Refolding, Purification, and Characterization of Human Erythropoietin Binding Protein Produced inEscherichia coli

Johnson, Dana L.; Middleton, Steven A.; McMahon, Frank J.; Barbone, Francis P.; Kroon, Daniel J.; Tsao, Eric; Lee, Woan Hwa; Mulcahy, Linda S.; Jolliffe, Linda K.

doi:10.1006/prep.1996.0014

Cited by 34 publications

(43 citation statements)

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“…Protein Expression and Characterization-The cloning, expression, protein recovery, and refolding of the EBP has been described in detail elsewhere (16). Briefly, the nucleotide sequence of the first 225 amino acids of the extracellular domain of the human EPOR (EBP) was cloned into plasmid pSAM3 that contains a synthetic pelB signal sequence.…”

Section: Methodsmentioning

confidence: 99%

“…The EBP protein was produced as insoluble protein localized to inclusion bodies. The EBP was recovered, solubilized, and refolded as described (16). The protein refolding process was monitored by high performance-size exclusion liquid chromatography (HP-SEC), and the active protein was detected by peak shift analysis through the addition of purified recombinant EPO.…”

Section: Methodsmentioning

confidence: 99%

“…Mutant EBPs created by site-specific mutagenesis were expressed in E. coli and purified as described for the wild-type EBP (16). Some mutant EBPs were purified by a modification of these methods in which the hydrophobic interaction chromatography step was replaced by a preparative high performance-size exclusion chromatography (HP-SEC) step.…”

Section: Methodsmentioning

confidence: 99%

“…Some mutant EBPs were purified by a modification of these methods in which the hydrophobic interaction chromatography step was replaced by a preparative high performance-size exclusion chromatography (HP-SEC) step. The purity of mutant and wild-type EBP was estimated by SDS-polyacrylamide gel electrophoresis and analytical HP-SEC as described (16). The concentrations of purified mutant and wild-type EBP were estimated using the experimentally determined extinction coefficient for the wild-type EBP of 2.3 absorbance units per mg/ml at 280 nm (16).…”

Section: Methodsmentioning

confidence: 99%

“…The functions of other amino acid residues were investigated based on their conservation among receptors of the cytokine receptor family or their homology to ligand binding determinants reported for other receptors of this family. Wildtype and mutant proteins were expressed in Escherichia coli as soluble EPO binding proteins (EBP), consisting of amino acids 1-225 of the extracellular domain of the mature EPOR (16), and analyzed for EPO binding in several different assay formats. Some mutant proteins were expressed as full-length receptor on the surface of COS cells and analyzed in receptor binding assays with 125 I-EPO.…”

mentioning

confidence: 99%

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Mutagenesis Studies of the Human Erythropoietin Receptor

Barbone¹,

Middleton²,

Johnson³

et al. 1997

Journal of Biological Chemistry

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Mutagenesis of the erythropoietin receptor (EPOR)permits analysis of the contribution that individual amino acid residues make to erythropoietin (EPO) binding. We employed both random and site-specific mutagenesis to determine the function of amino acid residues in the extracellular domain (referred to as EPO binding protein, EBP) of the EPOR. Residues were chosen for site-specific alanine substitution based on the results of the random mutagenesis or on their homology to residues that are conserved or have been reported to be involved in ligand binding in other receptors of the cytokine receptor family. Site-specific mutants were expressed in Escherichia coli as soluble EBP and analyzed for EPO binding in several different assay formats. In addition, selected mutant proteins were expressed as full-length EPOR on the surface of COS cells and analyzed for 125 I-EPO binding in receptor binding assays. Using these methods, we have identified residues that appear to be involved in EPO binding as well as other residues, most of which are conserved in receptors of the cytokine receptor family, that appear to be necessary for the proper folding and/or stability of the EPOR. We present correlations between these mutagenesis data and the recently solved crystal structure of the EBP with a peptide ligand.Erythropoietin (EPO) 1 is a glycoprotein hormone that functions as the primary regulator of erythropoiesis by binding a specific receptor (EPOR) on the surface of erythrocyte precursor cells, signaling their proliferation and differentiation into mature red blood cells (reviewed in Ref. 1). The human EPOR is a 484-amino acid glycoprotein comprised of extracellular and cytoplasmic domains of nearly equal size and a single transmembrane domain (2). The extracellular domain of the EPOR contains a 225-amino acid region referred to as the cytokine receptor homology (CRH) domain that shares conserved features with an expanding family of cytokine and growth factor receptors (3, 4) including the receptors for many interleukins (IL), colony stimulating factors, growth hormone (GH), thrombopoietin, leptin, interferons (IFN), and tissue factor among others. The CRH domains consist of two motifs of approximately 100 amino acids each that are structurally related to fibronectin type III domains. Based on this homology, Bazan (3, 4) proposed that the CRH domains consist of two motifs of seven ␤-strands each which adopt ␤-sheet structures with fibronectin type III-like or immunoglobulin (Ig)-like folds. These structural predictions have been confirmed by the solution of the crystal structures of the ligand-bound extracellular domains of the GH receptor and IFN-␥ receptor ␣ (IFN-␥R␣), the GH bound extracellular domain of the prolactin receptor, the extracellular domain of tissue factor, and most recently, the extracellular domain of the EPOR with a peptide ligand (5-10). Alignment of the CRH domains of this family of receptors based on their predicted ␤-strand secondary structural elements reveals several conserved characteristics (3, 4,...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Mutagenesis Studies of the Human Erythropoietin Receptor

Barbone¹,

Middleton²,

Johnson³

et al. 1997

Journal of Biological Chemistry

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Chemical and Enzymatic Synthesis of Sialylated Glycoforms of Human Erythropoietin

et al. 2021

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Recombinant human erythropoietin (EPO) is the main therapeutic glycoprotein for the treatment of anemia in cancer and kidney patients.T he in-vivo activity of EPO is carbohydrate-dependent with the number of sialic acid residues regulating its circulatory half-life.E PO carries three Nglycans and thus obtaining pure glycoforms provides am ajor challenge.W eh ave developed ar obust and reproducible chemoenzymatic approach to glycoforms of EPO with and without sialic acids.E PO was assembled by sequential native chemical ligation of two peptide and three glycopeptide segments.The glycopeptides were obtained by pseudoprolineassisted Lansbury aspartylation. Enzymatic introduction of the sialic acids was readily accomplished at the level of the glycopeptide segments but even more efficiently on the refolded glycoprotein. Biological recognition of the synthetic EPOs was shown by formation of 1:1c omplexes with recombinant EPO receptor.

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