KR101360375B1 - Recombinant E. coli producing soluble BMP-2 and method for producing soluble BMP-2 using the same - Google Patents

Abstract

The present invention relates to recombinant E. coli producing water-soluble BMP-2 and a method for producing water-soluble BMP-2 using the same. By using the recombinant protein production method according to the present invention, it is possible to produce BMP-2 having a water-soluble biological activity, and thus it can be utilized for therapeutic purposes such as bone formation and bone regeneration.

Description

[0001] The present invention relates to a recombinant E. coli producing BMP-2 and a method for producing the same using water-soluble BMP-2,

The present invention relates to recombinant E. coli producing water-soluble BMP-2 and a method for producing water-soluble BMP-2 using the same.

Bone regeneration proteins are multifunctional growth factors that induce various biological functions such as bone and cartilage formation, tooth formation, bone regeneration, and embryonic stem cell development . These proteins were first noticed in the fact that extracts from the removed parts of some bones in animals induce new bone formation . Twenty BMP proteins have been reported so far. As one of the proteins belonging to the TGF-b family as a class, bone regeneration promoting protein-2 (BMP-2) has a great effect on bone regeneration and bone recovery, (EH Riley et al., Bone morphogenetic protein-2: biology and applications, Clin. Orthop. Relat. Res. 324 (1996) 39-46).

BMP-2 in the context of fracture treatment requires a large amount of biologically active form of BMP-2 for various therapeutic purposes. The method of obtaining BMP-2 from direct isolation from bovine bone has problems due to low productivity and lack of reproducibility. Recombinant BMP-2 has so far been expressed through various expression systems including tobacco plant, CHO (chinese hamster ovary) cell, E. coli , etc. (JM Wozney et al., Wang, Novel regulators of bone formation: molecular clones and activities, Science 242 (1988) 1528-1534). Traditionally, Escherichia coli has attracted much attention in early testing of recombinant expression due to its advantages such as low cost production, rapid growth rate, and high efficiency expression of target protein. Despite these advantages, however, many heterologous proteins expressed in Escherichia coli are expressed in the form of an insoluble inclusion body, which has the problem of offsetting the existing advantages. Therefore, BMP-2, which is currently functionally active through solubilization of aggregates produced in Escherichia coli and then refolding again, (R. Ruppert et al., Human bone morphogenetic protein 2 contains a heparin-binding site which modifies its biological activity, Eur. J. Biochem. 237 (1996) 295-302). However, these refolding methods have disadvantages such as difficulty in increasing scale up, difficult time, and experience-based processes (LD Cabrita et al., Protein expression and Refolding-a practical guide to getting the most out of inclusion bodies, Biotechnol. Annu Rev. (2004) 10: 31-50). In order to overcome the problem of expressing aggregate-type proteins in such E. coli, approaches to fusing aggregation-tendency proteins at the C-terminus of high water-soluble expression-promoting proteins have been made (P. Braun et al., High throughput protein production for functional proteomics, Trends Biotechnol. 21 (2003) 383-388).

Therefore, the present inventors have made efforts to provide a method for producing a large amount of BMP-2 having biological activity. As a result, the present inventors have found that Escherichia coli lysyl tRNA synthetase (LysRS), which is a strong water-soluble derivative capable of expressing various flocculation- BMP-2 was fused to a recombinant expression vector, which was then expressed to produce BMP-2 having a water-soluble biological activity.

SUMMARY OF THE INVENTION

(a) preparing an expression vector comprising a LysRS gene as a fusion partner and a BMP-2 gene as a foreign protein;

(b) introducing the expression vector into a host cell to produce a transformant;

(c) culturing the transformant to induce expression of the recombinant protein and obtaining the same; And

(d) treating the recombinant protein with a protein-cleaving enzyme to isolate the foreign protein from the recombinant fusion protein.

(A) preparing an expression vector comprising a gene encoding a water-soluble expression-promoting protein and a gene encoding a foreign protein;

(b) introducing the expression vector into a host cell to produce a transformant; And

(c) culturing the transformant to induce expression of the recombinant fusion protein and obtaining the recombinant fusion protein.

