CN114133444A

CN114133444A - Preparation method of human BMP2 and analogues thereof

Info

Publication number: CN114133444A
Application number: CN202111413344.1A
Authority: CN
Inventors: 赵彦涛; 胡先同; 韩丽伟; 李利; 李岩峰; 郭继东; 王华东; 洪磊
Original assignee: Fourth Medical Center General Hospital of Chinese PLA
Current assignee: Fourth Medical Center General Hospital of Chinese PLA
Priority date: 2021-11-25
Filing date: 2021-11-25
Publication date: 2022-03-04

Abstract

The invention discloses a preparation method of human BMP2 and analogues thereof. The method comprises the following steps: firstly, introducing a coding gene of human BMP2 or analogues thereof into a recipient Escherichia coli cell; carrying out induction expression on the recombinant Escherichia coli cells, and then collecting and crushing to obtain inclusion bodies; denaturing the inclusion body to obtain denatured protein; renaturation and purification are carried out on the denatured protein to obtain the human BMP2 and the analogues thereof. The invention designs and constructs BMP2 analogues consisting of different sequences by taking escherichia coli as an expression system based on the theoretical sequence of BMP2, and finally obtains the production process of the mutant protein suitable for industrial production. The production process has the characteristics of simple operation, good process controllability and large-scale production.

Description

Preparation method of human BMP2 and analogues thereof

Technical Field

The invention belongs to the field of genetic engineering, and relates to prokaryotic recombinant expression, preparation, in-vitro activity detection and clinical application of a human BMP2 cytokine.

Background

Fracture repair is a slow and painful process, and currently, the research and development of drugs for promoting fracture repair are continuously carried out clinically. Bone morphogenetic proteins (Bone morphogenetic proteins) are a class of transforming production factors (TGF-. beta.) secreted by the Bone matrix. It was first proposed by Uris et al in 1965 and first isolated from the demineralized bone matrix of bovine bone in 1982. To date, a total of 8 family members (BMP2, BMP4, BMP5, BMP6, BMP7, BMP9, BMP12, BMP14) were found. Wherein, the BMP2 has obvious effect of inducing the differentiation of osteocyte so as to promote the ectopic fixation and the fracture repair.

The BMP2 protein has the typical characteristics of a TGF- β family of proteins: 7 cysteines, 6 of which form intramolecular disulfide bonds (15-80, 44-112, 48-114), and Cys78 in both molecules forming an intermolecular disulfide bond. Wozney first reported in 1982 to express BMP2 based on COS-1 cells. In 1992, Israel DI reported the expression of BMP2 on CHO cells. The Infuse approved for the treatment of degenerative disc disease by Medtronic corporation, usa in 2002 is a lumbar fusion device developed based on the expression of BMP2 by cells. The CHO expression protein can form a complete protein structure, but has low expression quantity and high production cost. At present, BMP2 is also reported based on escherichia coli expression and renaturation, but the characteristics of low renaturation efficiency and complex process exist generally. The complete BMP2 production process including the key steps of expression, renaturation, purification, preparation and the like, which is suitable for industrial production, is developed based on an escherichia coli expression system, and is favorable for reducing the production cost of BMP2, expanding clinical application and benefiting patients.

Disclosure of Invention

The invention aims to provide a preparation method of human BMP2 and analogues thereof.

A preparation method of human BMP2 and analogues thereof comprises the following steps:

(1) introducing coding genes of human BMP2 and analogues thereof into a receptor escherichia coli cell to obtain a recombinant escherichia coli cell;

(2) after the recombinant escherichia coli cells are subjected to induction expression, collecting the recombinant escherichia coli cells; breaking the recombinant escherichia coli cells to obtain an inclusion body;

(3) denaturing the inclusion body to obtain denatured protein;

(4) renaturation and purification are carried out on the denatured protein, and finally purified human BMP2 and analogues thereof are obtained.

The coding genes of the human BMP2 and the analogues thereof are BMP2-T, DS-BMP2-T respectively, and the nucleotide sequences of the coding genes are respectively shown in a sequence table SEQ ID NO: 1. SEQ ID NO: 3.

the human BMP2 and the analogues thereof are BMP2-T, DS-BMP2-T, and the amino acid sequences are respectively shown in the sequence table SEQ ID NO: 2. SEQ ID NO: 4.

when the prepared product is BMP 2-T:

in the step (3), the inclusion body denaturation buffer is 50mM of trihydroxymethyl aminomethane, 8M of urea,1mM of ethylene diamine tetraacetic acid, 20mM of dithiothreitol and pH 8.5;

in the step (4), the inclusion body renaturation buffer solution is 100mM of trihydroxymethyl aminomethane, 0.25M of sodium cyclamate, 2M of urea,2mM of ethylene diamine tetraacetic acid, pH8.5,1mM of cystine and 3mM of cysteine; the renaturation condition is a denaturation buffer solution, namely a renaturation buffer solution is 1:20, and the renaturation is carried out for at least 2 days at room temperature after 2 days at 4 ℃.

When the prepared product is DS-BMP 2-T:

the inclusion bodies in the step (3) contain misfolded target proteins; denaturing the crushed thallus supernatant containing the misfolded target protein to obtain BMP2 protein containing collagen binding polypeptide and fused with a TRX label in full length;

renaturation, purification, enzyme digestion and purification are carried out on the BMP2 protein containing the collagen binding polypeptide and the full-length fusion TRX label, and purified DS-BMP2-T protein is obtained;

the renaturation buffer solution in the step (4) is 100mM of trihydroxymethyl aminomethane, 0.75M of arginine hydrochloride, 0.5M of urea,1M of sodium chloride, 2mM of ethylene diamine tetraacetic acid, pH8.5, 3mM of cysteine and 1mM of cystine. The optimal renaturation method comprises the following steps: the denaturation solution was diluted in renaturation buffer at a ratio of 1:20 and renatured at 4 ℃ for 2 days and at room temperature for 2 days.

The invention has the beneficial effects that: the invention takes escherichia coli as an expression system, designs and constructs BMP2 analogues consisting of different sequences based on the theoretical sequence of BMP2, and compares the process cost, the activity and other related parameters. Finally obtaining the production process of the mutant protein suitable for industrial production. The production process has the characteristics of simple operation, good process controllability and large-scale production.

Drawings

FIG. 1 is a plasmid map of BMP 2-T.

FIG. 2 is a SDS-PAGE analysis of BMP2-T fermentation curves and fermentation product analysis.

FIG. 3 is a diagram of development and optimization of BMP2-T renaturation process.

FIG. 4 is a Q.FF purification and SDS-PAGE analysis of a BMP2-T renaturation sample.

Fig. 5 is a heparin purification chromatogram and SDS-PAGE analysis of the q.ff purified product of the BMP2-T sample.

FIG. 6 is a reverse phase purification chromatogram and SDS-PAGE analysis of a BMP2-T sample.

FIG. 7 is an SDS-PAGE analysis and a mass spectrometry analysis of a final purified sample of BMP 2-T.

FIG. 8 is a plasmid map of BMP 2.

FIG. 9 is a SDS-PAGE analysis of BMP2 fermentation curves and fermentation product analysis.

Figure 10 is a graph of sp.bb purification and SDS-PAGE analysis of denatured sample of BMP 2.

Fig. 11 is a graph of sp.bb purification and SDS-PAGE analysis of BMP2 renaturation samples.

Fig. 12 is a graph of the heparin purification of BMP2 sp.bb purified samples and the electrophoresis and mass spectrometry analysis of the final product.

FIG. 13 is a plasmid map of DS-BMP 2-T.

FIG. 14 is a Q.FF, Q.HP purification and SDS-PAGE profile of a DS-BMP2-T replicate sample.

FIG. 15 is a SDS-PAGE pattern of activation of purified samples of DS-BMP2-T and purification of the samples after activation.

FIG. 16 shows the in vivo activity of the final products of BMP2-T and DS-BMP 2-T.

Detailed Description

In order that the invention may be more fully understood, reference will now be made to the following description. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. pET-11a, pET-30a vectors and the like are all products of Novagen.

EXAMPLE 1 preparation of BMP2-T

(1) BMP2-T coding gene synthesis, vector construction and expression strain construction

1.1 the 290 th through 396 of gene coding BMP2 protein (283 th through 396 of unipro No. P12643) and introducing R291K mutation modification, under the premise of not changing amino acid sequence, replacing the codon with the codon of preference (high frequency use) of Escherichia coli so as to be suitable for expressing in Escherichia coli, and adding an initiation codon ATG before the optimized sequence, finally obtaining the optimized BMP2-T coding gene sequence as shown in sequence table SEQ ID NO: 1 is shown.

1.2 the sequence table SEQ ID NO: 1 was inserted between NdeI/BamHI cleavage sites of a vector pET11a (+) to obtain a recombinant plasmid (FIG. 1). In the recombinant plasmid, the inserted double-stranded DNA molecule is fused with partial DNA of the plasmid, and the expression sequence table SEQ ID NO: 2.

1.3 the recombinant plasmid is introduced into Escherichia coli BL21(DE3) by chemical transformation, and the strain with high expression level is selected by Amp antibody plate coating and monoclonal screening, and finally the BMP2-T expression strain with high expression level is obtained.

