Studies on the purification of thrombopoietin from kidney cell culture medium.
A thrombocytopoiesis-stimulating factor (TSF) has been 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 I). The proteins precipitated by 40% to 60% and 60% to 80% ammonium sulfate saturation increased the percent of sulfur 35 incorporation into platelets of assay mice (P less than 0.01). The ammonium sulfate-precipitated proteins that contained significant TSF activity were further refined on Sephadex G-75 columns (step II). The fraction containing the highest specific activity (greatest 35S incorporation into platelets of assay mice per milligram of protein) was further purified by diethylaminoethyl (DEAE)-cellulose column chromatography (step III). TSF activity was eluted from the columns between 0.3 and 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 four-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 to 8 minutes for the DEAE-HPLC, but longer times were found after SE-HPLC. TSF was further purified by additional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and SE-HPLC (step VI). The final product had significant TSF activity and represented a purification of approximately 500,000-fold. It was also shown that the isoelectric pH of partially purified TSF was 4.7 and the molecular weight of the more highly purified preparation was approximately 32,000. After extraction by a combination of chromatographic procedures, a single homogeneous product was obtained.
D000645:Ammonium Sulfate; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism; D002478:Cells, Cultured; D005591:Chemical Fractionation; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003470:Culture Media / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004622:Embryo, Mammalian; D006023:Glycoproteins / Q000302:isolation & purification*; D006801:Humans; D007525:Isoelectric Focusing; D007668:Kidney / