title: Soluble expression and partial purification of recombinant human erythropoietin from E. coli.
abstract: Human erythropoietin (hEpo) is an essential regulator of erythrocyte production that induces the division and differentiation of erythroid progenitor cells in the bone marrow into mature erythrocytes. It is widely used for the treatment of anemia resulting from chronic kidney disease, chemotherapy, and cancer-related therapies. Active hEpo, and hEpo analogs, have been purified primarily from mammalian cells, which has several disadvantages, including low yields and high production costs. Although an Escherichia coli (E. coli) expression system may provide economic production of therapeutic proteins, it has not been used for the production of recombinant hEpo (rhEpo) because it aggregates in inclusion bodies in the E. coli cytoplasm and is not modified post-translationally. We investigated the soluble overexpression of active rhEpo with various protein tags in E. coli, and found out that several tags increased the solubility of rhEpo. Among them maltose binding protein (MBP)-tagged rhEpo was purified using affinity and gel filtration columns. Non-denaturing electrophoresis and MALDI-TOF MS analysis demonstrated that the purified rhEpo had two intra-disulfide bonds identical to those of the native hEpo. An in vitro proliferation assay showed that rhEpo purified from E. coli had similar biological activity as rhEpo derived from CHO cells. Therefore, we report for the first time that active rhEpo was overexpressed as a soluble form in the cytoplasm of E. coli and purified it in simple purification steps. We hope that our results offer opportunities for progress in rhEpo therapeutics.
authors.affiliation: Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Seoul 138-736, South Korea.
authors.forename: Taeck-Hyun
authors.initials: TH
authors.lastname: Jeong
authors.affiliation: Department of Pharmacy, Sunchon National University, Suncheon, Jeonnam 540-742, South Korea.
authors.forename: Young-Jin
authors.initials: YJ
authors.lastname: Son
authors.affiliation: Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Seoul 138-736, South Korea.
authors.forename: Han-Bong
authors.initials: HB
authors.lastname: Ryu
authors.affiliation: Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Seoul 138-736, South Korea.
authors.forename: Bon-Kyung
authors.initials: BK
authors.lastname: Koo
authors.affiliation: Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Seoul 138-736, South Korea.
authors.forename: Seung-Mi
authors.initials: SM
authors.lastname: Jeong
authors.affiliation: Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Seoul 138-736, South Korea.
authors.forename: Phuong
authors.initials: P
authors.lastname: Hoang
authors.affiliation: Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Seoul 138-736, South Korea.
authors.forename: Bich Hang
authors.initials: BH
authors.lastname: Do
authors.affiliation: Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Seoul 138-736, South Korea.
authors.forename: Jung-A
authors.initials: JA
authors.lastname: Song
authors.affiliation: Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Seoul 138-736, South Korea.
authors.forename: Seon-Ha
authors.initials: SH
authors.lastname: Chong
authors.affiliation: Institute of Molecular and Cell Biology, Proteos, 61 Biopolis Drive, Singapore 138673, Singapore; Department of Biochemistry, National University of Singapore, 8 Medical Drive, Singapore 117597, Singapore; School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore.
authors.forename: Robert Charles
authors.initials: RC
authors.lastname: Robinson
authors.affiliation: Department of Physiology and Bio-Medical Institute of Technology, University of Ulsan College of Medicine, Seoul 138-736, South Korea. Electronic address: hchoe@ulsan.ac.kr.
authors.forename: Han
authors.initials: H
authors.lastname: Choe
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D049109:Cell Proliferation; D003001:Cloning, Molecular; D004921:Erythropoietin / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D058619:Maltose-Binding Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D012995:Solubility
chemical_list: D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; D004921:Erythropoietin
keywords: Escherichia coli expression system; Maltose binding protein (MBP); Therapeutic protein; rhEpo