1.4 the recombinant BMP2-T bacteria are inoculated in 500mL LB medium to prepare small-volume working seeds, when OD600 is 1.8, the culture is stopped and the seeds are put into a tank to be fermented at high density. High-density fermentation basal medium: 26.7g of glycerol, 40.0g of yeast powder, 1.8g of monopotassium phosphate, 1.8g of citric acid, 5mL of salt solution, 1mL of trace metal salt solution and 1mL of defoaming agent 204, distilled water is added to make up the volume of the solution to 1L, and the pH value is 7.2. The salt solution consists of water and solutes at concentrations of: 250g/L MgCl₂·6H₂O、100g/L CaCl₂·2H₂O, 100g/L KCl and 2M Citric acid (Citric acid). Trace metal salt solution: 6.8g ZnCl₂、54.0g FeCl₃·6H₂O、16.2g MnCl₂·4H₂O、2.2g CuSO₄·5H₂O、4.8g CoCl₂·6H₂O、0.024g(NH₄)₆Mo₇O₂₄·4H₂O, 0.2g KI, 119mL of 37% (vol.% concentration) concentrated hydrochloric acid, and distilled water to make up the volume of 1L. The feed consists of water and solutes at concentrations: 275g/L of glycerol and 225g/L of yeast powder.

1.5 pumping the working seeds and the sterilized culture medium into a sterilized fermentation tank through a peristaltic pump, and simultaneously adjusting the parameters of a fermentation system: the culture temperature is as follows: 37.0 ℃; oxygen dissolving and material supplementing linkage control: not starting; culture pH: 7.0(pH regulator: concentrated ammonia solution, 10% H)₂SO₄) (ii) a Initial dissolved oxygen after seed liquid inoculation:>90 percent; initial rotation speed after seed liquid inoculation: 500rpm; initial aeration after seed liquid inoculation: 15L/min. After the dissolved oxygen of the fermentation system is reduced to zero and begins to rise, the oxygen dissolving and feeding linkage is started, the initial linkage dissolved oxygen is controlled to be 25%, when the OD600nm value of the fermentation system is 15, IPTG is added to lead the concentration to be 1.0mM, then the second stage fermentation (induction expression) is carried out according to the following parameters, and the induction temperature is: 37.0 ℃; oxygen dissolving and material supplementing linkage control: 15 percent; pH at the induction fermentation stage: 7.0; and (3) induction fermentation time: for 5 hours. After the induction fermentation is finished, detecting the OD600nm value of the fermentation system, then centrifuging for 20min at 4 ℃ and 4000rpm, and collecting thalli obtained by the whole fermentation system. In a more specific experimental example, the fermentation profile obtained on a 20L fermenter basis is shown in FIG. 2.

1.6 collecting the thallus, resuspending the thallus with PBS buffer, (w/v is 1:10), resuspending for 10min with a high speed disperser 5000 rpm/min; then homogenizing and breaking the thalli under high pressure by a high-pressure homogenizer under the condition of cooling by cooling water, homogenizing for 3 times (the working pressure is 800bar), and centrifuging at 4000rpm for 30min to collect inclusion bodies.

1.7 the pellet was dissolved with an inclusion body dissolving solution (1 XPBS +2M Urea) at a dissolution ratio of 1:10 (w/v), then resuspend the inclusion body by stirring with a high speed disperser, put the well dispersed inclusion body solution on a magnetic stirrer to stir and dissolve for 1h, and then centrifuge at 4000rpm for 25min at 4 ℃ to collect the inclusion body.

1.8 the pooled inclusion body weight is resuspended in 1 × PBS in a 1:10 (w/v), then resuspending the inclusion bodies using high speed disperser agitation, placing the well dispersed inclusion body solution on a magnetic stirrer to stir 1.9BMP2-T inclusion bodies for renaturation process optimization and purification process optimization.

1.9.1 matrix design BMP2-T renaturation process optimization, 4 parallel denaturation conditions are adopted, each denaturation condition is carried out 4 parallel renaturation optimization, 16 renaturation comparison processes are totally carried out, and the denaturation conditions are as follows:

denaturation A: according to the following steps of 1:20, the denaturation buffer is: (50mM Tris,8M Urea,1mM EDTA,20mM DTT, pH8.5)

And (3) denaturation B: according to the following steps of 1:20, the denaturation buffer is: (50mM Tris,6M GuaHCl,1mM EDTA,20mM DTT, pH 8.5).

And (3) denaturation C: according to the following steps of 1:20, the denaturation buffer is: (50mM Tris,6M GuaHCl,1mM EDTA,50mM cysteine, pH 8.5).

And (3) denaturation D: according to the following steps of 1:20, the denaturation buffer is: (50mM Tris,8M urea,1mM EDTA,50mM cysteine, pH 8.5).

