Secretory production of erythropoietin and the extracellular domain of the erythropoietin receptor by Bacillus brevis: affinity purification and characterization.
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.
Nagao
M
M
Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Japan.
Inoue
K
K
Moon
S K
SK
Masuda
S
S
Takagi
H
H
Udaka
S
S
Sasaki
R
R
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
D017467:Receptors, Erythropoietin; D004921:Erythropoietin