Production method of recombinant human bone morphogenetic protein-7 mature peptide dimer
Through the titration regeneration and chromatography column purification methods, the problems of high production cost and low yield of BMP7 protein were solved, and high-purity and low-cost production of BMP7 dimer protein were achieved.
Patent Information
- Application Number
- CN202510113959.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-21
- Publication Date
- 2025-05-27
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
The prior art is difficult to effectively reduce the production cost of recombinant human bone morphogenetic protein-7 (BMP7), and eukaryotic cell culture methods face yield problems.
The high-purity recombinant human bone morphogenetic protein-7 mature peptide dimer was obtained by inclusion body expression, preliminary purification, post-denatment protein purification, protein regeneration and dimerization.
The production process of BMP7 protein is simplified, the production cost is reduced, and the yield of BMP7 protein is effectively improved, and a high-purity dimer protein is obtained.
Smart Images
Figure CN120040575A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of medical biotechnology, in particular to a method for producing a recombinant human bone morphogenetic protein-7 mature peptide dimer. Background Art
[0002] Bone morphogenetic proteins (BMPs) are the largest subgroup of the transforming growth factor-β (TGF-β) cytokine superfamily. They were originally discovered by their ability to induce ectopic bone formation in vivo, hence their name. To date, more than 20 members of the BMP subgroup have been identified. Among them, the biologically active BMP7 (formerly known as osteogenic protein-1 or OP1) is composed of two BMP7 monomers containing a cysteine knot, which dimerize to form a 148 amino acid long dimer.
[0003] BMP7 plays an important role in the development of eyes, kidneys and limbs of vertebrates. It also plays an important role in fracture repair, mainly in the reconstruction of non-fusion fractures of the spine and maxillofacial injuries. Bone morphogenetic proteins, including BMP7, need to fold into homologous or heterologous dimers to be biologically active. Therefore, only eukaryotic cells can produce active BMP7 proteins. Since a large amount of bone is required to isolate micrograms of this protein, the cost is high, and the production of BMP7 protein by eukaryotic cell culture also faces yield issues. In view of this, it is urgent to find a more promising industrial production method for BMP7 protein. Summary of the invention
[0004] The technical problem to be solved by the present invention is to provide a method for producing a recombinant human bone morphogenetic protein-7 mature peptide dimer.
[0005] In order to solve the above technical problems, the technical solution of the present invention is:
[0006] A method for producing a recombinant human bone morphogenetic protein-7 mature peptide dimer, wherein the recombinant dimer protein BMP7 is obtained by titration renaturation and chromatography column purification, and the specific steps are as follows:
[0007] (a) Inclusion body expression: The BMP7 recombinant expression plasmid was transformed into BL21 (DE3) Escherichia coli, and the positive transformants were cultured and collected;
[0008] (b) Preliminary purification of inclusion bodies
[0009] (b-1) Bacterial disruption: fully resuspend the inclusion bodies with bacterial disruption buffer and disrupt the bacteria under refrigeration conditions;
[0010] (b-2) Take the solution after cell lysis, centrifuge at low temperature, discard the supernatant to obtain the inclusion body precipitate, add the inclusion body washing buffer, and pipette the precipitate until it is completely dissolved;
[0011] (b-3) Centrifuge at low temperature, discard the supernatant, re-add the inclusion body washing buffer, pipette the precipitate until it is completely dissolved, and shake 2-3 times;
[0012] (b-4) Repeat step (b-3) 2-3 times;
[0013] (c) Purification of the denatured protein
[0014] (c-1) Inclusion body denaturation: Centrifuge at low temperature for 15-25 min to obtain the inclusion body precipitate, resuspend it completely with the denaturation buffer, centrifuge to separate the soluble and insoluble proteins, and take the supernatant;
[0015] (c-2) Filter the supernatant through a filter membrane, load it onto a pre-equilibrated SP cation exchange chromatography column, and continue to wash with the equilibration buffer until the baseline is reached after loading;
