CN117164696B - Production method of recombinant human bone morphogenetic protein-2 mature peptide dimer - Google Patents
Production method of recombinant human bone morphogenetic protein-2 mature peptide dimer Download PDFInfo
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention discloses a production method of recombinant human bone morphogenetic protein-2 mature peptide dimer, which comprises the following steps: (a) pretreatment of inclusion body denaturation; (b) protein purification after denaturation; (c) protein renaturation; (d) purification of BMP2 dimer protein after renaturation. The production method of the invention simplifies the production method process of the recombinant human bone morphogenetic protein-2 mature peptide dimer and improves the yield.
Description
Technical Field
The invention belongs to the technical field of medical biology, and particularly relates to a production method of a recombinant human bone morphogenetic protein-2 mature peptide dimer.
Background
In 1965, U.S. doctors Urist (Urist MR. Bone: formation by automatic induction. Science,1965, 150 (698): 893-899) found that decalcified bone interstitium had an ectopic osteogenic effect, and named the newly found protein having the effect of inducing formation of new bone as bone morphogenic protein (Bone Morphogenetic protein, BMP). Subsequently, numerous BMPs, other than BMP-1, have been discovered that are members of the TGF-beta superfamily, both genetically and proteolytically and biologically functionally very similar. Among them, the bone formation of BMP-2 is most studied, and the industrialization is most widely studied.
Human BMP-2 is 396 amino acid residues in its full length, has an osteogenic effect and is a mature peptide consisting of 114 amino acids with a glycosylation site at the asparagine residue at position 55, but whether it is glycosylated or not does not affect its biological activity. Furthermore BMP-2 is very hydrophobic and is almost insoluble in various solvents, its active form being in the form of dimers, in which the monomers each form 3 pairs of disulfide bonds, forming a pair of interchain disulfide bonds between the two monomers. The correct pairing of disulfide bonds directly affects their bioactivity and stability. In vitro and in vivo experiments demonstrated that BMP-2 has the ability to promote osteoblast differentiation and induce in vitro osteogenesis (Riley, EH; lane, JM; urist, MR, et al Bone morphogenetic protein-2:Biology and applications Clinoortho Relay Res.1996Mar.; 324): 39-46). However, the content of natural BMP-2 in various tissues is extremely low, and a large amount of BMP-2 is required to be produced by a gene recombination method.
The Chinese patent No. 102336829B discloses a method for producing mature peptide of recombinant human morphogenetic protein 2, which comprises loading the recombinant human morphogenetic protein-2 mature peptide solution with good renaturation on a balanced hydrophobic chromatographic column, then performing gradient elution with gradually reduced salt concentration by using an elution buffer solution, and collecting target peaks. To further increase the protein purity of the target peak, the purification method may be multi-step hydrophobic interaction chromatography or combined with ion exchange chromatography. The method has the advantages of simpler operation, lower cost, higher purification yield (more than 20%), higher purity (SDS-PAGE, HPLC and HPCE are all more than 97%), and the like, and is suitable for the production of recombinant human bone morphogenetic protein families. The invention discovers that the polarity of the mature peptide of the recombinant human bone morphogenetic protein-2 is increased by adopting high-concentration urea and DMF as cosolvent, and the combination degree with a hydrophobic medium is reduced in the hydrophobic chromatography process, so that the efficiency and the separation degree of protein purification are reduced. The invention also discovers that the purification efficiency and the separation degree of the protein can be effectively improved and the purity of the target protein is higher by adjusting the composition of the purification buffer solution, namely using urea and DMF with lower concentration as cosolvent and reducing the use amount of CHES. And the production cost can be reduced by more than 50 percent due to the reduction of the consumption of urea, DMF and CHES. Compared with the slow-release method (CN 200510050610.3), the purification method has the advantages of simpler operation, lower cost, higher purification yield (more than 20 percent), higher purity (more than 97 percent of SDS-PAGE, HPLC and HPCE) and the like. However, the production method of the invention is complex in process, and needs to perform 2-3 times of hydrophobic chromatography, and simultaneously perform 1 time of anion exchange chromatography and 1 time of cation exchange chromatography in combination. In addition, in the prepared buffer solution of the mature peptide of the recombinant human bone morphogenetic protein-2, 10-30mM 2-cyclohexylethane sulfonic acid (CHES), 0.2-1M urea and 2-10% N, N-dimethylformamide are needed, so that the components are complex and the clinical use risk is increased.
