JP3352429B2 - Expression system for producing recombinant human erythropoietin, method for purifying secreted human erythropoietin and use thereof - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates generally to the field of molecular biology of human erythropoietin. More particularly, the present invention relates to an expression system for producing biologically active recombinant human erythropoietin (rhEPO) and an improved method for purifying secreted rhEPOs.

[0002]

BACKGROUND OF THE INVENTION Human erythropoietin (hEPO) is
It is a glycoprotein having a molecular weight of 30-40 kD. In healthy adults, mature hEPO is produced in the kidney and secreted therefrom. Human erythropoietin mainly has the function of promoting the proliferation of erythropoietin cells in spleen, bone marrow and fetal liver cells and promoting the differentiation of erythrocytes. Natural human erythropoietin was initially prepared from urine of patients with aplastic anemia. The amount of human erythropoietin obtained from a patient's urine is very small, and conventional methods for purifying human erythropoietin are labor intensive and time consuming. Therefore, there is a need to develop a method for mass-producing and purifying human erythropoietin in a simple and economical manner.

[0003] Genetic engineering techniques have been developed for producing recombinant human erythropoietin (rhEPO) at high levels using mammalian cell lines as host cells. C encoding human erythropoietin (hEPO)
The DNA fragment was obtained from Jacob et al. (Jacob et.
al. , Nature, 313: 806-809,1.
985) and the nucleotide sequence was determined. In addition, Jacob and co-workers have transformed kidney fibroblast COS-1 cells under the control of the SV40 promoter with an expression vector into which a cDNA fragment encoding mature human erythropoietin has been introduced,
RhEPO is produced from the transformed kidney fibroblast COS-1 cells by transient expression thereof. Jacob et al. Have also determined the biological activity of the produced rhEPO.

[0004] US Patent Nos. 4,703,008 and 5,44
1,868, 5,547,993, 5,621,0
80 and 6,618,698 are 5.6 kb genomic DNAs containing the full-length human erythropoietin gene
And the use of the fragment inserted into an expression vector to transform monkey fibroblast COS-1 cells. RhEP from the transformed monkey fibroblast COS-1 cells
It was confirmed that O was temporarily secreted. In addition, stable transformed CHO cell lines capable of producing rhEPO have been obtained by the selection of methotrexate. E. coli E. coli. r in E. coli and yeast
A hEPO expression system has also been disclosed.

The baculovirus system in insect cell SF9 has been used to produce rhEPO (Quel).
le et al. , Blood 74 (2): 6
52-657, 1989). Although the yield of rhEPO from transformed SF9 cells is significantly improved (500,000 U / liter of culture), the molecular weight of the desired product obtained is lower than that of native human proteins due to low glycosylation. It is smaller than the molecular weight of lipopoietin. Mo
ri and co-workers (Gene 144 (2): 289)
-293, 1994) constructs a rhEPO expression vector having an interferon-α gene promoter, and converts the rhEPO expression vector into a B cell / leukemia BA.
Used to transform LL-1 cells. When transfected with Sendai virus, the transformed B-cell leukemia BALL-1 cells have higher levels of r than any of the transformants produced by previous researchers.
hEPO was produced.

[0006] A method for purifying rhEPO from a conditioned medium (conditioned medium) generally comprises the following steps. First, rhEPO is
DEAE cellulose column (Sherwood and
Goldwasher, Endocrinology
103 (3): 866-870, 1978) and DEAE Sephacel columns (Quelle
et al. , Blood 74 (2): 652-6.
57, 1989; Inoue et al. , Bi
logical and Pharmaceutical
al 17 (2): 180-184, 1994; B
en Ghanem et al. , Preparat
live Biochemistry 24 (2): 1
27-142, 1994) and selectively adsorbed or removed by passing through an ion exchange chromatography column.

[0007] Second, rhEPO is malt agglutinin-agarose (Qian et al., Blood).
68 (1): 258-262, 1986) and concanavalin A (ConA) -agarose (Quell).
e et al. , Blood 74 (2): 652.
-657, 1989). Third, C4 reverse phase HPLC was used to purify rhEPO homogeneously (Lange
et al. , Blood Cells 10 (2-
3): 305-314, 1984; Krystal
et al. , Blood 67 (1): 71-
79, 1986; Quelle et al. , Bl
wood 74 (2): 652-657, 1989;
noue et al. , Biological a
nd Pharmaceutical 17 (2):
180-184, 1994). US Patent 5,322,8
37 discloses a method using C4 reverse phase HPLC to uniformly prepare rhEPO having a specific activity of 120,000-160,000 U / mg.

[0008] An affinity column of immobilized monoclonal antibody was used to purify hEPO (or rhEPO). For example, rhEPO secreted from transformed lymphoblastoid cells was specifically adsorbed to an anti-hEPO monoclonal antibody-Sepharose 4B affinity column (Ben Ghane
m et al. , Preparative Bio
chemistry24 (2): 127-142,1
994). The adsorbed rhEPO fraction was eluted and then passed through a DEAE Sephacel ion exchange column. RhEPO of homogeneous product is about 5
Obtained in 0% yield.

[0009]

The use of recombinant DNA technology has increased the yield of rhEPO, but in order to purify the resulting rhEPO uniformly,
If a large amount of rhEPO needs to be prepared, a great deal of labor and a long time are still required. The prior art lacks effective means of producing large amounts of rhEPO in expression systems and is inadequate. In addition, the prior art requires a large amount of r
Effective means of purifying hEPO are lacking and inadequate. The present invention fulfills these long-standing needs and desires in the art.

[0010]

SUMMARY OF THE INVENTION The present invention provides a newly developed expression system for producing rhEPO. The present invention also provides a new method for purifying secreted rhEPO using a two-step column chromatography technique. Target protein rh
To express EPO, a PCR-amplified cDNA fragment encoding mature rhEPO was inserted into an expression vector under the control of the cytomegalovirus (CMV) promoter. Stably produce rhEPO in high yield,
Transformants (BHK-21 cells) having a secreting expression system were obtained by selection with the antibiotic G418. In order to purify the produced rhEPOs, first,
The target protein was precipitated from the conditioned medium using a salting out method. Glycoprotein contained in the precipitated portion was selectively adsorbed on the immobilized lectin resin. The bound glycoprotein was eluted with a buffer containing 0.5 M mannose, and a major component having a molecular weight of about 34 kD and a molecular weight of 35-45
rhEPOs containing a minor component of kD were obtained. 0.5
Another pool of strongly bound rhEPOs was obtained in an acidic buffer (pH 4.0) without being eluted with a buffer containing M mannose. These rhEPO isoforms were further purified by passing through a G-75 chromatography column.

The purification method disclosed in the present invention has the following advantages: (1) In the first step, the target protein rhEPO is
Concentrated by precipitation, thereby facilitating large-scale preparation of rhEPOs. (2) Reverse-phase HPLC required by conventional purification methods
Molecular weight of 35- which shows higher specific activity without using Chromatography or immunoaffinity column chromatography
It is possible to obtain a pool of rhEPOs of 45 kD and another pool of their isoforms with a molecular weight of 25-34 kD.

In one embodiment of the present invention, a cDNA fragment encoding human erythropoietin and pcD under the control of a cytomegalovirus (CMV) promoter.
An expression vector containing the NA3.1 vector is provided. Further, the cDNA fragment is preferably
SEQ ID No: 1 (SEQ ID NO: 1) and SEQI
It is produced by a PCR method using a primer selected from the group consisting of D No: 2 (SEQ ID NO: 2).

[0013] In another embodiment of the present invention, there is provided a cell line carrying the above expression vector. The cell line is preferably screened with the antibiotic G418. Further, the cell line is a BHK-
21 is preferred. Further, in another embodiment of the present invention, there is provided a method for producing human erythropoietin by culturing the above cell line in a conditioned medium and detecting production of human erythropoietin. Suitably, the method provides human erythropoietin produced.

Further, in another embodiment of the present invention,
A method for purifying human erythropoietin, comprising the steps of precipitating human erythropoietin from a sample, applying the precipitated human erythropoietin to an immobilized lectin column, and then eluting the human erythropoietin from a gel filtration column. Is provided. Preferably, the purified human erythropoietin obtained contains an isoform with a molecular weight of 35-45 kD and is about 90% pure. Further, in another embodiment of the present invention, there is provided a method of promoting the proliferation of hematopoietic cells by administering human erythropoietin produced by the above method to the cells. Further, in another embodiment of the present invention, there is provided a pharmaceutical composition comprising human erythropoietin produced by the above method and a pharmaceutically acceptable carrier. Other further aspects, features, and advantages of the present invention will be apparent from the following description of preferred embodiments of the invention, presented here for purposes of disclosure.

[0015]

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a cloned cD encoding human erythropoietin in a mammalian host cell.
The present invention relates to an expression system comprising an expression vector having an NA fragment. The cDNA fragment was amplified by polymerase chain reaction (PCR).
In fact, the template DN encoding human erythropoietin
The A fragment was extracted from a phage cDNA library. The two primers (SEQ ID Nos: 1-2) used in the PCR reaction were Jacob
And co-workers (Nature 313: 806-8)
09, 1985). A HindIII restriction site was introduced at the 5 'end of one primer and an XbaI restriction site was added at the 3' end of the other primer. The PC
The product of R contains the coding sequence for the signal peptide.
It is believed to be a 96 bp fragment. The PC
R was performed in a standard manner. The resulting reaction mixture was extracted with phenol / chloroform and used as a template for second land PCR. An approximately 600 bp fragment was obtained and identified by DNA sequencing.

CDN of human erythropoietin confirmed
The A fragment is a pcDNA3.1 vector (Nov
HindIII restriction site and X
Ligation between the baI restriction sites. In this way rhEP
A pcDNA3.1 expression vector producing O was prepared and named pcDNA3.1-HC. The restriction map is shown in FIG. Expression of the cloned cDNA was under the control of the CMV promoter. The constructed expression vector is used as a host strain. coli (NovaBlu
e) was transformed to produce a transformant.

The present invention also provides a mammalian cell line that secretes rhEPO. The above pcDNA3.1-
Using HC, electroporation was used to obtain baby hamster kidney fibroblast BHK-21 cells (ATCC CCL-
10) was transformed. The resulting BHK-21 transformants were screened with antibiotics G418 at various concentrations of 100-400 mg / ml. Antibiotic G41
8-resistant ones were collected and expanded. The clone secreting the highest level of rhEPO was identified as BHK21-pcDN.
A3.1-HC. BHK21-pcDNA
The biological activity of rhEPO secreted from 3.1-HC was determined by assessing its ability to promote the growth of erythroleukemia cell TF-1. In addition, Western blot and ELISA were used to confirm the immunochemical properties of the secreted rhEPO.

The present invention relates to BHK21-pcDNA3.1.
-Provides a new method of fractionating rhEPO isoforms secreted from cell lines such as HC without using reverse phase HPLC. In the method, the desired r
The hEPOs are first precipitated using a salting out method. Various amounts of ammonium sulfate (m / v) were added to the conditioned medium, and the fractionated pools were collected and analyzed by dot blotting. As a result, when the saturation of ammonium sulfate reached 50% to 80%, the target r was increased.
It was found that all of the hEPOs could be precipitated. Second, the collected fraction was incubated with the immobilized lectin resin to adsorb the glycoprotein containing the desired rhEPOs. The buffer used to release the bound proteins consisted of an increasing gradient of mannose up to 0.5M concentration. The tightly bound fraction
Thereafter, elution was carried out with an acidic buffer (pH 4.0). Both of the pools collected exhibited the biological activity and immunochemical properties of rhEPO. Third, each of the pools collected was concentrated and then Sephadex G
Passed through a -75 column. The mannose eluted fraction contains a pool of rhEPOs consisting of three major components with a molecular weight of about 34 kD and minor components with a molecular weight of 35-45 kD.
The rhEPO pool shows about 90% purity,
The specific activity reached 240,000 U / mg. This specific activity was equivalent to the specific activity on TF-1 cells calculated by the biological activity of rhEPO produced from CHO cells. The acid-eluted part has a molecular weight of 34 kD,
It contains three main rhEPO isoforms of 28 kD and 25 kD and can be purified uniformly by column chromatography on Sephadex G-75. These rhEPO isoforms show lower specific activity (80,000-12,400 U / mg) than rhEPOs obtained from the mannose eluting pool.

In one embodiment of the present invention, a cDNA fragment encoding human erythropoietin and pcDNA3.1 under the control of the cytomegalovirus promoter.
An expression vector containing the vector is provided. Further, the cDNA fragment is preferably SEQ ID NO:
ID No: 1 (SEQ ID NO: 1) and SEQ ID No:
2 (SEQ ID NO: 2) by a PCR method using primers selected from the group consisting of: In another embodiment of the present invention, there is provided a cell line carrying the above expression vector. The cell line is preferably
Screened by antibiotic G418. Further preferably, the cell line is BHK-21. Further, in another embodiment of the present invention, there is provided a method for producing human erythropoietin by culturing the above cell line in a conditioned medium and detecting the product. Preferably, human erythropoietin produced by this method is provided.

Further, in another embodiment of the present invention,
A method for purifying human erythropoietin, comprising the steps of precipitating human erythropoietin from a sample, applying the precipitated human erythropoietin to an immobilized lectin column, and then eluting the human erythropoietin from a gel filtration column. Is provided. The purified human erythropoietin obtained preferably contains an isoform with a molecular weight of 35-45 kD and is about 90% pure. Further, in another embodiment of the present invention, there is provided a method of promoting the proliferation of hematopoietic cells by administering human erythropoietin produced by the above method to the cells. Further, in another embodiment of the present invention, there is provided a pharmaceutical composition comprising human erythropoietin produced by the above method and a pharmaceutically acceptable carrier.

[0021]

The following examples are provided to illustrate various embodiments of the present invention and are not meant to limit the invention in any way. Example 1 Amplification of hEPO-encoding cDNA fragment The template cDNA used for PCR was human liver cDN
A library (ClonTech, catalog no. H
L3006a) was extracted with phenol / chloroform. Primers used for PCR were based on reported sequences (Jacob et al., Nature, 3
13: 806-809, 1985) and is shown below: EPOS: 5'-GCAAGCTTATGGGGGTG
CACGAATG-3 '(SEQ ID No: 1) EPOX: 5'-GCATCTAGATCATCTGTGT
CCCCTGTCCT-3 '(SEQ ID No:
2)

The primer EPOS contains a translation initiation codon followed by an introduced HindIII restriction site. Primer EPOX is a sequence at the end of the coding sequence terminating in a translation stop codon, in which Xba
An I restriction site has been added. The reaction mixture for the first round of PCR contains 1 ml of the previously prepared template cDN.
A, 1 ml of each of two primers (0.5 mM), 1
0 ml reaction buffer, 0.5 ml (1.25 units)
Ex Taq DNA polymerase (Takar
a, Japan) and sufficient water to match the final volume of 100 ml. The reaction mixture was heated to 95 ° C. for 5 minutes prior to a regular heating cycle. Subsequently, the reaction mixture was heat denatured at 95 ° C. for 1 minute, then at 50 ° C. for 1 minute and finally at 72 ° C. for 3 minutes. This heating cycle is 3
After 0 iterations, the product was incubated at 72 ° C. for 7 minutes to complete the final polymerization.
This PCR operation was performed under the control of a 'Touch Down' heating cycler device (Hybaid).
The resulting product was extracted and used as template D for the next PCR cycle.
Prepared for use as NA. Second round P
The heating cycle of the CR reaction is performed at an annealing temperature of 52 ° C.
The procedure was the same as the first PCR operation cycle, except that the procedure was changed to The obtained product was analyzed by 1.5% agarose gel electrophoresis. Amplification D having a size of about 600 bp
An NA fragment was obtained and was confirmed by DNA sequencing.

Example 2 Construction of hEPO Expression Vector (pcDNA3.1-HC) The gel-purified DNA fragment obtained in Example 1 and pc
Both DNA3.1 (Invitrogen) were cut with restriction enzymes HindIII and XbaI. Digested D
NA fragments were fractionated by electrophoresis on an agarose gel and extracted. The ligation mixture contained 50 ng of the PCR-amplified DNA fragment and 20 ng of the pcDNA3.1 vector. The ligation product was identified by DNA sequencing and was named pcDNA3.1-HC. The rhEPO expression vector pcDNA3.1-H
C was from pcDNA3.1-HC on July 22, 1999.
(ATCC assigned number: PTA
-420) was deposited internationally with the ATCC. The restriction map is shown in FIG. The pcDNA3.1-HC is converted to N
ovaBlue (Novagene) competent cells were transformed and clones resistant to both ampicillin (100 mg / ml) and tetracycline (25 mg / ml) were selected. FIG. 1 shows a pcDNA3.1 expression vector producing rhEPO, that is, pcD.
3 shows a restriction map of NA3.1-HC. The confirmed human erythropoietin cDNA fragment was replaced with pc
It was ligated between the restriction sites HindIII and XbaI of DNA3.1 (purchased from Novagene).

Example 3 RhEPO-Expressing Mammalian Host Cell BHK21-pcD
Cloning of NA3.1-HC The cell line BHK-21 (ATCC CCL-10) was grown in standard DMEM medium containing 10% fetal calf serum. To perform the transformation, 10 ⁷ BHK-21 cells were suspended in 0.8 ml of PBS buffer (10 mM phosphate buffer, pH 7.2) and 10 mg of cD
NA3.1-HC (Gene Pulser IIs)
system, BioRad). The resulting cell suspension was diluted with 10 ml of standard DMEM medium. The transformed BHK-21 cells were first plated in commonly used DMEM medium for the first 24 hours, followed by fresh DME containing G418 (400 mg / ml).
Cultured in M medium. Clones resistant to the antibiotic G418 were screened and expanded in the same medium.

Conditioned medium was collected from each clone and the biological activity of the secreted rhEPO was determined.
Erythroleukemia cell TF-1 (ATCC CRL-20
03; Kitamura et al. , J. et al. Ce
11 Physiol. 140: 323-334,
1989).
Usually, TF-1 cells are transformed with recombinant human GM-CSF (rh
GM-CSF, 2pg / ml, R & D System
m) in RPMI 1640 medium in the presence of m). 96
-10 ³ to perform the growth test on the well plate
TF-1 cells were grown in the absence of rhGM-CSF for 50
It was suspended in ml of RPMI 1640 medium, into which the conditioned medium collected from each of the above clonal cultures was added. Plates were incubated for 4 days under normal conditions. Subsequently, an XTT proliferation kit (Boehrin) was used to label viable TF-1 cells.
ger Mannheim), and the plates were incubated for an additional 12-16 hours. Auto E
Using a LISA reader (ANthos 2001)
The optical density (OD) of TF-1 cells incubated at a wavelength of 492 nm was measured. The background used as a control was the value of the optical density read at a wavelength of 620 nm. The clone showing the highest growth enhancing ability was screened and named BHK21-pcDNA3.1-HC. The BHK21-pcDNA3.1-HC contains 1
On July 22, 999, BHK21-pcDNA3.1-
HC (ATCC assigned number: P
TA-419) with the ATCC.

Example 4 Biological activity of rhEPO secreted by BHK21-pcDNA3.1-HC In a culture medium containing the antibiotic G418, the BHK21-pcDNA3.1-HC obtained in Example 3 above was added.
(2 × 10 ⁴ cells / ml medium) and grown for 7 days. The conditioned medium was collected, and the following tests were sequentially performed. R produced from transformed CHO cells
hEPO has an ED ₅₀ value of 0.1-0.5 units / ml ( ³
H-thymidine (assessed by the incorporation method) has the biological activity of enhancing TF-1 cell proliferation. T
The above standard rhEPO was used at 5.0, 2.5, 1.25, 0.63, for use in proliferation tests on F-1 cells.
0.32, 0.16, 0.08 and 0.04 units / m
diluted to 1. The XTT method described above was used. Optical density O
The standard curve for D _{492-620 nm} values (black circles) is shown in FIG. E
D ₅₀ values are converted to 0.5 units / ml. A two-fold serial dilution of conditioned medium was used for growth studies under the conditions described above. Optical density O measured
The curve of D _{492-620 nm} value ( _open circle) is shown in FIG. BHK
Rh secreted by 21-pcDNA3.1-HC
The biological activity of EPO is determined by the r secreted from CHO cells.
It was calculated to be 19.2 units / ml, equivalent to the biological activity of hEPO. FIG. 2 shows BHK21-pcDNA3.
1 shows the biological activity of rhEPO secreted by 1-HC. Closed circles indicate the obtained values after serial dilution of the medium.
Represents the OD value (standard) of hEPO. Open circles represent OD values of rhEPO secreted by BHK21-pcDNA3.1-HC. The biological activity of the latter is calculated to be 19.2 units / ml,
It was equivalent to the biological activity of rhEPO secreted from CHO cells.

Example 5 BHK21-pcDNA 3.1 Using ELISA Method
-Immunochemical properties of rhEPO secreted from HC BHK21-pcDNA3.1-r secreted from HC
An ELISA kit (purchased from R & D System) was used to identify the immunochemical properties of hEPO. C
200.0 standard rhEPO produced from HO cells,
Diluted to a concentration of 100.0, 50.0, 20.0, 5.0, 2.5 and 0.0 mIU / ml. ELISA was performed according to the instructions for use. A standard curve of rhEPO produced from CHO cells is shown in FIG. BHK2
1-pcDNA3.1 rhEPO secreted from HC
Was also serially diluted in the same manner as described above, and ELISA was performed.
The concentration data was corrected by extrapolating within the standard curve based on the read OD _{450-620 nm} value, and BHK21-pcDNA 3.1
The average concentration of rhEPO secreted from HC is 5,88
It was determined to be 6 mlU / ml. FIG. 3 shows an immunochemical characteristic curve of rhEPO secreted from CHO cells.

Although the biological activity of rhEPO secreted from BHK21-pcDNA3.1-HC was determined as described above, the biological activity obtained from enhancing TF-1 cell growth was obtained from ELISA. About three times higher than biological activity. Such differences are considered to be due to the following. That is, (1) used in the ELISA method,
A monoclonal antibody specific for standard rhEPO secreted from CHO cells was BHK21-pcDNA3.1-H.
RhEP secreted from different transformants such as C
That the presence of Os cannot be completely detected, and (2)
It is possible that unknown molecules exhibiting a synergistic effect with rhEPO which promotes the growth of TF-1 cells may be present in the conditioned medium.

Example 6 rHK secreted from BHK21-pcDNA3.1-HC
Purification of hEPO Condition collected as described in Example 4.
5% of all rhEPOs in 100 ml of medium
Salting out with 0-85% saturated ammonium sulfate gradient
it can. Centrifuge the precipitate (18,000 xg)
And the resulting pellet was used to bind 5 ml of ConA.
Buffer (1 mM MnCl_Two , CaCl_Two You
And MgCl_Two / 10 mM PIPES, pH 6.4)
Was dissolved. The obtained solution was mixed with 5 ml of ConA-Sep.
Suspend in harose and gently at room temperature for at least 2 hours
And incubated with shaking. Subsequently, the resulting blend
Apply the compound to the immobilized lectin column for ConA binding
Washed with 100 ml of buffer. The combined fractions are
Has a linear gradient of mannose reaching 0.5M
Elution with 50 ml of buffer
The combined fractions are transferred to an acidic buffer (0.1 M sodium acetate).
And 0.5M NaCl, pH 4.0).
Eluted fractions are analyzed on a 12% SDS-PAGE gel
And rhEPO was confirmed by dot blot
Was.

The fractions containing rhEPO eluted with the mannose gradient buffer or the acidic buffer were pooled and concentrated using a filter membrane (PM10 membrane, Amicon) until the final volume reached about 3 ml. The concentrated solution was applied to a column (16 mm × 100 mm) packed with Sephadex-G75 resin, and PBS buffer was added as an elution buffer. From the mannose elution pool, the main component rhE with a molecular weight of about 35 kD
PO and r of a trace component having a molecular weight of 36 kD-45 kD
A mixture consisting of hEPO could be obtained. The rhEPOs in this pool show an associated specific activity of 180,000-240,000 units / mg. Another pool (acid-
Eluted) each had a uniform molecular weight of 34 kD (124,
000 units / mg), and three major isoform components of rhEPO with molecular weights of 25 kD and 28 kD (80,000 units / mg). Fig. 4 shows the results of silver staining and Western blotting.
-5. FIG. 4 shows SDS-PAGE-silver staining of rhEPO of the same type as 34 kD and Western blotting. Its homogeneity is due to the acid-elution pool in Seph.
achieved through an adex G-75 column. FIG. 5 shows CHO cells (lane 1) and BHK21-pc
R secreted from DNA3.1-HC (lanes 2-4)
Figure 5 shows a Western blot of hEPO. The mannose elution pool was passed through a Sephadex G-75 column, and fractions containing rhEPO isoforms were sequentially collected. That portion is shown in lanes 2-4.

All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. As individual documents are specifically and individually embodied, these patents and documents are shown to be equally embodied herein. Those skilled in the art
It will be readily apparent that, as well as the particulars of the invention, it is well adapted to carry out its objects and achieve the stated results and advantages.
All of these examples, including the methods, procedures, procedures, molecules, and specific compounds described in the present invention, are representative of preferred embodiments, are exemplary, and limit the scope of the present invention. is not. Partial modifications and other uses will occur to those skilled in the art, which are also within the scope of the present invention, as defined in the appended claims.

