KR100486070B1 - Expression vector for human erythropoietin and pprcess for the mass-production of epo employing same - Google Patents

Abstract

The present invention relates to a vector for mass production of human erythropoietin (EPO) and a method for producing EPO using the same, wherein the DNA encoding the thioredoxin (Trx), a recognition site for protease and EPO can be obtained by culturing E. coli transformed with the expression vector of the present invention containing DNA encoding the EPO in sequence and mass-producing Trx-EPO fusion protein in a solubilized state and cleaving it with proteolytic enzymes. .

Description

Expression of human erythropoiesis-stimulating factor (EPO) vector and mass production method of EPO using same {EXPRESSION VECTOR FOR HUMAN ERYTHROPOIETIN AND PPRCESS FOR THE MASS-PRODUCTION OF EPO EMPLOYING SAME}

The present invention relates to a human erythropoietin (EPO) expression vector and a mass production method of EPO using the same.

EPO is a hormone that promotes red blood cell formation in higher organisms and is a glycoprotein composed of 166 amino acids and several types of sugars (Carnot, et al., Compt. Rend. , 143 , 384, 1906). In addition, EPO is an indispensable factor for the formation of red blood cells in the blood and is used for diagnosis and treatment of blood-related diseases such as anemia.

Natural human EPO is a monomeric acid glycoprotein with a total molecular weight of about 34 kDa and a protein molecular weight excluding sugar of about 18 kDa. In addition, it contains three N-linked sugar chains at the positions of amino acids Asn 24, Asn 38 and Asn 83 and one mucin-type O-linked sugar chain at the Ser 126 position. Because it plays an essential role, decreasing the sugar content of EPO sharply decreases its biological half-life, resulting in the loss of biological activity.

In normal people, EPO is maintained at about 15-30 mU, or 0.01 mM, per ml of blood (Gaarcia, JF, Lab. Clin. Med. , 99 , 624-635, 1982), but diseases such as aplastic anemia Patients suffering from EPA produce higher concentrations of EPO than normal people, so they may produce EPO separately from their blood or urine (White et al., Rec. Progr. Horm. Res. , 16 , 219, 1960; Espada et al. , Biochem.Med. , 3 , 475, 1970; Fisher, Pharmacol. Rev. , 24 , 459, 1972). However, this method has a disadvantage that the supply of raw materials for mass production of EPO is limited, and in the urine of normal people, not only the concentration of EPO is low, but also an inhibitor of activity requires a high purification technology to remove it. Patents 4,397,840, 4,303,650 and 3,865,810). In addition, methods for extracting EPO from sheep plasma have been developed, but it is known that sheep EPO may act as an antigen to humans (Goldwasser, Control cell. Dif. Develop. , Part A , 487-494, 1981). Sugimoto et al. Have reported a method for producing EPO by propagating human lymphocytes, including Namalva, BALL-1, NALL-1, TALL-1 and JBL, in an animal body (US Patent No. 4,377,513), since this method uses human tumor cells, studies to secure safety are required.

  In addition, methods for cloning human EPO genes and expressing them in animal cells (Korean Patent Publication Nos. 89-1011 and 93-5917) and yeast or insect cells (Korean Patent Publication No. 95-5988) have been developed. However, yeast or insect cells mainly produce high mannose type glycoproteins and have low glycosylation capacity and thus do not produce complex type glycoproteins such as human EPO. The structure is not suitable as a host cell for expressing human EPO because there is a risk of causing antigenicity in the human body. In addition, the production of human EPO using animal cells can produce the EPO most similar to the natural type, but the expression rate is very low, there is a disadvantage that the production cost is expensive.

In proteins, sugar chains contribute to biological activity by increasing the water solubility in vivo or by increasing the half-life in vivo through protection of the protein from chemical and physical attacks.

Of the four sugar chain structures of human EPO, the O-linked sugar chain at the Ser 126 position did not play any role in vivo or in vitro , and was removed when three N-linked sugar chains were removed. It was found that the activity in the body was drastically reduced but the activity in vitro was maintained (Masato H. et al., J. Biol. Chem. , 267 , 7703-7709, 1992). This fact indicates that sugar chains of human EPO do not participate in binding to receptors in vivo but only contribute to structural stability. Therefore, there have been many studies for mass production of EPO by producing E. coli, which has no glycosylation function, as a host cell, and then applying appropriate modifications to increase structural stability, but fail to express pure EPO in a solubilized state. It was. For example, Vossel et al. Expressed EPO in the form of a fusion protein in E. coli by conjugating 10 histidines to the amino terminus of EPO (Boissel JP et al., J. Biol. Chem. , 268 (21)). , 15983-93, 1993), Bill et al. Expressed the fusion protein with glutathione-S-transferase bound to the amino terminus of EPO using four E. coli expression vectors (Bill RM, et al., Biochem. , Biophys.Acta , 1261 , 35, 1995). However, EPO obtained by the above-described method is very low in the expression rate is difficult to obtain pure EPO, or very low water solubility is expressed in the form of inclusion bodies, there are many difficulties in mass production.

In order to develop a method for mass production of EPO protein from Escherichia coli, the present inventors transformed E. coli into a vector prepared by fusing a part of a highly soluble heterologous protein gene to the 5'-end of the EPO gene, and then culturing it. The present invention was completed by expressing a fusion protein in a solubilized state, and cutting the obtained fusion protein with a protease to produce an EPO protein having the same amino acid sequence as that of the natural form.

It is an object of the present invention to provide an expression vector and a microorganism transformed with the vector for expressing a human erythropoiesis stimulating factor in a solubilized state in E. coli.

Another object of the present invention is to provide a method for mass production of human erythroid stimulating factor by culturing the microorganism.

In accordance with the above object, in the present invention, DNA encoding thioredoxin as a vector for mass expression of human red blood cell stimulating factor (EPO) in a solubilized state in E. coli, a site for recognizing protease and EPO of SEQ ID NO: 1 It provides an expression vector containing the DNA coding in order.

The DNA encoding thioredoxin that may be used in the vector of the present invention may be all or part of the DNA encoding thioredoxin, and preferably the DNA encoding the 109 amino acid at the N-terminus of thioredoxin. Preferably SEQ ID NO: 7 may be used.

In addition, in the present invention, the DNA encoding the EPO can be any of DNAs encoding natural or variant EPO. In particular, the DNA represented by SEQ ID NO: 1 can be preferably used.

The protease recognition site in the vector of the present invention is included to easily separate the EPO from the expressed thioredoxin-EPO fusion protein, and known factors Xa, thrombin, and enterokinase recognition sites in the art. And enterokinase recognition sites are preferred. Enterokinase recognizes the Asp-Asp-Asp-Asp-Lys sequence (D4K, SEQ ID NO: 8) and cleaves its C-terminus, thus inserting DNA encoding the sequence into the vector results in EPO from the expressed fusion protein. It can be easily recovered.

To facilitate the purification of thioredoxin-EPO fusion proteins expressed from the vectors of the invention, the vectors of the invention may further comprise DNA encoding a histidine tag.

The DNAs can be easily introduced into one vector according to a conventional cloning method, and expression vectors can be prepared more conveniently using a vector pET 31a (Novagen) containing a histidine tag and a thioredoxin gene. have.

As a promoter in the vector of the present invention, any promoter commonly used for expression of heterologous proteins in Escherichia coli may be used, and examples thereof include a T7 promoter, a Tac promoter, and an arabinose promoter.

The vector of the present invention can be produced, for example, as follows.

First, a human kidney cDNA library (Clonetech Ca. No. HL5031t) was used as a template, and EPO cDNA was synthesized by conventional polymerase chain reaction (PCR) using primers of SEQ ID NO: 3 and SEQ ID NO: 4, After cleavage with Nco I and BamH I, the expression vector pTEPO was prepared by cloning the appropriate expression vector, for example, the Nco I- BamH I position of pET 31a (Novagen), which is an E. coli expression vector (FIG. 1).

When the thioredoxin-EPO fusion protein obtained by culturing E. coli transformed with the expression vector pTEPO was digested with protease, EPO was obtained by adding alanine and methionine in front of alanine, the first amino acid of human erythropoiesis. Site-directed mutagenesis was performed to remove the codon corresponding to the added alanine-methionine to prepare plasmid pThEPO expressing EPO having the same amino acid sequence as the native form.

In the present invention, E. coli, preferably E. coli BL21 (DE3) (Novagen), Top10 (Invitrogen) or XL-l blue (Novagen) with the expression vector can be expressed a vector containing the T7 promoter BL21 (DE3) can be transformed to obtain an E. coli transformant that expresses the thioredoxin-EPO fusion protein in a solubilized state. One of these E. coli transformants, HM 11215, was deposited as KCCM-10391 with the Korea Microorganism Conservation Center on June 21, 2002. When the obtained transformant is cultured under conditions suitable for expression, a thioredoxin-EPO fusion protein can be obtained in a solubilized state.

The thioredoxin-EPO fusion protein obtained above can be purified by affinity binding chromatography, preferably by nitrilotriacetic acid (Ni-NTA) affinity binding chromatography using affinity with histidine residues present in the fusion protein. . The purified fusion protein is cleaved with a suitable protease, preferably factor Xa, thrombin or enterokinase, which recognizes a recognition site present in the vector, and the same amino acid sequence as the native form (SEQ ID NO: 2) or mutation. Human EPO having a type amino acid sequence can be produced in large quantities.

Hereinafter, the specific configuration and operation of the present invention will be described with reference to Examples, but the scope of the present invention is not limited only to the following Examples.

Example 1 Construction of Human EPO Fusion Protein Expression Vectors

To obtain the human EPO cDNA gene, the oligonucleotide of SEQ ID NO: 3 designed to correspond to the 5'-terminus of human EPO and contain the Nco I restriction enzyme recognition sequence before alanine, the first amino acid of EPO, and to the 3'-terminus of EPO Oligonucleotides of SEQ ID NO: 4, corresponding to and designed to include stop codons and Bam HI restriction enzyme recognition sequences, were constructed. Human EPO cDNA by polymerase chain reaction (PCR) using a human kidney cDNA library (Clonetech Cat. No. HL5031t) as a template and oligonucleotides of SEQ ID NO: 3 and SEQ ID NO: 4 prepared above as primers Was synthesized.

For PCR, 1 μl of template, 1 μl of each primer (50 pmole / μl), 5 μl of 2 mM dNTP, 0.5 μl of Pfu DNA polymerase (Stratagen), and 5 μl of Pfu reaction buffer (100 mM Water was added to the reaction mixture of Tris-HCl (pH 8.3), 15 mM MgCl ₂ , 500 mM KCl, adjusted to a final volume of 50 μl, followed by a thermocycler (DNA thermocycler, Perkin-Elmer USA) at 94 ° C. After preliminary denaturation for 5 minutes, the reaction of 60 seconds at 94 ℃, 60 seconds at 60 ℃, 60 seconds at 72 ℃ was carried out 30 times.

The resulting PCR product was digested with restriction enzymes Nco I and BamH I and cloned into Nco I- BamH I cleavage site of plasmid pET32a (Novagen, Ca. No. 69015-3) containing a portion of the thioredoxin gene. The produced plasmid was named pTEPO.

When the plasmid pTEPO prepared above is expressed in E. coli, the fusion protein of thioredoxin and EPO is expressed, and when this is cleaved with protease, EPO is produced in which alanine and methionine are added in front of alanine, the first amino acid of EPO.

Therefore, a site-directed mutagenesis method using PCR was performed to prepare a vector expressing an EPO having the same amino acid sequence as the native form. The polymerase chain reaction was performed using pTEPO obtained above as a template and using SEQ ID NO: 5 and SEQ ID NO: 6 as primers.

PCR was performed using 2.5 units of Pfu turbo DNA polymerase (Stratagen, Ca. No. 200518) as polymerase and preliminarily denatured at 95 ° C for 5 minutes, followed by 30 seconds at 95 ° C, 60 seconds at 55 ° C, 68 The same procedure as described above was carried out except that the reaction for 2 minutes at 18 ° C. was performed 18 times.

The PCR product was recovered, and the restriction enzyme Dpn I was added to completely remove the existing plasmid. The E. coli XL-l blue (Novagen) strain was transformed with the obtained plasmid according to a conventional method, a DNA vector was recovered from the transformed strain, and the mutated nucleotide sequence was confirmed by DNA sequencing. Obtained plasmid pThEPO to produce. Subsequently, E. coli BL21 (DE3) was transformed with the vector pThEPO in the same manner to obtain the E. coli transformant HM 11215. Escherichia coli HM 11215 was deposited on June 21, 2002 with the accession number KCCM-10391 to the Korea Microbiological Conservation Center.

Example 2: Confirmation of the expression and availability of the fusion protein

The E. coli transformant HM 11215 obtained in Example 1 was inoculated in 5 ml of LB medium (5 g of yeast extract, 10 g of tryptone, 5 g / L of calcium chloride), and then cultured to obtain an absorbance of about 1 at 600 nm. When IPTG was added to incubate for 3 hours to induce the expression of the fusion protein. 1 ml of the obtained culture was taken and centrifuged at 12,000 x g for 5 minutes to obtain a cell precipitate. The resulting cell precipitate was dissolved in 100 μl of protein sample buffer (5% glycerin, 12 mM Tris-HCl, pH 6.8), 0.4% SDS, 0.05% bromophenol blue, 3 mM 2-mercaptoethanol and boiled water. After 5 minutes of reaction at 12,000 xg was centrifuged for 10 minutes to obtain a supernatant. 15% SDS polyacrylamide gel electrophoresis was performed on the obtained supernatant to confirm that the EPO fusion protein was expressed (FIG. 2). In FIG. 2, the first column is the molecular weight marker (Benchmarker, Invitrogen), the second column is the protein of the cell precipitate of the host cell used for expression, and the third column is the electrophoresis of the cell precipitate expressing the EPO fusion protein. .

To confirm whether the expressed EPO fusion protein was expressed in a solubilized state, 0.5 g of the cell precipitate of the transformant HM 11215 obtained by the above method was added to 20 ml of cell disruption buffer solution (10 mM NaH ₂ PO ₄ H ₂ O). (pH 8.0), 10 mM imidazole, 300 mM calcium chloride), and then lysozyme and Triton X-100 were added and left on ice for 30 minutes. The obtained cell solution was crushed using an ultrasonic crusher (Misonix XL2020, USA) and centrifuged at 20,000 μg for 15 minutes to obtain an insoluble and water soluble fraction. Each obtained fraction was subjected to 15% SDS polyacrylamide gel electrophoresis (FIG. 3). The first column of FIG. 3 is a cell precipitate obtained by centrifugation of the culture medium of the expressed host and mixed with a protein sample buffer, and the second column and the third column are the aqueous and insoluble fractions of the cell disruption solution. It was confirmed that about 70% of the expressed EPO fusion protein was in a solubilized state.

Example 3: Purification of EPO from Fusion Proteins

EPO fusion protein contains a histidine residue. Since histidine has a strong binding ability to nitrilotriacetic acid (Ni-NTA), the EPO fusion protein was purified by Ni-NTA affinity binding chromatography. First, 20 ml of Ni-NTA-immobilized resin (Novagen) was filled in a column, and then washed with a column binding buffer (the same composition as the cell disruption buffer solution). The solubilized EPO fusion protein fraction obtained in Example 2 was added to the column to bind and then washed sequentially with the same buffer and the above buffer with imidazol concentration increased to 50 mM. The EPO fusion protein bound to the column was eluted with the buffer containing 100 mM imidazole.