[0016] (c-3) Elute with the elution buffer, and collect the main peak to obtain the purified denatured BMP7 protein;
[0017] (d) Protein renaturation and dimerization
[0018] (d-1) Centrifuge the purified denatured BMP7 protein, dilute the supernatant after centrifugation into the renaturation solution for renaturation. The renaturation solution includes 10-100 mM sodium phosphate buffer, 0.1-0.5 M L-arginine (L-Arg), 1-2 mM cysteine, 0.1-0.5 mM cystine, 3-7 mM EDTA, 0.3-0.7 mM guanidine hydrochloride, pH = 8.0-8.5, protein concentration 0.1-0.2 mg / mL, and renaturation time 24-48 hours;
[0019] (d-2) Replace the buffer of the renatured protein by ultrafiltration. The buffer used for replacement is 10-100 mM sodium phosphate buffer, 0.1-0.5 M L-arginine, and pH is 6.8-7.2;
[0020] (e) Purification of the renatured BMP7 dimer protein
[0021] (e-1) Centrifuge the renatured BMP7 dimer protein at low temperature to obtain the supernatant, filter it through a filter membrane, load it onto a pre-equilibrated Q anion exchange chromatography column, and continue to wash with the equilibration buffer until the baseline is reached after loading;
[0022] (e-2) Elute with a gradient of gradually increasing salt concentration using the elution buffer, and collect the main peak;
[0023] (e-3) Dialyze the collected purified protein into the loading buffer;
[0024] (e-4) Filter the protein through a filter membrane and load it onto a pre-equilibrated butyl-Sepharose FF hydrophobic chromatography column. After loading, continue to wash with the equilibration buffer until the baseline is reached;
[0025] (e-5) Elute with an elution buffer using a gradually decreasing salt concentration gradient and collect the main peak.
[0026] Preferably, in the above production method of recombinant human bone morphogenetic protein-7 mature peptide dimer, the specific steps of inclusion body expression in step (a) are as follows:
[0027] (a-1) Transform the BMP7 recombinant expression plasmid into BL21(DE3) Escherichia coli, spread it on a resistant plate, place it in a 37°C constant temperature incubator for 12 h, and screen for positive transformants;
[0028] (a-2) Pick a single colony into 5-10 ml of LB medium containing the resistance as the seed solution, culture it at 37°C for 12-16 h, add the seed solution to the LB medium containing the resistance at a ratio of 1:50, culture it at 37°C until the OD600 reaches 1.0-1.2, then add 0.1-1 mM IPTG to induce inclusion body expression, and then continue to culture for 5-8 h to obtain the fermentation broth. Collect the bacteria at 6000-8000 rpm for 20 min.
[0029] Preferably, in the above production method of recombinant human bone morphogenetic protein-7 mature peptide dimer, the nucleotide sequence of the BMP7 recombinant expression plasmid is as shown in SEQ ID NO.1 in the sequence listing.
[0030] Preferably, in the above production method of recombinant human bone morphogenetic protein-7 mature peptide dimer, the LB medium containing the resistance is the LB medium containing 50-100 μg / ml ampicillin.
[0031] Preferably, in the above production method of recombinant human bone morphogenetic protein-7 mature peptide dimer, the specific steps of inclusion body primary purification treatment in step (b) are as follows:
[0032] (b-1) Bacterial lysis: Resuspend the bacteria collected in step (a-2) with the lysis buffer. Among them, the volume ratio of the fermentation broth to the lysis buffer is 8-12:1. Use a vortex oscillator to vortex for 30-60 s to fully resuspend the inclusion bodies; Use a high-pressure homogenizer to lyse the bacteria under refrigeration conditions, homogenize 3 times, with a pressure of 800 bar, centrifuge at 8000 rpm for 20 min to collect the bacteria. The lysis buffer contains 100 mM sodium phosphate buffer, 10 mM EDTA, 00 mM sodium chloride, and the pH is 6.8-7.2;
[0033] (b-2) Take the solution after cell disruption, centrifuge it at 6000 - 12000 rpm at 0 - 4 °C for 20 - 30 min, discard the supernatant to obtain the inclusion body precipitate, add the inclusion body washing buffer, pipette the precipitate until it is completely dissolved, shake it for 5 - 10 s every 10 - 15 min, and shake it 2 - 3 times; the inclusion body washing buffer contains 100 mM sodium phosphate buffer, 3 M urea, and the pH is 6.8 - 7.2;
[0034] (b-3) Centrifuge it at 6000 - 12000 rpm at 0 - 4 °C for 15 - 25 min, discard the supernatant, re-add the inclusion body washing buffer, pipette the precipitate until it is completely dissolved, shake it for 5 - 10 s every 10 - 15 min, and shake it 2 - 3 times;
[0035] (b-4) Repeat step (b-3) 2 - 3 times, with a total washing time of 2 - 3 hours.
[0036] Preferably, in the above production method of recombinant human bone morphogenetic protein-7 mature peptide dimer, the volume ratio of the fermentation broth to the cell lysis buffer in step (b-1) is 10:1.
[0037] Preferably, in the above production method of recombinant human bone morphogenetic protein-7 mature peptide dimer, the specific steps of protein purification after denaturation in step (c) are as follows:
[0038] (c-1) Inclusion body denaturation: After the pretreatment is completed, centrifuge it at 8000 - 12000 rpm at 0 - 4 °C for 15 - 25 min to obtain the inclusion body precipitate, resuspend it completely with the denaturation buffer, stir it at 0 - 4 °C and 60 - 100 rpm, with a denaturation time of 10 - 12 h, centrifuge it at 10000 - 14000 rpm at 0 - 4 °C for 15 - 24 min to separate the soluble and insoluble proteins, and take the supernatant; the denaturation buffer contains 10 - 100 mM sodium phosphate buffer, 7 - 8 M urea, 100 mM Tris (tris(hydroxymethyl)aminomethane), and the pH is 6.8 - 7.2;
[0039] (c-2) Filter the supernatant through a 0.45 μm filter membrane, load it onto a pre-equilibrated SP cation exchange chromatography column, and continue to wash it with the equilibration buffer until the baseline is reached after loading. The equilibration buffer contains 10 - 100 mM sodium phosphate buffer, 7 - 8 M urea, 100 mM Tris, and the pH is 6.8 - 7.2;
[0040] (c-3) Elute with elution buffer at elution mixing ratios of 10%, 20%, 30%, 40%, 50%, and 60%. Collect the main peak to obtain the purified denatured BMP7 protein. The elution buffer contains 10 - 100 mM sodium phosphate buffer, 5 - 7 M urea, 0.5 - 1 M NaCl, 100 mM Tris, and has a pH of 6.8 - 7.2.
[0041] Preferably, for the above production method of recombinant human bone morphogenetic protein-7 mature peptide dimer, the specific steps of step (d) protein renaturation and dimerization are as follows:
[0042] (d-1) Centrifuge the purified denatured BMP7 protein at 8000 rpm for 20 min. Dilute the supernatant after centrifugation into the renaturation solution at a ratio of 1:10 and perform renaturation at 2 - 4°C. The renaturation solution includes 10 - 100 mM sodium phosphate buffer, 0.1 - 0.5 M L-arginine, 1 - 2 mM cysteine, 0.1 - 0.5 mM cystine, 3 - 7 mM EDTA, 0.3 - 0.7 mM guanidine hydrochloride, pH = 8.0 - 8.5, with a protein concentration of 0.1 - 0.2 mg / mL and a renaturation time of 24 - 48 hours.
[0043] (d-2) Replace the buffer of the renatured protein by ultrafiltration. After the replacement of the protein solution is completed, continue to place it at 4°C for 48 hours. The buffer used for replacement is 10 - 100 mM sodium phosphate buffer, 0.1 - 0.5 M L-arginine, with a pH of 6.8 - 7.2.
[0044] Preferably, for the above production method of recombinant human bone morphogenetic protein-7 mature peptide dimer, the specific steps of step (e) purification of the renatured BMP7 dimer protein are as follows:
[0045] (e-1) Centrifuge the renatured BMP7 dimer protein at 10000 - 14000 rpm at a low temperature of 0 - 4°C for 15 - 25 min to obtain the supernatant, filter it through a 0.45 μm filter membrane, and load it onto a pre-equilibrated Q anion exchange chromatography column. After loading, continue to wash with the equilibration buffer until the baseline is reached. The equilibration buffer contains: 10 - 100 mM sodium phosphate buffer and 0.1 - 0.5 M L-arginine, with a pH of 6.8 - 7.2.
