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Purification of Biologically Active Human Erythropoietin-Binding Protein and Detection of its Binding Sites

Address correspondence to: Dr. Jong Y. Lee, P.O. Box 14945, Minneapolis, MN 55414, USA; tel 612 408 3125; fax 612 379 2467; e-mail leexx154{at}umn.edu.

To purify human erythropoietin-binding protein (Epo-bp), the recombinant vector JYL26 was constructed by inserting human Epo-receptor cDNA by PCR into a pGEX-2T plasmid vector with a thrombin cleavage site. EpoRex-th fusion protein, containing glutathione-S-transferase (GST) and Epo-bp, was purified by glutathione-affinity chromatography. Biologically active pure human Epo-bp (MW = 29 kDa) was then purified by Epo-agarose chromatography after cleaving off the GST by thrombin. Purified Epo-bp has a strong binding affinity to ¹²⁵I-Epo, and unlabeled Epo eliminates the binding (p <0.0001). Trypsin digested Epo-bp to ~20 kDa and completely eliminated the binding. Thus, Epo-bp ligand binding is specific and it may require an intact Epo-bp. Ligand-binding sites were detected using fluorescein-labeling in our new products, Epo-bp and anti-Epo-bp antibody ( Epo-bp), in various blood cell progenitors, including megakaryocytes, erythroblasts, normoblasts, and myeloblasts, while fluorescein-labeled pre-immune Fab-treated cells did not show any binding. Epo, Epo-bp, and their antibodies were measured in serum and plasma specimens by enzyme immunoassay methods developed in our laboratory; Epo in serum and plasma: 25.4 ± 2.17 and 24.2 ± 2.35; Epo-bp in serum and plasma: 24.2 ± 1.84 and 25.0 ± 1.26 mU/ml, respectively. These assays are simple, sensitive, and precise, compared to the conventional Epo radioimmunoassay, and are more environmentally friendly than assays that use radioactive reagents.

Keywords: Epo-receptor expression vector, Epo-binding site, Epo-bp immunoassay

This article has been cited by other articles:

				M. S. Lee, J. S. Lee, and J. Y. Lee Prevention of Erythropoietin-Associated Hypertension Hypertension, August 1, 2007; 50(2): 439 - 445. [Abstract] [Full Text] [PDF]