Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1998-12-07
2002-04-23
Kemmerer, Elizabeth C. (Department: 1646)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S471000, C435S069100, C530S412000, C530S413000, C530S417000, C530S418000
Reexamination Certificate
active
06376218
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATION
This patent application claims benefit of Taiwanese Patent Application No. 86120102, filed Dec. 31, 1997.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the field of molecular biology of human erythropoietin. More specifically, the present invention relates to an expression system for producing biologically active recombinant human erythropoietin (rhEPO) and an improved method for purifying the secreted rhEPOs.
2. Description of the Related Art
Human erythropoietin (hEPO) is a glycoprotein with molecular weight of 30-40 kD. In healthy adults, mature hEPO is generated in and secreted from kidney. Human erythropoietin mainly functions in enhancing the proliferation of erythropoietin cells in spleen, bone marrow and fetal hepatocytes, and the differentiation of erythrocytes.
Native human erythropoietin was originally prepared from urine of patients with aplastic anemia. The amount of human erythropoietin obtained from the patient's urine is rare, and a traditional process for purifying the human erythropoietin is laborious and time-consuming. Therefore, there is a demand to develop a process for producing and purifying a large amount of human erythropoietin in a simple and economical way.
Genetic engineering techniques using an mammalian cell line as a host for producing a high level of recombinant human erythropoietin (rhEPO) has been developed. A cDNA fragment encoding human erythropoietin was cloned and sequenced by Jacob et al. (
Nature,
313: 806-809, 1985). Further, Jacob and colleagues used an expression vector containing a cDNA fragment encoding mature human erythropoietin for transforming kidney fibroblast COS-1 cell under the control of SV40 promoter, and then producing rhEPO from the transformed kidney fibroblast COS-1 cell upon transient expression. Also, Jacob et al. determined the biological activity of produced rhEPO.
U.S. Pat. Nos. 4,703,008, 5,441,868, 5,547,993, 5,621,080, and 6,618,698 disclosed a 5.6 kb genomic DNA fragment containing a full length human erythropoietin gene which was inserted into an expression vector used for transforming monkey fibroblast COS-1 cells. The rhEPO transiently secreted from the transformed monkey fibroblast COS-1 cells could be detected. Further, a stably transformed CHO cell line capable of producing rhEPO could be obtained with the selection of methotrexate. A rhEPO-expressing system in
E. coli
and yeast was also disclosed.
A baculovirus system in insect SF9 cells was used for producing rhEPO (Quelle et al.,
Blood
74(2): 652-657, 1989). Although the yield of rhEPO from the transformed SF9 cells was greatly improved (500,000 U/liter culture), the molecular weight of target product was smaller than that of native human erythropoietin due to less glycosylation. Mori and colleagues (Gene 144(2): 289-293, 1994) constructed a rhEPO-expressing vector containing the promoter of interferon-&agr; gene and used the rhEPO-expressing vector for transforming B cell leukemia BALL-1 cells. When transfected with Sendei virus, the transformed B cell leukemia BALL-1 cells could produce rhEPO in a higher level than those transformants obtained by the former investigators.
The method for purifying rhEPO from conditioned medium usually comprises the following steps. Firstly, rhEPO was selectively adsorbed or excluded by passing through an ion-exchange chromatography column, such as DEAE cellulose column (Sherwood and Goldwasser,
Endocrinology
103(3): 866-870, 1978) and DEAE Sephacel column (Quelle et al.,
Blood
74(2): 652-657, 1989; Inoue et al.,
Biological and Pharmaceutical
17(2): 180-184, 1994; Ben Ghanem et al.,
Preparative Biochemistry
24(2): 127-142, 1994). Secondly, rhEPO was specifically adsorbed onto immobilized-lectin resin, such as wheat germ agglutinin-agarose (Qian et al.,
Blood
68(1): 258-262, 1986) and ConA-agarose (Quelle et al.,
Blood
74(2): 652-657, 1989). Thirdly, C4 reverse phase HPLC was usually employed for purifying rhEPO to homogeneity (Lange et al.,
Blood Cells
10(2-3): 305-314, 1984; Krystal et al.,
Blood
67(1): 71-79, 1986; Quelle et al.,
Blood
74(2): 652-657, 1989; Inoue et al.,
Biological and Pharmaceutical
17(2): 180-184, 1994). U.S. Pat. No. 5,322,837 disclosed a method of using C4 reverse phase HPLC for preparing rhEPO in homogeneity that exhibits a specific activity of 120,000-160,000 U/mg.
