Purification of biologically active human erythropoietin-binding protein and detection of its binding sites.
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 approximately 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 (alpha 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.
Lee
Jong Y
JY
Department of Medicine, University of Minnesota School of Medicine, P.O. Box 14945, Minneapolis, MN 55414, USA. leexx154@umn.edu
D000906:Antibodies / Q000097:blood; D001665:Binding Sites / Q000235:genetics; D002846:Chromatography, Affinity; D017931:DNA Primers; D005822:Genetic Vectors / Q000235:genetics; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007124:Immunoenzyme Techniques; D007457:Iodine Radioisotopes / Q000378:metabolism; D017467:Receptors, Erythropoietin / Q000097:blood / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification*
D000906:Antibodies; D017931:DNA Primers; D007457:Iodine Radioisotopes; D017467:Receptors, Erythropoietin; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase