KR102075256B1 - A method for isolation and purification of Macrophage colony stimulating factor - Google Patents

Abstract

The present invention relates to a method for isolating and purifying macrophage colony stimulating factors. The method for isolating and purifying macrophage colony stimulating factors (M-CSF) of the present invention provides high purity without the need for a large amount of buffers. It is very useful to obtain a dimeric form of macrophage colony stimulating factor (M-CSF).

Description

A method for isolation and purification of Macrophage colony stimulating factor

The present invention relates to a method for the isolation and purification of human macrophage colony stimulating factor (M-CSF) in dimeric form.

Macrophage colony stimulating factor (M-CSF, also known as CSF-1) was originally purified in 1977 in mouse L cell conditioned media and reported to stimulate the formation of macrophage colonies. It has been studied that M-CSF is involved in the proliferation, differentiation and survival of monocytes, macrophages and myeloid progenitor cells. Recent studies have also shown that M-CSF plays an important role in immune defense, bone metabolism, lipoprotein removal, fertilization, pregnancy and mammary gland development during normal and neonatal growth. M-CSF is produced by various cells such as osteoblasts, endothelial cells, fibroblasts, smooth muscle cells and macrophages.

 Macrophage colony stimulator (M-CSF) is a member of the type III protein tyrosine kinase receptor family that binds to colony stimulating factor receptor-1 (CSF-1R) and is encoded by the c46 fms tumor gene. The interaction between M-CSF and CSF-1R is known to initiate dimerization and autophosphorylation of CSF-1R leading to the activation of a series of downstream functional groups.

CSF-1R expression has been detected in numerous cells including hematopoietic stem cells, tissue macrophages, immature dendritic cells, osteoclasts and B cells. Interestingly, M-CSF has been shown to correlate with a variety of diseases, has been shown to be associated with a variety of cancers and autoimmune diseases, and has the potential to treat autoimmune diseases, tissue repair processes and cancers.

The amino acid residues of M-CSFα (256 amino acids), M-CSFβ (554 amino acids) and M-CSFγ (436 amino acids) isolated three cDNAs of different M-CSFs. Three isolated isoforms have identical N- and C-terminal regions. Previously cleaved human M-CSF (1-150 amino acids) was purified from E. coli and regenerated to form dimer proteins. This recombinant N-terminal non glycosylated region is completely biologically active and similar to glycosylated native M-CSF.

 However, the process of producing macrophage colony stimulator (M-CSF) is very complex and time consuming and requires sophisticated chromatography equipment.

In addition, macrophage colony stimulating factor (M-CSF) is a hematopoietic growth factor that stimulates proliferation and differentiation of monocytes. To be biologically active, M-CSF must form homodimers linked by disulfide bridges. However, the refolding step of recombinant M-CSF is not only extremely difficult but also very slow.

The present inventors have completed the present invention by establishing an efficient and simple method for isolating and purifying macrophage colony stimulating factor (M-CSF).

(0001) Yamanishi K, Takahashi M, Nishida T, Ohmoto Y, Takano M, et al. (1991) Renaturation, purification, and characterization of human truncated Macrophage colony stimulating factor expressed in Escherichia coli. J Biochem 109: 404-409. (0002) Pandit J, Bohm A, Jancarik J, Halenbeck R, Koths K, et al. (1992) Three-dimensional structure of dimeric human recombinant Macrophage colony stimulating factor. Science 258: 1358-1362.

The present invention establishes an efficient and rapid purification method for human macrophage colony stimulator (M-CSF), and an object of the present invention is to provide a method for purifying human macrophage colony stimulator (M-CSF).

In order to solve the above problems, the present invention

(a) cloning a fragment of the macrophage colony stimulating factor (M-CSF) encoding the amino acid represented by SEQ ID NO: 1 into a vector; (b) transforming the recombinant vector of step (a) to E. coli and culturing; (c) harvesting the culture of step (b) and lysing it with monomeric macrophage colony stimulating factor (M-CSF) using an internal buffer; (d) refolding the macrophage colony stimulating factor (M-CSF) in the dissolved monomer form of step (c) into a dialysis membrane using an external buffer; And (e) separating and purifying the macrophage colony stimulator (M-CSF) refolded in the dimeric form of step (d) from the monomeric form and purifying the macrophage colony stimulator (M). -CSF) provides a method for purifying.

The present invention also provides a dimeric form of macrophage colony stimulating factor (M-CSF) purified by the above method.

By using the method of isolation and purification of macrophage colony stimulating factor (M-CSF) of the present invention, macrophage colony stimulating factor (M-CSF) of high-purity dimer form can be obtained quickly without requiring a large amount of buffer. It's very useful.

1 shows a vector and purified M-CSF for expression of recombinant human M-CSF:
(A) the structure of the pET15b vector carrying human M-CSF (36-181 amino acids),
(B) Purification of M-CSF protein using Ni-NTA column,
Lane 1, uninduced E. coli Rosetta 2 (DE3) pLysS lysate; Lane 2, E. coli Rosetta 2 (DE3) pLysS lysate after IPTG induction; Lane 3 is M-CSF protein purified using Ni-NTA column,
(C) detection of purified M-CSF protein by Western blotting using anti-His antibody,
Lane 1, uninduced E. coli Rosetta 2 (DE3) pLysS lysate; Lane 2, E. coli Rosetta 2 (DE3) pLysS lysate after IPTG induction; Lane 3 is M-CSF protein purified using Ni-NTA column.
2 is a diagram showing the results of analyzing the non-reduction of M-CSF protein refolding by SDS-PAGE:
(A) SDS-PAGE analysis of refolded M-CSF
Lane 1, M-CSF before dialysis; Lanes 2-13, M-CSF after dialysis against the indicated refolding buffer.
(B) Detected graph of refolded M-CSF protein by western blotting using anti-His antibody
Lane 1, M-CSF before dialysis; Lanes 2-13, M-CSF after dialysis against the indicated refolding buffer.
Figure 3 shows the results of purification of dimerized M-CSF using an ion exchange column:
(A) SP-sepharose fraction; (B) DEAE-sepharose fraction; (C) Q-sepharose fraction.
4 shows the properties of the dimer M-CSF:
(A) photograph confirming the reduction of dimer M-CSF to monomeric M-CSF in the presence of DTT,
(B) detection of reduced M-CSF by western blotting using anti-His antibody,
(C) MALS analysis of purified dimer M-CSF using Q-sepharose.
5 shows the biological activity of the purified M-CSF dimer:
(A) shows the results of MTT analysis of BMM treated with recombinant M-CSF dimer for each concentration.
(B) Pictures of TRAP-positive multinuclear cells differentiated by treatment with purified M-CSF and RANKL
(C) It is a graph showing the expression of NFATc1 and its target genes (TRAP, CTSK and OSCAR) induced by purified M-CSF and RANKL.

The present inventors have found that human macrophage colony stimulator (M-CSF) plays an important role in the proliferation and differentiation of mononuclear phagocytes in hematopoietic stem cells. Various protocols for expression and purification have been reported, but the refolding process is very difficult and very time consuming. In order to solve this problem, M was partially cut from M-CSF designated as SEQ ID NO: 2 from Escherichia coli . -CSF (amino acids 36-181 aa, SEQ ID NO: 1) were labeled with histidine, expressed and purified using Ni-nitrilotriacetic (NTA) under denaturing conditions (with internal buffer). Then, redox refolding buffer (external buffer) mixed with reduced glutathione (GSH), glutathione disulfide (GSSG) and L-arginine (external buffer) accurately refolded the protein without M-CSF aggregation and Q Chromatography on -sepharose column selectively purified M-CSF dimer from monomer form, and the dimer nature of purified M-CSF was confirmed using multiple light scattering combined with high performance liquid chromatography.

In addition, cell proliferation and osteoclast differentiation tests using bone marrow-derived macrophages completed the present invention by demonstrating that recombinant M-CSF is active in vitro and in vivo.

Hereinafter, the purification method according to the present invention will be described in detail step by step.

(A) cloning a fragment of the macrophage colony stimulating factor (M-CSF) encoding the amino acid represented by SEQ ID NO: 1 to the vector. Recombinant Escherichia coli used in this step can be used without limitation as long as it expresses a human macrophage colony stimulating factor. More preferably, total RNA was extracted from reverse transcribed human T lymphocyte dendritic (CEM) cells. Human M-CSF fragments encoding amino acids 36-181 (SEQ ID NO: 1) can be amplified by PCR and bound to the NdeI and BamHI sites of the pET-15b vector, but are not limited thereto.

(b) transforming the recombinant vector of step (a) to E. coli and culturing. Recombinant vectors containing human macrophage colony stimulating factor (SEQ ID NO: 1) can be transformed into E. coli and cultured in a medium, but is not limited thereto.

(c) harvesting the culture of step (b) and lysing it with macrophage colony stimulating factor (M-CSF) in monomeric form using an internal buffer.

The internal buffer may be urea (Urea), sodium chloride (NaCl) and Tris (Tris), urea (Urea) does not have the structure of the protein, because it can be dissolved in large quantities in an inert structure in large quantities The monomeric form of macrophage colony stimulator (M-CSF) can be dissolved.

(d) The macrophage colony stimulating factor (M-CSF) in the dissolved monomer form of step (c) is refolded using an external buffer in a dialysis membrane.

The monomer-type macrophage colony stimulating factor (M-CSF) in which the internal buffer and the external buffer are exchanged buffers through a dialysis membrane (dialysis bag) having a micropore (pore) monomer-type macrophage colony stimulating factor (M-CSF) may be refolded in dimeric form.

The external buffer may include reduced glutathione (GSH), glutathione disulfide (GSSG), and L-arginine (L-arginine), and in one preferred embodiment of the present invention, the buffer described in Table 2 may be the most preferred. GRx (100 mM Tris pH 7.3, 1 mM EDTA, 0.1 mM GSSG, 0.5 mM GSH, 250 mM L-arginine).

(e) separating and purifying the macrophage colony stimulating factor (M-CSF) refolded in the dimeric form of step (d) from the monomeric form.

In step (d), the macrophage colony stimulating factor (M-CSF) is not an active dimer form (M-CSF dimeric form), and further purified by cation exchange chromatography to form a dimer. In this step, the purity of macrophage colony stimulating factor (M-CSF) is increased.

The functional group of the cation exchange chromatography used in the present invention is not only weak cation carboxymethyl (CM-) and carboxy (C-) but also strong cation sulfo (S). -), Sulfomethyl (SM-), sulfoethyl (SE-), sulfopropyl (SP-) and phospho (P-) can be used in various ways, and the column resins Sepharose (Sepharose), Sepha Sepax, agarose, Sephacel, Polystyrene, Polyacrylate, Cellulose and Toyoperl may be used in various ways. In one aspect, the purification method of the present invention can perform cation exchange chromatography using a Q-Sepharose column.

In addition, the purification method according to the present invention can quickly obtain a high-purity dimer form of macrophage colony stimulating factor (M-CSF) can be expected to be higher productivity than the conventional purification method when used industrially.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only intended to illustrate the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

Example 1 Experimental Materials and Methods

<1-1> Cleaved human M- Of CSF Cloning

Total RNA was extracted from human T lymphocytes (CEM) cells reverse transcribed with M-MLV reverse transcriptase (Promega) using the Easy-spin (DNA free) Total RNA Extraction Kit (iNtRON) and according to the manufacturer's instructions. Human M-CSF fragments encoding amino acids 36-181 (SEQ ID NO: 1) were amplified by PCR and bound to the NdeI and BamHI sites of the pET-15b vector.

<1-2> human M- Of CSF  Expression

PET15b M-CSF (36-181aa, SEQ ID NO: 1) constructs grown in 50 mL of Luria-Bertani (LB) medium containing ampicillin (100 μg / mL) were transferred to Escherichia coli Rosetta 2 (DE3) pLysS cells (Novagen ) Was incubated overnight at 37 ℃. The cultures were placed in 1 L of LB medium with ampicillin (100 μg / mL) and incubated at 37 ° C. at 600 nm until the optical density reached 0.6-0.8, followed by 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG). ) Was added. After 4 more hours of incubation, the cells were harvested by centrifugation and the pellets were stored at -20 ° C.

<1-3> M- CSF  Purification in monomer form

The bacterial pellet was resuspended in 30 ml of lysis buffer (0.5 M NaCl, 5 mM imidazole, 20 mM Tris, pH 7.9). The bacterial cell suspension was sonicated for 3 minutes at 1 minute intervals and Triton X-100 (Sigma) was added to the suspension (final concentration 1%). The suspension was further dissolved by shaking at 4 ° C. for 1 hour. After centrifugation at 12,000 rpm for 30 minutes at 4 ° C, the supernatant was discarded and the insoluble pellet was dissolved by stirring with 20 ml of binding buffer (6 M urea, 0.5 M NaCl, 5 mM imidazole, 20 mM Tris, pH 7.9). Centrifuged at 12,000 rpm for 2 hours at room temperature and 30 minutes at 4 ° C.

The purified supernatant was mixed with 2 ml of Ni-NTA affinity resin (Millipore) by shaking culture at 4 ° C. overnight. After incubation, the resin was placed in a column and washed with 20 ml of binding buffer. The protein was then eluted with 20 ml of elution buffer (6 M Urea, 0.5 M NaCl, 20 mM imidazole, 20 mM Tris pH 7.9). The eluted protein was dialyzed at 4 ° C. in dialysis buffer (6 M Urea, 0.5 M NaCl, 20 mM Tris pH 7.9) to remove excess imidazole.

<1-4> M- CSF  Monomer form Refolding

To analyze the optimal buffer conditions for refolding M-CSF, eluted M-CSF was dialyzed at 4 ° C. for 24 hours using various kinds of refolding buffers:

Dxx (100 mM Tris pH 7.3, 1 mM EDTA, 1 mM dithiothreitol (DTT))

DxP (100 mM Tris pH 7.3, 1 mM EDTA, 1 mM DTT, 0.055% polyethylene glycol (PEG))

DRx (100 mM Tris pH 7.3, 1 mM EDTA, 1 mM DTT, 250 mM L-arginine)

DRP (100 mM Tris pH 7.3, 1 mM EDTA, 1 mM DTT, 250 mM L-arginine, 0.055% PEG)

DSx (100 mM Tris pH 7.3, 1 mM EDTA, 1 mM DTT, 200 mM sucrose)

DSP (100 mM Tris pH 7.3, 1 mM EDTA, 1 mM DTT, 200 mM sucrose, 0.055% PEG)

Gxx (100 mM Tris pH 7.3, 1 mM EDTA, 0.1 mM GSSG, 0.5 mM GSH),

GxP (100 mM Tris pH 7.3, 1 mM EDTA, 0.1 mM GSSG, 0.5 mM GSH, 0.055% PEG)

GRx (100 mM Tris pH 7.3, 1 mM EDTA, 0.1 mM GSSG, 0.5 mM GSH, 250 mM L-arginine)

GRP (100 mM Tris pH 7.3, 1 mM EDTA, 0.1 mM GSSG, 0.5 mM GSH, 250 mM L-arginine, 0.055% PEG)

GSx (100 mM Tris pH 7.3, 1 mM EDTA, 0.1 mM GSSG, 0.5 mM GSH, 200 mM sucrose)

GSP (100 mM Tris pH 7.3, 1 mM EDTA, 0.1 mM GSSG, 0.5 mM GSH, 200 mM sucrose, 0.055% PEG)

<1-5> M- CSF Dimer  Tablets in the form

After dialysis against refolding buffer GRx (external buffer), mix with 2 ml of SP Sepharose, Q Sepharose or DEAE Sepharose (GE healthcare) and load each Sepharose on a gravity flow column (BIO-RAD) to 4 ° C. Rotate overnight at. After washing with BC buffer (200 mM Tris pH 7.3, 0.2 mM EDTA, 20% glycerol), the bound protein was washed with 2 ml BC buffer (200 mM Tris pH 7.3, 0.2 mM EDTA, 20% glycerol, 100-1000 mM KCl) three times. Each fraction was analyzed by non-reducing SDS PAGE. BC100 and BC200 fractions were combined and dialyzed against PBS (140 mM NaCl, 2.7 mM KCl, 6.5 mM Na2HPO, 1.5 mM KH2PO, pH7.4) at 4 ° C. for 24 hours.

<1-6> Multi angle Mining  Method MALS )

The molecular weight of M-CSF was determined by performing analysis by high performance liquid chromatography (HPLC, Shimadzu) and MALS (Wyatt Technology Corporation) spectroscopy at the Korea Basic Science Research Institute. Combined HPLC equipment and MALS spectrometer combined to perform an integrated analysis technique. HPLC was performed with 5 mg / mL of protein: flow rate 0.5 mL / min was maintained at Superdex 75 Increase 10/300 GL (GE Healthcare). Differential refractive index spectra were obtained using Optilab T-rEX (Wyatt Technology Corporation). The weight average molar mass was calculated from the elution data using ASTRA 6 software (Wyatt Technologies).

<1-7> Cell Proliferation Assay

Bone marrow derived macrophages (BMM) were isolated from 8 week old male C57BL / 6 mice as described previously and seeded in 96 well tissue culture plates at a density of 2 × 10 3 cells supplemented with MEM-α (GE Health care). 10% fetal bovine serum (Merk) and 22.5, 45 or 90 ng / mL of M-CSF were administered for 1-3 days. MTT assays using Cell Proliferation KIT 1 (Roche Diagnostics GmbH) were performed according to manufacturer's recommendations to investigate the biological activity of M-CSF.

<1-8> osteoclast formation

To generate osteoclasts, BMM was added 2 × 10 ⁴ in MEM-α with 10% FBS, M-CSF (30 ng / ml) and Receptor activator of nuclear factor kappa-Β ligand (RANKL) (100 ng ml). Cells were cultured in 48-well tissue culture plates. After incubation for 3 days, cells were fixed and stained using acidic phosphatase, leukocyte kit (Sigma) according to the manufacturer's recommendations. TRAP-positive multinucleated cells were observed under light microscopy.

<1-9> Quantitative Reverse Transcripion PCR  ( qRT - PCR )

Total RNA was extracted from cells after 3 days incubation with M-CSF and RANKL and reverse transcribed by M-MLV reverse transcriptase (Promega). Real-time PCR was performed using an IQTM SYBR Green Supermix (Bio-Rad) and a CFX connect ^™ real-time PCR detection system. Relative mRNA levels were normalized to β-Actin mRNA levels. All reactions were performed in three replicates and the data presented are the average of three independent experiments. Target gene expression was quantified using the primers in Table 1 below.

gene primer SEQ ID NO: β-Actin 5'-GCAAGTGCTTCTAGGCGGAC-3 ' 3 5'-AAGAAAGGGTGTAAAACGCAGC-3 ' 4 CTSK 5'-ACGGAGGCATTGACTCTGAAGATG-3 ' 5 5'-GGAAGCACCAACGAGAGGAGAAAT-3 ' 6 NFATc1 5'-CTCGAAAGACAGCACTGGAGCAT-3 ' 7 5'-CGGCTGCCTTCCGTCTCATAG-3 ' 8 OSCAR 5'-CTGCTGGTAACGGATCAGCTCCCCAGA-3 ' 9 5'-CCAAGGAGCCAGAACCTTCGAAACT-3 ' 10

<1-10> SDS-PAGE and Western blotting

The protein sample was either 2X reduced sample buffer (100 mM Tris pH 6.8, 20% glycerol, 4% SDS, 0.04% bromophenol blue, 10% β-mercaptoethanol) or 5X non-reducing sample buffer (250 mM Tris pH 6.8, 50% glycerol, 10% SDS, 0.05% bromophenol blue) and denatured at 95 ℃ for 10 minutes. Samples were analyzed by 15% SDS-PAGE and stained with Coomassie Brilliant Blue G-250 or transferred to polyvinylidene difluroide membranes (Millipore) and Western blotted using anti-His antibody (Thermo Fisher).

< Example  2> Experimental results

<2-1> M- Of CSF  Expression and Purification

As a first step in the purification of biologically active M-CSF proteins, RT-PCR was used to clone the human M-CSF gene from human T lymphoblastoid (CEM) cells, followed by human M-CSF construct (amino acids 36-181). ) Was introduced into the pET15b vector containing 6 histidine residues at the N-terminus (FIG. 1A). Protein was expressed in E. coli Rosetta 2 (DE3) pLysS. A 16kDa band was detected via Reducing-SDS-PAGE (FIG. 1B, lane 2), which was smaller than the expected size (20 kDa). Western blotting was performed with anti-His antibodies to determine if this fast-moving protein was His-tagged M-CSF. As shown in FIG. 1C (lane 2), it was confirmed that the 16 kDa protein corresponds to His-tagged M-CSF. Since all His-tagged M-CSF was found in inclusion bodies, the protein was purified under denaturing conditions using a Ni-nitrilotriacetic (NTA) column (Figure 1A, lane 3).

<2-2> M- CSF Dimeric Refolding  And ion exchange column  refine

Since the biologically active M-CSF has proven to be a disulfide bond dimer, in order to attempt to regenerate the monomeric form using the 12 redox refold buffers (Table 2) described in Examples 1-4, M-CSF dimerization levels were analyzed after dialysis using two reducing agents: othiothreitol (DTT) and glutathione (GSH / GSSG).

As a result, glutathione (GSH / GSSG) was more efficient at promoting disulfide bond formation (dimer) and rearrangement (FIG. 2).

However, most of the M-CSF proteins precipitated due to aggregation during the refolding process. Arginine and sucrose are well known protein stabilizers that help prevent aggregation. In order to increase the yield of correctly refolded M-CSF, these reagents were added to the redox refold buffer. Sucrose showed that L-arginine significantly reduced M-CSF aggregation (lanes 6, 7, 12 and 13), while having little effect (lanes 2, 4, 5, 10 and 11). Polyethylene glycol (PEG) is known to significantly improve protein refolding by reducing aggregation. However, the addition of PEG had no effect on refolding (FIG. 2).

Based on these results, redox refolding buffer containing GSH / GSSG and L-arginine was optimal for refolding of M-CSF dimer, so GRx (lane 10) was used as an external buffer.

Biologically active dimer M-CSF was analyzed by ion-exchange chromatography columns set to separate from inactive monomer form. Both monomers and dimers flowed through the SP-sepharose strong cation column without binding (FIG. 3A). Some dimers were retained in the DEAE-sepharose weak anion-exchange column, but most are still present in the unbound fraction (Figure 3B). When a Q-sepharose strong anion-exchange column was used, more than 95% of M-CSF dimers were specifically retained and purified without any monomers (FIG. 3C).

<2-3> M- CSF Dimeric  Properties and Biological Activities

Purified dimers were analyzed using SDS-PAGE in the presence or absence of reducing agent DTT to determine whether the material purified using Q-sepharose was indeed a dimer composed of M-CSF monomers. As shown in FIG. 4A, the M-CSF dimer was quite pure and could be reduced to the M-CSF monomer using DTT. This result was confirmed by western blotting using anti-His antibody (FIG. 4B). Based on the theoretical molecular weight of the monomeric M-CSF fragment (20 kDa), the apparent molecular weight of the dimer (30 kDa) differed from the expected (40 kDa). To resolve this discrepancy, the absolute molecular weight of the dimer M-CSF was determined by performing multiple angle light scattering (MALS) combined with high performance liquid chromatography.The absolute molecular weight of the dimer M-CSF was 40 kDa ( 4C), the faster migration of M-CSF in SDS-PAGE was large due to the acid isoelectric point of 5.41.

The biological activity of purified M-CSF was assessed through its ability to grow bone marrow-derived macrophages (BMMs). As a result of MTT assay, BMM cells increased exponentially even at low concentrations of purified M-CSF (22.5 ng / ml) (FIG. 5A).

In addition, the effect of purified M-CSF on osteoclast formation by RANKL was evaluated. TRAP staining in FIG. 5B confirmed that significant amounts of multinuclear osteoclasts differentiated with BMM in the presence of M-CSF and RANKL. With TRAP staining, the expression of NFATc1 and its target genes (TRAP, CTSK and OSCAR) were very high during osteoclast formation (FIG. 5C). These observations indicate that the purified recombinant M-CSF is biologically active and excellent in inducing proliferation of BMM in vivo.

So far I looked at the center of the preferred embodiment for the present invention. Those skilled in the art will appreciate that the present invention can be implemented in a modified form without departing from the essential features of the present invention. Therefore, the disclosed embodiments should be considered in descriptive sense only and not for purposes of limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope will be construed as being included in the present invention.

<110> Chungbuk National University Industry-Academic Cooperation Foundation <120> A method for isolation and purification of Macrophage colony          stimulating factor <130> PN1804-134 <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 146 <212> PRT <213> Artificial Sequence <220> The amino acid sequence truncated in M-CSF (36-181 aa) <400> 1 Ser Glu Tyr Cys Ser His Met Ile Gly Ser Gly His Leu Gln Ser Leu   1 5 10 15 Gln Arg Leu Ile Asp Ser Gln Met Glu Thr Ser Cys Gln Ile Thr Phe              20 25 30 Glu Phe Val Asp Gln Glu Gln Leu Lys Asp Pro Val Cys Tyr Leu Lys          35 40 45 Lys Ala Phe Leu Leu Val Gln Asp Ile Met Glu Asp Thr Met Arg Phe      50 55 60 Arg Asp Asn Thr Pro Asn Ala Ile Ala Ile Val Gln Leu Gln Glu Leu  65 70 75 80 Ser Leu Arg Leu Lys Ser Cys Phe Thr Lys Asp Tyr Glu Glu His Asp                  85 90 95 Lys Ala Cys Val Arg Thr Phe Tyr Glu Thr Pro Leu Gln Leu Leu Glu             100 105 110 Lys Val Lys Asn Val Phe Asn Glu Thr Lys Asn Leu Leu Asp Lys Asp         115 120 125 Trp Asn Ile Phe Ser Lys Asn Cys Asn Asn Ser Phe Ala Glu Cys Ser     130 135 140 Ser gln 145 <210> 2 <211> 554 <212> PRT <213> Artificial Sequence <220> <223> Macrophage colony stimulating factor, M-CSF <400> 2 Met Thr Ala Pro Gly Ala Ala Gly Arg Cys Pro Pro Thr Thr Trp Leu   1 5 10 15 Gly Ser Leu Leu Leu Leu Val Cys Leu Leu Ala Ser Arg Ser Ile Thr              20 25 30 Glu Glu Val Ser Glu Tyr Cys Ser His Met Ile Gly Ser Gly His Leu          35 40 45 Gln Ser Leu Gln Arg Leu Ile Asp Ser Gln Met Glu Thr Ser Cys Gln      50 55 60 Ile Thr Phe Glu Phe Val Asp Gln Glu Gln Leu Lys Asp Pro Val Cys  65 70 75 80 Tyr Leu Lys Lys Ala Phe Leu Leu Val Gln Asp Ile Met Glu Asp Thr                  85 90 95 Met Arg Phe Arg Asp Asn Thr Pro Asn Ala Ile Ala Ile Val Gln Leu             100 105 110 Gln Glu Leu Ser Leu Arg Leu Lys Ser Cys Phe Thr Lys Asp Tyr Glu         115 120 125 Glu His Asp Lys Ala Cys Val Arg Thr Phe Tyr Glu Thr Pro Leu Gln     130 135 140 Leu Leu Glu Lys Val Lys Asn Val Phe Asn Glu Thr Lys Asn Leu Leu 145 150 155 160 Asp Lys Asp Trp Asn Ile Phe Ser Lys Asn Cys Asn Asn Ser Phe Ala                 165 170 175 Glu Cys Ser Ser Gln Asp Val Val Thr Lys Pro Asp Cys Asn Cys Leu             180 185 190 Tyr Pro Lys Ala Ile Pro Ser Ser Asp Pro Ala Ser Val Ser Pro His         195 200 205 Gln Pro Leu Ala Pro Ser Met Ala Pro Val Ala Gly Leu Thr Trp Glu     210 215 220 Asp Ser Glu Gly Thr Glu Gly Ser Ser Leu Leu Pro Gly Glu Gln Pro 225 230 235 240 Leu His Thr Val Asp Pro Gly Ser Ala Lys Gln Arg Pro Pro Arg Ser                 245 250 255 Thr Cys Gln Ser Phe Glu Pro Pro Glu Thr Pro Val Val Lys Asp Ser             260 265 270 Thr Ile Gly Gly Ser Pro Gln Pro Arg Pro Ser Val Gly Ala Phe Asn         275 280 285 Pro Gly Met Glu Asp Ile Leu Asp Ser Ala Met Gly Thr Asn Trp Val     290 295 300 Pro Glu Glu Ala Ser Gly Glu Ala Ser Glu Ile Pro Val Pro Gln Gly 305 310 315 320 Thr Glu Leu Ser Pro Ser Arg Pro Gly Gly Gly Ser Met Gln Thr Glu                 325 330 335 Pro Ala Arg Pro Ser Asn Phe Leu Ser Ala Ser Ser Pro Leu Pro Ala             340 345 350 Ser Ala Lys Gly Gln Gln Pro