Blood, 15 June 2002, Vol. 99, No. 12, pp. 4400-4405
HEMATOPOIESIS
Purification and characterization of the yeast-expressed
erythropoietin mutant Epo (R103A), a specific inhibitor of human
primary hematopoietic cell erythropoiesis
Suzanne Burns,
Murat O. Arcasoy,
Li Li,
Elizabeth Kurian,
Katri Selander,
Peter D. Emanuel, and
Kevin W. Harris
From the South Texas Veterans Health Care System, and
Department of Medicine, Division of Hematology, University of Texas
Health Science Center, San Antonio, TX; Department of Medicine,
Division of Hematology-Oncology, Duke University Medical Center,
Durham, NC; and Department of Medicine, University of Alabama at
Birmingham and Birmingham Veterans Affairs Medical Center, Birmingham.
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
125I-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.
