A recombinant BMP2, soluble expression method and application thereof

Abstract

The invention provides a recombinant BMP2, a soluble expression method and an application thereof. The recombinant human bone morphogenetic protein BMP2 with a protein tag is soluble expressed in an engineering strain Escherichia coli BL21 (DE3) and separated and purified. The BMP2 gene is codon-optimized, and different protein tags are applied to the N-termini of the protein peptide segments, so that soluble expression can be achieved in Escherichia coli BL21 (DE3). The recombinant fusion protein is purified by nickel column affinity chromatography. The soluble expression ratio of the target protein is high, which further promotes the development of BMP2 with biological activity and related products.

Description

Technical Field

[0001] The present invention relates to the technical field of bone morphogenetic protein expression, and in particular to a recombinant BMP2, a soluble expression method and application thereof. Background Art

[0002] Bone morphogenetic protein (BMP) is one of the osteogenic signaling molecules in the transforming growth factor β (TGF-β) family and is an osteoinductive factor that stimulates osteogenesis and cartilage regeneration (Urist MR, Lietze A, Mizutani H, et al. A bovine low molecular weight bone morphogenetic protein (BMP) fraction. [J]. Clin Orthop Relat Res, 1982, 162 (162): 219-232.). Diao Aipo and others from Tianjin University of Science and Technology cloned BMP2 and BMP7 mature peptide gene fragments into insect cell-baculovirus protein expression vectors in patent CN201110120294.8, expressed them in insect cell system and separated and purified them with nickel column, and obtained about 40mg of BMP2 or BMP7 mature peptide protein per liter of insect cell culture fluid. They also expressed rhBMP7-2 heterodimer in Escherichia coli BL21 (DE3) in patent CN201410334579.5, and the protein concentration of inclusion body was 98μg / mL after dialysis renaturation. In patent CN201510964178.2, Jiang Hui increased the production of rhBMP2 in Escherichia coli by modifying ribosome binding sites, reaching about 136.3mg / L.

[0003] It can be seen that the expression yield of BMP2 in the prior art is not ideal. Therefore, it is necessary to propose a method for improving the soluble expression of BMP2 to promote the development and industrial application of biologically active BMP2 and related products. Summary of the invention

[0004] In view of the deficiencies of the prior art, the present invention aims to provide a recombinant BMP2, a soluble expression method and application thereof, so as to at least solve one of the many problems arising in the above-mentioned prior art.

[0005] One of the purposes of the present invention is to use codon optimization to molecularly transform and artificially construct the hBMP-2 gene, and to optimize the codons of the amino acid sequence from position 283 to position 396 of the hBMP-2 sequence, a total of 114 amino acids, and replace them with codons preferred by Escherichia coli. The nucleotide coding sequence of BMP2 is obtained by the method of whole gene synthesis as shown in SEQ ID NO: 1.

[0006] Another object of the present invention is to provide the use of the above-mentioned BMP2 in protein production and purification, which can achieve a higher soluble expression ratio of the target protein BMP2.

[0007] Another object of the present invention is to provide a soluble expression method of the above-mentioned BMP2, comprising the following steps: constructing a BMP2 fusion protein expression vector carrying a protein tag, transforming the expression vector into Escherichia coli BL21 (DE3) for inducing expression, and then isolating and purifying to obtain a fusion protein containing BMP2.

[0008] According to an embodiment of the present invention, the protein tag is one of SUMO protein, Cytb5 protein and DsbC protein with 8×(His-Glu) purification tag; the nucleotide sequence of the fusion protein is one of SEQ ID NO: 2-4. Preferably, the fusion protein containing BMP2 in the separated and purified product is one of fusion proteins SUMO-BMP2, Cytb5-BMP2 and DsbC-BMP2.

[0009] According to an embodiment of the present invention, the fusion protein is connected to pET21a(+) to form a plasmid.

[0010] According to an embodiment of the present invention, the separation and purification adopts nickel column affinity chromatography.

[0011] Compared with the prior art, the present invention has the following effects:

[0012] 1. The present invention recombinantly constructs a codon-optimized BMP2 gene, and by adding different protein tags to the N-terminus of the BMP2 peptide segment, the soluble expression of BMP2 is improved, the denaturation, dissolution and renaturation of inclusion bodies are avoided, and the bacterial cell fragmentation liquid is centrifuged, the supernatant is collected, and affinity chromatography is performed using a nickel column to obtain a BMP2 fusion protein containing a protein tag, which simplifies the downstream processing technology and is suitable for industrial applications.

[0013] 2. The BMP2 soluble expression method provided by the present invention has high yield and high soluble expression ratio of the target protein, which has effectively promoted the industrialization of BMP2-related products. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1The diagrams are of the expression plasmids pET21a(+)-SUMO-BMP2, pET21a(+)-Cytb5-BMP2, and pET21a(+)-DsbC-BMP2 used in the present invention.

[0015] Figure 2 The figure is a gel electrophoresis diagram of the SUMO-BMP2, Cytb5-BMP2 and DsbC-BMP2 fusion proteins of the present invention after being expressed in Escherichia coli.

[0016] Figure 3 This is a gel electrophoresis diagram of the SUMO-BMP2, Cytb5-BMP2 and DsbC-BMP2 fusion proteins of the present invention after being expressed in Escherichia coli and separated and purified by a nickel column.

[0017] Figure 4 This is a gel electrophoresis diagram of the DsbC-BMP2 fusion protein of the present invention after being expressed in Escherichia coli and separated and purified by a nickel column.

[0018] Figure 5 This is a gel electrophoresis diagram of the Escherichia coli engineered bacteria containing plasmid pET21a(+)-DsbC-BMP2 after expression in a fermenter. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical solution and beneficial technical effect of the present invention clearer, the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods. It should be understood that the specific implementation methods described in this specification are only for explaining the present invention, not for limiting the present invention.

[0020] The present invention uses methods such as codon optimization and gene full synthesis to achieve molecular modification and artificial assembly of the hBMP-2 gene, and obtains a gene that can efficiently express human bone morphogenetic protein containing a protein tag. According to the amino acid sequence from position 283 to position 396 of the NP_001191.1 sequence published on the National Center for Biotechnology Information (NCBI, http: / / www.ncbi.nlm.nih.gov / ), a total of 114 amino acids, codon optimization is performed and replaced with codons preferred by Escherichia coli, and a nucleotide coding sequence of BMP2 is obtained by a full gene synthesis method as shown in SEQ ID NO 1.

[0021] The present invention provides expression vectors pET21a(+)-SUMO-BMP2, pET21a(+)-Cytb5-BMP2 and pET21a(+)-DsbC-BMP2 of BMP2 fusion proteins carrying different protein tags, wherein SUMO protein, Cytb5 protein and DsbC protein with 8×(His-Glu) purification tags are used as protein tags to form fusion proteins with BMP2 respectively, and a protease recognition site (amino acid sequence: ENLYFQ ↑S, “↑” is a protease cutting site) of TEV Protease (Tobacco etch virus protease) is inserted between the protein tag and BMP2, and the nucleotide sequences corresponding to the three fusion proteins (SUMO-BMP2, Cytb5-BMP2 and DsbC-BMP2) are codon optimized, and the nucleotide sequences of the fusion proteins are as shown in SEQ ID NO: 2, 3, and 4, respectively, were connected to the cloning sites NdeI and XhoI of the plasmid pET21a(+) to form the recombinant plasmids pET21a(+)-SUMO-BMP2, pET21a(+)-Cytb5-BMP2, and pET21a(+)-DsbC-BMP2.

[0022] In order to solve the cumbersome protein separation and purification process of BMP2 in inclusion body form, the present invention provides an Escherichia coli genetic engineering strain Escherichia coli BL21 (DE3) / pET21a (+) -SUMO-BMP2, pET21a (+) -Cytb5-BMP2, pET21a (+) -DsbC-BMP2 that can soluble express BMP2 fusion proteins containing protein tags. After shake flask culture and fermentation tank culture, bacterial cells are collected, and the supernatant after the bacterial cells are crushed contains fusion proteins. After separation and purification by nickel column affinity chromatography, fusion proteins SUMO-BMP2 (molecular weight of about 28kDa), Cytb5-BMP2 (molecular weight of about 28kDa), and DsbC-BMP2 (molecular weight of about 40kDa) are obtained.

[0023] To achieve the above object, the technical solution adopted by the present invention is a method for effectively improving the soluble expression of BMP2 in E.coli, comprising the following steps:

[0024] (1) Construction of expression plasmids for BMP2 fusion proteins containing protein tags

[0025] According to the amino acid sequence published by NCBI (NP_001191.1), the protein tag at the N-terminus of BMP2 can be SUMO protein, Cytb5 protein, DsbC protein, respectively forming fusion proteins SUMO-BMP2, Cytb5-BMP2, DsbC-BMP2, and there is a TEV Protease protease cleavage site between BMP2 and the protein tag. The nucleotide sequence of the fusion protein was codon optimized, and after full gene synthesis, it was connected to the pET21a (+) expression vector. The corresponding plasmid map is shown in Figure 1 ( Figure 1 A is pET21a(+)-SUMO-BMP2, Figure 1 B is pET21a(+)-Cytb5-BMP2, Figure 1 C is pET21a(+)-DsbC-BMP2).

[0026] (2) Efficient expression of fusion proteins SUMO-BMP2, Cytb5-BMP2, and DsbC-BMP2 in BL21(DE3)

[0027] The expression plasmid described in step (1) was transformed into E. coli BL21 (DE3) competent cells, spread on ampicillin-resistant LB solid plates for overnight culture, single colonies were picked and inoculated into LB culture medium for culture as seed liquid, and then transferred to fresh sterile LB culture medium, cultured until the OD600 of the bacterial liquid was 0.6-0.8, and the inducer IPTG was added to induce expression, and then the bacteria were collected by centrifugation. After washing the bacteria, they were ultrasonically broken in an ice bath and centrifuged.

[0028] (3) Isolation and purification of BMP2 fusion protein containing protein tag

[0029] The nickel column was balanced with a balance buffer, and the supernatant sample of the bacterial disruption solution was passed through a microporous filter membrane. The flow rate was controlled to allow the sample to fully combine with the chromatography column filler. Then, the volume flow rate ratio of the elution buffer was adjusted in turn in the purifier, and the imidazole concentration was used for washing to obtain the separated and purified BMP2 fusion protein.

[0030] Example 1 Synthesis of BMP2 containing protein tags and construction of expression plasmid

[0031] The amino acid sequence of human bone morphogenetic protein-2 (BMP2) was obtained from the National Center for Biotechnology Information (NCBI, http: / / www.ncbi.nlm.nih.gov / ), according to the BMP2The amino acid sequence from position 283 to position 396 of the amino acid sequence of the precursor protein (NP_001191.1), a total of 114 amino acids, was optimized and replaced with codons preferred by Escherichia coli. The optimized nucleotide sequence is shown in SEQ ID NO: 1. In addition, SUMO protein, Cytb5 protein, and DsbC protein with 8× (His-Glu) purification tags were used as protein tags to form fusion proteins with BMP2 respectively. The corresponding nucleotide sequences were codon-optimized. When the corresponding genes were synthesized by GenScript Biotech Co., Ltd., restriction endonuclease NdeI and XhoI restriction sites were introduced upstream and downstream respectively. The full gene PCR product and the Escherichia coli expression vector pET21a (+) plasmid were double-digested with NdeI and XhoI, and the recombinant plasmid pET21a (+)-SUMO-BMP2 was constructed by in vitro ligation. pET21a(+)-Cytb5-BMP2, pET21a(+)-DsbC-BMP2, the ligation product was heat-shock transformed into Escherichia coli competent Top10, spread on LB solid plates containing 50 μg / mL ampicillin, cultured overnight at 37°C, picked a single colony and inoculated it into LB medium (containing 50 μg / mL ampicillin), cultured overnight at 37°C 220rpm, centrifuged at 6000rpm, extracted the plasmid and sent it to Sangon Biotech Co., Ltd. for sequencing.

[0032] Example 2 High-efficiency expression of fusion proteins SUMO-BMP2, Cytb5-BMP2, and DsbC-BMP2 in BL21 (DE3)

