Purification and biological activity of a recombinant human erythropoietin produced by lymphoblastoid cells.
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.
Ben Ghanem
A
A
Institut National de Transfusion Sanguine, Paris, France.
Winchenne
J J
JJ
Lopez
C
C
Chrétien
S
S
Dubarry
M
M
Craescu
C T
CT
Le Caer
J P
JP
Casadevall
N
N
Rouger
P
P
Cartron
J P
JP
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
D011994:Recombinant Proteins; D004921:Erythropoietin