[0032]

As described in detail above, the present invention relates to a cDNA fragment encoding human erythropoietin, a plasmid vector, an expression vector containing a cytomegalovirus promoter, and a cell line containing the expression vector. A method for producing human erythropoietin by culturing a cell line, and an improved purification method thereof. According to the present invention, 1) an expression system for acidifying recombinant human erythropoietin with a novel expression vector, A method for purifying secreted human erythropoietin is provided. 2) In the first step, the target protein rhE
The POs are concentrated by precipitation, whereby rhEP
3) RhEPO having a higher specific activity and a molecular weight of 35-45 kD exhibiting higher specific activity without using reverse phase HPLC or immunoaffinity column chromatography which is required in conventional purification methods. Pools and their isoforms, molecular weight 25-3
An extraordinary effect is obtained, such as being able to obtain another pool of 4 kD.

[0033]

[Sequence List] SEQUENCE LISTING <110> Li-Wei Hsu; Su-Chen Chaug <120> An expression system for producing recombinant human erythropoietin, a method for purifying the secreted human erythropoietin and uses the reof <130> 10XP-ROC < 160> 2 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <400> 1 gcaagcttat gggggtgcac gaatg 25

<210> 2 <211> 27 <212> DNA <213> Artificial Sequence <400> 2 gcatctagat catctgtccc ctgtcct 27

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a pcDNA3.1 expression vector, pcc.
1 shows a restriction map of DNA3.1-HC.

FIG. 2 shows BHK21-pcDNA3.1-HC.
1 shows the biological activity of rhEPO secreted by E. coli.

FIG. 3. RhEPO secreted from CHO cells.
1 shows the immunochemical characteristic curve of

FIG. 4 shows a 34 kD isotype of rhEPO SDS-
PAGE-silver staining and western blotting.

FIG. 5 shows CHO cells (lane 1) and BHK
FIG. 2 shows a Western blot of rhEPO secreted from 21-pcDNA3.1-HC (lanes 2-4).

──────────────────────────────────────────────────続 き Continuation of front page (56) References JP-A-2-115196 (JP, A) JP-A-62-149624 (JP, A) JP-A-7-138292 (JP, A) JP-A-2-115 475 (JP, A) JP-A-3-95199 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 15/09 C07K 1/16 C07K 14/505 C12P 21/02 BIOSIS (DIALOG) WPI (DIALOG)

Claims (6)

(57) [Claims] 1. The recombinant human erythropoietin (rhEPO) secreted from a transformant is applied to a two-step column chromatography to give a molecular weight of 35-45 kD.
To obtain a pool comprising a pool Oyo originator these isoforms in which the molecular weight 25-34KD rhEPO containing rhEPO, a method of purifying the rhEPO, then step described; (1) transformed cells From the sample obtained from the culture
RhEP with saturation of ammonium sulfate from 50% to 80%
Precipitating the O compound, (2) applying the precipitated the rhEPO such a column of immobilized lectin, a molecular weight of about (3) mannose gradient buffer
RhEPO as main component having 35 kD and molecular weight 36 kD
Mixing of trace components rhEPO with -45 kD
Eluting the rhEPO-containing pool containing the substance ; (4) After elution with the above-mentioned mannose, it is further firmly bound.
The combined fractions are eluted with an acidic buffer to give a molecular weight of 34.
Three main rhEPO eyes of kD, 28 kD and 25 kD
Eluting the pool containing isoform, (5) molecular weight by the step of (3) - (4) 35
Pools containing -45 kD rhEPO and their pools
RhEPO with a molecular weight of 25-34 kD which is an isoform
To obtain a pool containing, concentrating the respective (6) collected pool, then applying the rhEPO containing pooled gel filtration column, and eluting the said rhEPO, and characterized in that it consists of A method for purifying rhEPO. 2. Purified r from said mannose elution pool
2. The method of claim 1, wherein the hEPO contains an isoform having a molecular weight between 35 kD and 45 kD. 3. Purified r from said mannose elution pool
The method of claim 1, wherein hEPO has a purity of about 90%. 4. The transformed cell is BHK21-pcDN.
A3.1-HC (ATCC; PTA-419),
The method of claim 1. 5. To elute the pool containing rhEPO mannose solution continuous gradient up to 0.5M concentration, method of claim 1. 6. A pool containing the isoform of rhEPO is eluted with an acidic buffer (approximately pH 4.0) .
The method of claim 1.