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, Kevin W. Harris

Research output: Contribution to journalArticle

9 Citations (Scopus)

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 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.

Original languageEnglish (US)
Pages (from-to)4400-4405
Number of pages6
JournalBlood
Volume99
Issue number12
DOIs
StatePublished - Jun 15 2002

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Erythropoiesis
Erythropoietin
Yeast
Purification
Yeasts
Erythropoietin Receptors
Animals
Animal Models
Erythroid Precursor Cells
Thalassemia
Pichia
Sickle Cell Anemia
Cell growth
Amino Acid Substitution
Alanine
Arginine
Assays
Bone

ASJC Scopus subject areas

  • Hematology

Cite this

Purification and characterization of the yeast-expressed erythropoietin mutant Epo (R103A), a specific inhibitor of human primary hematopoietic cell erythropoiesis. / Burns, Suzanne; Arcasoy, Murat O.; Li, Li; Kurian, Elizabeth; Selander, Katri; Emanuel, Peter D.; Harris, Kevin W.

In: Blood, Vol. 99, No. 12, 15.06.2002, p. 4400-4405.

Research output: Contribution to journalArticle

Burns, Suzanne ; Arcasoy, Murat O. ; Li, Li ; Kurian, Elizabeth ; Selander, Katri ; Emanuel, Peter D. ; Harris, Kevin W. / Purification and characterization of the yeast-expressed erythropoietin mutant Epo (R103A), a specific inhibitor of human primary hematopoietic cell erythropoiesis. In: Blood. 2002 ; Vol. 99, No. 12. pp. 4400-4405.
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