Chinese patent No. 104151419B discloses a preparation method for expressing recombinant human bone morphogenetic protein-2 (recombinanth. Mu. man bone morphogenetic protein-2, rhBMP-2) in Escherichia coli in the form of inclusion body, which comprises the following main steps: homogenizing under high pressure to break thallus cells, rinsing inclusion bodies, regulating different pH values, denaturing urea to dissolve the inclusion body proteins, speed-controlling renaturation of the inclusion body proteins, centrifuging, concentrating by an ultrafiltration membrane, filtering, sterilizing, freeze-drying to obtain a finished product, and the like. The preparation method of the invention utilizes the renaturation by regulating different pH values and controlling the urea concentration change speed for several times in the rinsing process, and can prepare the rhBMP-2 with high purity and high activity by the ultrafiltration membrane technology. Meanwhile, the chromatography step can be omitted, the product quality requirement can be met through the modern ultrafiltration membrane technology, the working hours are greatly shortened, and the production cost is reduced. The invention can obtain the rhBMP-2 pure product with high efficiency, simplicity and low cost. According to the method, in the aspect of removing the impurity protein and impurities, chloroform, acetone, ethanol and other solvents harmful to human are not required to be used for traditional impurity removal, and meanwhile, the step of removing the impurity protein by a high-temperature heating method is omitted, so that not only is energy saved, but also the damage of high Wen Duichong groups of human BMP-2 is avoided. The method of the invention has simple operation and low production cost, is suitable for large-scale industrial production, and realizes the process method for producing the recombinant human BMP-2 with low cost. However, the recombinant human bone morphogenetic protein-2 prepared by the invention is a monomeric protein with a molecular weight of 13kDa, and has low biological activity and lower clinical application prospect.
Chinese patent No. CN104447977B discloses a method for purifying recombinant human bone morphogenetic protein-2. The method comprises the following steps: 1) Cracking inclusion bodies containing rhBMP-2 protein to obtain inclusion body cracking liquid; 2) Sequentially carrying out affinity chromatography and cation exchange chromatography on the inclusion body lysate, and separating and purifying to obtain a denatured recombinant human bone morphogenetic protein-2 solution; 3) Renaturation is carried out on the denatured rhBMP-2 protein to obtain a renaturated rhBMP-2 protein crude product; 4) And sequentially carrying out affinity chromatography and gel filtration chromatography separation and purification on the renaturated rhBMP-2 protein crude product to obtain a renaturated rhBMP-2 protein pure product. The method provided by the invention is used for purifying rhBMP-2 protein, the purity of the prepared product is more than 95%, the protein yield is 9.4%, endotoxin and DNA residues meet the quality requirements of biological medicines, and the method is suitable for industrial scale production; meanwhile, the method also provides reference for research and development and production of similar recombinant protein medicaments. However, the production method of the invention has complex process, and the recombinant human bone morphogenetic protein-2 needs to be subjected to 1 affinity chromatography and 1 ion exchange chromatography after denaturation, and then needs to be subjected to 1 affinity chromatography and 1 gel filtration chromatography after renaturation. And the affinity chromatographic column used in the production method is a heparin column, so that the heparin column has low loading capacity and influences the protein purification yield. The method requires shake culture at 4-10deg.C for 4-7 days, and takes longer time.
The Chinese patent application CN107312789A discloses a preparation method of recombinant human bone morphogenetic protein, which is characterized by comprising the following steps: adopting a codon optimization software to preliminarily optimize a cDNA sequence of the hBMP-2 according to the using frequency of the escherichia coli codons, and further replacing partial nucleotide codes according to empirical value calculation to obtain an optimized DNA sequence of the hBMP-2 gene; the method comprises the steps of preparing an escherichia coli rhBMP-2 strain by intercepting cDNA of rhBMP-2 for encoding a carboxyl terminal amino acid peptide of the rhBMP-2, obtaining BMP inclusion bodies through fermentation and process optimization, and preparing high-purity rhBMP-2 through separation, purification and renaturation. The renaturation link of the fusion protein dimer with the activity of the recombinant human bone morphogenetic protein-2 prepared by the patent application requires more than 7 days, the time is long, and the buffer solution of the recombinant human bone morphogenetic protein-2 obtained by renaturation contains 0.8-M urea, has complex components and increases the clinical use risk.
Chinese patent No. CN104961820B discloses a method for recovering recombinant human bone morphogenetic protein-2 by circulating chromatography. The invention provides a renaturation method of recombinant human bone morphogenetic protein-2, which comprises the following steps: 1) Denaturing the recombinant human bone morphogenetic protein-2 inclusion bodies to obtain denatured proteins; 2) Carrying out affinity chromatography renaturation on the denatured protein, and collecting eluent to obtain a renatured recombinant human bone morphogenetic protein-2 dimer; the amino acid sequence of the recombinant human bone morphogenetic protein-2 is sequence 2 in a sequence table. Experiments prove that the method for renaturating rhBMP-2 protein has high protein concentration, less reagent consumption and renaturation rate of more than 70 percent. The affinity chromatographic column used in the production method of the recombinant human bone morphogenetic protein-2 mature peptide is a heparin column, the loading capacity of the heparin column is low, the protein purification yield is affected, and the buffer solution of the recombinant human bone morphogenetic protein-2 obtained by renaturation contains 3M urea, so that the components are complex, and the clinical use risk is increased.
The invention aims to solve the problems that in the existing production method of the recombinant human bone morphogenetic protein-2 mature peptide dimer, multiple times of chromatography are needed, a low-load heparin column is relied on, buffer solution is of non-neutral pH, the components are complex, the renaturation link takes a long time and the like, and provides a new production method of the recombinant human bone morphogenetic protein-2 mature peptide dimer by adding inclusion body denaturation pretreatment, protein concentration optimization, renaturation buffer solution pH and formula optimization, chromatographic column optimization selection and the like.
Disclosure of Invention
For the above reasons, the present invention aims to provide a method for producing recombinant human bone morphogenic protein-2 mature peptide dimers. Specifically, in order to achieve the purpose of the present invention, the present invention adopts the following technical scheme:
one aspect of the present invention relates to a method for producing a recombinant human bone morphogenic protein-2 mature peptide dimer comprising the steps of:
(a) Pretreatment of inclusion body denaturation
(1) Centrifuging the solution after bacterial disruption at 6000-12000 rpm at 0-4deg.C for 15-25 min, discarding inclusion body precipitate obtained from supernatant, adding inclusion body washing buffer, blowing the precipitate until it is completely dissolved, oscillating for 5-10 s every 10-15 min, and oscillating for 2-3 times; the inclusion body washing buffer solution comprises 10-100mM sodium phosphate buffer solution and 1M urea, and the pH value is 6.8-7.2.
(2) Centrifuging at 6000-12000 rpm at 0-4deg.C for 15-25 min, discarding supernatant, adding inclusion body washing buffer again, blowing to completely dissolve precipitate, oscillating for 5-10 s every 10-15 min, and oscillating for 2-3 times;
(3) Repeating the step (2) for 1-2 times, and washing for 1-2 hours in total;
(b) Protein purification after denaturation
(1) Inclusion body denaturation: after the pretreatment is completed, obtaining inclusion body sediment by centrifuging at a low temperature of between 6000 and 12000 rpm and between 0 and 4 ℃ for 15 to 25 minutes, completely re-suspending the inclusion body sediment by using a denaturation buffer solution, stirring at a low temperature of between 0 and 4 ℃ for over night by 60 to 100 rpm, centrifuging at a low temperature of between 10000 and 14000 rpm for 15 to 24 minutes by using a low temperature of between 0 and 4 ℃, and separating soluble proteins and insoluble proteins, and taking supernatant; the denaturation buffer comprises 10-100mM sodium phosphate buffer, 5-7M urea and has a pH of 6.8-7.2.