According to another embodiment of the present invention, there is provided a method for producing an expression vector comprising: (a) preparing an expression vector comprising a gene encoding a water-soluble expression-promoting protein and a gene encoding a foreign protein;

(b) introducing the expression vector into a host cell to produce a transformant;

(c) culturing the transformant to induce expression of the recombinant fusion protein and obtaining the same; And

(d) separating the recombinant fusion protein from the recombinant fusion protein by treating the recombinant fusion protein with a protein-cleaving enzyme.

In another embodiment, the present invention provides a recombinant fusion protein in which a water-soluble expression-promoting protein and a foreign protein are fused and an exogenous protein having a tag in which a water-soluble expression-promoting protein is cleaved from the recombinant fusion protein.

In the present invention, the water-soluble expression-promoting protein may be LysRS or RBD, and the exogenous protein may be BMP-2.

In addition, the expression vector may be characterized in that a nucleotide encoding a protein cleavage enzyme recognition site is linked between a gene encoding a water-soluble expression-promoting protein and a gene encoding a foreign protein, and the water-soluble expression- And the host cell may be characterized by being linked to the N-terminus of the protein, and the host cell may be characterized by being E. coli.

In another embodiment, the present invention provides an expression vector for producing a recombinant fusion protein comprising a gene encoding a water-soluble expression-promoting protein and a gene encoding a foreign protein, and a microorganism transformed with the expression vector.

As used herein, the term "exogenous protein" refers to any protein that a person skilled in the art intends to produce in large quantities and which is capable of expression in a transformant by inserting a nucleotide encoding the protein into a recombinant expression vector.

The term "recombinant fusion protein" as used herein refers to a protein to which another protein is linked or another amino acid sequence is added at the N-terminus or C-terminus of the original foreign protein sequence.

The term "expression vector" as used herein is a linear or circular DNA molecule consisting of a fragment encoding a polypeptide of interest operably linked to an additional fragment provided in the transcription of an expression vector. Such additional fragments include promoter and termination coding sequences. The expression vector also includes one or more cloning start points, one or more selection markers, and the like. Expression vectors are generally derived from plasmid or viral DNA or contain both elements.

As used herein, the term " a water-soluble expression-promoting protein " refers to peptides reported to be capable of expressing a fusion protein in a water-soluble form and includes GST (Glutathione S transferase), maltose binding protein, ubiquitin, thioredoxin, The present invention is not limited thereto, and any water-soluble expression-promoting protein that is generally known can be used without limitation.

By using the recombinant protein production method according to the present invention, it is possible to produce BMP-2 having a water-soluble biological activity, and thus it can be utilized for therapeutic purposes such as bone formation and bone regeneration.

1 is a schematic diagram of a BMP-2 fusion protein (LysRS-BMP-2).
Fig. 2 shows the results of expression of BMP-2 and LysRS-BMP-2 in E. coli.
FIG. 3 shows the results of purification using nickel affinity chromatography of LysRS-BMP-2.
Figure 4 shows the result of SDS-PAGE analysis of isolated LysRS-BMP-2.
Figure 5 shows the results of efficient isolation of BMP-2 from LysRS-BMP-2 by treatment with TEV.
FIG. 6 shows the results of efficient separation of BMP-2 by treatment with TEV in the cell lysate state.
FIG. 7 shows the results of separation and purification of BMP-2 isolated by TEV treatment in cell lysate through nickel affinity chromatography.
Figure 8 shows ALP activity of isolated BMP-2 based on cell staining.
Figure 9 shows ALP activity of isolated BMP-2 using spectroscopic assay.

Preparation of materials

Restriction enzymes for DNA recombination were purchased from NEB (UK), and recombinant human BMP-2 for use as a control was purchased from the R & D system (USA). Bone marrow cell line (C2C12) was obtained from the American Type cell culture collection. DMEM and FBS were purchased from Gibco.