[0033] The recombinant plasmids pET21a(+)-SUMO-BMP2, pET21a(+)-Cytb5-BMP2, and pET21a(+)-DsbC-BMP2 of Example 1 were transformed into Escherichia coli BL21 (DE3) competent cells, spread on LB solid plates containing 50 μg / mL ampicillin, and cultured overnight at 37°C. After a single colony of an appropriate size grew, a single colony was picked as a template, and colony PCR identification was performed using PCR primers of the target gene sequence. The total reaction volume was 10 μL, and the PCR product was identified by 1% agarose gel electrophoresis. The correct recombinant transformants were identified and inoculated into LB liquid medium containing 50 μg / mL ampicillin, cultured overnight at 37°C 220 rpm as seed solution, and then transferred to 500 mL shake flasks containing 100 mL LB medium (final ampicillin concentration of 50 μg / mL) at a 1% inoculum (volume ratio) for culture. The OD 600When the concentration of isopropyl-β-D-thiogalactoside reaches 0.6-0.8, isopropyl-β-D-thiogalactoside (the final concentration of IPTG is 0.2-1.0mmol / L) is added, and the expression is induced in a shake flask for 12h-16h. The fermentation broth is centrifuged to collect the bacteria, and ultrasonically broken in an ice bath. After 12% SDS-PAGE analysis, only a small amount of target protein is found in the cell debris precipitate, and the fusion protein is mainly present in the supernatant in a soluble form. Gel electrophoresis Figure 2 As shown, in each lane, 1-2: supernatant and precipitate of the bacterial cell lysate of BL21 (DE3) without the target plasmid; 3-5: total protein, supernatant and precipitate of the bacterial cell lysate containing plasmid pET21a (+) -SUMO-BMP2; 6-8: total protein, supernatant and precipitate of the bacterial cell lysate containing plasmid pET21a (+) -Cytb5-BMP2; 9-11: total protein, supernatant and precipitate of the bacterial cell lysate containing plasmid pET21a (+) -DsbC-BMP2.

[0034] Example 3 Isolation and purification of BMP2 fusion protein containing protein tag

[0035] 100 mL of fermentation broth was collected by centrifugation at 6000 rpm for 5 min, and the engineered bacteria were resuspended in 20 mL of equilibrium buffer Buffer A (20 mmol / L Tris, 500 mmol / L sodium chloride, pH 7.5), ultrasonically disrupted in an ice bath, centrifuged at 10000 rpm for 30 min at 4°C, and the cell debris precipitate was discarded. The supernatant was collected as the sample to be separated and purified, filtered through 0.22 μm, and affinity chromatography was performed on a nickel column (column volume of 5 mL). The sample volume was 10 mL, the sample flow rate was 1 mL / min, the elution flow rate was 1 mL / min, the imidazole concentration of the elution buffer was 100 mmol / L, 300 mmol / L, and 500 mmol / L, respectively. Each eluted sample was collected, and the results of 12% SDS-PAGE analysis were shown in Figure 2. Figure 3 As shown, in lanes 1 to 4: supernatant of bacterial cell disruption solution containing plasmid pET21a(+)-SUMO-BMP2, nickel column flow-through, 100mmol / L imidazole elution, 500mmol / L imidazole elution; 5 to 9: supernatant of bacterial cell disruption solution containing plasmid pET21a(+)-Cytb5-BMP2, nickel column flow-through, 100mmol / L imidazole elution, 300mmol / L imidazole elution, 500mmol / L imidazole elution. 10 to 14: supernatant of bacterial cell disruption solution containing plasmid pET21a(+)-DsbC-BMP2, nickel column flow-through, 100mmol / L imidazole elution, 300mmol / L imidazole elution, 500mmol / L imidazole elution.

[0036] The elution concentration conditions of the supernatant of BL21(DE3) / pET21(+)-DsbC-BMP2 bacterial cell disruption solution were optimized. The sample volume was 10 mL, the sample flow rate was 1 mL / min, the elution flow rate was 1 mL / min, and the proportion of elution buffer Buffer B (20 mmol / L Tris, 500 mmol / L sodium chloride, 500 mmol / L imidazole, pH 7.5) was adjusted so that the concentrations of the eluents were 50 mmol / L, 100 mmol / L, 150 mmol / L, 200 mmol / L, 300 mmol / L, and 500 mmol / L imidazole, respectively. Each elution sample was collected, and the results of 12% SDS-PAGE analysis were as follows: Figure 4 shown.

[0037] Example 4 Induced expression of engineered bacteria Escherichia coli BL21 (DE3) / pET21a (+) -DsbC-BMP2 in a 5L fermenter

