title: Engineering of erythropoietin receptor for use as an affinity ligand.
abstract: Recombinant human erythropoietin receptor (rhEPOR) has applicability as an affinity ligand for purification of recombinant human erythropoietin (rHuEPO) because of its specific binding to rHuEPO. For application of rhEPOR as a ligand for purification of rHuEPO, soluble rhEPOR was expressed in the periplasm of Escherichia coli and engineered by directed evolution through random mutagenesis and integration of mutations. From the screening of random mutagenesis, we identified an amino acid mutation (H114Y) contributing to rHuEPO binding and four amino acid mutations (R76S, A132D, A162D, and C181Y) contributing to expression of soluble rhEPOR. However, the rHuEPO that binds to engineered rhEPOR having H114Y mutation is difficult to dissociate from the engineered rhEPOR. Therefore, H114Y mutation was not suitable for the construction of the rhEPOR ligand. As a rhEPOR ligand, engineered rhEPOR containing four amino acid mutations (EPORm4L) was constructed by integration of mutations except for H114Y. The expression of EPORm4L (127mgl(-1) of culture medium) was markedly increased in comparison with wild-type rhEPOR (2mgl(-1) of culture medium). Small-scale affinity chromatography demonstrated that EPORm4L worked as an affinity ligand for purification of rHuEPO.
authors: Sagami Chemical Research Institute, Ayase, Kanagawa 252-1193, Japan. Electronic address: hatayama@sagami.or.jp.
authors: Kouta
authors: K
authors: Hatayama
authors: Sagami Chemical Research Institute, Ayase, Kanagawa 252-1193, Japan; Tosoh Corporation, Ayase, Kanagawa 252-1123, Japan.
authors: Teruhiko
authors: T
authors: Ide
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015202:Protein Engineering; D017467:Receptors, Erythropoietin / Q000097:blood / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
chemical_list: D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins
keywords: Affinity chromatography; Directed evolution; Erythropoietin; Erythropoietin receptor; Ligand