The extracellular domain of the human erythropoietin receptor: expression as a fusion protein in Escherichia coli, purification, and biological properties.
We developed an efficient production system of the soluble extracellular domain of the human erythropoietin receptor (sEPO-R) and characterized the binding of erythropoietin (EPO) with the purified recombinant protein. The sEPO-R, fused to the maltose binding protein (MBP), was expressed as a soluble protein in the periplasm of Escherichia coli (E. coli) and did not accumulate in inclusion bodies. After lysis of the bacteria by an osmotic shock, the fusion protein was purified by affinity chromatography on amylose followed by size exclusion chromatography (SEC). Specific binding of 125I-labelled EPO to the sEPO-R was demonstrated by competitive and saturation binding assays. A single affinity class (Kd = 0.25 nM) of the binding site was evident by Scatchard analysis. This value is similar to the Kd observed between EPO and the EPO-R of high affinity present on human erythroid progenitors. The complex has a molecular size corresponding to a 1:1 complex of EPO and the fusion protein.
Ahaded
A
A
Institut National de la Transfusion Sanguine (INTS), Paris, France.
Winchenne
J J
JJ
Cartron
J P
JP
Lambin
P
P
Lopez
C
C
D018528:ATP-Binding Cassette Transporters; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004921:Erythropoietin / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D006801:Humans; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D011485:Protein Binding; D017467:Receptors, Erythropoietin / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D017467:Receptors, Erythropoietin; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D004921:Erythropoietin