KR100502999B1 - Recombinant human G-CSF/GM-CSF fusion protein and it's coding gene - Google Patents

Abstract

The present invention relates to a recombinant human G-CSF / GM-CSF fusion protein and a gene encoding the same. The present invention relates to a recombinant human G-CSF / GM- that can exert both a function of a G-CSF protein and a GM-CSF protein. By providing a CSF fusion protein, it provides an excellent single fusion protein that can be used more conveniently when the function of G-CSF protein and GM-CSF protein is required, including in vivo or ex vivo expansion of bone marrow hepatocytes. It works.

Description

Recombinant human G-CSF / GM-CSF fusion protein and it's coding gene

The present invention relates to a recombinant protein and a gene encoding the same, and more particularly, to a recombinant human G-CSF / GM-CSF fusion protein and a gene encoding the same.

Colony-stimulating factor (CSF) is a cytokine that stimulates expansion and differentiation of bone marrow hepatocytes. Different CSFs act on myeloid cells at various stages of development and promote the formation of specific colonies of various strains. GM-CSF and G-CSF are hematopoeictic growth factors that promote differentiation of bone marrow cells and are also active factors of mature bone marrow cells.

Recently, recombinant GM-CSF protein and G-CSF protein have been studied to treat various diseases including cancer and hematopoietic diseases (Buchsel PC, Forgey A, Grape FB, Hamann SS.Granulocyte macrophage colony-stimulating factor: current practice and novel approaches.Clin J Oncol Nurs 2002 Jul-Aug; 6 (4): 198-205 Related Articles, Links), Recombinant G-CSF and GM-CSF have been renamed under the trade names 'Filgrastima' and 'Sargramostim' in the United States. Each is known.

GM-CSF acts on bone marrow cells to increase inflammatory leukocyte colonies, including granulocytes and macrophages (Staynov, DZ, DJ Cousins, and TH Lee. 1995. A regulatory element in the Promoter of the human Granulocyte- Macrophage Colony Stimulating Factor gene that has related sequences in other T-cell expressed cytokine genes.Proc. Natl. Acad. Sci. 92: 3606-3610.). It also exhibits activity as an in vitro macrophage activating element, stimulating various antimicrobial activities and enabling inflammatory responses (Jarmin, DI, RJ Nibbs, T. Jamieson, JS de Bono and GJ Graham. 1999. Granulocyte macrophage colony-stimulating factor and interleukin-3 regulate chemokine and chemokine receptor expression in bone marrow macrophages.Exp . Hematol. 27: 1735-1745 . ; Bischof, RJ, D. Zafiropoulos, JA Hamilton and IK Campbell. 2000. Exacerbation of acute inflammatory arthritis by the colony-stimulating factors CSF-1 and granulocyte macrophage (GM) -CSF: evidence of macrophage infiltration and local proliferation.Clin.Exp . Immunol. 119: 361-367.). GM-CSF has been used to promote bone marrow recovery after cancer chemotherapy and has been used to recover bone marrow stem cells for transplantation (Keller, JR, SEW Jacobsen, KT Sill, LR Ellingsworth, and FW Ruscetti. 1991. Stimulation of granulopoiesis by transforming growth factor β: Synergy with Granulocyte / Macrophage Colony Stimulating Factor.Proc.Natl.Acad.Sci . 88: 7190-7194 . ; Aglietta, M., F. Montemurro, F. Fagioli, C. Volta, B Botto, M. Cantonetti, V. Racanelli, L. Teofili, R. Ferrara, S. Amadori, GL Castoldi, F. Dammacco and A. Levis. 2000. Short term treatment with Escheria coli recombinant human granulocyte-macrophage-colony stimulating factor prior to chemotherapy for Hodgkin disease Cancer 88:. 454-460).. It is on the rise in clinical research for the treatment of various diseases, including infectious diseases, hematopoietic diseases and cancer, and has become one of the most clinically successful applications as a biological therapy (Janik, JE, LL Miller, WC Kopp). , DD Taub, H. Dawson, D. Stevens, P. Kostboth, BD Curti, KC Conlon, BK Dunn, SE Donegan, R. Ullrich, WG Alvord, BL Gause and DL Longo. 1999. Treatment with tumor necrosis factor-alpha and granulocyte-macrophage colony-stimulating factor increases epidermal Langerhans' cell numbers in cancer patients.Clin . Immunol. 93: 209-221 . ; Groenewegen, G. and GC de Gast. 1999. GM-CSF can cause T cell activation; results .. of sequential chemo-immunotherapy Eur J. Cancer 35 suppl 3:... S23-24 .; Jones, TC 1999. Use of granulocyte-macrophage colony stimulating factor (GM-CSF) in prevention and treatment of fungal infections Eur J Cancer 35 Suppl. 3: S8-10.). It also showed potential as a prospective vaccine adjuvant (Kapoor, D., SR Aggarwal, NP Singh, V. Thakur, SK Sarin. 1999. Granulocyte-macrophage colony stimulating factor enhances the efficacy of hepatitis B virus vaccine in previously unvaccinated . haemodialysis patients J. Viral Hepat 6: .... 405-409 .; Anandh, U., B. Bastani and S. Ballal 2000. Granulocyte-Macrophage Colony Stimulating Factor as an adjuvant to Hepatitis B vaccination in maintenance hemodialysis patients Am J. Nephrol. 20: 53-56 . ; McClay EF.Adjuvant therapy for patients with high-risk malignant melanoma.Semin Oncol. 2002 Aug; 29 (4): 389-99.).

G-CSF acts on bone marrow hepatocytes to promote differentiation of granulocytes. It has been used extensively for clinical transplantation since inducing hematopoietic stem cell movement and enhancing bone marrow hepatocyte colonies (Petit I, Szyper-Kravitz M, Nagler A, Lahav M, Peled A, Habler L, Ponomaryov T, Taichman RS). , Arenzana-Seisdedos F, Fujii N, Sandbank J, Zipori D, Lapidot T. G-CSF induces stem cell mobilization by decreasing bone marrow SDF-1 and up-regulating CXCR 4. Nat Immunol 2002 Jul; 3 (7): 687- 94; de la Rubia J, Regadera A, Martin G, Cervera J, Sanz G, Martinez J, Jarque I, Garcia I, Andreu R, Moscardo F, Jimenez C, Molla S, Benlloch L, Sanz M. Second mobilization and collection of peripheral blood progenitor cells in healthy donors is associated with lower CD34 (+) cell yields.J Hematother Stem Cell Res. 2002 Aug; 11 (4): 705-9.). G-CSF interacts with immune cells in the periphery and is also used in clinical studies for the treatment and prevention of various diseases, including cancer as well as hematopoietic and autoimmune diseases (CasTaqna L, Bertuzzi A, Nozza A, Siracusano L, Balzarotti M, Magagnoli M, Sarina B, Timofeeva I, Sinnone M, Grimoldi MG, Fare M, Santoro A. Reduced intensity conditioning regimen followed by glycosylated G-CSF mobilized PBSCT in patients with solid tumors and malignant lymphomas.Bone Marrow Transplant. 2002 Aug; 30 (4): 207-14 .; Ott MG, Merget-Millitzer H, Ottmann OG, Martin H, Bruggenolte N, Bialek H, Seger R, Hossle JP, Hoelzer D, Grez M. Mobilization and Transduction of CD34 + Peripheral Blood Stem Cells in Patients with X-Linked Chronic Granulomatous Disease.J Hematother Stem Cell Res 2002 Aug; 11 (4): 683-94; Adams JR, Lyman GH, Djubegovic B, Feinglass J, Bennett CL.G- CSF as prophylaxis of febrile neutropenia in SCLC.Expert Opin Pharmacother 2002 Sep; 3 (9): 12 73-81; Hanna NH, Gordon MS, Fife K, Sandler AB.Phase I trial of topotecan plus vinorelbine with / without filgrastim (G-CSF) in patients with refractory malignancies. Am J Clin Oncol 2002 Aug; 25 (4): 337-9; Smith MA, Smith JG. Clinical experience with the use of rhG-CSF in secondary autoimmune neutropenia. Clin Lab Haematol 2002 Apr; 24 (2): 93-7).