[0038] The engineered bacteria Escherichia coli BL21(DE3) / pET21a(+)-DsbC-BMP2 was inoculated into 10 mL LB medium (final concentration of ampicillin was 50 μg / mL), cultured at 37°C with a shaker at 220 rpm for 10 h, used as a primary seed solution, and then transferred to 50 mL LB medium (final concentration of ampicillin was 50 μg / mL) at a 1% inoculation amount, cultured at 37°C with a shaker at 220 rpm for 8 h, and transferred to 2 L fermentation medium (yeast powder 20 g / L, peptone 10 g / L, potassium dihydrogen phosphate 13.5 g / L, magnesium sulfate 1 g / L, glycerol 8 g / L) at a 2.5% inoculation amount, and fed-batch fermentation was performed. 500mL feed medium (yeast powder 80g / L, peptone 40g / L, potassium dihydrogen phosphate 13.5g / L, magnesium sulfate 4g / L, glycerol 400g / L), control the dissolved oxygen value at 25%-45%, the culture temperature at 37°C during the bacterial growth stage, pH = 6.8-7.0, when the bacterial density OD600 reaches 50, cool to 25°C, add inducer IPTG (final concentration 0.4mmol / L), and end the culture after 25h of induction. SDS-PAGE analysis is as follows Figure 5 As shown, Figure 5 Lanes 1-3 in A are the total protein, supernatant, and precipitate of bacterial cell disruption solution; Figure 5Lane 1 in B is BL21 (DE3) without the target plasmid, lane 2 is BL21 (DE3) / pET21a (+) -DsbC-BMP2 without the inducer, and lanes 3-14 are the bacteria (diluted 5 times) at 1h, 3h, 5h, 7h, 9h, 13h, 15h, 17h, 19h, 21h, 23h, and 25h of fermentation induction, respectively. The engineered bacteria were cultured in a 5L fermenter, and the bacterial density OD600 of the fermenter culture reached 110-115. The protein concentration of the bacterial cell fragmentation liquid was determined by the Bradford method and combined with the grayscale analysis of ImageJ software. Each liter of fermentation liquid cultured in the fermenter contained approximately 572.4mg of DsbC-BMP2 fusion protein, and the soluble expression ratio of the target protein could reach 87.8%.