[0046] (e-2) Perform gradient elution with a gradually increasing salt concentration using the elution buffer at elution mixing ratios of 30% and 40%. Collect the main peak. The elution buffer is 10 - 100 mM sodium phosphate buffer, 0.1 - 0.5 M L-arginine, and 0.5 - 1 M NaCl, with a pH of 6.8 - 7.2.
[0047] (e-3) Dialyze the collected purified protein into the loading buffer, which is 10 - 100 mM sodium phosphate buffer, 0.1 - 0.5 M L-arginine, and 1 - 1.5 M (NH 4 ) 2 SO 4 , with a pH of 6.8 - 7.2;
[0048] (e-4) Filter the protein through a 0.45 μm filter membrane and load it onto a pre-equilibrated butyl FF hydrophobic chromatography column. After loading, continue to wash with the equilibration buffer until the baseline is reached. The equilibration buffer is 10 - 100 mM sodium phosphate buffer, 0.1 - 0.5 M L-arginine, and 1 - 1.5 M (NH 4 ) 2 SO 4 , with a pH of 6.8 - 7.2;
[0049] (e-5) Elute with a gradient of gradually decreasing salt concentration using the elution buffer, and the elution mixing ratios are 30%, 40%, and 50%. Collect the main peak. The elution buffer is 10 - 100 mM sodium phosphate buffer, 0.1 - 0.5 M L-arginine, with a pH of 6.8 - 7.2.
[0050] The above production method of recombinant human bone morphogenetic protein-7 mature peptide dimer has a total time of no more than 72 hours; the buffers used for replacement do not contain urea and / or 2-cyclohexylethanesulfonic acid; the molecular weight of the obtained recombinant human bone morphogenetic protein-7 mature peptide is between 35 - 38 kDa.
[0051] Beneficial effects:
[0052] The above production method of recombinant human bone morphogenetic protein-7 mature peptide dimer obtains a highly pure recombinant BMP7 dimer protein through renaturation and chromatography column purification, simplifies the production process of recombinant human bone morphogenetic protein-7 mature peptide dimer, reduces the production cost of BMP7 protein, and effectively improves the yield of BMP7 protein. Description of the drawings
[0053] Figure 1 It is a gel electrophoresis photograph of BMP7 inclusion body protein after expression and fragmentation, which shows that most of the BMP7 protein is expressed in the form of inclusion bodies in the precipitate.
[0054] Figure 2 It is a gel electrophoresis photograph of BMP7 inclusion body protein before denaturation treatment, which shows that through the preliminary purification treatment of inclusion bodies, the vast majority of irrelevant proteins in the inclusion bodies have been removed.
[0055] Figure 3It is a gel electrophoresis photograph of the purified BMP7 protein after denaturation, indicating that the BMP7 inclusion body protein after inclusion body pretreatment can successfully obtain a high-purity target protein through one-step chromatography after denaturation.
[0056] Figure 4 It is a gel electrophoresis photograph of the renatured BMP7 denatured protein, indicating that a BMP7 mature peptide dimer with high biological activity can be obtained after the BMP7 denatured protein is renatured by the method of the present invention.
[0057] Figure 5 It is a gel electrophoresis photograph of the purified BMP7 dimer protein, indicating that the BMP7 mature peptide dimer in the renatured protein can be purified through two-step chromatography. Detailed implementation manners
[0058] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the specific implementation manners.
[0059] Example 1
[0060] A production method of a recombinant human bone morphogenetic protein-7 mature peptide dimer, the specific steps are as follows:
[0061] (a) Inclusion body expression
[0062] (a-1) Transform the BMP7 recombinant expression plasmid (nucleotide sequence as shown in SEQ ID NO.1 in the sequence listing) into BL21(DE3) Escherichia coli, coat it on a resistant plate, and place it in a 37°C constant temperature incubator for 12 h to screen positive transformants.
[0063] (a-2) Pick a single colony into 10 ml of LB medium containing 80 μg / ml ampicillin (in LB medium: 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of sodium chloride) as the seed liquid, culture it at 37°C for 16 h, add the above seed liquid to 500 ml of LB medium containing ampicillin at a ratio of 1:50, culture it at 37°C until the OD600 is 1.0, then add 0.5 mM IPTG to induce inclusion body expression, and then continue to culture for 5 h to obtain a fermentation broth, and collect the bacteria at 8000 rpm for 20 min. The expression result is as Figure 1 shown.