Under the above denaturation conditions, the denatured sample was stirred at room temperature for 4h, and then centrifuged at 15000rpm/4 ℃ for 30min to collect the supernatant for renaturation studies.

1.9.2 the renaturation conditions are as follows:

renaturation conditions 1: 100mM Tris,0.5M Arg-HCl,0.5M urea,1M NaCl,2mM EDTA,3mM Cysteine,1mM Cysteine, pH 8.5; the proportion is as follows: 1:40.

Renaturation conditions 2: 100mM Tris,0.5M Arg-HCl,2M urea,2mM EDTA,3mM Cysteine,1mM Cystine, pH 8.5; the proportion is as follows: 1:40.

Renaturation conditions 3: 100mM Tris,0.5M Sucralose (sodium cyclamate), 2M urea,2mM EDTA,3mM Cysteine,1mM Cysteine, pH 8.5; the proportion is as follows: 1:40.

Renaturation conditions 4: 100mM Tris,0.25M Sucralose (sodium cyclamate), 2M urea,2mM EDTA,3mM Cysteine,1mM Cysteine, pH 8.5; the proportion is as follows: 1:40.

The renaturation sample is diluted, and then renaturated at 4 ℃ for 2 days and then at room temperature for 2 days.

1.9.3 after renaturation was completed, the renaturation sample was changed to 15mM Tris,0.5M urea, pH8.5, 25mM cyclohexylaminoethanesulfonic acid, 5% dimethylformamide by gel filtration chromatography with Sephadex G25 as the chromatography filler. The optimal renaturation conditions were confirmed based on the comparison of SDS-PAGE analysis (FIG. 3), and were as follows: and (3) denaturation conditions: the denaturation buffer solution is 50mM of trihydroxymethyl aminomethane, 8M of urea,1mM of ethylene diamine tetraacetic acid, 20mM of dithiothreitol and pH8.5, the denaturation proportion is inclusion body, the denaturation buffer solution is 1g to 20mL, and the denaturation time is 4 hours; renaturation conditions are as follows: 100mM Tris,0.25M cyclamate, 2M urea,2mM EDTA, pH8.5,1mM cystine, 3mM cysteine; denaturation buffer renaturation buffer ═ 1:20, renaturation was carried out at 4 ℃ for 2 days and then at room temperature for at least 2 days.

1.10 based on the above renaturation conditions, 5g of inclusion bodies were denatured in a denaturation volume of 100mL in a condition of 50mM Tris,8M urea,1mM EDTA,20mM dithiothreitol and pH8.5, and renatured in a renaturation volume of 2L. Firstly, capturing protein by the renaturation sample by adopting an anion exchange chromatography, using Q Sepharose FF as a chromatography filler, and using a Buffer A as a balance solution: 15mM Tris,0.5M Urea,25mM cyclohexylaminoethanesulfonic acid, 5% dimethylformamide, pH 8.5; the eluent was Buffer B: 15mM Tris,0.5M urea,25mM cyclohexylaminoethanesulfonic acid, 5% dimethylformamide, 1M sodium chloride, pH 8.5. Firstly, balancing a chromatographic column by using a balance solution, diluting the renaturation solution by using Buffer A to 10mS/cm, then loading, continuously washing by using the balance solution Buffer A after loading until ultraviolet reaches a base line, then carrying out gradient elution with gradually increased salt concentration by using a mixed solution of the balance solution and an eluent, wherein the mixing ratio is 10%, 20%, 30% and 50% of Buffer B, and respectively collecting corresponding main peak proteins. In a more specific example, a total of 470mg of purified protein was collected based on 2L of renaturation solution, and the protein concentration was 0.5mg/mL (for chromatogram and electrophoretogram, see FIG. 4).

1.11 Secondary purification of captured samples Heparin affinity purification was used, and the equilibration and elution were run through anion exchange chromatography (Buffer A and Buffer B). Firstly, a chromatographic column is balanced by using a gentle Buffer solution, a sample is diluted by one time by using Buffer A, then the sample is loaded, after the sample loading is finished, the balance solution is continuously used for flushing until ultraviolet reaches a base line, then, an eluent Buffer B is used for carrying out linear elution with gradually increased salt concentration in 10 column volumes for elution, and a main peak is collected. In a more specific example, a total of 44.1mg of purified protein was collected based on 235mL (117.5mg) of capture protein, at a protein concentration of 1.47mg/mL (chromatograms and electropherograms are shown in FIG. 5).