The combination of G-CSF protein and GM-CSF protein can produce better results in the treatment of various diseases and in the production of progeitor cells from peripheral blood (PBPC) from peripheral blood. GM-CSF protein and G-CSF protein promoted clonal expansion of purified CD34 (+) megakaryocytes in vitro from normal human peripheral blood. PBPC has been effectively remoblized by the simultaneous help of G-CSF protein and GM-CSF protein, demonstrating that binding of G-CSF protein and GM-CSF protein may be an effective way to activate PBPC. . In addition, PBPC migration by erythropoietin (EPO) was also greatly improved by the continuous treatment of GM-CSF protein and G-CSF protein, improving the efficiency of PBPC aggregation.

The combination of GM-CSF protein and G-CSF protein with other cytokines greatly increased bone marrow hepatocytes from human umbilical cord blood (UCB) samples, providing an effective method for collecting hematopoietic stem cells in vitro.

Both GM-CSF protein and G-CSF protein were produced from cancer cells and prolonged the survival of inflammatory cells, suggesting that mutual binding could be applied in the treatment of cancer. This finding indicates that G-CSF proteins and GM-CSF proteins can bind simultaneously and sequentially with cells to increase their activity, which enhances the potential importance of recombinant G-CSF / GM-CSF fusion proteins.

However, there is no known gene linked to the human G-CSF gene and the human GM-CSF gene and a fusion protein expressed therefrom.

Accordingly, an object of the present invention is to provide a gene produced by linking a human G-CSF gene and a human GM-CSF gene.

It is also an object of the present invention to prepare a G-CSF / GM-CSF fusion protein prepared from the gene.

In order to achieve the above object, the present invention provides a polynucleotide consisting of the DNA sequence of SEQ ID NO: 1.

The present invention also provides a polypeptide consisting of the amino acid sequence of SEQ ID NO: 2.

Before ligation of cDNAs of human G-CSF and GM-CSF into a single construct, the inserted cDNA and linker can be amplified by PCR using the following primers.

G-CSF is amplified using primers 31 and 32, and GM-CSF uses primers 33 and 39. GS linker fragment (Olafsena, T., Rasmussena, IB , Norderhaugb, L., Bruland, S. and Sandliea, I. (1998) IgM secretory tailpiece drives multimerisation of bivalent scFv fragments in eukaryotic cells Immunotechnology 4:. 141-153. ) Amplification uses primers 34 and 35.

On the other hand, the combination of G-CSF, GM-CSF and linker sequences is performed using Splicing Overlapped Extension (SOEing) technology (Horton RM, Cai ZL, Ho SN, Pease LR. Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction.Biotechniques . 1990 8 (5): 528-35.). Through the first SOEing reaction, DNA fragments of the G-CSF and the linker and the linker and the GM-CSF can be combined by the following procedure.

First, the G-CSF and linker fragments are annealed and bound together using Taq polymerase without using any primers under a cycle of denaturing, extending.

Add primers 31 and 35 after the reaction and amplify with cycles of denaturation, annealing and expansion to obtain G-CSF / linker fragments.

On the other hand, using similar procedures and primers 34 and 39, linker / GM-CSF fragments can also be obtained.

Amplified G-CSF / linker and linker / GM-CSF fragments were purified from 1% agarose gels and used in the second SOEing reaction for the preparation of G-CSF / linker / GM-CSF DNA fragments. G-CSF / Linker and Linker / GM-CSF DNA fragments are annealed and bound together using Taq polymerase under a cycle of denaturation and expansion without the use of any primers. After the reaction primers 31 and 39 are added and amplified in a cycle of denaturation, annealing and expansion.

Through the above procedure, a single amplified DNA fragment can be prepared from PCR amplification products of human G-CSF and GM-CSF. The first SOEing with G-CSF fragment and linker, and GM-CSF and linker, respectively The reaction results in amplification of DNA fragments that are slightly longer than G-CSF and GM-CSF fragments, confirming that the first SOEing reaction was successful. In addition, a second SOEing with a G-CSF / linker and a linker / GM-CSF fragment yields a G-CSF / linker / GM-CSF fragment and the prepared G-CSF / linker / GM-CSF DNA fragment. Cloned into pGEMT and subcloned to the Nco I and Not I positions of the pET-22b (+) vector to prepare pEThGGMCSF constructs expressing the G-CSF / GM-CSF fusion protein. Further analysis of the pEThGGMCSF construct shows that the open reading frame has the GS linker sequence and the entirety of the randomly coupled human G-CSF and GM-CSF coding regions.

Linearly expanded structures are found at the 3 'end of the human G-CSF protein and the 5' end of the human GM-CSF protein, respectively, allowing independent structures of G-CSF and GM-CSF in fusion proteins.

Expression of the G-CSF / Linker / GM-CSF fusion protein is performed by IPTG induction after transformation of the construct with E. coli BL21 (DE). That is, cells are cultured in medium supplemented with glucose and ampicillin and transferred to glucose-free medium supplemented with IPTG and ampicillin to induce expression of the cloned G-CSF / GM-CSF fusion protein.

After induction, cells were harvested, Kim JK, Tsen, MF, Ghetie, V. and Ward, ES (1994a) Identifying amino acid residues that influence plasma clearance of murine IgG1 fragments by site-directed muTakenesis.Eur . J. Immunol. 24 , 542-547 . ; McClay EF. Adjuvant therapy for patients with high-risk malignant melanoma. Semin Oncol. 2002 Aug; 29 (4): 389-99. Proteins are extracted from the cytoplasm and cell inclusion bodies.

As a result, a 40 kDa recombinant human G-CSF / GM-CSF fusion protein (rhG-CSF / GM-CSF) can be extracted from the cell inclusion body, even though the construct was expressed in a pelB leader sequence to obtain a protein expressed in the periplasm. fusion protein is expressed in the cell inclusion body, which may mean that an insoluble protein aggregate is formed by overexpression in E. coli cells.

The expressed fusion protein corresponds to the size of bound human G-CSF and human GM-CSF and can be identified using Eliza and Western blot.

On the other hand, since the rhG-CSF / GM-CSF fusion protein in urea solution is denatured due to the high concentration of urea, it needs to be restored before the measurement of biological activity, and simple dialysis of the protein does not induce precipitation of the protein. The protein can be dissolved well, which will mean that the protein is folded back into its natural form.

Colony-stimulating activity of the dialyzed rhG-CSF / GM-CSF fusion protein can be examined by assaying colony formation with bone marrow cells derived from leukemia patients, and the purified 40 kDa protein significantly increased colony formation. Can be confirmed. This supports the fact that 40kDa proteins from urea samples not only act as colony stimulating elements but can also be folded back into biologically active forms by simple dialysis.

The colony forming units of the medium treated with the fusion protein are higher than the medium treated with the same number of recombinant G-CSF or GM-CSF, respectively, although the activity of the fusion protein is only in the G-CSF and GM-CSF. Although it is still unknown whether it is a side effect or more than just adding it, the results indicate that G-CSF and GM-CSF act independently in the fusion protein as fusion proteins, and G-CSF and GM- The binding of CSFs indicates that they do not interfere with each other.

In addition, because the diverse cell population constitutes myeloid cells, and thus many differentiated cells respond to G-CSF or GM-CSF, whether G-CSF and GM-CSF in the fusion protein work together on the same cell or not. It is unclear whether they act individually, but both CSFs in the fusion protein show colony-stimulating activity of G-CSF and GM-CSF even though they are bound by a short GS linker of Gly-Gly-Gly-Gly-Ser. The facts can be confirmed.

Hereinafter, the configuration of the present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited only to the following examples.

Example 1 Preparation of G-CSF / GM-CSF Gene Linked with Recombinant Human G-CSF Gene and GM-CSF Gene

Oligonucleotide was purchased from 'Bionix (Seoul, Korea)' as a material and restriction enzyme was purchased from 'Kosco (Seoul, Korea)'. Culture medium for E. coli expression was purchased from 'Difco Ltd (Sparks, MD, USA)', other chemicals were 'Sigma Aldrich Co. Ltd (St. Louis, MI, USA). Ni-NTA purification system was purchased from Qiagen Co. (Hilden, Germany).

Plasmids pGEMT (Promega, Madsion, WI, USA) and pET-22b (+) (Novagen, Darmstadt, Germany) were used as the material of the expression construct. E. coli strains DH-5α and BL21 (DE) were used for plasmid stability and expression of recombinant proteins. DNA and protein manipulations were performed by standard procedures (Garland, 1987; Bradley, 1980), and PCR was performed by manufacturer's instructions using Taq DNA polymerase (Promega). Human GM-CSF cDNA was developed by the University of Texas at Southwestern Medical School's Dr. David Carbone 'and human G-CSF cDNA were purchased from the R & D system (Minneapolis. MN, USA).

The insert cDNA and linker were amplified using the following primers before ligation of human G-CSF and GM-CSF cDNA into a single construct.

Human G-CSF was amplified using primers 31 and 32, and human GM-CSF used primers 33 and 39. GS linker fragment (Olafsena, T., Rasmussena, IB , Norderhaugb, L., Bruland, S. and Sandliea, I. (1998) IgM secretory tailpiece drives multimerisation of bivalent scFv fragments in eukaryotic cells Immunotechnology 4:. 141-153. Amplification was performed using primers 34 and 35.

PCR reaction was carried out using Taq polymerase by the following amplification conditions. One cycle consisting of denaturing at 95 ° C. for 4 minutes, annealing at 50 ° C. for 2 minutes, and extension at 72 ° C. for 3 minutes, followed by 1 cycle at 95 ° C. 45 cycles consisted of denaturation for minutes, annealing at 50 ° C. for 2 minutes, and elongation at 72 ° C. for 3 minutes.

After 10 min extension at 72 ° C., the amplified DNA fragments were purified from 1% agarose gel.

On the other hand, the mutual coupling of G-CSF, GM-CSF and linker sequences was performed using a Splicing Overlapped Extension (SOEing) technique, as shown in Figure 1a.

First, through the first SOEing reaction, DNA fragments containing G-CSF / linker and linker / GM-CSF were prepared according to the following scheme.

First G-CSF and linker fragments were annealed and bound together using Taq polymerase without any primers after 10 cycles consisting of denaturation at 95 ° C. for 1 minute and extension at 72 ° C. for 4 minutes. .

After the reaction, primers 31 and 35 were added and amplified by performing 30 cycles consisting of denaturation at 95 ° C. for 30 seconds, annealing at 60 ° C. for 30 seconds and extension for 1 minute at 72 ° C., resulting in G- CSF / linker sections were made.

Using the same procedure and primers 34 and 39 above, a linker / GM-CSF fragment was obtained.

Amplified G-CSF / linker and linker / GM-CSF fragments were purified from 1% agarose gel and used in the second SOEing reaction below for the preparation of G-CSF / linker / GM-CSF DNA fragments (FIG. 1A). ).

First, the G-CSF / Linker and Linker / GM-CSF DNA fragments were annealed and after 10 cycles consisting of denaturation at 95 ° C. for 1 minute and expansion at 72 ° C. for 4 minutes without the use of any primers, Taq polymerase was used to interconnect each other. Primers 31 and 39 were then added and amplified by 30 cycles consisting of annealing at 95 ° C. for 30 seconds, restoring at 60 ° C. for 30 seconds, and expansion at 72 ° C. for 1 minute.

As a result, a polynucleotide composed of the DNA sequence of SEQ ID NO: 1 was obtained.

After reaction, approximately 1.1 kb of DNA fragment prepared above was isolated from 1% agarose gel (FIG. 1B), cloned into pGEMT vector, and subsequently contained pel B leader, His.Taq and T7 promoters. Was cloned into the pET-22 (+) vector.

That is, 1.1 kb DNA fragments were first cloned into pGEMT and subcloned into the Nco I and Not I positions of the pET-22b (+) vector to prepare pEThGGMCSF constructs expressing the G-CSF / GM-CSF fusion protein.

Example 2 Preparation of Proteins from the Gene Prepared in Example 1

Expression of the G-CSF / Linker / GM-CSF fusion protein was performed by IPTG induction after transformation of the construct with E. coli BL21 (DE). 25 mL of glucose-free medium supplemented with IPTG and ampicillin to induce expression of cloned G-CSF / GM-CSF fusion proteins after culturing cells in 50 mL of medium supplemented with glucose and ampicillin Moved to.

The cells were recovered after 5 hours of induction, etc. Kim (Kim JK, Tsen, MF, Ghetie, V. and Ward, ES (1994a) Identifying amino acid residues that influence plasma clearance of murine IgG1 fragments by site-directed muTaqenesis. Eur Peripheral cytoplasm as described by J. Immunol. 24 , 542-547 . ; McClay EF.Adjuvant therapy for patients with high-risk malignant melanoma.Semin Oncol. 2002 Aug; 29 (4): 389-99. Proteins were extracted from space, cytoplasm, cell inclusion bodies. Cell pellets were subjected to osmotic shock for 40 minutes in TES buffer (0.5 M sucrose, 0.1 mM EDTA, 0.2 M Tris-Cl, pH 7.4) for extraction of periplasmic protein. Residual cell pellets were sonicated using sonication buffer (0.25 M NaCl, 0.05 M Tris, pH 7.5), and insoluble residues were urea denaturing buffer (8 M urea, 0.1 M NaH ₂ PO ₄ , 0.01 M Tris, pH 8.0).

The extracted protein was subjected to Ni-NTA agarose purification as directed by the manufacturer (Qiagen).

As a result of the experiment with the above materials and methods, it was confirmed that 40 kDa recombinant human G-CSF / GM-CSF fusion protein (rhG-CSF / GM-CSF) was expressed in the urea sample (FIG. 2). Although the construct had a pel B leader sequence to obtain a protein expressed in the periplasm, the fusion protein was expressed in the cell inclusion body, which formed insoluble protein aggregates by overexpression in E. coli cells. Meant.

Meanwhile, the present inventors prepared recombinant G-CSF and GM-CSF proteins using pET-22b (+) vectors, respectively. GM-CSF was expressed in the cell inclusion body, and G-CSF was expressed in the cytoplasm. It could be confirmed (Fig. 2). However, CSF / GM-CSF fusion proteins were expressed in cell inclusion bodies even though GM-CSF was bound to G-CSF expressed in the cytoplasm.

In addition, expressed fusion proteins corresponded to the size of bound human G-CSF and human GM-CSF, and were identified using Eliza and Western blot.

Experimental Example 1: Functional Analysis of the Protein Prepared in Example 2

Colony stimulating activity of the purified protein was examined as described by Garland (1987), and all experiments were performed in three replicates in a 24 well plate. Each of 2 × 10 ⁶ myeloid cells extracted from leukemia patients was briefly suspended in 1 mL of DMEM / 10% FCS medium and then plated into each well. 8 μg or 16 μg of Ni-NTA purified protein was added to each cell well and allowed to stand by 2.2% low melting agarose. The wells were incubated at 37 ° C. in humid air containing 5% CO ₂ . A week later colony numbers were counted at five random sites in each well and the mean number of colonies was compared with each other.

On the other hand, in order to investigate the colony-stimulating activity of the recombinant G-CSF / GM-CSF fusion protein, rhG-CSF / GM-CSF fusion protein in 8M urea solution denatured due to the high concentration of urea before the measurement of biological activity It needed to be restored. Simple dialysis of the protein did not induce precipitation of the protein, but it dissolves well, which may mean that the protein is folded back into its natural form.

Colony-stimulating activity of the dialyzed rhG-CSF / GM-CSF fusion protein was examined by assaying colony formation with bone marrow cells derived from leukemia patients. The addition of purified 40 kDa protein 8 and 16 μg / mL Compared with the negative control significantly increased colony formation (FIG. 3), which indicates that the 40 kDa protein from the urea sample not only acts as a colony stimulating element but also can be folded back into the biologically active form by simple dialysis. It meant.

Colony forming units of media treated with the fusion protein were higher than media treated with the same number of recombinant G-CSF or GM-CSF, respectively (FIGS. 2 and 3). Although it is ambiguous whether the activity of the fusion protein is only a side effect of G-CSF and GM-CSF or more than just addition, the experimental results show that G-CSF and GM-CSF act independently of the fusion protein as the fusion protein. It was shown that the binding of G-CSF and GM-CSF did not interfere with each other.

On the other hand, since a diverse cell population constitutes bone marrow cells, and thus many differentiated cells respond to G-CSF or GM-CSF, whether G-CSF and GM-CSF in the fusion protein work together on the same cell or not. It is unclear whether they act individually, but both CSFs showed colony-stimulating activity of G-CSF and GM-CSF in fusion proteins even though bound by the short GS linker of Gly-Gly-Gly-Gly-Ser.

As described above, the polypeptide consisting of the amino acid sequence of SEQ ID NO: 2 encoded from the polynucleotide consisting of the DNA sequence of SEQ ID NO: 1 of the present invention can be extracted from the cell inclusion body, the function of the G-CSF protein and GM-CSF As both proteins can function, they can be provided as one simpler fusion protein when the functions of G-CSF protein and GM-CSF protein are needed, including in vivo or ex vivo expansion of bone marrow hepatocytes. It is a very useful invention in the engineering industry.

Figure 1a is a schematic diagram showing the construction of human G-CSF / linker / GM-CSF fusion DNA.

1B is a photograph showing the PCR amplification products of human G-CSF / GM-CSF bound with human G-CSF, GM-CSF and linker sequences.

2 is a photograph showing SDS-PAGE analysis of purified recombinant human G-CSF, GM-CSF and G-CSF / GM-CSF fusion proteins.

3 is a graph showing colony stimulating activity of purified, folded G-CSF / GM-CSF fusion proteins.

<110> YOUN, Hyun Joo KIM, Jin-Kyoo LEE, Dong seok Inje University <120> Recombinant human G-CSF / GM-CSF fusion protein and it's coding gene <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 930 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (1) .. (930) <400> 1 atg gcc acc ccc ctg ggc cct gcc agc tcc ctg ccc cag agc ttc ctg 48 Met Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu 1 5 10 15 ctc aag tgc tta gag caa gtg agg aag atc cag ggc gat ggc gca gcg 96 Leu Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala 20 25 30 ctc cag gag aag ctg tgt gcc acc tac aag ctg tgc cac ccc gag gag 144 Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu 35 40 45 ctg gtg ctg ctc gga cac tct ctg ggc atc ccc tgg gct ccc ctg agc 192 Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser 50 55 60 tcc tgc ccc agc cag gcc ctg cag ctg gca ggc tgc ttg agc caa ctc 240 Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu 65 70 75 80 cat agc ggc ctt ttc ctc tac cag ggg ctc ctg cag gcc ctg caa ggg 288 His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Gln Gly 85 90 95 ata tcc ccc gag ttg ggt ccc acc ttg gac aca ctg cag ctg gac gtc 336 Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val 100 105 110 gcc gac ttt gcc acc acc atc tgg cag cag atg gaa gaa ctg gga atg 384 Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met 115 120 125 gcc cct gcc ctg cag ccc acc cag ggt gcc atg ccg gcc ttc gcc tct 432 Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser 130 135 140 gct ttc cag cgc cgg gca gga ggg gtc ctg gtt gct agc cat ctg cag 480 Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln 145 150 155 160 agc ttc ctg gag gtg tcg tac cgc gtt cta cgc cac ctt gcg cag ccc 528 Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro 165 170 175 ggt gga ggc gga tcg gca ccc gcc cgc tcg ccc agc ccc agc acg cag 576 Gly Gly Gly Gly Ser Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln 180 185 190 ccc tgg gag cat gtg aat gcc atc cag gag gcc cgg cgt ctc ctg aac 624 Pro Trp Glu His Val Asn Ala Ile Gln Glu Ala Arg Arg Leu Leu Asn 195 200 205 ctg agt aga gac act gct gct gag atg aat gaa aca gta gaa gtc atc 672 Leu Ser Arg Asp Thr Ala Ala Glu Met Asn Glu Thr Val Glu Val Ile 210 215 220 tca gaa atg ttt gac ctc cag gag ccg acc tgc cta cag acc cgc ctg 720 Ser Glu Met Phe Asp Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu 225 230 235 240 gag ctg tac aag cag ggc ctg cgg ggc agc ctc acc aag ctc aag ggc 768 Glu Leu Tyr Lys Gln Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly 245 250 255 ccc ttg acc atg atg gcc agc cac tac aag cag cac tgc cct cca acc 816 Pro Leu Thr Met Met Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr 260 265 270 ccg gaa act tcc tgt gca acc cag act atc acc ttt gaa agt ttc aaa 864 Pro Glu Thr Ser Cys Ala Thr Gln Thr Ile Thr Phe Glu Ser Phe Lys 275 280 285 gag aac ctg aag gac ttt ctg ctt gtc atc ccc ttt gac tgc tgg gag 912 Glu Asn Leu Lys Asp Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu 290 295 300 cca gtc cag gag gtc acc 930 Pro Val Gln Glu Val Thr 305 310 <210> 2 <211> 310 <212> PRT <213> Homo sapiens <400> 2 Met Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu 1 5 10 15 Leu Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala 20 25 30 Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu 35 40 45 Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser 50 55 60 Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu 65 70 75 80 His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Gln Gly 85 90 95 Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val 100 105 110 Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met 115 120 125 Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser 130 135 140 Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln 145 150 155 160 Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro 165 170 175 Gly Gly Gly Gly Ser Ala Pro Ala Arg Ser Pro Ser Pro Ser Thr Gln 180 185 190 Pro Trp Glu His Val Asn Ala Ile Gln Glu Ala Arg Arg Leu Leu Asn 195 200 205 Leu Ser Arg Asp Thr Ala Ala Glu Met Asn Glu Thr Val Glu Val Ile 210 215 220 Ser Glu Met Phe Asp Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu 225 230 235 240 Glu Leu Tyr Lys Gln Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly 245 250 255 Pro Leu Thr Met Met Ala Ser His Tyr Lys Gln His Cys Pro Pro Thr 260 265 270 Pro Glu Thr Ser Cys Ala Thr Gln Thr Ile Thr Phe Glu Ser Phe Lys 275 280 285 Glu Asn Leu Lys Asp Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu 290 295 300 Pro Val Gln Glu Val Thr 305 310

Claims (2)

Polynucleotide consisting of the DNA sequence of SEQ ID NO: 1. Polypeptide consisting of the amino acid sequence of SEQ ID NO: 2.