PURIFICATION OF COLONY-STIMULATING FACTOR BY AFFINITY-CHROMATOGRAPHY

  • 1 January 1982
    • journal article
    • research article
    • Vol. 60  (1) , 238-244
Abstract
Whether L-cell-derived colony-stimulating factor (CSF) could be purified by a single-step affinity chromatographic technique was determined. A quantity of 100 .times. 106 U of purified anti-CSF was coupled to cyanogen bromide activated Sepharose 4B; colony assays revealed complete binding of the antibodies to the gel. Three 10-l pools of serum-free L-cell CSF were concentrated by ultrafiltration, applied to the gel and eluted with a low pH, high molarity buffer. Recovery of CSF ranged from 68-100% with > 1000-fold decreases in protein content. Specific activity of the purified CSF ranged from 2.8-5.9 .times. 107 U of CSF/mg protein. Following iodination, each purified pool of CSF revealed a major 63,000-dalton peak of radioactivity that comigrated with CSF activity in SDS[sodium dodecyl sulfate]-acrylamide gels. Several smaller peaks of 50,000 and 40,000 MW were also detected. Approximately 2/3 of the purified CSF was adherent to concanavalin-A with elution by a competing sugar. Electrophoretic mobility was retarded by incubation with neuraminidase. These chromatographic studies confirm that CSF is a glycoprotein but also suggest variable degrees of glycosylation of the molecule. This chromatographic technique should prove useful in the rapid purification of large quantities of CSF for physiologic and biochemical characterization.