098] 3. Enzymatic cleavage of tag

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

[0100] 4. Refinement (key deviation)

[0101] After the enzyme cleavage reaction, no ammonium sulfate precipitation was performed. The enzyme cleavage solution was all loaded into a dialysis bag with a molecular weight cut-off of 10 kDa. The dialysis bag was 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 solution was changed every 4-6 hours, and dialysis was performed for a total of 3 times, for a total of about 20 hours. This step aimed to remove small molecules and salts by diffusion, but it was not possible to effectively separate the Trx-His tag and TEV enzyme, which had a similar molecular weight to the target protein.

[0102] 5. Lyophilization

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

[0104] The comparative example finally obtained about 6.9 mg of lyophilized powder from 0.5 g of wet bacteria. But the product is a mixture of target protein, Trx-His tag and TEV enzyme.

[0105] Detection method:

[0106] Protein concentration determination: BCA protein quantitative kit was used, and the operation was strictly in accordance with the instruction.

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

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

[0109] All examples and comparative examples were detected by the above method, and the results are shown in Table 1.

[0110] Table 1 Recombinant human enamel matrix protein preparation and activity data

[0111]

[0112] Regarding sequence optimization: Comparative Example 1 resulted in severe aggregation of the protein to form inclusion bodies due to the failure to remove the signal peptide, making it almost impossible to obtain active protein through the soluble route. This is in sharp contrast to Example 1, which achieved efficient soluble expression by removing the signal peptide. This difference demonstrates the importance of removing the signal peptide and sequence optimization step, which is the key to solving the problem of soluble expression of enamel matrix protein in prokaryotic expression system, and brings a revolutionary improvement in yield.

[0113] Regarding the tag and enzyme cutting system: Comparative Example 2 uses the traditional GST / thrombin system, which not only has a yield of only about half of Example 1, but also has the risk of label and expensive protease residues, resulting in product purity and safety problems, and high cost. The Trx-His / TEV enzyme system used in Example 1, combined with subsequent specific salt precipitation purification, successfully achieved high yield, high purity and low cost production. This proves that the combination of label and enzyme cutting, purification of the present application is a synergistic whole, and its overall technical effect is much better than the prior art solution.

[0114] Regarding the necessity of the purification step: Comparative Example 3 omitted the key salting-out step, resulting in a sharp decrease in the purity of the final product to 65% and containing a large amount of impurity proteins, and its anti-inflammatory effect was significantly worse than Example 1. This proves that the purification step of removing the tag and protease by salting-out at a specific concentration is essential to obtain a high-purity, high-activity end product.

[0115] Regarding the optimization of process parameters: the three examples achieved fine-tuning of purity while maintaining high yield and high activity by adjusting the imidazole concentration in affinity chromatography. This shows that the parameter range provided by the present application is effective, and the process has certain flexibility and robustness.

[0116] In summary, the present application successfully solves the long-standing industry problems of low yield, poor purity, high cost, and complex process in the preparation of recombinant human enamel matrix protein by the synergistic combination of sequence optimization (signal peptide removal) and specific tag, enzyme digestion, and purification process at the two core levels.

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

[0118] The above describes the present application and its embodiments, which are not restrictive, and the drawings shown are only one of the embodiments of the present application, and the actual application is not limited thereto. In summary, if a person skilled in the art is inspired by it, without departing from the purpose of the present application, without creatively designing similar ways and embodiments of the technical solution, they should all belong to the protection scope of the present application.

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, which was then transformed into the expression host bacteria to obtain a recombinant strain; (2) Induced expression: The recombinant strain was cultured and the tagged fusion protein was induced to be expressed; (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; The amino acid sequence of the human ameliorative matrix protein for removing the signal peptide in step (1) is as follows: 。 2. The method according to claim 1, characterized in that, 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).

3. The method according to claim 1, characterized in that, 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.

4. The method according to claim 1, characterized in that, In step (3), the affinity chromatography medium is Ni-NTA affinity chromatography medium.

5. The method according to claim 4, characterized in that, 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.

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

7. 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.

8. 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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