Recombinant human erythropoietin: purification and analysis of carbohydrate linkage.
Erythropoietin was purified to homogeneity from the culture medium of a baby hamster kidney cell line stably transfected with a human erythropoietin gene. A three-step procedure was used, which included affinity chromatography, ion-exchange chromatography, and reverse-phase chromatography. Purity of the protein was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino-terminal sequence analysis. Overall recovery was 35%. The biological activity of purified recombinant erythropoietin was similar to that of the native hormone in vitro. The purified recombinant hormone contained N-linked carbohydrate at residues 24, 38, and 83, and and O-linked carbohydrate at residue 126.
Broudy
V C
VC
Department of Medicine, University of Washington, Seattle 98195.
Tait
J F
JF
Powell
J S
JS
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002241:Carbohydrates / Q000032:analysis*; D002460:Cell Line; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D003470:Culture Media / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004921:Erythropoietin / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D007668:Kidney / Q000378:metabolism; D010455:Peptides / Q000032:analysis; D011994:Recombinant Proteins / Q000096:biosynthesis*; D014162:Transfection
D000596:Amino Acids; D002241:Carbohydrates; D003470:Culture Media; D010455:Peptides; D011994:Recombinant Proteins; D004921:Erythropoietin