[0064] (b) Preliminary pretreatment of inclusion bodies: Resuspend the collected bacterial cells in step (a-2) with lysis buffer. The volume ratio of the fermentation broth to the lysis buffer is 10:1. Vortex for 60 s using a vortex oscillator to fully resuspend the inclusion bodies. Lyse the bacteria using a high-pressure homogenizer under refrigeration conditions, homogenize 3 times, with a pressure of 800 bar, 8000 rpm, and centrifuge to collect the bacterial cells at 20 min. The lysis buffer contains 100 mM sodium phosphate buffer, 10 mM EDTA, 100 mM sodium chloride, and the pH is 6.8 - 7.2. Take the solution after cell lysis, centrifuge at 8000 rpm at 0 - 4 °C for 25 min, discard the supernatant to obtain the inclusion body precipitate, add the inclusion body washing buffer, pipette the precipitate until completely dissolved, shake for 10 s every 15 min, and shake 3 times. The inclusion body washing buffer contains 100 mM sodium phosphate buffer, 3 M urea, and the pH is 6.8 - 7.2. Centrifuge at 8000 rpm at 0 - 4 °C for 20 min, discard the supernatant, re-add the inclusion body washing buffer, pipette the precipitate until completely dissolved, shake for 10 s every 15 min, and shake 3 times. Repeat the steps 2 times, with a total washing time of 2 - 3 hours.
[0065] (c) Purification of denatured protein: After the pretreatment is completed, centrifuge at 8000 rpm at 0 - 4 °C for 20 min to obtain the inclusion body precipitate, resuspend it completely with the denaturation buffer, stir at 0 - 4 °C and 100 rpm, with a denaturation time of 12 h, centrifuge at 10000 rpm at 0 - 4 °C for 20 min to separate the soluble and insoluble proteins, and take the supernatant. The denaturation buffer contains 50 mM sodium phosphate buffer, 7 M urea, 100 mM Tris, and the pH is 6.8 - 7.2. The result after denaturation is as Figure 2 shown. Filter the supernatant after denaturation through a 0.45 μm filter membrane and load it onto a pre-equilibrated SP cation exchange chromatography column. After loading, continue to wash with the equilibration buffer until reaching the baseline. The equilibration buffer contains 50 mM sodium phosphate buffer, 7 M urea, 100 mM Tris, and the pH is 6.8 - 7.2. Elute with the elution buffer, and the elution mixing ratios are 10%, 20%, 30%, 40%, 50%, 60%. Collect the main peak to obtain the purified denatured BMP7 protein. The elution buffer contains 50 mM sodium phosphate buffer, 7 M urea, 1 M NaCl, 100 mM Tris, and the pH is 6.8 - 7.2. The result of the purified BMP77 denatured protein is as Figure 3 shown.
[0066] (d) Protein Renaturation and Dimerization: Centrifuge the purified denatured BMP7 protein at 8000 rpm for 20 min. Dilute the supernatant after centrifugation into the renaturation solution at a ratio of 1:10 and perform renaturation at 4°C. The renaturation solution includes 50 mM sodium phosphate buffer, 0.5 M L-arginine, 1 mM cysteine, 0.1 mM cystine, 3 mM EDTA, 0.7 mM guanidine hydrochloride, pH = 8.0 - 8.5, protein concentration 0.2 mg / mL, and renaturation time 48 hours. Replace the buffer of the renatured protein by ultrafiltration, and continue to place the protein solution at 4°C for 48 hours after the replacement is completed. The buffer used for replacement contains: 50 mM sodium phosphate buffer, 0.5 M L-arginine, pH 6.8 - 7.2; The gel electrophoresis result after protein renaturation is as Figure 4 shown.