(2) Filtering the supernatant with a 0.45 μm filter membrane, loading the filtered supernatant on a balanced cation exchange chromatographic column, and continuously flushing the supernatant with a balance buffer solution to reach a base line after loading, wherein the balance buffer solution comprises 10-100mM sodium phosphate buffer solution, 5-7M urea and the pH value is 6.8-7.2;
(3) Eluting with gradient with gradually increased salt concentration of elution buffer solution containing 10-100mM sodium phosphate buffer solution, 5-, M urea and 0.5-1M NaCl at pH of 6.8-7.2, and collecting main peak to obtain purified BMP2 denatured protein;
(c) Protein renaturation
(1) Purified BMP2 denatured protein 2-4 o C under the condition ofRenaturation, wherein the renaturation solution comprises 10-100mM sodium phosphate buffer solution, 0.1-0.5M L-Arg,1-2mM cysteine, 0.1-0.5mM cystine, 3-7mM EDTA,0.3-0.7mM guanidine hydrochloride, pH=6.8-7.2), protein concentration of 0.08-0.12mg/mL and renaturation time of 10-15 hours;
(2) And (3) carrying out buffer replacement on the renaturated protein by ultrafiltration to concentrate the protein concentration to 8-12 times of the original concentration to obtain the renaturated BMP2 dimer protein, wherein the replaced buffer comprises the following components: 10-100 of mM sodium phosphate buffer solution, 0.1-0.5M L-Arg and pH value of 6.5-7.2;
(d) Purification of BMP2 dimer protein after renaturation
(1) Centrifuging the renatured BMP2 dimer protein at 10000-14000 rpm for 15-25 min at 0-4 ℃ to obtain a supernatant, filtering with a 0.45 mu m filter membrane, loading the supernatant on a cation exchange chromatographic column which is well balanced, and continuing to rinse the supernatant with a balance buffer solution until the supernatant reaches a base line after loading, wherein the balance buffer solution comprises: 10-100mM sodium phosphate buffer and 0.1-0.5M L-Arg, and the pH is 6.8-7.2;
(2) Eluting with an elution buffer comprising 10-100mM sodium phosphate buffer, 0.1-0.5M L-Arg and 0.5-1M NaCl at pH 6.8-7.2 with a gradient of increasing salt concentration to collect the main peak;
(3) Subjecting the purified protein to buffer displacement by ultrafiltration and concentrating the protein concentration to 4-6 times the original concentration, the displaced buffer comprising: 10-100 parts of mM sodium phosphate buffer solution, 0.1-0.5 part of M L-Arg and pH of 6.8-7.2.
In a preferred embodiment of the invention, the post-breaker solution is a solution containing a bacterial engineering bacterium encoding a mature peptide sequence of recombinant human bone morphogenic protein-2.
In a preferred embodiment of the present invention, the mature peptide sequence of the recombinant human bone morphogenic protein-2 is shown in SEQ No.1 of the sequence Listing.
In a preferred embodiment of the invention, the total time of the preparation process does not exceed 24 hours.
In a preferred embodiment of the invention, none of the post-displacement buffers comprises urea and/or 2-cyclohexylethane sulfonic acid.
In a preferred embodiment of the invention, the recombinant human bone morphogenic protein-2 mature peptide prepared by said method has a molecular weight between 24 and 26 kDa.
