STUDIES ON THE PURIFICATION OF THROMBOPOIETIN FROM KIDNEY-CELL CULTURE-MEDIUM

  • 1 January 1985
    • journal article
    • research article
    • Vol. 106  (2) , 162-174
Abstract
A thrombocytopoiesis-stimulating factor (TSF) was purified from human embryonic kidney (HEK) cell culture medium. In the initial purification step, crude HEK cell culture medium was fractionated with saturated ammonium sulfate (step 1). The proteins precipitated by 40%-60% and 60%-80% ammonium sulfate saturation increased the % of 35S incorporation into platelets of assay mice (P < 0.01). The ammonium sulfate-precipitated proteins that contained significant TSF activity were further refined on Sephadex G-75 columns(step III). The fraction containing the highest specific activity (greatest 35S incorporation into platelets of assay mice/mg of protein) was further purified by DEAE cellulose column chromatography (step III). TSF activity was eluted from the columns between 0.3-1.0 mol/L NaCl. Additional Sephadex chromatography of post-DEAE-chromatographic preparations further increased the purity of the TSF (step IV). TSF from this 4-step procedure was further processed on a DEAE-high-performance liquid chromatography (HPLC) column (step Va) or size exclusion (SE)-HPLC columns (step Vb). After HPLC, the activity was localized in a region corresponding to a retention time of 6-8 min for the DEAE-HPLC, but longer times were found after SE-HPLC. TSF was further purified by additional sodium dodecylsulfate-polyacrylamide gel electrophoresis and SE-HPLC (step VI). The final product had significant TSF activity and represented a purification of .apprx. 500,000-fold. The isoelectric pH of partially purified TSF was 4.7. The MW of the more highly purified preparation was .apprx. 32,000. After extraction by a combination of chromatographic procedures, a single homogenous product was obtained.