High-level expression and purification of a recombinant human erythropoietin produced using a baculovirus vector

摘要: 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 baculovirus, was accomplished suspension culture using reduced levels serum media supplements experimentally determined to provide optimum factor production (500,000 U/L). Purification this virtual homogeneity (greater than or equal 99%) via simple three-step procedure involving isocratic elution from DEAE-Sephacel, reverse-phase high performance liquid chromatography (HPLC) on C4 medium, single-step purified hormone concanavalin A agarose. Overall, an 890-fold recovery 80% assayed vitro. Biologically, is highly active, possessing specific activity vitro 200,000 U/mg protein. Chemically, (molecular weight [mol wt] 26,200) appears exceptionally uniform its oligosaccharide constitution (30%) contrasted heterogeneously glycosylated erythropoietins derived mammalian cells (mol wt 30,000 38,000; 40% 50% complex-type oligosaccharide). Thus, insect cell line comprises not only abundant source readily studies mechanism action surface receptor, but also represents uniquely homogeneous form that should prove advantageous direct structural analyses.