Advantageous effects
The production method of the invention can remove irrelevant proteins to the greatest extent by carrying out pretreatment before the denaturation of the inclusion bodies, improve the purity of the inclusion bodies of the target proteins, and obtain the high-purity denatured proteins by one-time chromatography after the denaturation of the high-purity inclusion bodies; in the renaturation process, factors such as protein purity, protein concentration, pH of a renaturation buffer solution, composition and concentration of a reducing agent and an oxidizing agent in the renaturation buffer solution directly influence the renaturation condition of the modified protein, and the time required by renaturation is shortened to a great extent by optimizing the factors; in the protein chromatography process, a composite weak cation exchange medium is selected, the multimodal ligand can provide ion interaction, hydrogen bond and hydrophobic interaction, so that the efficient separation and purification of denatured protein and renaturated protein can be realized, and the separation of denatured protein and renaturated protein dimer can be realized by replacing a heparin column, thereby simplifying the production method and process of the recombinant human bone morphogenetic protein-2 mature peptide dimer and improving the yield. In particular, the method of the invention has at least 1 or all of the following advantages:
(1) The production method has simple process, and can realize the production of the recombinant human bone morphogenetic protein-2 mature peptide by carrying out denaturation pretreatment, protein concentration optimization, renaturation buffer pH and formula optimization, chromatographic column optimization selection and 1 time of chromatography before and after denaturation.
(2) The buffer solution of the recombinant human bone morphogenetic protein-2 mature peptide prepared by the invention only contains PBS buffer solution and 0.1-0.5M arginine, does not contain urea, 2-cyclohexyl ethane sulfonic acid (CHES) and the like, and has simple components, low cost and high clinical use safety.
(3) The recombinant human bone morphogenetic protein-2 mature peptide prepared by the invention is dimer protein with the molecular weight of about 24-26kDa, and has high biological activity.
(4) The method of the invention uses cation exchange chromatographic columns for 2 times of chromatography, and the chromatographic columns have high load and strong capturing capability, and can improve the protein yield.
(5) The invention improves the purity of the denatured protein, optimizes the protein concentration, optimizes the pH and formula of the renaturation buffer solution, controls the temperature and the like, and only takes about 24 hours in the renaturation link of the recombinant human bone morphogenetic protein-2 mature peptide.
(6) The final buffer solution of the recombinant human bone morphogenetic protein-2 mature peptide dimer prepared by the invention has the pH of 6.8-7.2, reduces the loss of calcium ions and is beneficial to bone regeneration.
Drawings
Fig. 1: gel electrophoresis photographs of BMP2 inclusion body protein after denaturation pretreatment, which demonstrate that most of the extraneous proteins in inclusion bodies have been removed by inclusion body denaturation pretreatment;
fig. 2: gel electrophoresis pictures of the BMP2 protein after denaturation, which show that the BMP2 inclusion body protein after inclusion body pretreatment is denatured and then high-purity target protein is successfully obtained through one-time chromatography;
fig. 3: gel electrophoresis photographs after the renaturation of the BMP2 denatured protein show that the BMP2 denatured protein can obtain a BMP2 mature peptide dimer with high biological activity after the renaturation of the BMP2 denatured protein according to the scheme of the invention;
fig. 4: gel electrophoresis photographs of the purified BMP2 dimer protein illustrate that the mature peptide dimer of BMP2 in the renatured protein can be purified by one-time chromatography.
Detailed Description
In order to further understand the present invention, a technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Unless otherwise specified, all reagents involved in the examples of the present invention are commercially available products and are commercially available.
Example 1
(1) Pretreatment of inclusion body denaturation: taking a bacterial engineering bacterium post-disruption solution containing a mature peptide sequence (the peptide sequence is shown as SEQ No.1 and the nucleotide sequence is shown as SEQ ID No. 2) of the coded recombinant human bone morphogenetic protein-2, centrifuging the engineering bacterium at 8000rpm for 20 min at low temperature, and discarding the supernatant; adding proper volume of inclusion body washing buffer solution (10 mM sodium phosphate buffer solution, 1M urea, pH of 6.8-7.2) into inclusion body precipitate obtained by the centrifugation, blowing the precipitate until the precipitate is completely dissolved, performing voltex (high frequency oscillation) for 5-10 s every 15 min, and repeating the operation for 0.5 hours (voltex 2 times); centrifuging at 8000rpm for 20 min at low temperature, discarding supernatant, adding equal volume of inclusion body washing buffer (1/10 of the volume of fermentation broth) again, blowing to completely dissolve precipitate, performing Votex (high frequency oscillation) 5-10 s every 15 min, and repeating operation for 0.5 hr (Votex 2 times). The gel electrophoresis results of the BMP2 inclusion body protein denaturation are shown in fig. 1, totaling 1.5 hours of washing.
