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 do main of the human erythropoietin receptor (sEPO-R) and characterized the bi nding of erythropoietin (EPO) with the purified recombinant protein. The sE PO-R, fused to the maltose binding protein (MBP), was expressed as a solubl e protein in the periplasm of Escherichia coli (E. coli) and did not accumu late in inclusion bodies. After lysis of the bacteria by an osmotic shock, the fusion protein was purified by affinity chromatography on amylose follo wed by size exclusion chromatography (SEC). Specific binding of I-125-label led EPO to the sEPO-R was demonstrated by competitive and saturation bindin g assays. A single affinity class (K-d = 0.25 nM) of the binding site was e vident by Scatchard analysis. This value is similar to the K-d observed bet ween EPO and the EPO-R of high affinity present on human erythroid progenit ors. The complex has a molecular size corresponding to a 1:1 complex of EPO and the fusion protein.