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Purification and characterization of the yeast-expressed erythropoietin mutant Epo (R103A), a specific inhibitor of human primary hematopoietic cell erythropoiesis.

Publication ,  Journal Article
Burns, S; Arcasoy, MO; Li, L; Kurian, E; Selander, K; Emanuel, PD; Harris, KW
Published in: Blood
June 15, 2002

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.

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Published In

Blood

DOI

ISSN

0006-4971

Publication Date

June 15, 2002

Volume

99

Issue

12

Start / End Page

4400 / 4405

Location

United States

Related Subject Headings

  • Yeasts
  • Recombinant Proteins
  • Receptors, Erythropoietin
  • Mutation
  • Immunology
  • Humans
  • Hematopoietic Stem Cells
  • Erythropoietin
  • Erythropoiesis
  • Erythroblasts
 

Citation

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Burns, S., Arcasoy, M. O., Li, L., Kurian, E., Selander, K., Emanuel, P. D., & Harris, K. W. (2002). Purification and characterization of the yeast-expressed erythropoietin mutant Epo (R103A), a specific inhibitor of human primary hematopoietic cell erythropoiesis. Blood, 99(12), 4400–4405. https://doi.org/10.1182/blood.v99.12.4400
Burns, Suzanne, Murat O. Arcasoy, Li Li, Elizabeth Kurian, Katri Selander, Peter D. Emanuel, and Kevin W. Harris. “Purification and characterization of the yeast-expressed erythropoietin mutant Epo (R103A), a specific inhibitor of human primary hematopoietic cell erythropoiesis.Blood 99, no. 12 (June 15, 2002): 4400–4405. https://doi.org/10.1182/blood.v99.12.4400.
Burns, Suzanne, et al. “Purification and characterization of the yeast-expressed erythropoietin mutant Epo (R103A), a specific inhibitor of human primary hematopoietic cell erythropoiesis.Blood, vol. 99, no. 12, June 2002, pp. 4400–05. Pubmed, doi:10.1182/blood.v99.12.4400.
Burns S, Arcasoy MO, Li L, Kurian E, Selander K, Emanuel PD, Harris KW. Purification and characterization of the yeast-expressed erythropoietin mutant Epo (R103A), a specific inhibitor of human primary hematopoietic cell erythropoiesis. Blood. 2002 Jun 15;99(12):4400–4405.

Published In

Blood

DOI

ISSN

0006-4971

Publication Date

June 15, 2002

Volume

99

Issue

12

Start / End Page

4400 / 4405

Location

United States

Related Subject Headings

  • Yeasts
  • Recombinant Proteins
  • Receptors, Erythropoietin
  • Mutation
  • Immunology
  • Humans
  • Hematopoietic Stem Cells
  • Erythropoietin
  • Erythropoiesis
  • Erythroblasts