These tools will no longer be maintained as of December 31, 2024. Archived website can be found here. PubMed4Hh GitHub repository can be found here. Contact NLM Customer Service if you have questions.


Pubmed for Handhelds

PUBMED FOR HANDHELDS

Search MEDLINE/PubMed

  • Title: Purification of biologically active human erythropoietin-binding protein and detection of its binding sites.
    Author: Lee JY.
    Journal: Ann Clin Lab Sci; 2007; 37(1):63-70. PubMed ID: 17311871.
    Abstract:
    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.
    [Abstract] [Full Text] [Related] [New Search]