[0067] (e) Purification of Renatured BMP7 Dimer Protein: Obtain the supernatant by centrifuging the renatured BMP7 dimer protein at 10000 rpm at low temperature of 4°C for 20 min, filter it through a 0.45 μm filter membrane, and load it onto a pre-equilibrated Q anion exchange chromatography column. After loading, continue to wash with the equilibration buffer until reaching the baseline. The equilibration buffer contains: 50 mM sodium phosphate buffer and 0.5 M L-arginine, pH 6.8 - 7.2; Perform gradient elution with gradually increasing salt concentration using the elution buffer, and the elution mixing ratio is 30%, 40%. Collect the main peak. The elution buffer contains 50 mM sodium phosphate buffer, 0.5 M L-arginine and 1 M NaCl, pH 6.8 - 7.2; Dialyze the collected purified protein into the loading buffer. The loading buffer contains 50 mM sodium phosphate buffer, 0.3 M L-arginine and 1 M (NH 4 ) 2 SO 4 , pH 6.8 - 7.2; Filter the protein through a 0.45 μm filter membrane and load it onto a pre-equilibrated butyl FF hydrophobic chromatography column. After loading, continue to wash with the equilibration buffer until reaching the baseline. The equilibration buffer contains: 50 mM sodium phosphate buffer and 0.5 M L-arginine, 1 M (NH 4 ) 2 SO 4 , pH 6.8 - 7.2; Perform gradient elution with gradually decreasing salt concentration using the elution buffer, and the elution mixing ratio is 30%, 40%, 50%. Collect the main peak. The elution buffer contains 50 mM sodium phosphate buffer, 0.5 M L-arginine, pH 6.8 - 7.2; Concentrate the collected protein 5 times by ultrafiltration and dialyze it into the storage buffer. The storage buffer contains 50 mM acetic acid and 2% trehalose. The purification result is as Figure 5As shown, through the analysis by Image J software, the purity of the recombinant human bone morphogenetic protein-7 mature peptide dimer reaches over 95%.
[0068] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, which are all regarded as the protection scope of the present invention.
Claims
1. A method for producing a recombinant human bone morphogenetic protein-7 mature peptide dimer, characterized in that: The recombinant dimeric protein BMP7 was obtained by titration renaturation and chromatography column purification, and the specific steps are as follows: (a) Inclusion body expression: The BMP7 recombinant expression plasmid was transformed into BL21 (DE3) Escherichia coli, and the positive transformants were cultured and collected; (b) Preliminary purification of inclusion bodies (b-1) Bacterial disruption: resuspend the inclusion bodies with bacterial disruption buffer and disrupt the bacteria under refrigeration conditions; (b-2) taking the solution after the lysis of the bacteria, centrifuging at low temperature, discarding the supernatant to obtain the inclusion body precipitate, adding the inclusion body washing buffer, and blowing the precipitate until it is completely dissolved; (b-3) Centrifuge at low temperature, discard the supernatant, re-add inclusion body washing buffer, pipette the precipitate until it is completely dissolved, and shake 2-3 times; (b-4) Repeat step (b-3) 2-3 times; (c) Protein purification after denaturation (c-1) Denaturation of inclusion bodies: centrifuge at low temperature for 15-25 min to obtain inclusion body precipitates, resuspend completely with denaturation buffer, centrifuge to separate soluble and insoluble proteins, and take the supernatant; (c-2) The supernatant is filtered with a filter membrane and loaded onto a well-equilibrated SP cation exchange chromatography column. After loading, the column is continuously rinsed with an equilibrium buffer to reach the baseline; (c-3) eluting with elution buffer, collecting the main peak to obtain purified BMP7 denatured protein; (d) Protein refolding and dimerization (d-1) centrifuging the purified denatured BMP7 protein, and diluting the supernatant after centrifugation into a refolding solution for refolding, wherein the refolding solution includes 10-100 mM sodium phosphate buffer, 0.1-0.5 M L-arginine, 1-2 mM cysteine, 0.1-0.5 mM cystine, 3-7 mM EDTA, 0.3-0.7 mM guanidine hydrochloride, pH = 8.0-8.5, protein concentration 0.1-0.2 mg / mL, and refolding time 24-48 hours; (d-2) performing buffer replacement on the renatured protein by ultrafiltration, wherein the buffer used for replacement is 10-100 mM sodium phosphate buffer, 0.1-0.5 M L-arginine, and pH is 6.8-7.2; (e) Purification of BMP7 dimer protein after renaturation (e-1) The refolded BMP7 dimer protein was centrifuged at low temperature to obtain the supernatant, which was filtered with a filter membrane and loaded onto a well-equilibrated Q anion exchange chromatography column. After loading, the column was continuously rinsed with the equilibration buffer to reach the baseline; (e-2) eluting with a gradient of elution buffer with gradually increasing salt concentration to collect the main peak; (e-3) dialyzing the collected purified protein into loading buffer; (e-4) The protein is filtered through a filter membrane and loaded onto a equilibrated butyl-agarose gel FF hydrophobic chromatography column. After loading, the column is continuously rinsed with an equilibration buffer to reach the baseline; (e-5) Elution was performed using an elution buffer with a gradient of gradually decreasing salt concentration to collect the main peak.