Ala Asp Val Thr Gly Thr Ala Leu Pro         355 360 365 Arg Val Gly Pro Val Arg Pro Thr Gly Gln Asp Trp Asn His Thr Pro     370 375 380 Gln Lys Thr Asp His Pro Ser Ala Leu Leu Arg Asp Pro Pro Glu Pro 385 390 395 400 Gly Ser Pro Arg Ile Ser Ser Leu Arg Pro Gln Gly Leu Ser Asn Pro                 405 410 415 Ser Thr Leu Ser Ala Gln Pro Gln Leu Ser Arg Ser His Ser Ser Gly             420 425 430 Ser Val Leu Pro Leu Gly Glu Leu Glu Gly Arg Arg Ser Thr Arg Asp         435 440 445 Arg Arg Ser Pro Ala Glu Pro Glu Gly Gly Pro Ala Ser Glu Gly Ala     450 455 460 Ala Arg Pro Leu Pro Arg Phe Asn Ser Val Pro Leu Thr Asp Thr Gly 465 470 475 480 His Glu Arg Gln Ser Glu Gly Ser Phe Ser Pro Gln Leu Gln Glu Ser                 485 490 495 Val Phe His Leu Leu Val Pro Ser Val Ile Leu Val Leu Leu Ala Val             500 505 510 Gly Gly Leu Leu Phe Tyr Arg Trp Arg Arg Arg Ser His Gln Glu Pro         515 520 525 Gln Arg Ala Asp Ser Pro Leu Glu Gln Pro Glu Gly Ser Pro Leu Thr     530 535 540 Gln Asp Asp Arg Gln Val Glu Leu Pro Val 545 550 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> b-Actin forward primer <400> 3 gcaagtgctt ctaggcggac 20 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> b-Actin reverse primer <400> 4 aagaaagggt gtaaaacgca gc 22 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> CTSK forward primer <400> 5 acggaggcat tgactctgaa gatg 24 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> CTSK reverse primer <400> 6 ggaagcacca acgagaggag aaat 24 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> NFATc1 forward primer <400> 7 ctcgaaagac agcactggag cat 23 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> NFATc1 reverse primer <400> 8 cggctgcctt ccgtctcata g 21 <210> 9 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> OSCAR forward primer <400> 9 ctgctggtaa cggatcagct ccccaga 27 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> OSCAR reverse primer <400> 10 ccaaggagcc agaaccttcg aaact 25

Claims (7)

(a) cloning a fragment of a macrophage colony stimulating factor (M-CSF) encoding the amino acid sequence of SEQ ID NO: 1 into a vector;
(b) transforming the recombinant vector of step (a) to E. coli and culturing;
(c) Harvesting the culture of step (b), using the internal buffer containing urea (Urea), sodium chloride (NaCl) and Tris (Cris) monomeric macrophage colony stimulating factor (M-CSF) Dissolving with;
(d) adding the dissolved monomeric macrophage colony stimulating factor (M-CSF) in the dialysis membrane of step (c) to reduced glutathione (GSH), glutathione disulfide (GSSG) and L-arginine (L-) refolding using an external buffer containing arginine; And
(e) The macrophage colony stimulating factor (M-CSF) refolded in the dimer form of step (d) was subjected to monomer exchange chromatography using a Q-sepharose column. Selectively purifying the M-CSF dimer from the form; a method for purifying a macrophage colony stimulator (M-CSF) of a dimer. delete The method of claim 1,
The external buffer of step (d) is a refolding buffer.

delete delete delete delete

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