Purification of GCT cell-derived human colony-stimulating factors.
We have purified human-active colony-stimulating factors from the human cell line, GCT, using sequential ultrafiltration; cation exchange, gel permeation, and reverse-phase high performance liquid chromatography (RHPLC); and ion-exchange HPLC. Activity eluted from sodium dodecyl sulfate-polyacrylamide gels with a peak at 17,500 daltons. Similar results were obtained by processing 35S-methionine-labeled conditioned medium, which showed a labeled band in the same region of activity. This purified HPLC fraction, which had a specific activity of greater than 1 x 10(7) colonies/mg protein, stimulated neutrophil colonies at day 7 and neutrophil, neutrophil-macrophage, and eosinophil colonies at day 14 of culture, suggesting that it contained both granulocyte (G-CSF) and granulocyte-macrophage (GM-CSF) colony-stimulating factors. It also promoted the growth of erythroid progenitors, and the GM-CSF fraction purified by hydrophobic chromatography had erythroid-enhancing activity. Separation of G-CSF from GM-CSF was accomplished by the addition of trifluoroacetic acid to the mobile phase at the reverse-phase HPLC step.
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003115:Colony-Stimulating Factors / Q000032:analysis / Q000302:isolation & purification*; D016179:Granulocyte Colony-Stimulating Factor; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D006133:Growth Substances / Q000032:analysis / Q000302:isolation & purification; D018219:Histiocytoma, Benign Fibrous / Q000473:pathology; D006801:Humans; D014407:Tumor Cells, Cultured / Q000032:analysis*
D003115:Colony-Stimulating Factors; D006133:Growth Substances; D016179:Granulocyte Colony-Stimulating Factor; D016178:Granulocyte-Macrophage Colony-Stimulating Factor