1.12 final purification of the final product high purity protein was obtained by reverse phase chromatography with POROS50R2 as the chromatographic packing and the balance: 0.1% TFA, 5% ACN; the eluent is: 0.1% TFA, 95% ACN. Firstly, balancing a chromatographic column by using a balance liquid, then loading, continuously washing by using the balance liquid after loading until ultraviolet reaches a base line, then eluting by using a mixed solution of the balance liquid and an eluent in an elution method of linearly increasing the concentration of an organic solvent in 10 column volumes, and collecting a main peak. In a more specific example, a total of 16.2mg of purified protein was collected based on 15mL (22mg) of affinity purified protein, at a protein concentration of 0.81mg/mL (chromatograms and electropherograms are shown in FIG. 6).

1.13 reverse phase purification of samples for formulation and possible lyophilization. The method specifically comprises the following steps: 1) removing organic solvent from the sample collected by reverse phase chromatography by gel chromatography (desalting with G25) to 80mM glycine pH4.0 buffer solution, and making into preparation; 2) removing the organic solvent from the sample collected by the reverse phase chromatography in a freeze-drying mode, dissolving the freeze-dried sample by using 80mM glycine pH4.0 buffer solution, and preparing the preparation; 3) samples collected by reverse phase chromatography were replaced by ultrafiltration concentration to 80mM glycine pH4.0 buffer and further concentrated by ultrafiltration to complete the formulation. The operation of the lyophilized formulation was as follows: and combining the samples with the purity meeting the requirements, freeze-drying the samples until the water content and the organic solvent residue meet the requirements, sealing the samples, and freezing and storing the samples. The formulation of the preparation buffer solution can be as follows: 5.0mg/mL sucrose, 25mg/mL glycine, 3.7mg/mL glutamic acid, 0.1mg/mL sodium chloride, 0.1mg/mL polysorbate, pH 4.5. The final formulation product was either frozen in liquid form (-20 ℃) or lyophilized to solid form and stored at low temperature (4 ℃). The electrophoresis and mass spectrometry analysis results of the final product are shown in FIG. 7, the molecular weight of the final product is detected by liquid mass spectrometry, and the detection result shows that the molecular weight of the final product is 24246.6Da and is very close to the theoretical molecular weight of 24248.1Da of the protein dimer. The above data are sufficient to demonstrate that the final BMP2-T protein obtained based on this process exists as a dimer. In this example, the overall recovery of BMP2-T protein expression and purification is shown in Table 1.

TABLE 1 calculation of BMP2-T protein expression and purification recovery

EXAMPLE 2 preparation of BMP2

2.1 BMP2 encoding gene synthesis, vector construction and expression strain construction

Replacing the 283-396 position of a gene for coding BMP2 protein (283-396 position of unipro No. P12643) with a codon preferred by escherichia coli on the premise of not changing an amino acid sequence so as to be suitable for expressing in the escherichia coli, and adding an initiation codon ATG before the optimized sequence to obtain the optimized gene sequence of BMP2, wherein the sequence of the gene is shown in a sequence table SEQ ID NO: 3, respectively.

The sequence table SEQ ID NO: 3 was inserted between the NcoI/XhoI cleavage sites of the vector pET-28a (+) to obtain a recombinant plasmid (FIG. 8). In the recombinant plasmid, the inserted double-stranded DNA molecule is fused with partial DNA of the plasmid, and the expression sequence table SEQ ID NO: 4.

The recombinant plasmid is introduced into Escherichia coli BL21(DE3) by chemical transformation, and strains with high expression level are selected by kana resistant plate coating and monoclonal screening, and finally BMP2 expression strains with high expression level are obtained.

2.2 fermentation and Inclusion Collection of the strains

The fermentation curves of the fermentation process and the inclusion body collection process of the bacterial cells are the same as that of the BMP2-T process, and the fermentation curve of the 20L fermentation tank is shown in FIG. 9.

2.3 BMP2 Inclusion body purification, renaturation and purification

Active BMP2 protein was obtained by a two-fold dilution renaturation method, in which the inclusion bodies were first solubilized with a denaturing buffer consisting of: 100mM Tris,8M urea,1mM EDTA,20mM dithiothreitol, pH8.0, in a denaturation ratio of inclusion body to denaturation buffer of 1g to 20mL overnight, followed by centrifugation at 15000rpm for 20 minutes to collect the supernatant for protein purification under subsequent denaturation conditions. The chromatographic packing is SP sepharose Big beads, and the equilibrium Buffer is Buffer C: 20mM tris,8M urea, pH 6.0; elution Buffer D: 20mM Tris,8M urea,1M NaCl, pH 6.0. Firstly, a chromatographic column is balanced by using a balance solution, a sample is diluted according to a denatured protein solution and a balance Buffer solution in a ratio of 1:2, then the sample is loaded, after the sample loading is finished, the sample is continuously washed by using balance solution Buffer C until ultraviolet reaches a base line, then gradient elution with gradually increased salt concentration is carried out by using a mixed solution of the balance solution and an eluent, the sodium chloride concentration is increased from 0M to 1M in 2 column volumes, and a protein main peak is collected (a purification chromatogram and an electrophoresis chart of the denatured sample are shown in figure 9). After pooling the protein peaks of interest, the buffer was replaced by gel filtration chromatography to 50mM Tris,6M guanidine hydrochloride, pH8.0, and dithiothreitol was added to a final concentration of 20mM and denatured overnight.

