High-level expression and purification of a recombinant human
erythropoietin produced using a baculovirus vector
FW Quelle, LF Caslake, RE Burkert and DM Wojchowski
Department of Molecular and Cell Biology, Pennsylvania State University,
University Park 16802.
Conditions presently have been established for the high-level expression
and simplified purification of recombinant human erythropoietin produced in
Spodoptera frugiperda cells. Expression, as mediated by infection with a
recombinant baculovirus, was accomplished in suspension culture using
reduced levels of serum and media supplements experimentally determined to
provide optimum levels of factor production (500,000 U/L). Purification of
this recombinant human erythropoietin to virtual homogeneity (greater than
or equal to 99%) was accomplished via a simple three-step procedure
involving isocratic elution from DEAE-Sephacel, reverse-phase high
performance liquid chromatography (HPLC) on a C4 medium, and the
single-step elution of purified hormone from concanavalin A agarose.
Overall, an 890-fold purification was accomplished with a recovery of 80%
as assayed in vitro. Biologically, this purified erythropoietin is highly
active, possessing a specific activity in vitro of 200,000 U/mg protein.
Chemically, this erythropoietin (molecular weight [mol wt] 26,200) appears
exceptionally uniform in its oligosaccharide constitution (30%) as
contrasted with heterogeneously glycosylated erythropoietins derived from
mammalian cells (mol wt 30,000 to 38,000; 40% to 50% complex-type
oligosaccharide). Thus, human erythropoietin as presently produced in an
insect cell line comprises not only an abundant source of highly active,
readily purified hormone for studies of its mechanism of action and cell
surface receptor, but also represents a uniquely homogeneous form that
should prove advantageous for direct structural analyses.
Volume 74,
Issue 2,
pp. 652-657,
08/01/1989
Copyright © 1989 by The American Society of Hematology
