title: Purification and characterization of the yeast-expressed erythropoietin mutant Epo (R103A), a specific inhibitor of human primary hematopoietic cell erythropoiesis.
abstract: A drug that specifically inhibits erythropoiesis would be clinically useful. The erythropoietin (Epo) mutant Epo (R103A) could potentially be used for this purpose. Epo (R103A) has a single amino acid substitution of alanine for arginine at position 103. Because of this mutation, Epo (R103A) is only able to bind to one of the 2 subunits of the erythropoietin receptor (EpoR) homodimer and is thus a competitive inhibitor of Epo activity. To produce large quantities of Epo (R103A) to test in animal models of thalassemia and sickle cell disease, we expressed and purified recombinant Epo (R103A) from the yeast Pichia pastoris. Using this method milligram quantities of highly purified Epo (R103A) are obtained. The yeast-expressed Epo (R103A) is properly processed and glycosylated and specifically inhibits Epo-dependent cell growth and (125)I-Epo binding. Epo (R103A) does not, however, directly induce apoptosis in 32D cells expressing EpoR. Epo (R103A) inhibits erythropoiesis of human CD34(+) hematopoietic cells and completely blocks erythroid burst-forming unit formation in normal human bone marrow colony assays. Yeast-expressed Epo (R103A) is a specific inhibitor of primary erythropoiesis suitable for testing in animal models.
authors.affiliation: South Texas Veterans Health Care System, and Department of Medicine, Division of Hematology, University of Texas Health Science Center, San Antonio, TX, USA.
authors.forename: Suzanne
authors.initials: S
authors.lastname: Burns
authors.forename: Murat O
authors.initials: MO
authors.lastname: Arcasoy
authors.forename: Li
authors.initials: L
authors.lastname: Li
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Kurian
authors.forename: Katri
authors.initials: K
authors.lastname: Selander
authors.forename: Peter D
authors.initials: PD
authors.lastname: Emanuel
authors.forename: Kevin W
authors.initials: KW
authors.lastname: Harris
mesh_terms: D001667:Binding, Competitive; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D003001:Cloning, Molecular; D004900:Erythroblasts / Q000166:cytology / Q000187:drug effects; D004920:Erythropoiesis / Q000187:drug effects*; D004921:Erythropoietin / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects*; D006801:Humans; D009154:Mutation; D017467:Receptors, Erythropoietin / Q000378:metabolism; D011994:Recombinant Proteins; D015003:Yeasts / Q000235:genetics*
chemical_list: D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins; D004921:Erythropoietin