2.4 renaturation of the denatured sample by the first step of dilution, the renaturation solution can be: 50mM tris, 1M sodium chloride, 0.75M cyclohexylaminoethanesulfonic acid, 1mM ethylenediaminetetraacetic acid, pH8.5,1mM oxidized glutathione, 2mM reduced glutathione. Renaturation solution 1:50, room temperature renaturation for 3 days. After the first renaturation step, the renaturation solution is diluted by 20mM Tris, 4M urea and pH8.0 buffer solution according to the proportion of 1:4 and is renatured for 3 days at room temperature.

2.5 after renaturation is finished, cation (SP.BB) chromatography purification is carried out on the renaturation sample, an equilibrium buffer solution (20mM PB, 4M Urea, pH6.0) and an elution buffer solution (20mM PB, 4M Urea,1M NaCl, pH6.0) are carried out, after loading is finished, the balance buffer solution is used for flushing until the conductivity and ultraviolet stability are achieved, a linear elution method is adopted, the salt concentration is gradually increased to 1M from 0M in 15 column volumes, and a target protein peak is collected. In a more specific example, 100mL of renaturation sample was obtained based on 4.3g of inclusion bodies, and a total of 43mL of purified protein was collected at a protein concentration of 0.43 mg/mL. (purification chromatograms and electrophorograms of denatured samples are shown in FIG. 10).

2.6 the purified sample is further purified by heparin affinity, the chromatographic packing is heparin sepharose, the sample is loaded after the chromatographic column is balanced by a balance liquid consisting of 20mMPB, 4M urea and pH8.0, after the loading is finished, the sample is washed by a balance buffer solution until the electric conductivity and the ultraviolet stability are achieved, the gradient elution method is adopted for elution (0%, 30% and 50% of eluents are sequentially adopted), and corresponding main peaks are respectively collected (the purification chromatogram is shown in figure 11). In a more specific example, a total of 30mL of purified protein was collected based on 15mg of capture protein, with a protein concentration of 0.5 mg/mL.

2.7 final purification of the final product high purity protein was obtained by reverse phase chromatography with POROS50R2 as the chromatographic packing and the balance: 0.1% TFA, 5% ACN; the eluent is: 0.1% TFA, 95% ACN. Firstly, balancing a chromatographic column by using a balance liquid, then loading, continuously washing by using the balance liquid after loading until ultraviolet reaches a base line, then eluting by using a mixed solution of the balance liquid and an eluent in an elution method of linearly increasing the concentration of an organic solvent in 10 column volumes, and collecting a main peak. Based on 10.36mg of capture protein, a total of 7mL of purified protein was collected, with a protein concentration of 1.48 mg/mL.

2.8 liquid formulations of the final product and possible methods of lyophilization formulation are as described above for example BMP 2-T. Buffer was replaced to formulation buffer by means of gel chromatography (desalting with G25): 5.0mg/mL sucrose, 25mg/mL glycine, 3.7mg/mL glutamic acid, 0.1mg/mL sodium chloride, 0.1mg/mL polysorbate, pH4.5, final protein concentration 1.5 mg/mL. The final formulation product was either frozen in liquid form (-20 ℃) or lyophilized to solid form and stored at low temperature (4 ℃). The electrophoresis results of the final product are shown in FIG. 11. And (3) detecting the molecular weight of the final product by liquid-mass spectrometry, wherein the detection result shows that the molecular weight of the final product is 26057.0Da and is very close to the theoretical molecular weight of 26058.16Da of the protein. Based on this data, it is well demonstrated that the final BMP2 protein obtained based on the above process exists in the form of a dimer. (the electrophoretogram and mass spectrometry results are shown in FIG. 12).

EXAMPLE 3 preparation of DS-BMP2-T

3.1 Synthesis of DS-BMP2-T encoding Gene, vector construction and construction of expression Strain

The 290-396 gene encoding the BMP2 protein (unipro No. P12643, 283-396) was modified by introducing the R291K mutation. In order to improve the binding force of the BMP2 mutant to collagen in a bone matrix, a bone-binding polypeptide (with the sequence of DSSDSSDSSDSSDSSDSS) is introduced into the N terminal of the protein on the basis of a core protein sequence, a connecting sequence (with the sequence of GSGS) is introduced into a connecting region of the bone-binding polypeptide and a BMP2-T sequence, and in order to mention the expression of the protein, a TRX tag (thioredoxin protein) form is expressed through fusion at the N terminal, and finally high expression of the protein is obtained. The final expression protein sequence is shown as SEQ ID NO: and 6. On the premise of not changing the amino acid sequence, the codon is replaced by a codon preferred by escherichia coli (high-frequency use) so as to be suitable for expression in the escherichia coli, and finally, the optimized coding gene sequence of the DS-BMP2-T is shown as a sequence table SEQ ID NO: 5, respectively. The sequence table SEQ ID NO: 5, inserting the double-stranded DNA molecule artificially synthesized by the base complementary pairing principle into KpnI/XhoI enzyme cutting sites of a vector pET-32a (+) to obtain the recombinant plasmid. In the recombinant plasmid, the inserted double-stranded DNA molecule is fused with a part of DNA of the plasmid itself to form a fusion gene. The recombinant plasmid (FIG. 13) is introduced into Escherichia coli BL21(DE3) by chemical transformation, and strains with high expression level are selected by means of kana antibody plate coating and monoclonal screening, and finally, the DS-BMP2-T recombinant expression strain with high expression level is obtained.

3.2 cell fermentation and cell disruption methods were the same as BMP2-T example 1.

3.3 renaturation of the DS-BMP2-T inclusion bodies

Firstly, adding solid urea into the bacteria-breaking supernatant to the final concentration of 8M and the dithiothreitol concentration of 20mM, denaturing at room temperature for 4 hours, then centrifuging at 15000rpm for 20 minutes, and taking the supernatant to obtain a denatured liquid.

Renaturation is carried out by adopting a dilution method, and the renaturation solution comprises the following components: 100mM Tris, 0.75M arginine hydrochloride, 0.5M urea,1M sodium chloride, 2mM EDTA, pH8.5, 3mM cysteine,1mM cystine. The denaturation solution was diluted in renaturation buffer at a ratio of 1:20 and renatured at 4 ℃ for 2 days and at room temperature for 2 days.

3.3 purification of DS-BMP2-T

After renaturation is finished, concentrating the renaturation solution by using a 10kDa ultrafiltration membrane package and replacing the buffer solution to Q FF balance solution: (15mM Tris,0.5M Urea,25mM CHES, 5% DMF, pH 8.5); the method comprises the steps of firstly using an equilibrium chromatography column, then loading, continuously washing with an equilibrium solution after loading until ultraviolet reaches a baseline, then using a mixed solution of the equilibrium solution and an eluent (15mM Tris,0.5M Urea,25mM CHES, 5% DMF,1M NaCl, pH8.5) to carry out linear elution, specifically increasing the salt concentration from 0M to 0.5M in 10 column volumes, then increasing the salt concentration from 0.5M to 1M in 4 column volumes, and respectively collecting protein main peaks. In a more specific example, 8000mL of renaturation sample was obtained based on 400mL of disrupted supernatant of cells, and 225mg of purified protein was collected at a protein concentration of 7.5mg/mL (chromatogram and electrophoresis chart shown in FIG. 14).

3.4 EK activation, purification and preparation of DS-BMP2-T

In order to improve the efficiency of protein folding and enzyme digestion, the purified protein can be used for enzyme digestion by replacing a buffer solution by a gel chromatography method and collecting a purified product, wherein the gel chromatography packing can be Sephadex G25, and the buffer solution is: (15mM Tris,0.5M Urea,25mM CHES, 5% DMF, pH 8.5). Based on 37.5mg of purified protein, a total of 33mg of purified protein was collected. Directly carrying out EK enzyme digestion on the DS-BMP2-T precursor collected by G25, wherein the optimized enzyme digestion conditions are as follows: the protein concentration is 1mg/mL, the enzyme digestion ratio (mass ratio is 1:500), and the enzyme digestion is carried out overnight at 25 ℃.

Then carrying out anion exchange chromatography on the enzyme digestion sample, wherein the chromatographic packing is Q sepharose High Performance, and the equilibrium solution is as follows: (15mM Tris,0.5M Urea,25mM CHES, 5% DMF, pH 8.5); the eluent is: (15mM Tris,0.5M Urea,25mM CHES, 5% DMF,1M NaCl, pH 8.5); firstly, balancing a chromatographic column by using a balance liquid, then, loading a digestion sample, continuously washing by using the balance liquid after the loading is finished until ultraviolet reaches a base line, then, linearly eluting by using a mixed solution of the balance liquid and an eluent, specifically, increasing the salt concentration from 0M to 0.5M in 6 column volumes, and respectively collecting protein main peaks. After digestion and purification, a total of 10.5mL of purified protein was collected, with a protein concentration of 0.61mg/mL (FIG. 15). The final product was purified in reverse phase, liquid formulations and possibly lyophilized formulations as referred to BMP 2-T.

Example 4 in vitro Activity Studies of BMP2 and analogs thereof

The difference of different administration amounts on the chondrogenesis inducing capability of the cells is indirectly reflected by detecting the content of alkaline phosphatase induced and expressed by BMP2 and analog molecules thereof.

Experimental materials: MC3T3-E1 cell, DMEM + 10% FBS + 1% double antibody, BMP2 control (0.5mg/mL, Hangzhou nine-source gene), BMP2-T (lyophilized powder 1.5mg), cell lysate (Biyuntian), BCA kit (Biyuntian), ALP detection kit (Biyuntian)

5.1 inoculation of MC3T3-E1 cells in 24-well plates, 5X 10⁴One/well, put at 37 ℃/5% CO₂Culturing in an incubator for 24 hours;

5.2 discard original culture medium, and gently rinse cells 2-3 times with PBS, positive control, BMP2-T, BMP2, DS-BMP2-T with administration concentration of 10 μ g/mL, negative control added with complete culture medium, put at 37 deg.C/5% CO₂Continuously culturing for 3 days in the incubator;

5.3 discarding the original culture medium, replacing the new medicament, continuing to culture for 3 days in the same steps;

5.4 discarding the culture medium, rinsing the PSB, adding 150 mu L of cell lysate, standing for 3-5min, collecting the cell lysate, centrifuging at 12000rpm for 5min, and collecting the supernatant.

5.5 Total protein amount was determined according to BCA kit and ALP assay kit Activity of ALP.

The ALP viability calculation equation is as follows: ALP/BCA ═ DEA/mg protein

5.6 by the above experimental approach, the activity of the finally obtained samples of BMP2, BMP2-T, DS-BMP2-T, respectively, was 101.6%, 125.1% and 130.4% of the activity of the control, using commercial BMP2 as control (representing a significant difference compared to the commercial BMP2 control group, P < 0.05).

EXAMPLE 5 in vivo Activity data of BMP2 and its analogs

BMP2 and its analogs were tested for osteogenic activity in Balb/c-nu/nu nude mice (male, about 20 g).

6.1 weighing 4 parts of calcined bone powder, 1g of calcined bone powder each, and irradiating for sterilization for later use.

6.2 taking 500 mu g of each of DS-BMP2-T, BMP2-T and commercial BMP2, diluting to 1mL, filtering and sterilizing, mixing with calcined bone powder respectively, incubating overnight at 4 ℃, freeze-drying, and storing at 4 ℃ for later use. The blank calcined bone powder without the complex factor was used as a Control (Control).

6.3 operation process: a nude mouse was anesthetized with sodium pentobarbital (0.3%) and the triceps muscle of the hind limb was sterilized with iodophor, an incision was made along the tibia, the skin and the fascia were incised, the muscle space was separated, and 10mg of the material was implanted into the muscle space. The muscles and skin were sutured. Sterilizing with iodophor, placing into cage, breeding in single cage, and feeding with normal diet and drinking water.

6.4 materials were obtained 28d after dosing: after euthanasia, the nude mice were cut open to the skin of the operative area, and the material and surrounding muscles were removed and fixed with formalin for 24 h. After fixation the material was decalcified with 10% EDTA 14 d.

6.5 dehydrating and embedding the decalcified material, and finally carrying out paraffin section.

6.6 Paraffin sections were HE stained, photographed under microscopic examination and new bone area calculated.

In this example, the in vivo experiments and HE staining showed no new bone formation (0%) in the control group, and the new bone areas (BV/TV) in the commercially available BMP2 control group, BMP2 group, BMP2-T group and DSBMP2-T group were 71%, 72%, 82% and 85%, respectively (FIG. 16).

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

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Claims

1. A preparation method of human BMP2 and analogues thereof is characterized by comprising the following steps:

(3) denaturing the inclusion body to obtain denatured protein;

2. The method for preparing human BMP2 and its analogs according to claim 1, wherein the coding genes of human BMP2 and its analogs are BMP2-T, DS-BMP2-T, respectively, and the nucleotide sequences thereof are represented by SEQ ID NO: 1. SEQ ID NO: 3.

3. the method for preparing human BMP2 and its analogs according to claim 1, wherein said human BMP2 and its analogs are BMP2-T, DS-BMP2-T, respectively, and the amino acid sequences thereof are represented by SEQ ID NOs of the sequence Listing: 2. SEQ ID NO: 4.

4. the process for preparing BMP2 of human origin and analogs thereof according to claim 1, wherein when the product is BMP 2-T:

5. The process for preparing BMP2 of human origin and analogs thereof according to claim 1, wherein when the product is DS-BMP 2-T: