KR20030085337A - Vector for expressing human BMP-2 and the process for mass preparation of BMP-2 - Google Patents

Abstract

PURPOSE: A human derived BMP-2 protein expression vector and a method for producing BMP-2 protein using the same vector are provided, thereby effectively mass-producing the BMP-2 protein having improved bone formation and bone induction activities. CONSTITUTION: A pair of primers specific to human derived BMP-2 gene are provided. A method for producing an Escherichia coli mutant producing the human derived BMP-2 protein comprises the steps of: (1) PCR amplifying the human derived BMP-2 gene using a pair of primers; (2) inserting the amplified human derived BMP-2 gene into a vector pGEM-T to produce a recombinant shuttle vector pBMP-T; (3) inserting the BMP-2 gene contained in the recombinant shuttle vector into an E. coli expression vector pRSET to produce a recombinant expression vector pBMP-RSET; (4) cloning the recombinant expression vector pBMP-RSET to E. coli BL21 pLysS to produce E. coli BMP-RSET(KCTC 18099P). A method for producing the BMP-2 protein is characterized by culturing E. coli BMP-RSET(KCTC 18099P).

Description

Vector for expressing human JPM-2 and the process for mass preparation of JPM-2 protein expression vector

The present invention relates to a BMP-2 protein expression vector derived from humans and a method for mass production of BMP-2 using the same. More specifically, to prepare a primer for selectively amplifying a human-derived BMP-2 (Bone Morphogenetic Protein-2) gene; Producing a recombinant expression vector by cloning the BMP-2 gene into an expression vector modified so that the BMP-2 gene obtained using the primer is controlled by a T7 promoter in the vector; The present invention relates to a method for producing E. coli mutant strains capable of producing recombinant BMP-3 protein having excellent bone inducing ability by introducing into transformed E. coli, and producing a large amount of recombinant BMP-2 protein therefrom.

Born Morphogenetic protein (BMP) is a member of the TGFβ-superfamily. When the bone matrix of the demineralized bone was inserted into the muscle of the rat, ectopic bone formation was observed at the site where the bone matrix was inserted. Based on these experiments, it has been demonstrated that in order for bone to grow, it must contain a substance that induces the differentiation of undifferentiated cells of the cell-producing cell population in the bone matrix. Bone: formation and autoinduction. Science 150: 893-899 (1965). This protein component present in the bone matrix was named BMP [Urist MR, Strates BS. Bone morphogenetic protein. J. Dental Res 50: 1392-1406 (1971).

Subsequently, studies have been attempted to separate BMP proteins from animal bones of various species for the purpose of treating bone diseases, but pure BMP has not been purified. However, in the process, it has been found that the mixture containing BMP has excellent bone inducing ability, thereby increasing the possibility of using BMP as a therapeutic agent for bone related diseases.

Meanwhile, BMP-1,2,3 and 2b (now BMP-4) genes have been cloned [Wozney JM et al ., Novel regulations of bone formation: molecular clones and activities. Science 242: 1528-1534 (1988)], after which the genes of the BMP family have been cloned and at least 13 BMPs have been discovered.

Among them, BMP-2, as well as to strongly induce allograft sex (autologous) and heterogeneous tissue property (heterologous) bone formation in in vivo, osteoblast cells (preosteoblast) or undifferentiated stem cells in in vitro (stem cell) Is known as a potent inducer of bone formation that differentiates into osteoblasts. In addition, when BMP-2 is injected into muscle in vivo , ectopic bone formation occurs, and when BMP-2 is treated to C2C12 cells, which are mouse premyoblastic cell lines, these cells become muscle cells. It has been found that the differentiation of the furnace into expression and expression of the marker gene of osteoblasts.

The BMP-2 protein consists of a total of 396 amino acids and has a molecular weight of 44.7 KDa. BMP-2 is not made active from the outset, as is the case with other growth factors or peptide hormones. That is, BMP-2 protein peptides translated from mRNA are relatively short secretory leaders located at the N-terminal, propeptide and C-terminal occupying the largest portion. It consists of three parts of a mature peptide located at the secret leader sequence, which is removed after the protein is secreted, and is a propeptide moiety that usually has 3 to 4 glycosylation sites. Is removed when the protein is activated. The mature peptide located at the C-terminus has seven Cysteine residues involved in disulfide bonds, and the closeness of the proteins belonging to the TGF-βsuperfamily is determined based on the similarity of the amino acid sequence of this region. Done.

The active form of BMP-2 consists of 114 amino acids and has a molecular weight of about 12.9 KDa. They usually form dimers of mature form, some of which contain propeptides. While these mature peptides may form homodimers, they often form heterodimers with other BMP protein types, and in some cases these heterodimers are more than homodimers. It also shows strong activity.

