Phosphorylatable and epitope-tagged human erythropoietins: utility and purification of native baculovirus-derived forms.
The hematopoietic glycopeptide erythropoietin (EPO) is a prime regulator of red cell production in mammals, yet the precise nature of its interaction with specific cell surface receptors is poorly understood. Towards defining domains of EPO that are involved in receptor activation, we have developed (i) conditions for the expression of recombinant human EPO (rhEPO) at high levels in SF9 cells using modified 2- and 5-liter stirred reactors, (ii) a two-step procedure for the purification of this EPO without denaturation, and (iii) forms of EPO tagged with either a hemagglutinin influenza virus epitope or a consensus sequence for in vitro phosphorylation. Compared to EPO expressed in mammalian cells, rhEPO from SF9 cells in N-glycosylated with simple, neutral oligosaccharides of limited size, yet as purified presently using nondenaturing procedures, possesses exceptionally high in vitro activity (> or = 500,000 U/mg). Thus, this form of EPO should prove advantageous for direct physicochemical analyses. Regarding epitope-tagged and phosphorylatable EPOs, forms modified at the amino terminus (Ala1) fully retained receptor binding and in vitro biological activities. In contrast, forms modified at the carboxy terminus (Cys161) were inactive and did not compete for receptor binding, indicating that integrity of this domain is essential for receptor recognition. For active amino-terminal-modified forms, the specific binding of MAb 12CA5 to native HAI-EPO and the utility of 32P-labeled PHOS-EPO in receptor binding and internalization studies also were demonstrated. The development of these unique, highly active forms of human EPO should advance studies of essential interactions between this cytokine and its cell surface receptor.
D000595:Amino Acid Sequence; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D000939:Epitopes; D004921:Erythropoietin / Q000031:analogs & derivatives* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral; D006801:Humans; D008958:Models, Molecular; D015335:Molecular Probes; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017467:Receptors, Erythropoietin / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism
D000939:Epitopes; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D006389:Hemagglutinins, Viral; D015335:Molecular Probes; D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins; D004921:Erythropoietin