2. The method for producing recombinant human bone morphogenetic protein-7 mature peptide dimer according to claim 1, characterized in that: The specific steps of inclusion body expression in step (a) are: (a-1) The BMP7 recombinant expression plasmid was transformed into BL21 (DE3) Escherichia coli, spread on a resistant plate, placed in a 37°C constant temperature incubator for 12 h, and positive transformants were screened; (a-2) Pick a single colony into 5-10 ml of LB medium containing resistance as a seed solution, culture at 37°C for 12-16 h, add the seed solution to LB medium containing resistance at a ratio of 1:50, culture at 37°C until OD600 is 1.0-1.2, then add IPTG 0.1-1 mM to induce inclusion body expression, then continue to culture for 5-8 h to obtain fermentation broth, and collect bacteria at 6000-8000 rpm for 20 min.
3. The method for producing recombinant human bone morphogenetic protein-7 mature peptide dimer according to claim 1 or 2, characterized in that: The nucleotide sequence of the BMP7 recombinant expression plasmid is shown in the sequence table SEQ ID NO.
1.
4. The method for producing recombinant human bone morphogenetic protein-7 mature peptide dimer according to claim 2, characterized in that: The LB medium containing resistance is an LB medium containing 50-100 μg / ml ampicillin.
5. The method for producing recombinant human bone morphogenetic protein-7 mature peptide dimer according to claim 1 or 2, characterized in that: The specific steps of the preliminary purification of inclusion bodies in step (b) are as follows: (b-1) disrupting the bacteria: resuspending the bacteria collected in step (a-2) with a bacterial disruption buffer, wherein the volume ratio of the fermentation broth to the bacterial disruption buffer is 8-12:1, and vortexing for 30-60 seconds using a vortex oscillator to fully resuspend the inclusion bodies; using a high-pressure homogenizer to disrupt the bacteria under refrigeration conditions, homogenizing 3 times, at a pressure of 800 bar, 8000 rpm, and centrifuging for 20 minutes to collect the bacteria, wherein the bacterial disruption buffer contains 100 mM sodium phosphate buffer, 10 mM EDTA, 100 mM sodium chloride, and a pH of 6.8-7.2; (b-2) taking the solution after the lysis of bacteria, centrifuging at 6000-12000 rpm, 0-4°C for 20-30 min, discarding the supernatant to obtain the inclusion body precipitate, adding inclusion body washing buffer, blowing the precipitate until it is completely dissolved, and oscillating for 5-10 s every 10-15 min, 2-3 times; the inclusion body washing buffer contains 100 mM sodium phosphate buffer, 3 M urea, and a pH of 6.8-7.2; (b-3) Centrifuge at 6000-12000 rpm and 0-4°C for 15-25 min, discard the supernatant, re-add inclusion body washing buffer, pipette the precipitate until it is completely dissolved, and oscillate for 5-10 s every 10-15 min, 2-3 times; (b-4) Repeat step (b-3) 2-3 times, washing for a total of 2-3 hours.
6. The method for producing recombinant human bone morphogenetic protein-7 mature peptide dimer according to claim 5, characterized in that: In the step (b-1), the volume ratio of the fermentation liquid to the bacterial lysis buffer is 10:
1.