Fabrication of protein expression plasmids

The expression vectors pGE-BMP-2 and pGE-LysRS-BMP-2 capable of expressing the fusion proteins of BMP-2 and LysRS-BMP-2 were prepared by PCR using specific primers, respectively. The primer set for the direct form of BMP-2 is as follows. 5'-GTCA ATT AAT ATG ATG CAA GCC AAA CAC AAA CAG CG-3 '(front primer), 5'-GTC ACT AAG CTT GCG ACA CCC ACA ACC CTC C-3' (back primer). PCR products were digested with AseI / HindIII and the DNA fragments were ligated with pGE-lysRS (SI Choi et al., Protein solubility and folding enhancement by interaction with RNA, PLoS ONE 3 (2008) e2677 ) As an NdeI / HindIII site to construct a plasmid in the form of pGE-BMP-2. In the case of LysRS-BMP-2, 5'-GTC ACT GGT ACC CAA GCC AAA CAC AAA CAG CG-3 'was used as a front primer and the rear primer used was the same as that of direct BMP-2. The PCR product was digested with KpnI / HindIII and the thus obtained DNA fragment was inserted into KpnII / HindIII site in pGE-lysRS-3 to prepare a plasmid in the form of pGE-LysRS-BMP-2. The pGE-lysRS-3 plasmid was a modification of pGE-lysRS, which was in the form of a cassette of LysRS-protein cleaving enzyme TEV cleavage site-histidine tag-MCS, MCS was KpnI-BamHI-EcoRV-SalI-HindIII . Protein expression of these plasmids is regulated by the T7 promoter.

Protein expression and separation purification

Escherichia coli transformed with each plasmid A single colony of the HMS174 (DE3) plysE (Novagen) strain was inoculated into 2 ml LB containing chloramphenicol at a concentration of 50 μg / ml and ampicillin antibiotic at a concentration of 30 μg / ml, Lt; / RTI > 1 ml of the culture was diluted in fresh 20 ml of LB containing the same concentration of antibiotic. Expression of the BMP-2 protein was induced by incubation with 1 mM IPTG at a cell density of 0.5 at OD600 for 3 hours. 10 ml of cells were harvested by centrifugation and dissolved in 0.3 ml PBS. After sonication of the cells, the proteins were divided into total (total), soluble (soluble) and insolublec (insoluble) portions by centrifugation at 4 ° C and 12,000g for 10 minutes and analyzed by SDS-PAGE SI Choi et al., Protein solubility and folding enhancement by interaction with RNA, PLoS ONE 3 (2008) e2677).

BMP-2 isolated from LysRS-BMP-2 and LysRS-BMP-2 by TEV treatment with protein-cleaving enzyme was isolated and purified by nickel chromatography. Next, the nickel column was pre-wetted and homogenized with a buffer of the condition [50 mM Tris-HCl (pH 8.0), 500 mM NaCl and 10% glycerol], and the protein solution of the same buffer condition was applied. After washing with 34.5 mM Imidazole buffer, the proteins were separated using imidazole buffer increasing gradually from 123 mM to 300 mM. The separated proteins were collected and dialyzed using the following storage buffer: 50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 2 mM? -Mercaptoethanol and 0.1% Tween-20.

Cutting of Fusion Proteins via Tobacco Vaccine (TEV) Protein Cleavage Enzyme

Recombinant protein cleavage enzyme TEV (Invitrogen) was added to the cell lysate containing isolated LysRS-BMP-2 or LysRS-BMP-2 according to manufacturer's instructions in the following buffer and temperature conditions [50 mM Tris-Cl (pH 8.0), 0.5 mM EDTA, 1 mM DTT, 30 ° C] for two hours.

Alkaline phosphatase (ALP) experiments

The biological activity of BMP-2 was evaluated using a method of measuring BMP-2 in C2C12 cells followed by measurement of ALP activity using cell staining and spectroscopic methods. The ALP kit was used using cytotoxicity. Simply inoculated 1X0 ⁵ C2C12 cells in 24 well plates and cultured for one day. After washing through PBS, cells were treated with BMP-2 (recombinant BMP-2 according to the present invention or commercially available BMP-2 for use as a control) in DMEM culture medium (FBS final concentration 5%). Two days later, C2C12 cells were fixed with Citrate-Acetone-Formaldehyde fixative solution and stained with alkaline solution for 30 minutes at room temperature in the dark. The stained cells were confirmed by photographs.

ALP activity was measured using a TRACP & ALP kit (Takara) according to the manufacturer's method. For stereoscopic spectrometer (spectroscopic) method and then inoculated with 4X0 ³ C2C12 cells in 96 well plates and cultured for one day. After washing with PBS, the BMP-2 mixture was treated. Two days later, the cells were treated with extraction buffer for 5 minutes at 4 ° C and then incubated at 37 ° C for 1 hour in an ALP buffer containing pNPP (p-nitro-phenyl phosphate) substrate. The reaction was quenched with 0.9N NaOH and the absorbance was measured at 405 nm

Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Expression and Purification of BMP-2 Fusion Protein in E. coli

BMP-2 was fused to the C-terminus of LysRS to form LysRS-BMP-2 for the water-soluble expression of BMP-2 protein in E. coli cytoplasm. In addition, a site recognized by the protein cleavage enzyme TEV and six histidine tags were designed as shown in FIG. 1 by introducing them as a linking peptide between LysRS and BMP-2. These linked peptides were introduced for BMP-2 cleavage from LysRS through one purification using nickel affinity chromatography and subsequent cleavage of the proteolytic enzyme TEV. BMP-2 and LysRS-BMP-2 were expressed at 37 ° C. The degree of water solubility of the expressed BMP-2 and LysRS-BMP-2 proteins was analyzed by SDS-PAGE. The LysRS-BMP-2 fusion protein was more than 90% soluble (Figure 2), whereas the direct form of BMP-2 was almost completely water-insoluble. These results indicate that LysRS dramatically increases the solubility of BMP-2 in fusion form while maintaining high expression levels. This also means that the method of fusing BMP-2 to LysRS is an effective method for expressing BMP-2 in a soluble form in the E. coli system.

After confirmation of aqueous expression, the LysRS-BMP-2 fusion protein was expressed in 1 L culture and purified through a single step nickel affinity chromatography at a progressive imidazole concentration (123 mM to 300 mM) 3). Separated tablet form was analyzed by SDS-PAGE and fractions # 24 to # 35 were collected and dialyzed using a storage buffer. As shown in FIG. 4, the purity of LysRS-BMP-2 was about 90% and the overall separation and purification efficiency was about 9.6 mg / L by SDS-PAGE.

Purification of BMP-2 isolated from fusion protein

As a result of the preliminary experiments, the isolated LysRS-BMP-2 fusion protein did not show any activity, suggesting that the large LysRS structure of 55 kDa in the fusion form has functional activity, but dimer of BMP-2 required for biological activity. And that it would have had a negative impact on formation. It was tested whether the protein-cleaving enzyme TEV could specifically and efficiently cleave the purified LysRS-BMP-2 fusion protein. 5, it was confirmed that 14.4 kDa of BMP-2 was efficiently cleaved from the fusion protein by treatment with the protein-cleaving enzyme TEV. In addition, we examined the ability of the LysRS-BMP-2 fusion protein to cleave by the action of the protein cleavage enzyme TEV in the cell lysate (cell extract) state before the protein separation and purification step. As a result, the TEV enzyme efficiently cleaved the fusion protein even in a cell lysate state (Fig. 6). Next, BMP-2 released from TEV enzyme treatment in cell lysate was separated and purified by nickel affinity chromatography in one step as shown in FIG. The purified and purified BMP-2 protein was used for the activity test. The purity of the protein was found to be approximately 50% by SDS-PAGE analysis, and it was confirmed that this was due to the mixing of 65 kDa protein (Fig. 7).

Functional activity of recombinant BMP-2

BMP-2 is well known to promote the expression of alkaline phosphatase (ALP; orthophosphoric monoester phosphohydrolyase) and ALP activity has also been well used as a typical marker of bone formation. Therefore, since the ALP activity can be an important test for verifying the biological activity obtained through proper folding of BMP-2, the activity of the recombinant BMP-2 according to the present invention can be artificially introduced into the inclusion body in E. coli And compared with commercially available BMP-2 (conveniently referred to as std-BMP-2) obtained by refolding.

C2C12 cells were treated with std-BMP-2 and recombinant BMP-2 at concentrations of 7.3, 14.6, 29.3, 58.6, 117.2, and 234.4 nM, respectively. As a result of applying cell staining, the ALP activity was proportional to the BMP-2 concentration as shown in FIG. Recombinant BMP-2 according to the present invention showed a clear activity although it was about 2-4 times lower than std-BMP-2. Considering the overall purity of the protein, the biological activity of the recombinant BMP-2 protein appears to be in the range of 50-100% relative to the control std-BMP-2 (FIG. 7). As a control, cells were treated with only water, only with storage buffer, with LysRS only as a water soluble partner, and no ALP activity was found here. These results indicate that the final BMP-2 protein obtained by the method fused to LysRS has been correctly folded into a biologically active form.

For more quantitative analysis, ALP activity in C2C12 cells treated with BMP-2 at 4.9, 9.8, 19.5, 39.1, 78.1, and 156.3 nM, respectively, was also confirmed by spectroscopic measurements. As can be seen from FIG. 9, the results were consistent with those obtained by the cytotoxicity assay. From these results, it was found that the recombinant BMP-2 obtained by the LysRS fusion method showed significant functional activity, and it was confirmed that the LysRS fusion method can produce biologically active BMP-2.

[Comparative Experimental Example 1]

1-1: Expression of IL20-BMP-2 Fusion Protein and Measurement of BMP-2 Activity

BMP-2 was fused to IL20 (secretion enhancer) and expressed and purified in yeast. The biological activity of BMP-2 was measured in the same manner as in Example 3, and BMP-2 was found to have little activity 10).

1-2: Expression of DsbA-BMP-2 Fusion Protein and IL1RA-BMP-2 Fusion Protein and Measurement of BMP-2 Activity

DsbA-BMP-2 fusion protein and IL1RA-BMP-2 fusion protein were expressed and purified. The biological activity of BMP-2 was measured in the same manner as in Example 3, and BMP-2 showed little activity (FIG. 11).

[Comparative Experimental Example 2]

Expression and activity measurement of RBD-BMP-2 fusion protein

BMP-2 was fused to the human lysyl tRNA synthetase N-terminal domain (RBD) and expressed and purified. The biological activity of BMP-2 was measured. As a result, the degree of water solubility of the isolated BMP-2 was slightly lower than that of the case using LysRS (FIG. 12), and the separated BMP-2 showed biological activity as in the case of using LysRS (Fig. 13). In addition, BMP-2 is a disulfide-rich protein, and PDI, which is known to promote the formation of disulfide bonds, was also fused with RBD. As a result, it was confirmed that BMP-2 was activated by RBD regardless of PDI even when PDI was fused together with RBD (FIG. 14).

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (14)

(a) [gene encoding a water-soluble expression-promoting protein] - [nucleotide coding for a protein cleavage enzyme recognition site] - [gene encoding 1 to 6 histidine] - [gene encoding BMP-2] To produce an expression vector linked as described above;
(b) introducing the expression vector into a host cell to produce a transformant; And
(c) culturing the transformant to induce expression of the recombinant fusion protein and obtaining the recombinant fusion protein.
The method according to claim 1,
Wherein the water-soluble expression-promoting protein is LysRS or RBD.
delete delete The method according to claim 1,
Wherein the water-soluble expression-promoting protein is linked to the N-terminus of the BMP-2 protein.
The method according to claim 1,
Wherein the host cell is Escherichia coli.
(a) [gene coding for a water-soluble expression-promoting protein] - [nucleotide coding for a protein-cleaving enzyme recognition site] - [gene coding for 1 to 6 histines] - [gene encoding BMP-2] To produce an expression vector linked as described above;
(b) introducing the expression vector into a host cell to produce a transformant;
(c) culturing the transformant to induce expression of the recombinant fusion protein and obtaining the same; And
(d) separating the BMP-2 protein from the recombinant fusion protein by treating the recombinant fusion protein with a protein-cleaving enzyme.
[Gene coding for a water-soluble expression-promoting protein] - [nucleotide coding for a protein cleavage enzyme recognition site] - [gene coding for 1 to 6 histines] - [gene coding for BMP-2] An expression vector for producing a recombinant fusion protein.
9. A microorganism transformed with the expression vector of claim 8.





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