(2) Protein purification after inclusion body denaturation: after the pretreatment is completed, obtaining inclusion body sediment by low-temperature centrifugation at 8000rpm for 20 min, completely suspending by using denaturation buffer (10 mM sodium phosphate buffer, 5M urea and pH of 6.8-7.2), stirring overnight at low speed of 4 ℃ (60-100 rpm), and separating soluble and insoluble proteins by low-temperature centrifugation at 12000 rpm for 20 min, wherein the target proteins are mainly present in the supernatant; filtering the supernatant with 0.45 μm filter membrane, loading on balanced MMC ion exchange chromatography column, and washing with balance buffer solution (the balance buffer solution comprises 10-100mM sodium phosphate buffer solution and 6M urea, and pH is 7.0) until reaching baseline; the main peak was collected by one-step elution with elution buffer (elution buffer contains 10mM sodium phosphate buffer, 6M urea and 0.55M NaCl, pH 7.0). Gel electrophoresis after purification of the denatured BMP2 protein is shown in FIG. 2.
(3) Protein renaturation: purified BMP2 denatured protein 2-4 o Renaturation under the condition of C (10 mM sodium phosphate buffer, 0.1M L-Arg, 1mM cysteine, 0.1mM cystine, 5mM EDTA,0.5mM guanidine hydrochloride, pH 7.0), protein concentration 0.08-0.12mg/mL, renaturation time 12 hours; renaturated egg by ultrafiltrationBuffer replacement was performed on the white (post-replacement buffer: 10mM sodium phosphate buffer, 0.1L-Arg, pH 7.0) and the protein concentration was concentrated to 10-fold the original concentration (completed within 12 hours). Gel electrophoresis after renaturation of BMP2 denatured proteins is shown in fig. 3.
(4) Purifying BMP2 dimer protein after renaturation: filtering the supernatant with 0.45 μm filter membrane after renaturation by low-temperature centrifugation at 12000 rpm for 20 min, loading on a balanced cation exchange chromatographic column, and continuing to rinse with a balance buffer solution until reaching a base line after loading (the balance buffer solution comprises 10 sodium phosphate buffer solution and 0.1L-Arg, and the pH value is 7.0); eluting with an elution buffer in one step, and collecting the main peaks (elution buffer contains 10mM sodium phosphate buffer, 0.1M L-Arg and 0.65M NaCl, pH 7.0); the purified protein was subjected to buffer displacement by ultrafiltration (post-displacement buffer: 10. 10mM sodium phosphate buffer, 0.1M L-Arg, pH 7.0), and the protein concentration was concentrated to 5-fold the original concentration. The purification results of BMP2 dimer protein are shown in fig. 4.
The foregoing describes preferred embodiments of the present invention, but is not intended to limit the invention thereto. Modifications and variations to the embodiments disclosed herein may be made by those skilled in the art without departing from the scope and spirit of the invention.