A comparison of the amino acid sequences of the BMP genes identified so far revealed that all BMP subfamily except BMP-1 belong to the TGF-βsuperfamily. Recently, the growth and differentiation factor (GDF) family has been found to belong to the TGF-β superfamily. Among the various proteins belonging to the TGF-βsuperfamily, BMP-2 is the basic type that best preserves the amino acid sequence and its function. The limb bud determines the embryo-ventral axis of embryos during the development of higher animals. , tooth bud, hair, gland and suture formation not only act as an important mediator of epithelial mesenchymal interaction, but also to osteoblast differentiation It is an important extracellular signaling substance that plays a wide range of roles.

Despite the useful effects of these BMP-2 proteins, studies on human BMP-2 are still insignificant, although the amount of BMP-2 is present in cells involved in bone formation and in other cells. This is because there is a limit to the human sample from which bone can be obtained even if it is very small and a considerable amount is present in the bone.

In recent years, various studies have been conducted focusing on the availability of BMP-2 as described above. By applying genetic engineering method, DNA of human BMP-2 is cloned into animal CHO cells and expressed. Although BMP-2 protein is produced, its expression is so small that it is sold only for expensive experiments, and there has been a continuous demand for mass production of BMP-2 for medical use.

As described above, in order to mass-produce the BMP-2 protein, which is useful because it has excellent bone induction ability, but has difficulty in mass production, the present inventors have prepared a primer capable of selectively amplifying the BMP-2 gene and using the PCR. By carrying out the method, the cloning site of the human-derived BMP-2 gene was correctly designed and amplified. In addition, a recombinant expression vector was prepared by modifying an expression vector such that the amplified human-derived BMP-2 gene is regulated by a T7 promoter in the vector, and cloning the gene, thereby introducing the transformed E. coli mutant strain. After the preparation of the present invention, it was found that the E. coli mutant of the present invention can produce a large amount of recombinant BMP-2 protein.

Accordingly, an object of the present invention is to provide a method for producing a large amount of recombinant BMP-2 protein derived from human using E. coli.

Another object of the present invention is to provide an E. coli mutant strain that produces a recombinant BMP-2 protein.

Still another object of the present invention is to provide a recombinant BMP-2 protein expressed in the E. coli mutant strain and a method for purifying the same.

1 is a nucleotide sequence of a primer capable of selectively amplifying a human-derived BMP-2 gene.

RSETF: forward primer, RSETB: backward primer

Figure 2 is a photograph of the electrophoresis of the result after the amplification of the BMP-2 gene derived from humans by performing PCR using the primer of FIG.

Lane 1: size marker

Lanes 2 and 3: amplified mature BMP-2 gene

3 is a result of electrophoresis after cleavage of a recombinant shuttle vector pBMP-T prepared by cloning a PCR-amplified human BMP-2 gene fragment into a shuttle vector.

Lane 1: λ / Hind III size markers for DNA size identification

Lane 2: recombinant vector pBMP-T

Lane 3: cleavage of the recombinant vector pBMP-T with restriction enzymes SalI and NcoI

Lane 4: cleavage of the recombinant vector pBMP-T with restriction enzymes BamHI and EcoRI

Lanes 5, 6, 7, 8, 9 and 10: DNA from other transformants was digested with restriction enzymes SalI / NcoI and BamHI / EcoRI

4 is a cleavage map of a recombinant shuttle vector pBMP-T prepared by cloning a PCR-amplified human BMP-2 gene fragment into a shuttle vector.

5 is a result of electrophoresis of the resultant after cleavage of the recombinant expression vector pBMP-RSET prepared by cloning the BMP-2 gene derived from human to the E. coli expression vector.

Figure 6 is a cleavage map of the recombinant expression vector pBMP-RSET produced by cloning the BMP-2 gene derived from human to E. coli expression vector.

Figure 7 is a time-based OD measurement results of E. coli mutant strains expressing human BMP-2 gene.

8 is a result of measuring the time-dependent expression of recombinant BMP-2 protein of E. coli mutant strains expressing human-derived BMP-2 gene using SDS-PAGE and Western blot.

9 is a diagram showing the result of the first purification of recombinant BMP-2 protein from the expressed E. coli mutant cell lysate using ion exchange resin. Here, the part represents the fraction from which the recombinant BMP-2 protein was isolated.

10 is a result of confirming the presence or absence of the recombinant BMP-2 protein by performing a SDS-PAGE and Western blot a part of the first purified fraction.

11 is a result of confirming the presence of recombinant BMP-2 protein by performing the first purified fractions purified by a nickel column and subjected to SDS-PAGE and Western blot.

The present invention relates to a human-derived BMP-2 protein expression vector and a method for mass production of BMP-2 using the same, preparing a primer for selectively amplifying a human-derived BMP-2 gene; Preparing a recombinant expression vector in which the obtained BMP-2 gene is modified to be controlled by the T7 promoter in the vector; It is characterized by providing a recombinant BMP-2 protein having excellent bone inducing ability and E. coli mutant producing the same by introducing it into E. coli and transforming it.

Hereinafter, the present invention will be described in more detail.

E. coli mutant strain producing a recombinant BMP-2 protein derived from humans provided in the present invention;

(1) PCT amplification of a BMP-2 gene derived from humans using a special primer prepared in the present invention;

(2) inserting the PCR-amplified human BMP-2 gene into a pGEM-T vector to produce a recombinant shuttle vector pBMP-T;

(3) preparing a recombinant expression vector pBMP-RSET by inserting a human-derived BMP-2 gene included in the recombinant shuttle vector into an E. coli expression vector pRSET;

(4) preparing the E. coli strain Escherichia coli BMP-RSET by introducing the recombinant expression vector pBMP-RSET into E. coli BL21 pLysS;

It is prepared by a method comprising a.

Human BMP-2 gene is used as the BMP-2 gene of the present invention.

In the present invention, the PCR primers used to obtain human-derived BMP-2 genes are prepared by the present inventors with reference to existing known sequences so as to accurately design and amplify the gene region to be cloned.

In the present invention, pRSET was used as an E. coli expression vector for cloning a human-derived BMP-2 gene. The pRSET includes a T7 promoter region that regulates expression of the gene, and in addition to the T7 promoter region, six histidine- Since it has a tag, it enables partial purification of the recombinant protein produced after expression, thereby advantageously purifying only the desired recombinant BMP-2 protein in high yield.

In general, Escherichia coli used to express foreign genes is E. coli BL21 (DE3) with T7 RNA polymerase. However, the strain is expressed at the basal level even if the expression of the T7 polymerase is not controlled to an appropriate level, so that the growth of the cells when E. coli produces harmful proteins, such as slowing the proliferation of cells There have been many problems in production. Thus, in the present invention, E. coli BL21 pLysS was used as a host E. coli for the expression of BMP-2 gene derived from humans. This strain has the advantage that the expression of T7 RNA polymerase is strongly regulated compared to the existing E. coli, Therefore, there is a very advantageous advantage in mass production.

The method for highly efficient purification of human-derived recombinant BMP-2 protein produced from the BMP-2 producing E. coli mutant strain provided by the present invention is as follows.

(1) crushing E. coli BMP-RSET of E. coli mutant strain, and then purifying the cell eluate on a column packed with CM Sepharose; And

(2) concentrating the first purified eluate using a cutoff membrane; And

(3) second purification by passing the concentrate through a nickel or copper-containing resin;

Characterized in that configured to include.

In order to demonstrate that the E. coli mutant strain provided by the present invention produces recombinant BMP-2 derived from the human BMP-2 gene, biochemical and molecular biological experiments such as PCR and Western blot are performed to carry out recombinant BMP at the molecular level. Characterization of -2 was analyzed. E. coli mutant strain provided by the present invention can be provided as an ideal expression system for mass production, unlike higher biological cells.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited thereto.

Example 1 Amplification and Cloning of BMP-2 Gene from Humans

For amplification of BMP-2 genes derived from humans, BMP-2 from the cDNA library of the BMP-2 gene (Genetics Institute, USA), with reference to the nucleotide sequences contained in the gene bank (NIH, USA), a gene sequencing database. Primers specific for were prepared. The sequence is shown in FIG. In the primer sequence of FIG. 1, the underlined portions are restriction enzyme cleavage sequences and the shaded portions represent the beginning and end of the mature BMP-2 gene, respectively.

PCR was performed using the primers above to amplify the BMP-2 gene derived from humans (MJ thermal cycler PTC 100). At this time, PCR was performed for 30 cycles in the order of denaturation (94 ° C, 1 minute), annealing (55 ° C, 2 minutes), extension (72 ° C, 3 minutes), and final extension was performed at 72 ° C for 10 minutes. .

Electrophoresis of the PCR-amplified BMP-2 gene is shown in FIG. 2. 2, it can be seen that BMP-2 gene derived from human was selectively amplified by PCR using the primer.

Example 2 Preparation of Recombinant Shuttle Vector pBMP-T

The PCR-amplified human BMP-2 gene was inserted into pGEM-T easy vector and introduced into E. coli JM109 to prepare a recombinant shuttle vector pBMP-T for cloning BMP-2 gene.

The human-derived BMP-2 gene fragment PCR-amplified in Example 1 was inserted into a pGEM-T easy vector and introduced into the E. coli JM109 prepared by culturing at 37 ° C. by the calcium chloride method (CaCl ₂ method). E. coli was transformed.

As a result, various transformants were prepared, among which transformants without defects were selected, and the recombinant was prepared to confirm whether the BMP-2 gene of human origin was inserted into the pGEM-T easy vector. Vector pBMP-T was digested with restriction enzymes SalI / NcoI and BamHI / EcoRI and then electrophoresed and the results are shown in FIG. 3.

3, it can be seen that the human-derived BMP-2 gene is completely inserted into the vector. The recombinant shuttle vector prepared from such a transformant was named pBMP-T, and a cleavage map thereof is shown in FIG. 4.

Example 3: Preparation of Recombinant Expression Vector pBMP-RSET

A recombinant expression vector pBMP-RSET was prepared for expressing a human-derived BMP-2 gene included in the recombinant shuttle vector pBMP-T in Escherichia coli. To this end, pRSET, an E. coli expression vector having a T7 promoter region and six histidine tags, was used.

From the recombinant shuttle vector pBMP-T, a 350 kb full gene including a startcodon and a signal sequence of BMP-2 was prepared, and the E. coli in the same direction as the T7 promoter was prepared. Inserted into the recombinant expression vector pBMP-RSET was prepared. That is, since the cleavage site by BamHI and EcoRI exists in the primer prepared in the present invention, the recombinant pBMP-T vector is cleaved with BamHI and EcoRI and then eluted, and this is purified by treating the same restriction enzyme as described above. The recombinant expression vector pBMP-RSET was prepared by mixing with one pRSET vector, binding with T4 conjugated enzyme, and then introducing the transformant into Escherichia coli.

The recombinant expression vector pBMP-RSET was treated with a restriction enzyme to confirm the presence of the BMP-2 gene. The recombinant expression vector pBMP-RSET was digested with restriction enzymes BamHI and EcoRI , followed by electrophoresis. As a result, the presence of 350 bp human BMP-2 gene was present (FIG. 5). The cleavage map of the prepared expression vector pBMP-RSET is shown in FIG. 6.

Example 4: Escherichia coli mutant strain E.coli Preparation of BMP-RSET

The recombinant expression vector pBMP-RSET was introduced into Escherichia coli BL21 (DE3) pLysS cultured at 37 ° C. to prepare E. coli mutant strains producing recombinant BMP-2 protein.

After centrifugation of 10 ml of the E. coli BL21 (DE3) pLysS cell suspension (OD ₆₀₀ = 0.5), the pellet from which the supernatant was removed was resuspended in 1/2 volume of 100 mM calcium chloride (CaCl ₂ ). This was again centrifuged to remove the supernatant and resuspended by adding 1/10 volume of 100 mM calcium chloride.

10 μl of the ligation mixture containing the BMP-2 gene was added to the competent cells thus prepared and allowed to stand for 1 hour on ice. Heat shock was applied at 42 ° C. for 90 seconds to transform the BMP-2 gene into E. coli cells. Then, the mixture was allowed to stand for 5 minutes on ice, and then 10 ml of LB medium (tryptone 10g, yeast 5g, sodium chloride 5g, total 1L) was added and shaken at 37 ° C for 1 hour. 200 μl of these cells were taken, plated in LB-AP agar medium, and cultured at 37 ° C. to prepare an E. coli mutant strain into which the BMP-2 gene was introduced.

The above E. coli mutant strain was named Escherichia coli (BMP-RSET), and was deposited on March 11, 2002 to the Korea Biotechnology Research Institute gene bank with the accession number KCTC 18099P.

Example 5: Expression and Identification of Recombinant BMP-2

The recombinant BMP-2 protein was expressed and identified from the E. coli BMP-RSET.

LB medium containing 10 g tryptone, 5 g calcium chloride, and 5 g yeast extract was used as a medium for culturing E. coli BMP-RSET. After inoculating the E. coli BMP-RSET E. coli mutant strain in the LB medium, the culture was shaken at 37 ℃ and inoculated again in fresh LB medium in 1/100 volume and incubated for about 90 minutes at 30 ℃. When the absorbance of the culture solution was measured to be OD ₆₀₀ = 0.6, 500 µM of IPTG was added thereto, and then shaken again for about 5 hours, and the cultured cells were harvested by centrifugation.