7. The method for producing recombinant human bone morphogenetic protein-7 mature peptide dimer according to claim 1, characterized in that: The specific steps of protein purification after denaturation in step (c) are as follows: (c-1) Denaturation of inclusion bodies: After pretreatment, centrifuge at 8000-12000 rpm and 0-4°C for 15-25 min to obtain inclusion body precipitates, resuspend completely with denaturation buffer, stir at 0-4°C and 60-100 rpm, denaturation time 10-12 h, centrifuge at 10000-14000 rpm and 0-4°C for 15-24 min to separate soluble and insoluble proteins, and take the supernatant; the denaturation buffer contains 10-100 mM sodium phosphate buffer, 7-8 M urea, 100 mM Tris, pH 6.8-7.2; (c-2) The supernatant was filtered with a 0.45 μm filter membrane and loaded onto a equilibrated SP cation exchange chromatography column. After loading, the column was continuously rinsed with an equilibration buffer to reach the baseline. The equilibration buffer contained 10-100 mM sodium phosphate buffer, 7-8 M urea, 100 mM Tris, and had a pH of 6.8-7.
2. (c-3) Elution was performed with elution buffer at a mixing ratio of 10%, 20%, 30%, 40%, 50%, and 60%, and the main peak was collected to obtain purified denatured BMP7 protein. The elution buffer contained 10-100 mM sodium phosphate buffer, 5-7 M urea and 0.5-1 M NaCl, 100 mM Tris, and a pH of 6.8-7.
2.
8. The method for producing recombinant human bone morphogenetic protein-7 mature peptide dimer according to claim 1, characterized in that: The specific steps of the protein renaturation and dimerization in step (d) are: (d-1) The purified denatured BMP7 protein was centrifuged at 8000 rpm for 20 min, and the supernatant after centrifugation was diluted into a refolding solution at a ratio of 1:10, and refolded at 2-4°C, wherein the refolding solution includes 10-100 mM sodium phosphate buffer, 0.1-0.5 M L-arginine, 1-2 mM cysteine, 0.1-0.5 mM cystine, 3-7 mM EDTA, 0.3-0.7 mM guanidine hydrochloride, pH = 8.0-8.5, protein concentration 0.1-0.2 mg / mL, and refolding time 24-48 hours; (d-2) The renatured protein was subjected to buffer replacement by ultrafiltration. After the replacement of the protein solution, it was allowed to stand at 4° C. for 48 hours. The buffer used for replacement was 10-100 mM sodium phosphate buffer, 0.1-0.5 M L-arginine, and pH 6.8-7.
2.
9. The method for producing recombinant human bone morphogenetic protein-7 mature peptide dimer according to claim 1, characterized in that: The specific steps of purifying the BMP7 dimer protein after renaturation in step (e) are as follows: (e-1) The renatured BMP7 dimer protein was centrifuged at 10000-14000 rpm at 0-4°C for 15-25 min to obtain the supernatant, filtered with a 0.45 μm filter membrane, and loaded onto a well-equilibrated Q anion exchange chromatography column. After loading, the column was continuously rinsed with an equilibration buffer to reach the baseline. The equilibration buffer contained: 10-100 mM sodium phosphate buffer and 0.1-0.5 M L-arginine, with a pH of 6.8-7.2; (e-2) eluting with an elution buffer having a gradually increasing salt concentration gradient, the elution mixing ratio is 30% and 40%, and the main peak is collected, the elution buffer is 10-100 mM sodium phosphate buffer, 0.1-0.5 M L-arginine and 0.5-1 M NaCl, and the pH is 6.8-7.2; (e-3) dialyzing the collected purified protein into a loading buffer, wherein the loading buffer comprises 10-100 mM sodium phosphate buffer, 0.1-0.5 M L-arginine and 1-1.5 M (NH4)2SO4, with a pH of 6.8-7.2; (e-4) The protein was filtered with a 0.45 μm filter membrane and loaded onto a butyl FF hydrophobic chromatography column that had been equilibrated. After loading, the column was rinsed with an equilibration buffer to reach the baseline. The equilibration buffer was 10-100 mM sodium phosphate buffer and 0.1-0.5 M L-arginine, 1-1.5 M (NH4)2SO4, and pH was 6.8-7.
2. (e-5) using an elution buffer to perform elution with a gradient of gradually decreasing salt concentration, the elution mixing ratio is 30%, 40%, and 50%, and the main peak is collected. The elution buffer is 10-100 mM sodium phosphate buffer, 0.1-0.5 M L-arginine, and the pH is 6.8-7.2.