High-level expression and purification of a recombinant human erythropoietin produced using a baculovirus vector.
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.
Quelle
F W
FW
Department of Molecular and Cell Biology, Pennsylvania State University, University Park 16802.
Caslake
L F
LF
Burkert
R E
RE
Wojchowski
D M
DM
D000818:Animals; D002460:Cell Line; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004921:Erythropoietin / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D005822:Genetic Vectors; D006801:Humans; D007304:Insect Viruses / Q000235:genetics*; D007915:Lepidoptera / Q000235:genetics / Q000378:metabolism; D051379:Mice; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; D004921:Erythropoietin