KR20140136593A - Soluble expression and purification method of active recombinant human Dkk2 - Google Patents

Abstract

The present invention relates to a soluble expression and a purification method of recombinant protein human Dkk2 having biological activity using the PDI tag of human. The present invention provides a recombinant expression vector including genes coding human protein disulfide isomerase (hPDI) and human Dickkopf2 (hDkk2) genes. In addition, provided are a recombinant microorganism transformed by the recombinant expression vector, a manufacturing method of the hDkk2 recombinant protein (rhDkk2) and the rhDkk2 manufactured by the method.

Description

(Soluble expression and purification method of active recombinant human Dkk2)

The present invention relates to a water-soluble expression and purification method of a human Dkk2 recombinant protein having a biological activity using a human PDI tag.

The Wingless-type (Wnt) signaling mechanism is regulated by the amount of β-catenin, the main protein that regulates Wnt / β-catenin signaling, and is involved in various processes in embryogenesis and cell homeostasis It involves multiple developmental processes, including cell proliferation, differentiation and migration in the form. Mutations in the Wnt protein, a glycolipoprotein, often cause multiple cancers and degenerative diseases. For this reason, it has been shown that Wnt, such as secreted Frizzled related proteins (sFRPs), Wnt inhibitory protein (WIF), Shisa proteins, Norrin, Wise / SOST and Dickkopf (Dkk) Inhibitors of signaling mechanisms are being studied for anti-cancer and anti-angiogenesis.

The human Dkk (hDkk) family is one of the antagonists corresponding to the Wnt signaling mechanism and consists of four members (Dkk1, 2, 3, 4) with 10 cysteine- Conserved domains (Cys1 and Cys2) with a few residues and negatively regulate Wnt signaling in some cancer cells. Among the hDKK family, Dkk2 is a potent inhibitor of Wnt signaling that binds to both the LRP5 / 6 and Kremen receptor (Krm) -like Dkk1 to produce a frizzled receptor (FzR) -Wnt-low density lipoprotein receptor related protein 5 / 6 (LRP5 / 6). The Wnt signaling inhibitory function of Dkk2 induces apoptosis and regulates cell cycle-related genes in renal cancer cells. Therefore, Dkk2 as well as other Wnt antagonists can be used as cancer therapeutic agents and cancer diagnostic biomarkers.

However, despite attempts to isolate the water-soluble Wnt protein from Escherichia coli for use as a therapeutic protein, Dkk2 with palmitoylation and hydrophobic properties due to the conserved cysteine- As with proteins, it was impossible to purify into a water-soluble form. Glutathione S-transferase (GST), thioredoxin (Trx) and maltose binding protein (GST) have been used to render Wnt proteins insoluble and to improve their yield. MBP) were used. Recently, the present inventors have discovered that human protein disulfide isomerase (DNF), one of the strong water soluble tags, has dual functions including disulfide isomerization and chaperon and anti-chaperon analogues. hPDI) was used for the aqueous expression and purification of secreted proteins from E. coli .

On the other hand, International Patent Publication No. 2010-075194 discloses a method for producing a water-soluble active Dkk through thioredoxin (Trx) -Dkk2c fusion protein using thioredoxin as a solubilization molecule. However, when Dkk2 of the present invention is isolated There is no mention of the human PDI tagging system.

It is an object of the present invention to provide a recombinant expression vector comprising a gene encoding a human protein disulfide isomerase (hPDI) and a human Dickkopf2 (hDkk2) gene.

It is another object of the present invention to provide a recombinant microorganism transformed with the recombinant expression vector.

It is still another object of the present invention to provide a method for producing the hDkk2 recombinant protein (rhDkk2) and a hDkk2 recombinant protein (rhDkk2) produced by the production method.

In order to achieve the above object, the present inventors confirmed the expression level of recombinant human Dkk2 (recombinant human Dkk2; rhDkk2) with various tags and selected rhDkk2 tagged with hPDI. The present inventors first purified water-soluble rhDkk2, identified by Western blotting and MALDI-TOR MS analysis, and confirmed biological activity using HEK293 cells, and completed the present invention.

The present invention provides a recombinant expression vector comprising a gene encoding human protein disulfide isomerase (hPDI) and a human Dickkopf2 (hDkk2) gene. Specifically, the hDkk2 gene is represented by SEQ ID NO: 1, and the hPDI gene is represented by SEQ ID NO: 2.

In the present invention, " vector " means a DNA molecule that is replicated by itself, which is used to carry the clone gene (or another fragment of the clone DNA).

In the present invention, an "expression vector" means a recombinant DNA molecule comprising a desired coding sequence and a suitable nucleic acid sequence necessary for expressing a coding sequence operably linked in a particular host organism. The expression vector may preferably comprise one or more selectable markers. The marker is typically a nucleic acid sequence having a property that can be selected by a chemical method, and includes all genes capable of distinguishing a transformed cell from a non-transformed cell. Examples include, but are not limited to, antibiotic resistance genes such as ampicilin, kanamycin, G418, Bleomycin, hygromycin, chloramphenicol, It can be selected appropriately.

The gateway vector system used in the present invention is composed of an entry vector and a destination vector. The entry vector is a vector having attL1 and attL2 at both ends of the target gene. The destination vector is a vector containing attR1 and attR2. The entry vector and the target vector are subjected to LR reaction by attR1 and attR2 of the target vector and attL1 and attL2 of the entry vector. In this process, the target gene contained in the entry vector is transferred to the destination vector. attR1 and attR2 are replaced with attB1 and attB2 sequences.

The present invention also provides a recombinant microorganism transformed with the recombinant expression vector. Specifically, the microorganism is characterized by being Escherichia coli, more specifically, Escherichia coli BL21 (DE3).

The present invention also relates to a method for producing a recombinant microorganism, which comprises culturing the recombinant microorganism in a medium to express an hPDI tag-hDkk2 recombinant protein; Treating the tobacco etch virus (TEV) protease to remove the hPDI tag; And recovering the hDkk2 recombinant protein from which the hPDI tag has been removed. The present invention also provides a method for producing hDkk2 recombinant protein (rhDkk2).

Meanwhile, although the tobacco etch virus (TEV) protease is used in the embodiment of the present invention, the protease may be a thrombin such as thrombin, Factor Xa, Ubiquitin-specific protease, Furin, Genease I or Proteinase K can be applied to the present invention. , But is not limited thereto.

In addition, the present invention provides the hDkk2 recombinant protein (rhDkk2) produced by the above production method.

The issues related to the genetic engineering techniques used in the present invention are described in Sambrook et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2001) Frederick M. Ausubel et al., Current Protocols in Molecular Biology Volume 1,2,3, John Wiley & Sons, Inc. (1994)).

The present invention relates to a water-soluble expression and purification method of a human Dkk2 recombinant protein using a human PDI tag, and a method for separating and purifying a Dkk2 protein having enhanced solubility and stability in E. coli through tagging with the hPDI domain. The Dkk2 protein thus obtained is expected to play an important role as a therapeutic agent for anti-cancer and anti-angiogenesis.

Fig. 1 shows a process for producing an expression vector for overexpression of rhDkk2. (A) shows the process of constructing a rhDkk2 protein expression vector having several tags through a gateway cloning system. (B) a recombinant protein. Each protein has several tags and TEV recognition sites (arrows).
2 is E. coli Expression of rhDkk2 in BL21 (DE3). Each 10 ug of protein was loaded onto a 10% tris-tricine SDS-PAGE gel. M, molecular weight size markers; C, total cell lysate before 0.5mM IPTG induction; T, total cell lysate after induction; S, supernatant of cell lysate after induction.
Figure 3 is for the separation of rhDkk2. (A) rhDkk2 was isolated from E. coli through His affinity, ion exchange and gel filtration column chromatography. Lane 4 shows that hPDI-rhDkk2 was cleaved after treatment with TEV protease. After the anion exchange column, the gel filtration column was used to separate rhDkk2. M: molecular weight marker; lane 1, Escherichia coli cell lysate before IPTG induction; lane 2: supernatant of cell lysate after induction; lane 3: 1 ^st separation of hPDI-rhDkk2 fusion protein (84.6 kDa); lane 4: hPDI tagging with TEV protease (hPDI: 53.8 kDa, Dkk2: 25.1 kDa); lane 5: 2 ^nd separation of rhDkk2 by anion exchange chromatography. (B) hPDI-rhDkk2. (C) gel filtration chromatogram (left) and 10% tris-tricine SDS-PAGE analysis (right) for final isolated rhDkk2. SDS-PAGE gels of the final isolated rhDkk2 were identified with Coomassie blue (left-hand gel) and silver-stained (right-hand gel) solutions. Finally, isolated rhDkk2 was identified by western blot analysis with anti-Dkk2 antibody (insert box).
Fig. 4 shows MALDI-TOF MS analysis results of rhDkk2. To confirm rhDkk2, the reduced sample was precipitated with 10% trichloroacetic acid (TCA) in a final concentration of 10 mM dithiolthreitol (DTT). All cysteine residues of the precipitated protein were transformed into 25 mM iodoacetamide. Samples were loaded onto a 14.4% tris-tricine SDS-PAGE gel. After dyeing the gel, each protein band associated with the predicted protein size was cut out and treated with trypsin at 37 < 0 > C overnight. After extracting the trypsin-treated sample from the gel, MALDI-TOF MS analysis (Voyager-DE ^TM STR) was performed to identify the separated proteins.
Figure 5 shows the biological activity of rhDkk2. (A) HEK293 cells were treated with separate rhDkk2 for 24 h. Each protein was treated at 0, 10, 100, 250, and 500 ng / ml, respectively. A total cell lysate of 20 ug protein was loaded onto a 10% tris-tricine SDS-PAGE gel. Downstream regulatory proteins in Wnt signaling were detected by Western blot. (B) Western blot analysis. Protein levels were calculated as GAPDH concentrations as a standard method.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited by these examples.

< Example  1> Gateway In cloning (Gateway cloning)  Of expression vector with multiple tags

The DNA sequence of hDkk2 (GeneBank: BC075078.2) encoding 259 amino acids was synthesized by the gene synthesis service (Source BioScience Lifesciences, UK, Clone ID 30915595). In order to remove the DNA sequence encoding the signal transduction peptide and amplify the gene, the following two primers were used. And forward 5'-AGTTCG GGTACC AAACTCAACTCCATCAAGTCC-3 'and reverse 5'-CCTCAA TCTAGA TCAAATTTTCTGACACACATG-3' , underlined part represents a restriction enzyme site, Kpn I and Xba I, respectively. The amplified DNA was subcloned into pDONR207 vector having N-terminal tobacco etch virus recognition site (TEVrs; ENLYFQG), and pENTR-Dkk2 was obtained. (pDEST-His6), pHGGWA (pDEST-GST), and pHXGWA (pDEST-GST) were constructed through LR recombination (Invitrogen, CA, USA) in order to construct an expression vector containing several tags for hDkk2 over- -TRX), pHNGWA (pDEST-NusA), pHMGWA (pDEST-MBP) and pDEST-hPDIb'a ', respectively (Figure 1A). The final constructs coding for the hDkk2 fusion protein with multiple tags were confirmed by DNA sequencing (Macrogen, Inc., Daejon, Korea). To cut the tag, a TEV recognition site was inserted between the tag and the target protein (Fig. 1B).

< Example  2> hPDI - rhDkk2 Manifestation of

RhDkk2 of several tagged plasmids was isolated from competent E. coli And transformed with BL21 (DE3) (Norvagen) host cells. Single colonies were cultured at 37 占 폚 in 2 ml LB medium containing 50 ug / ml ampicillin. When the optical density (OD) of the culture solution reached from 0.4 to 0.5 at 600 nm, final 0.5 mM isopropyl-beta -D-thiogalactoside (IPTG) was added for expression of the fusion protein And added to the culture medium. Thereafter, the culture conditions were changed to 180 rpm and 30 캜. Bacterial cells were further cultured for an additional 3 hours. The cultured cells were harvested by centrifugation at 13,000 rpm for 1 minute and then the cell pellet was resuspended in 200 uL lysis buffer (50 mM Tris-Cl, pH 8.0, 0.5 mM EDTA, 5% glycerol (v / v) ). After breaking the cells by sonication, the expression and solubility levels of the fusion proteins were analyzed by 10% tris-tricine SDS-PAGE and calculated with the ImageJ program ( http://imagej.nih.gov/ij ).

All structures with multiple tags overexpressed the rhDkk2 protein, but only rhDkk2 with the MBP, NusA and hPDI tags were expressed water-soluble (FIG. 2). Of these, strains with hPDI-rhDkk2 showed high solubility and stability (Table 1). Therefore, for purification, a strain with hPDI-rhDkk2 was finally selected and cultured.

Expression and solubility levels of rhDkk2 with multiple tags Tagging protein Expression level (%) Solubility (%) rhDkk2 rhDkk2 His6- (3.1 kDa) 98.7 3.7 Trx- (15.7 kDa) 80.6 17.2 GST- (30 kDa) 85.1 32.4 MBP- (43.4 kDa) 79.7 75.7 NuSA- (57.8 kDa) 90.6 21.6 hPDI- (58.3 kDa) 91.9 91.9

< Example  3> rhDkk2 Separation and purification of

To isolate soluble rhDKK2 from E. coli , the hPDI-TEVrs-rhDkk2 construct was transformed into E. coli BL21 (DE3). Single colonies were pre-inoculated in 2 mL LB medium containing 50 ug / ml ampicillin and incubated overnight at 37 ° C. Pre-cultured cells at a final concentration of 0.5% were inoculated into 1 L of fresh LB medium containing 50 ug / ml ampicillin and cultured under the same conditions as above. When cells reached 0.5-0.7 at OD600, a final concentration of 0.5 mM IPTG was added to the culture medium and further incubated for 18 h at 18 C to improve the level of soluble expression of the fusion protein. The cultured cells were harvested by centrifugation at 3500 rpm for 30 minutes and then the cell pellet was resuspended in lysis buffer (50 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, 5 mM) at a cell dry weight ratio of 20 mL / % glycerol (v / v)). Cell lysates were sonicated 20 times for cell destruction and centrifuged at 18,000 rpm for 20 minutes. The supernatant was filtered through a 0.45 um Whatman ^TM syringe filter (GE Healthcare, Bucks, UK) and eluted with binding buffer (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5% glycerol (v / A pre-packed 5 mL HisTrap ^™ FF column (GE Healthcare, Bucks, UK) was used. All column work was performed on the AKTA explorer system (Amersham Pharmacia Biotech, Bucks, UK). To remove unbound proteins, the HisTrap ^™ FF column was washed with binding buffer in 2-4 column volumes (CV) and washed with 8-10 CV of wash buffer (50 mM Tri- HCl, pH 8.0, 500 mM NaCl, 110 mM imidazole, 5% glycerol (v / v)). The hPDI-rhDkk2 fusion protein was then eluted with elution buffer (50 mM Tri-HCl, pH 8.0, 500 mM NaCl, 200 mM imidazole, 5% glycerol (v / v)). The fractions containing the hPDI-rhDkk2 fusion protein were pooled and the fractions containing the hPDI-rhDkk2 fusion protein were dissolved in buffer A (50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 5% glycerol (v / v) The dialysis membrane of the cut-off 12-14 kDa (Viskase Companies, Inc, USA) was dialyzed three times for 2 hours at 4 ° C to dilute the imidazole and NaCl. Next, the separated total proteins were subjected to TEV protease treatment at a TEV protease ratio of 20: 1 (w / w) to cleave the fusion protein. The cleavage was carried out at 18 [deg.] C for 14-16 hours. After proteolytic cleavage, the cleavage reaction mixture was filtered through a 0.45 um Whatman ^™ syringe filter and pre-packed 5 mL HiTrap SP ^™ column (GE Healthcare, Bucks, UK) equilibrated with buffer A to separate rhDkk2 through anion exchange column UK). The column was washed with 4-6 CV of buffer A and then eluted with a linear gradient to buffer B (1 M NaCl in buffer A). The fractions containing rhDkk2 were concentrated to 1 mL using a centrifugation filter with a molecular weight cut-off of 10 kDa (Millipore Corp., MA, USA) or higher, and the supernatant was collected by centrifugation at 13,000 rpm for 5 minutes. For further separation, the collected samples were collected on a Superdex 75 16/60 (GE) column pre-equilibrated with storage buffer (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10 mM DTT, 5% glycerol Healthcare, Bucks, UK). Finally, the isolated rhDkk2 was concentrated and analyzed by 10% tris-tricine SDS-PAGE. Protein concentration in the separation process was measured by Bradford assay using BSA as standard method. The results are shown in Fig. The final yield of rhDkk2 was 0.073 mg / g (dry weight of E. coli cells) and the purity was 94% (Table 2).

Separation step volume
(ml) Wet weight
(Wet weight)
(g) Total protein
(mg / g) water
(%) Bacterial culture 200 0.98 - - Supernatant 20 - 81.6 - 1 ^st purification 42.5 - 4.03 - TEV protease treatment 42.5 - 5.03 - 2 ^nd purification 0.9 - 0.39 - Gel filtration 1.3 - 0.07 94

< Example  4> MALDI - TOF MS  Identification of Recombinant Fusion Proteins by Analysis

10 mM DTT was added to the separated rhDKK2 and then 10% trichloroacetic acid (TCA, 6.1 N, Sigma-Aldrich Corp.) was added to the sample to confirm the rhDKK2 isolated by MALDI-TOF MS analysis . After that, it was quickly left on ice for 5 minutes. After centrifugation of the sample at 12,000 rpm for 10 minutes, the precipitated sample was resuspended in 50 μl iodoacetamide (IAA, Sigma-Aldrich Corp.) 9 uL IA buffer (0.5 M Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 2% SDS, 5% glycerol) and then reacted in the dark at room temperature for 30 minutes. 12 uL of protein sample was loaded onto 10% tris-tricine SDS-PAGE gel. The band associated with rhDkk2 was excised from the gel and treated with 20 uL trypsin solution overnight. All 1 uL protein reactants were mixed in an equal volume of a-cyano-4-hydroxycinnamic acid (10 mg / mL) solution and MALDI-TOF MS was performed using Voyager-DE ™ STR (Applied Biosystems). Data analysis was performed using Data Explorer TM software (Applied Biosystems). The masses of trypsin-treated peptides were all consistent. The isolated rhDkk2 was identified by Western blot (Fig. 3C insert box) and MALDI-TOF MS analysis (Fig. 4).

< Example  5> Triton ( Triton ) X-114 phase separation and Endotoxin ( endotoxin)  analysis

To remove endotoxin from the separate rhDkk2, a final concentration of 1% Triton X-114 was added to the sample and the mixed sample was reacted at 4 占 폚 for 30 minutes. After quickly transferring to 37 ° C, it was centrifuged at 12,000 rpm for 10 minutes. The aqueous phase of the upper layer containing rhDkk2 was carefully collected and used for endotoxin analysis.

Endpoint Chromogenic Limulus Amebocyte Lysate (LAL) test (Lonza, Basel, Switzerland) was performed to determine the amount of residual endotoxin in the aqueous phase containing rhDkk2. Samples at a concentration of 1 ug / mL were dispensed at 50 占 폚 in pre-warmed endotoxin-microplate wells at 37 占 and the same volume of LAL reagent was added to each well. Plates were incubated at 37 占 폚 for 10 minutes, and a pre-heated 100 占 퐇 chromogenic substrate solution was added to each well and reacted at 37 占 폚 for 6 minutes. After this, 100 ul stopping reagent (25% acetic acid (v / v)) was added and analyzed at 405 nm using a spectrophotometer. After the absorbance analysis, the endotoxin concentration was calculated using a standard curve obtained from the endotoxin standard solution (1.0, 0.5, 0.25, 0.1 EU / ml).

As a result, the amount of endotoxin in the isolated protein was higher than 2 EU / ug before treatment with Triton X-114, but the endotoxin level was lower than 0.15 EU / ug after treatment (Table 3).

 Treatment with Triton X-114 The endotoxin level of the isolated protein (EU / ug) Before processing 2.449 After processing 0.115

< Example  6> rhDkk2 Activity measurement

To measure the biological activity of rhDkk2, it was treated with HEK293 cells showing normal Wnt signal transduction. HEK293 cells were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS), sodium bicarbonate (Sigma # S8875), MEM sodium pyruvate (Gibco, # 11360-070) MEM non-essential amino acids (MEM NEAA) (Gibco, # 11140-050) and antibiotics (100 U / ml penicillin G, 100 ug / ml streptomycin sulfate) Were cultured in a minimal essential medium (MEM) (Gibco, # 61100-061) in an incubator containing 5% CO ₂ at 37 ° C. 4-5 × 10 ⁵ cells were plated on a 6-well tissue culture test plate (SPL life When cell confluency was 70 to 80% except for FBS medium, rhDkk2 was cultured for 24 hours at concentrations of 0, 10, 100, 250 and 500 ng / ml, respectively. The whole cell lysate was collected with NP-40 lysis buffer (150 mM sodium chloride, 10% NP-40, 50 mM Tris inhibitor) Gt; SDS-PAGE. &Lt; / RTI &gt;

For Western blot analysis, the gel was transferred to a PVDF membrane (Pall Corp.) and the membrane was blocked with blocking solution (5% skim milk powder, 10 mM Tris-Cl, pH 7.4, 150 mM NaCl). For rhDkk2 detection, membranes were reacted overnight at 4 ° C in blocking solution with anti-Dakk2 antibody (1: 200, Abcam, Ab38594). The membrane was then rinsed four times with TBST (10 mM Tris-Cl, pH 7.4, 150 mM NaCl, 0.05% Tween 20) for 10 minutes. The washed membrane was reacted with peroxidase-labeled anti-rabbit IgG (1: 5000, Abclon, AbC-5003) dissolved in TBST buffer for 1 hour at room temperature and rinsed 4 times with TBST. After washing, the membranes were analyzed using picoEPD (Elpis biotech, Daejeon, Korea).

In Wnt signaling, the level of expression of rhDkk2-related protein was determined by measuring the biological activity of rhDkk2. β-catenin (1: 1000, Cell signaling, # 9562), c-Myc (1: 1000, Sigma, M4439), cyclin D1 (1: 1000, cell signaling, # 2978), GAPDH : 1000, Ab frontier, LF-PA0209) and β-actin (1: 2000, Ab frontier, LF-PA0018) were used.

As a result, when rhDkk2 was treated in HEK293 cells, all downstream-related proteins of the Wnt signaling mechanism were decreased (FIG. 5). Thus, the present inventors could confirm the function of rhDkk2 as an inhibitor corresponding to the Wnt signal transduction-like human Dkk1.

<110> University of Ulsan Foundation for Industry Cooperation <120> Soluble expression and purification method of active recombinant          human Dkk2 <130> ADP-2013-0143 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 681 <212> DNA <213> Homo sapiens <400> 1 aaactcaact ccatcaagtc ctctctgggc ggggagacgc ctggtcaggc cgccaatcga 60 tctgcgggca tgtaccaagg actggcattc ggcggcagta agaagggcaa aaacctgggg 120 caggcctacc cttgtagcag tgataaggag tgtgaagttg ggaggtattg ccacagtccc 180 caccaaggat catcggcctg catggtgtgt cggagaaaaa agaagcgctg ccaccgagat 240 ggcatgtgct gccccagtac ccgctgcaat aatggcatct gtatcccagt tactgaaagc 300 atcttaaccc ctcacatccc ggctctggat ggtactcggc acagagatcg aaaccacggt 360 cattactcaa accatgactt gggatggcag aatctaggaa gaccacacac taagatgtca 420 catataaaag ggcatgaagg agacccctgc ctacgatcat cagactgcat tgaagggttt 480 tgctgtgctc gtcatttctg gaccaaaatc tgcaaaccag tgctccatca gggggaagtc 540 tgtaccaaac aacgcaagaa gggttctcat gggctggaaa ttttccagcg ttgcgactgt 600 gcgaagggcc tgtcttgcaa agtatggaaa gatgccacct actcctccaa agccagactc 660 catgtgtgtc agaaaatttg a 681 <210> 2 <211> 1461 <212> DNA <213> Homo sapiens <400> 2 gacgcaccgg aagaagagga tcatgtcctg gttctgcgca aaagcaactt cgcggaagca 60 ctggccgcac ataaatatct gctggtggaa ttttatgctc cttggtgcgg tcattgcaaa 120 gccctggccc cggagtacgc caaagccgca ggtaaactga aagctgaagg tagcgaaatc 180 cgcctggcaa aggttgatgc tacggaagag agtgacctgg cgcagcagta tggtgtccgc 240 ggctatccta caattaaatt cttccgtaac ggtgataccg catctccaaa agaatatacc 300 gctggtcgcg aggcggacga tattgttaac tggctgaaga aacgcactgg ccctgccgca 360 accaccctgc ctgatggcgc tgctgccgaa agcctggtcg aaagtagcga agttgccgtc 420 attggtttct ttaaggatgt agaatctgac agtgccaaac agtttctgca agcggcagag 480 gctatcgatg acatcccgtt cggcatcacc tctaacagtg acgtattcag taaataccaa 540 ctggataaag acggcgttgt gctgttcaag aaatttgatg aaggtcgcaa caactttgag 600 ggtgaggtga ccaaggagaa cctgctggat tttattaagc acaaccaact gccgctggtt 660 attgaattta cagaacaaac ggcgccgaaa attttcggcg gtgagattaa aacacatatc 720 ctgctgtttc tgccgaagag cgtttctgat tacgatggta aactgagtaa ttttaaaacc 780 gccgcagaat ctttcaaagg taagattctg ttcattttca ttgatagcga ccacacggac 840 aatcagcgta tcctggagtt ctttggtctg aagaaagagg aatgcccggc tgtgcgtctg 900 attacgctgg aagaggaaat gacaaagtac aagccggaga gcgaggaact gactgcagaa 960 cgtatcaccg aattttgtca tcgtttcctg gaggggaaga ttaagccgca tctgatgagc 1020 caggaactgc cggaggattg ggacaaacag ccagtgaaag ttctggtggg gaagaatttt 1080 gaagatgtgg ccttcgatga gaagaagaat gtgtttgtgg agttctacgc cccgtggtgt 1140 gggcactgta aacagctggc gccgatctgg gacaaactgg gcgaaacgta taaagatcac 1200 gaaaatattg tgatcgcgaa aatggattct accgcgaatg aagtagaagc ggtaaaagta 1260 cactcttttc cgacgctgaa attctttcca gcgagcgcgg atcgtactgt cattgattat 1320 aatggcgaac gtactctgga cggctttaag aaatttctgg aaagcggcgg ccaggatggc 1380 gcgggcgatg atgatgacct ggaagacctg gaagaagcgg aggaaccaga catggaggag 1440 gatgacgacc agaaagcggt c 1461 <210> 3 <211> 229 <212> PRT <213> Homo sapiens <400> 3 Gly Gly Thr Lys Leu Asn Ser Ile Lys Ser Ser Leu Gly Gly Glu Thr   1 5 10 15 Pro Gly Gln Ala Ala Asn Arg Ser Ala Gly Met Tyr Gln Gly Leu Ala              20 25 30 Phe Gly Gly Ser Lys Lys Gly Lys Asn Leu Gly Gln Ala Tyr Pro Cys          35 40 45 Ser Ser Asp Lys Glu Cys Glu Val Gly Arg Tyr Cys His Ser Pro His      50 55 60 Gln Gly Ser Ser Ala Cys Met Val Cys Arg Arg Lys Lys Lys Arg Cys  65 70 75 80 His Arg Asp Gly Met Cys Cys Pro Ser Thr Arg Cys Asn Asn Gly Ile                  85 90 95 Cys Ile Pro Val Thr Glu Ser Ile Leu Thr Pro His Ile Pro Ala Leu             100 105 110 Asp Gly Thr Arg His Arg Asp Arg Asn His Gly His Tyr Ser Asn His         115 120 125 Asp Leu Gly Trp Gln Asn Leu Gly Arg Pro His Thr Lys Met Ser His     130 135 140 Ile Lys Gly His Glu Gly Asp Pro Cys Leu Arg Ser Ser Asp Cys Ile 145 150 155 160 Glu Gly Phe Cys Cys Ala Arg His Phe Trp Thr Lys Ile Cys Lys Pro                 165 170 175 Val Leu His Gln Gly Glu Val Cys Thr Lys Gln Arg Lys Lys Gly Ser             180 185 190 His Gly Leu Glu Ile Phe Gln Arg Cys Asp Cys Ala Lys Gly Leu Ser         195 200 205 Cys Lys Val Trp Lys Asp Ala Thr Tyr Ser Ser Lys Ala Arg Leu His     210 215 220 Val Cys Gln Lys Ile 225

Claims (9)

A recombinant expression vector comprising a gene encoding human protein disulfide isomerase (hPDI) and a human Dickkopf2 (hDkk2) gene. 2. The recombinant expression vector according to claim 1, wherein the hDkk2 gene is represented by SEQ ID NO: 2. The recombinant expression vector according to claim 1, wherein the hPDI gene is represented by SEQ ID NO: 2. A recombinant microorganism transformed with the recombinant expression vector according to claim 1. The recombinant microorganism according to claim 4, wherein the microorganism is E. coli. The recombinant microorganism according to claim 5, wherein the Escherichia coli is BL21 (DE3). Culturing the recombinant microorganism according to claim 4 in a medium to express an hPDI tag-hDkk2 recombinant protein;
Treating the tobacco etch virus (TEV) protease to remove the hPDI tag; And
And recovering the hDkk2 recombinant protein from which the hPDI tag has been removed. A recombinant protein of hDkk2 produced by the production method according to claim 7 (rhDkk2). 9. The hDkk2 recombinant protein (rhDkk2) according to claim 8, wherein the hDkk2 recombinant protein is represented by SEQ ID NO: 3.

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