Enterokinase buffer solution (20 mM Tris-HCl (pH 7.4), 50) with Sephadex G-25 resin (Phramacia biotech) to cleave the partially purified EPO fusion protein with enterokinase (Novagen) in this manner. After substitution with mM NaCl, 2 mM CaCl ₂ ), 20 U of enterokinase (Novagen) was added per 1.5 mg of the fusion protein, followed by reaction at 25 ° C. for 16 hours to cleave the fusion protein. An enterokinase cleavage reaction solution was cut onto a Ni-NTA column to obtain only free EPO. The column was equilibrated with equilibration buffer (50 mM NaH ₂ PO ₄ , pH 8.0, 0.3 M NaCl, 10 mM Imidazol) followed by dropping of the enterokinase cleavage reaction solution. When EPO is cleaved with enterokinase, there is no histidine tag, so when the column is washed with the same buffer as the equilibration buffer, only free EPO is eluted, and the cleaved fusion protein and the cleaved Trx protein bind to the metal ion of the column. Only free EPO can be obtained.

Western blotting according to the Hollow et al. (Ed Harlow and David Lane, Antibodies , A Laborative Manual , Cold Spring Harbor Laboratory, 1988) using the samples obtained in each step of the purification process of Examples 2 and 3 as follows (Western blotting) was performed. Wet the nitrocellulose membrane (Amersham, USA) in blotting buffer (170 mM glycine, 25 mM Tris-HCl (pH 8), 20% methanol) and then 15% SDS-PAGE gel using a blot kit Blotting was performed for 1 hour. The nitrocellulose membrane was then left in a 0.5% skim milk (DIFCO, USA) solution for 1 hour and PBST solution (0.14M NaCl, 2.7) containing rabbit anti-EPO antibody (R & D systems, USA) and 0.05% Tween 20. mM KCl, 1.5 mM KH 2 PO 4, 8.1 mM Na 2 HPO 4, 3% BSA) and reacted at room temperature for 1 hour. After the reaction was completed, the membrane was washed three times with PBST solution to remove unreacted antibody, and then added to horseradish peroxidase (HRP) -bound goat anti-rabbit IgG (Amersham, USA) PBST solution diluted 5,000-fold and at room temperature. The reaction was carried out for a time. The membrane was then washed with PBST solution and developed by addition of a peroxidase substrate kit (Amersham, USA) solution (FIG. 4). 4 is a water precipitate obtained by transforming E. coli MH11215 cells in cultured E. coli MH11215 cells, and the third column is an EPO fusion protein fraction in a Ni-NTA column. The fraction which did not bind to the column after passage, the fourth and fifth columns were the fractions washed with 10 mM and 50 mM imidazole, respectively, and the sixth column was thioredoxin-EPO eluted from the Ni-NTA column. The fusion protein fraction, column 7 is a fraction cut with enterokinase, column 8 is a fraction of EPO purified by passing through a Ni-NTA column once more. From the above results, it was confirmed that the natural EPO protein was purified purely according to the method of the present invention.

According to the method of the present invention, human erythropoiesis-stimulating factor having the same amino acid sequence or variant amino acid as the natural type can be produced in large quantities using E. coli.

Figure 1 shows the manufacturing process of E. coli expression vector pThEPO containing human EPO cDNA,

2 is a photograph of SDS-polyacrylamide gel confirming that the human EPO fusion protein is expressed in a solubilized state in E. coli,

Figure 3 is a photograph of Western blot analysis of the samples obtained in each step of the purification method for obtaining purified EPO from the human EPO fusion protein expressed in E. coli.

<110> HANMI PHARM. IND. CO., LTD. <120> Expression vector for human erythropoietin and process for the mass-production of EPO employing same <130> 200207-0048 <160> 8 <170> KopatentIn 1.71 <210> 1 <211> 501 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (1) .. (498) <400> 1 gcc cca cca cgc ctc atc tgt gac agc cga gtc ctg gag agg tac ctc 48 Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu 1 5 10 15 ttg gag gcc aag gag gcc gag aat atc acg acg ggc tgt gct gaa cac 96 Leu Glu Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His 20 25 30 tgc agc ttg aat gag aat atc act gtc cca gac acc aaa gtt aat ttc 144 Cys Ser Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe 35 40 45 tat gcc tgg aag agg atg gag gtc ggg cag cag gcc gta gaa gtc tgg 192 Tyr Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val Trp 50 55 60 cag ggc ctg gcc ctg ctg tcg gaa gct gtc ctg cgg ggc cag gcc ctg 240 Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu 65 70 75 80 ttg gtc aac tct tcc cag ccg tgg gag ccc ctg cag ctg cat gtg gat 288 Leu Val Asn Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp 85 90 95 aaa gcc gtc agt ggc ctt cgc agc ctc acc act ctg ctt cgg gct ctg 336 Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu 100 105 110 cct tgg cag aag gaa gcc atc tcc cct cca gat gcg gcc tca gct gct 384 Pro Trp Gln Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala 115 120 125 cca ctc cga aca atc act gct gac act ttc cgc aaa ctc ttc cga gtc 432 Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val 130 135 140 tac tcc aat ttc ctc cgg gga aag ctg aag ctg tac aca ggg gag gcc 480 Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala 145 150 155 160 tgc agg aca ggg gac aga tg a 501 Cys Arg Thr Gly Asp Arg 165 <210> 2 <211> 166 <212> PRT <213> Homo sapiens <400> 2 Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu 1 5 10 15 Leu Glu Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His 20 25 30 Cys Ser Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe 35 40 45 Tyr Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val Trp 50 55 60 Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu 65 70 75 80 Leu Val Asn Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp 85 90 95 Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu 100 105 110 Pro Trp Gln Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala 115 120 125 Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val 130 135 140 Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala 145 150 155 160 Cys Arg Thr Gly Asp Arg 165 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Primer for cloning EPO <400> 3 gccgccatgg ccccaccacg cctcatctgt 30 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer for cloning EPO <400> 4 gggggatcct atcatctgtc ccctgtc 27 <210> 5 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Primer for cloning EPO <400> 5 ggtaccgacg acgacgacaa ggccccacca cgcctcatct gt 42 <210> 6 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Primer for cloning EPO <400> 6 acagatgagg cgtggtgggg ccttgtcgtc gtcgtcggta cc 42 <210> 7 <211> 327 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide coding for N-terminal 109 a.a of Thioredoxin <400> 7 atgagcgata aaattattca cctgactgac gacagttttg acacggatgt actcaaagcg 60 gacggggcga tcctcgtcga tttctgggca gagtggtgcg gtccgtgcaa aatgatcgcc 120 ccgattctgg atgaaatcgc tgacgaatat cagggcaaac tgaccgttgc aaaactgaac 180 atcgatcaaa accctggcac tgcgccgaaa tatggcatcc gtggtatccc gactctgctg 240 ctgttcaaaa acggtgaagt ggcggcaacc aaagtgggtg cactgtctaa aggtcagttg 300 aaagagttcc tcgacgctaa cctggcc 327 <210> 8 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Enterokinase recognition site <400> 8 Asp Asp Asp Asp Lys 1 5

Claims (10)

Plasmid pThEPO with a cleavage map as described in FIG. delete delete delete delete delete E. coli transformed with the plasmid of claim 1. The method of claim 7, wherein E. coli HM 11215 (KCCM-10391). 1) culturing the transformed E. coli of claim 7 under conditions suitable for expression of human erythropoiesis-stimulating factor; 2) purifying the fusion protein by affinity binding chromatography from the cultured cell disruption of Escherichia coli; And 3) separating the human erythrocyte forming stimulating factor after cleaving the purified protein with proteolytic enzymes. delete