title: Secretory production of erythropoietin and the extracellular domain of the erythropoietin receptor by Bacillus brevis: affinity purification and characterization.
abstract: Bacillus brevis secretes a large amount of cell wall proteins into the culture medium. For construction of Bacillus brevis expression-secretion vectors of human erythropoietin (EPO) and the extracellular domain of mouse erythropoietin receptor (sEPOR), cDNA for each mature form was inserted into a plasmid containing the promoter region and the signal-peptide encoding region of a cell wall protein. Culture supernatants of transformants were affinity purified using a monoclonal antibody-fixed gel for EPO and and EPO-fixed gel for sEPOR. The affinity purification efficiently removed unwanted proteins, giving samples with sufficiently high purity to analyze amino acid sequences of N-terminal regions and biological activities. Combination of this secretory production and affinity purification may facilitate isolation of a large amount of pure EPO and sEPOR, and is useful for further understanding the molecular mechanism of interaction between EPO and EPOR.
authors.affiliation: Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Nagao
authors.forename: K
authors.initials: K
authors.lastname: Inoue
authors.forename: S K
authors.initials: SK
authors.lastname: Moon
authors.forename: S
authors.initials: S
authors.lastname: Masuda
authors.forename: H
authors.initials: H
authors.lastname: Takagi
authors.forename: S
authors.initials: S
authors.lastname: Udaka
authors.forename: R
authors.initials: R
authors.lastname: Sasaki
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001407:Bacillus / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D004921:Erythropoietin / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D017467:Receptors, Erythropoietin / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D017467:Receptors, Erythropoietin; D004921:Erythropoietin