Claims (5)
1. A method for producing a recombinant human bone morphogenic protein-2 mature peptide dimer comprising the steps of:
(a) Pretreatment of inclusion body denaturation
(1) Centrifuging the solution after bacterial disruption at 6000-12000 rpm at 0-4deg.C for 15-25 min, discarding inclusion body precipitate obtained from supernatant, adding inclusion body washing buffer, blowing the precipitate until it is completely dissolved, oscillating for 5-10 s every 10-15 min, and oscillating for 2-3 times; the inclusion body washing buffer solution comprises 10-100mM sodium phosphate buffer solution and 1M urea, and the pH value is 6.8-7.2;
(2) Centrifuging at 6000-12000 rpm at 0-4deg.C for 15-25 min, discarding supernatant, adding inclusion body washing buffer again, blowing to completely dissolve precipitate, oscillating for 5-10 s every 10-15 min, and oscillating for 2-3 times;
(3) Repeating the step (2) for 1-2 times, and washing for 1-2 hours in total;
(b) Protein purification after denaturation
(1) Inclusion body denaturation: after the pretreatment is completed, obtaining inclusion body sediment by centrifuging at a low temperature of between 6000 and 12000 rpm and between 0 and 4 ℃ for 15 to 25 minutes, completely re-suspending the inclusion body sediment by using a denaturation buffer solution, stirring at a low temperature of between 0 and 4 ℃ for over night by 60 to 100 rpm, centrifuging at a low temperature of between 10000 and 14000 rpm for 15 to 24 minutes by using a low temperature of between 0 and 4 ℃, and separating soluble proteins and insoluble proteins, and taking supernatant; the denaturation buffer solution comprises 10-100mM sodium phosphate buffer solution, 5-7M urea and pH value of 6.8-7.2;
(2) Filtering the supernatant with a 0.45 μm filter membrane, loading the filtered supernatant on a balanced cation exchange chromatographic column, and continuously flushing the supernatant with a balance buffer solution to reach a base line after loading, wherein the balance buffer solution comprises 10-100mM sodium phosphate buffer solution, 5-7M urea and the pH value is 6.8-7.2;
(3) Eluting with gradient with gradually increased salt concentration of elution buffer solution containing 10-100mM sodium phosphate buffer solution, 5M urea and 0.5-1M NaCl at pH of 6.8-7.2, and collecting main peak to obtain purified BMP2 denatured protein;
(c) Protein renaturation
(1) Purified BMP2 denatured protein 2-4 o Renaturation is carried out under the condition of C, wherein the renaturation solution comprises 10-100mM sodium phosphate buffer solution, 0.1-0.5M L-Arg,1-2mM cysteine, 0.1-0.5mM cystine, 3-7mM EDTA,0.3-0.7mM guanidine hydrochloride, pH=6.8-7.2, protein concentration of 0.08-0.12mg/mL and renaturation time of 10-15 hours;
(2) The renatured protein is subjected to buffer replacement by ultrafiltration, the protein concentration is concentrated to 8-12 times of the original concentration, so as to obtain the renatured BMP2 dimer protein, and the buffer used for replacement comprises the following components: 10-100 of mM sodium phosphate buffer solution, 0.1-0.5M L-Arg and pH value of 6.8-7.2;
(d) Purification of BMP2 dimer protein after renaturation
(1) Centrifuging the renatured BMP2 dimer protein at 10000-14000 rpm for 15-25 min at 0-4 ℃ to obtain a supernatant, filtering with a 0.45 mu m filter membrane, loading the supernatant on a cation exchange chromatographic column which is well balanced, and continuing to rinse the supernatant with a balance buffer solution until the supernatant reaches a base line after loading, wherein the balance buffer solution comprises: 10-100mM sodium phosphate buffer and 0.1-0.5M L-Arg, and the pH is 6.8-7.2;
(2) Eluting with an elution buffer comprising 10-100mM sodium phosphate buffer, 0.1-0.5M L-Arg and 0.5-1M NaCl at pH 6.8-7.2 with a gradient of increasing salt concentration to collect the main peak;
(3) Subjecting the purified protein to buffer displacement by ultrafiltration and concentrating the protein concentration to 4-6 times the original concentration, the buffer for displacement comprising: 10-100 parts of mM sodium phosphate buffer solution, 0.1-0.5 part of M L-Arg and pH of 6.8-7.2.
2. The method according to claim 1, wherein the post-breaker solution is a solution containing a bacterial engineering bacterium encoding a mature peptide sequence of recombinant human bone morphogenic protein-2.
3. The production process according to claim 1, wherein the total time of the production process is not more than 24 hours.
4. The production process according to claim 1, wherein the buffer for displacement does not contain urea and/or 2-cyclohexylethane sulfonic acid.
5. The method of claim 1, wherein the recombinant human bone morphogenic protein-2 mature peptide has a molecular weight of between 24 and 26 kDa.
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