[0039] The present invention is not limited to what is described in the specification and implementation modes, and therefore additional advantages and modifications can be easily realized by those skilled in the art. Therefore, without departing from the spirit and scope of the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details, representative devices, and illustrative examples shown and described herein. Sequence Listing <110> Yi Xiang, Zhongkang Pharmaceutical Group (Huangshi) Co., Ltd. <120> A recombinant BMP2, soluble expression method and application thereof <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 342 <212> DNA <213> BMP2 (Artificial Sequence) <400> 1 caagctaaac acaaacagag aaagaggcta aagtcgagct gcaaacgtca tccgttgtac 60 gttgacttct ctgatgtagg ttggaatgat tggattgtgg ctccgccagg ttatcacgcc 120 ttctactgcc acggcgagtg cccgtttccg ctcgcggacc acttgaattc caccaatcat 180 gcgatcgtgc agaccctggt caacagcgtt aactccaaaa tccgaaagc atgttgtgtt 240 ccgacggaac tgagcgcgat tagcatgctg tacctggacg agaacgaaaa ggtggtgctg 300 aagaactatc aagatatggt tgtcgaggc tgcggttgcc gc 352 <210> 2 <211> 723 <212> DNA <213> SUMO-BMP2(Artificial Sequence) <400> 2 atgtcacatg aacacgagca cgaacacgaa cacgagcatg agcacgagca cgaggggcatg 60 agcgattcgg aagtgaatca agaagccaag ccggaggtga agccggaggt tagccgagg 120 acccatatta acctgaaagt tagcgacggc tctagcgaga tcttctcaa atcaagaag 180 accactccgt tgcgccgtct gatggaagcg ttgctaac gtcagggtaa agaatggat 240 tccttgcgct ttctgtacga cggcattcgt acccaggcag atcaacgcc agaggacctg 300 gatatggaag acacgacat catcgaagcg catcgtgaac agatggtgg ttccgagaac 360 ttatattcc aaagcggatc ccaagctaaa cacaacaga gaagaggct aaagtcgagc 420 tgcaaacgtc atccgttgta cgttgacttc tctgatgtag gttggaatga ttggattgtg 480 gctccgccag gttatcacgc cttctactgc cacggcgagt gcccgtttcc gctcgcggac 540 cacttgaatt ccaccaatca tgcgatcgtg cagaccctgg tcaacagcgt taactccaaa 600 atcccgaaag catgttgtgt tccgacggaa ctgagcgcga ttagcatgct gtacctggac 660 gagaacgaaa aggtggtgct gaagaactat caagatatgg ttgtcgaggg ctgcggttgc 720 cgc 747 <210> 3 <211> 723 <212> DNA <213> cytb5-BMP2 (Artificial Sequence) <400> 3 atgtcacatg aacacgaaca cgagcacgaa cacgaacagg agcacgagca cgagggcatg 60 120 gacagcaagt ctacgtgggt tattctgcat cataaggtgt acgacctgac caaattcctg 180 gaagagcacc cgggtggcga ggaagttctc ggtgagcaag cgggtggcga tgctaccgaa 240 aattttgaag atgtcggtca tagcactgat gcgcgtgaac tgagcaagac ctatatcatc 300 ggcgagttgc acccggatga ccgctccaag atcgccaaac cgagcgaaac ccttgagaac 360 ctgtatttcc agtcgggatc ccaagctaaa cacaagaga gaagaggct aaagtcgagc 420 tgcaaacgtc atccgttgta cgttgacttc tctgatgtag gttggaatga ttggattgtg 480 gctccgccag gttatcacgc cttctactgc cacggcgagt gcccgtttcc gctcgcggac 540 cacttgaatt ccaccaatca tgcgatcgtg cagaccctgg tcaacagcgt taactccaaa 600 atcccgaaag catgttgtgt tccgacggaa ctgagcgcga ttagcatgct gtacctggac 660 gagaacgaaa aggtggtgct gaagaactat caagatatgg ttgtcgaggg ctgcggttgc 720 cgc 747 <210> 4 <211> 1077 <212> DNA <213> Dsbc-BMP2(Artificial Sequence) <400> 4 atgggacatg aacacgagca cgaacacgaa cacgagcacg agcacgagca cgaaggtagc 60 gcggcaattc aacagacctt agcaaaaatg ggtattaaaa gcagcgacat ccagccggcg 120 ccggtcgcgg gtatgaac cgttttgacc aacagcggcg tcctgtacat cactgacgac 180 ggcaaacaca tcattcaagg tccgatgtat gatgtgtctg gcactgcgcc ggtgaatgtt 240 accaataaaa tgctgctgaa gcagttgaat gccctggaaa aggagatgat cgtgtacaaa 300 gcgccacaag aaaagcatgt tattacggtg tttaccgata tcacgtgcgg ttactgccat 360 aaactgcatg agcagatggc tgactataac gctctgggca ttaccgtgcg ctatctggcc 420 ttcccgcgtc agggtctgga ctccgatgca gaaaaggaga tgaaggcgat ctggtgcgca 480 aaagataaaa acaaagcgtt cgatgacgtg atggcgggca agagcgttgc tccggcgagc 540 tgtgatgtgg acatcgccga tcattatgtt ctcggcgttc agttgggggt ttctggtacg 600 ccggctgtag tcctgtccaa cggcaccttg gttccgggtt accagcctcc gaaggagatg 660 aaggaatttc tggacgaaca ccaaaaaatg acctcgggca agggttcgga aaacctgtac 720 ttccaaagcg gatcccaagc taaacacaaa cagagaaaga ggctaaagtc gagctgcaaa 780 cgtcatccgt tgtacgttga cttctctgat gtaggttgga atgattggat tgtggctccg 840 ccaggttatc acgccttcta ctgccacggc gagtgcccgt ttccgctcgc ggaccacttg 900 aattccacca atcatgcgat cgtgcagacc ctggtcaaca gcgttaactc caaaatcccg 960 aaagcatgtt gtgttccgac ggaactgagc gcgattagca tgctgtacct ggacgagaac 1020 gaaaaggtgg tgctgaagaa ctatcaagat atggttgtcg agggctgcgg ttgccgc 1111

Claims

1. A method for increasing the soluble expression of BMP2, characterized in that: The steps include: S1. Constructing a BMP2 fusion protein expression vector carrying a protein tag using a protein tag with an 8× (His-Glu) purification tag and recombinant BMP2; The nucleotide sequence of the recombinant BMP2 is shown in SEQ ID NO: 1; The protein tag is DsbC protein; S2. The expression vector obtained in S1 is transformed into competent E. coli for induction expression, and the bacteria are collected after shake flask culture and fermentation culture, and the supernatant is taken after separation and purification to obtain the fusion protein containing BMP2, thereby achieving soluble expression of BMP2.

2. The soluble expression method according to claim 1, characterized in that: The nucleotide sequence of the BMP2 fusion protein is shown in SEQ ID NO:

4.

3. The soluble expression method according to claim 1, characterized in that: The BMP2 fusion protein expression vector carrying the protein tag is formed by connecting pET21a(+).

4. The soluble expression method according to claim 1, characterized in that: The separation and purification adopts nickel column affinity chromatography.

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