One of the big problems when using E. coli to produce foreign proteins is that they form inclusion bodies in which the protein is expressed in large quantities but most of them are inactive. The reason for this is attributable to an excessive concentration of IPTG, a protein expression inducer. Therefore, in the present invention, it is cultured under the conditions as described above to suppress the production of the inclusion body.

In order to confirm that the BMP-2 protein was expressed from the cultured E. coli BMP-RSET, the E. coli mutant strain was lysed with a cell lysis buffer (Cell Lysis Buffer, 50 mM Tris-HCl, 5% 2-mercaptoethanol, 0.4%). w / v SDS, containing 10 mM EDTA, pH8.0), and then subjected to Coomassie-blue Polyacrylamide gel running using the crushing solution. Western blots were then performed using anti-BMP-2 Lf monoclonal antibodies (R & D systems).

The measurement results of the OD measurement according to the incubation time of the E. coli mutant strain expressing the BMP-2 gene are shown in FIG. 7. Hourly Optical Density (OD) values are shown in Table 1 below.

time One 2 3 4 5 6 7 8 9 10 11 12 OD 0.01 0.03 0.13 0.6 1.26 2.4 3.96 4.88 4.89 5.77 5.42 4.59

In addition, the results of measuring the expression level of BMP-2 protein over time of the E. coli mutant strain expressing BMP gene by SDS-PAGE and Western blot are shown in FIG. 8. As a result, it can be seen that the recombinant BMP-2 production rate of the sample 2 hours after induction was increased.

Example 6: Purification of Recombinant BMP-2

The harvested E. coli BMP-RSET mutant was harvested in 0.1 mM sodium phosphate buffer, and the cells were disrupted using a French press, and the cell eluate was CM Sepharose (Ion exchange resin). After the first purification using a column filled with sepharose) and concentrated using a membrane (membrane) cut off of 10 KDa.

As a result of SDS-PAGE and Western blot of the protein purified first using a CM Sepharose column, an ion exchange resin, it can be seen that BMP-2 protein is separated from fractions 38 to 43 (FIG. 9). .

The concentrated BMP-2 protein thus concentrated was subjected to secondary purification using a nickel resin. Since the gene encoding the histidine protein is contained in the recombinant expression vector pRSET, the recombinant protein also contains six histidine proteins at the N-terminus. Therefore, purification can be easily carried out by passing through a resin containing nickel or copper.

The first purified protein was passed through a nickel resin and washed three times or more with a phosphate buffer (20 mM sodium phosphate, 500 mM NaCl, pH 6.0), followed by 50, 100, 200, 350, 500 mM Proteins were isolated using a buffer in which sequentially added imidazole solution.

The recombinant BMP-2 protein purified as described above was subjected to Coomassie-blue Polyacrylamide gel running, followed by anti-BMP-2 (anti-BMP-2 Lf) monoclonal antibody. Western blot was performed. As a result, it can be seen that recombinant BMP-2 was purified to 80% purity (FIG. 10). Here, the arrow parts represent the recombinant BMP-2 protein parts.

As can be seen through the above embodiment, the present invention is produced from E. coli mutant and E. coli mutant strains capable of producing a large amount of human-derived recombinant BMP-2 protein having excellent bone formation and bone induction ability, and a pharmaceutical composition for bone formation It is a very useful invention because it has an excellent effect of providing a recombinant BMP-2 protein that can be added and used as an active ingredient.

Claims (5)

A primer of the following sequence specific for a BPM-2 gene from human origin. In the method for producing E. coli mutant strain producing BMP-2 protein, (1) PCT amplifying a human-derived BMP-2 gene using the primer of claim 1; (2) inserting the PCR-amplified human BMP-2 gene into a pGEM-T vector to produce a recombinant shuttle vector pBMP-T; (3) preparing a recombinant expression vector pBMP-RSET by inserting a human-derived BMP-2 gene included in the recombinant shuttle vector into an E. coli expression vector pRSET; 4, by introducing the recombinant expression vector pBMP-RSET in Escherichia coli (Escherichia coli) BL21 pLysS E. coli mutant strain Escherichia coli (Escherichia coli) to prepare a BMP-RSET; Method for producing E. coli mutant strains to produce human-derived BMP-2 protein, characterized in that comprises a. The BMP-2 protein of human origin, characterized in that manufacturing by the method of 2, wherein the substrate producing E. coli mutant strain Escherichia coli (Escherichia coli) BMP-RSET ( KCTC 18099P). The E. coli mutant strain of the base material 3 Escherichia coli (Escherichia coli) recombinant BMP-2 proteins, characterized in that production from the BMP-RSET. A composition for bone formation and induction, comprising the recombinant BMP-2 protein according to claim 4 as an active ingredient.