An immobilized monoclonal antibody affinity column was used for the purification of hEPO (or rhEPO). For example, rhEPO secreted from transformed lymphoblastoid cells was specifically adsorbed onto an anti-hEPO monoclonal antibody-Sepharose 4B affinity column (Ben Ghanem et al.,
Preparative Biochemistry
24(2): 127-142, 1994). The bound rhEPO fraction was eluted, and then passed through a DEAE-Sephacel ion exchange column. A homogeneous product of rhEPO could be obtained with a recovery rate of about 50%.
Although the use of recombinant DNA technology has improved the yield of rhEPO, purification of rhEPO to homogeneity is still laborious and time-consuming, especially when large scale preparation of rhEPO is required.
The prior art is deficient in the lack of effective means of producing a large quantity of rhEPO in an expression system. Further, the prior art is deficient in the lack of effective means of purifying large scale amounts of rhEPO. The present invention fulfills this long-standing need and desire in the art.
SUMMARY OF THE INVENTION
The present invention provides a newly developed expression system for producing rhEPO. Also provided is a novel method of purifying the secreted rhEPOs using a two-step column chromatography technique. For expression of the target protein rhEPO, a PCR-amplified cDNA fragment encoding mature rhEPO was inserted into an expression vector under control of the cytomegalovirus (CMV) promoter. A transformant (BHK-21 cell) harboring the expression vector stably producing and secreting rhEPO with a high yield was obtained under the selection with antibiotic G418. For purification of the rhEPOs, the target proteins were first precipitated from conditioned medium using a salting-out technique. The glycoproteins in the precipitated portion were selectively adsorbed onto immobilized-lectin resin. When bound glycoproteins were eluted with a buffer containing 0.5 M mannose, a pool of rhEPOs was obtained containing major components with molecular weight of around 34 kD and minor components with molecular weight of 35-45 kD. Another pool of rhEPOs persistently bound without being eluted out with the buffer containing 0.5 M mannose could be stripped with an acidic buffer (pH 4.0). The isoforms of rhEPO could be further purified by passing through a G-75 chromatography column.
The purification method disclosed in the present invention provides the following advantages: (1) the target protein rhEPOs are concentrated by precipitation in the first step whereby a large scale preparation of rhEPOs becomes easier, and (2) a pool of rhEPOs with molecular weight of 35-45 kD exhibiting higher specific activity and their isoforms, another pool of rhEPOs with molecular weight of 25-34 kD can be obtained without using reverse phase HPLC or immuno-affinity column chromatography as required by conventional purification methods.
In one embodiment of the present invention, there is provided an expression vector containing a cDNA fragment encoding human erythropoietin and pcDNA3.1 vector under the control of cytomegalovirus promoter. Preferably, the cDNA fragment is produced by PCR using the primers selected from the group consisting of SEQ ID No: 1 and SEQ ID No: 2.
In another embodiment of the present invention, there is provided a cell line harboring the expression vector. Preferably, the cell line is screened by antibiotic G418 resistance. More preferably, the cell line is BHK-21.
In yet another embodiment of the present invention, there is provided a method of producing human erythropoietin by culturing the above mentioned cell line in a conditioned medium and then detecting the production of human
Chang Su-Chen
Hsu Li-Wei
Adler Benjamin Aaron
Kemmerer Elizabeth C.
Research Development Foundation
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