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.

[1]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[2]  J. Kollár,et al.  Purification and characterization of decorin core protein expressed in Escherichia coli as a maltose-binding protein fusion. , 1996, Analytical biochemistry.

[3]  T. Arakawa,et al.  Dimerization of the extracellular domain of the erythropoietin (EPO) receptor by EPO: one high-affinity and one low-affinity interaction. , 1996, Biochemistry.

[4]  L. Jolliffe,et al.  Refolding, purification, and characterization of human erythropoietin binding protein produced in Escherichia coli. , 1996, Protein expression and purification.

[5]  I. Poola,et al.  Ligand binding kinetics of a soluble full-length murine erythropoietin receptor. , 1995, Biochemical and biophysical research communications.

[6]  T. Taniguchi.  Cytokine signaling through nonreceptor protein tyrosine kinases. , 1995, Science.

[7]  I. Kerr,et al.  Jaks and Stats in signaling by the cytokine receptor superfamily. , 1995, Trends in genetics : TIG.

[8]  C. Tournamille,et al.  Efficient production of biologically active human recombinant proteins in human lymphoblastoid cells from integrative and episomal expression vectors. , 1994, Gene.

[9]  J. L. Le Caer,et al.  Purification and biological activity of a recombinant human erythropoietin produced by lymphoblastoid cells. , 1994, Preparative biochemistry.

[10]  N. Casadevall,et al.  Preparation and biological properties of monoclonal antibodies against human erythropoietin. , 1993, Hybridoma.

[11]  S. Jones,et al.  The extracytoplasmic domain of the erythropoietin receptor forms a monomeric complex with erythropoietin. , 1993, Blood.

[12]  M. Koury,et al.  The molecular mechanism of erythropoietin action. , 1992, European journal of biochemistry.

[13]  S. Masuda,et al.  Production and ligand-binding characteristics of the soluble form of murine erythropoietin receptor. , 1992, Biochemical and biophysical research communications.

[14]  J. Winkelmann,et al.  Ligand binding properties of the human erythropoietin receptor extracellular domain expressed in Escherichia coli. , 1992, The Journal of biological chemistry.

[15]  C. Tournamille,et al.  Cloning of the gene encoding the human erythropoietin receptor. , 1991, Blood.

[16]  K. Clauser,et al.  Dimerization of the extracellular domain of the human growth hormone receptor by a single hormone molecule. , 1991, Science.

[17]  V. Broudy,et al.  Erythropoietin receptor characteristics on primary human erythroid cells. , 1991, Blood.

[18]  N. Casadevall,et al.  Solubilization and hydrodynamic characteristics of the erythropoietin receptor. Evidence for a multimeric complex. , 1990, European journal of biochemistry.

[19]  L. Zon,et al.  Erythropoietin receptor. Subunit structure and activation. , 1990, The Journal of clinical investigation.

[20]  K. Arai,et al.  Cloning and expression of a gene encoding an interleukin 3 receptor-like protein: identification of another member of the cytokine receptor gene family. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[21]  A. D’Andrea,et al.  Human erythropoietin receptor: cloning, expression, and biologic characterization. , 1990, Blood.

[22]  L. Deaven,et al.  The gene for the human erythropoietin receptor: analysis of the coding sequence and assignment to chromosome 19p. , 1990, Blood.

[23]  H. Lodish,et al.  Friend spleen focus-forming virus glycoprotein gp55 interacts with the erythropoietin receptor in the endoplasmic reticulum and affects receptor metabolism. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[24]  H. Lodish,et al.  Activation of cell growth by binding of Friend spleen focus-forming virus gp55 glycoprotein to the erythropoietin receptor , 1990, Nature.

[25]  B. Slatko,et al.  An Escherichia coli vector to express and purify foreign proteins by fusion to and separation from maltose-binding protein. , 1988, Gene.

[26]  Chu di Guana,et al.  Vectors that facilitate the expression and purification of foreign peptides in Escherichia coli by fusion to maltose-binding protein. , 1988 .

[27]  P. Mayeux,et al.  The erythropoietin receptor of rat erythroid progenitor lens. Characterization and affinity cross-linkage. , 1987, The Journal of biological chemistry.

[28]  S. Krantz,et al.  Identification of the receptor for erythropoietin by cross-linking to Friend virus-infected erythroid cells. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[29]  H. Towbin,et al.  Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[30]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[31]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[32]  R. Dernick,et al.  Simplified method for silver staining of proteins in polyacrylamide gels and the mechanism of silver staining , 1985 .