title: Purification and biological activity of a recombinant human erythropoietin produced by lymphoblastoid cells.
abstract: A recombinant human erythropoietin (rH-EPO) was obtained from the culture supernatants of human B-lymphoblastoid cells transfected by the human EPO gene. rH-EPO was purified by a two-step method based on immunoaffinity and ion exchange chromatography. The first step was achieved by an anti-EPO monoclonal antibody (Mab). This Mab, immobilized on Sepharose 4B, allowed a 410-fold purification of the protein. The second step consisted of ion exchange chromatography on DEAE Sephacel. The combination of these two steps results in a highly purified rH-EPO with a global yield of about 50%; the specific activity of the protein was 176,000 IU/A280. The NMR spectrum was characteristic for a well structured, single-conformation protein. The purified protein was analyzed by SDS-PAGE and isoelectric focusing. The biological activity of purified rH-EPO was measured in vivo, by the incorporation of 59Fe into red blood cells (RBC) of polycythemic mice and in vitro by the proliferative response of an EPO-dependent cell line. The purified protein expressed in lymphoblastoid cells of human origin had the same biological activity as that of urinary EPO and rH-EPO produced in other mammalian cells.
authors.affiliation: Institut National de Transfusion Sanguine, Paris, France.
authors.forename: A
authors.initials: A
authors.lastname: Ben Ghanem
authors.forename: J J
authors.initials: JJ
authors.lastname: Winchenne
authors.forename: C
authors.initials: C
authors.lastname: Lopez
authors.forename: S
authors.initials: S
authors.lastname: Chrétien
authors.forename: M
authors.initials: M
authors.lastname: Dubarry
authors.forename: C T
authors.initials: CT
authors.lastname: Craescu
authors.forename: J P
authors.initials: JP
authors.lastname: Le Caer
authors.forename: N
authors.initials: N
authors.lastname: Casadevall
authors.forename: P
authors.initials: P
authors.lastname: Rouger
authors.forename: J P
authors.initials: JP
authors.lastname: Cartron
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism*; D016466:CHO Cells; D002455:Cell Division; D002460:Cell Line; D002845:Chromatography; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D004921:Erythropoietin / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D006801:Humans; D007158:Immunologic Techniques; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D017421:Sequence Analysis; D014162:Transfection
chemical_list: D011994:Recombinant Proteins; D004921:Erythropoietin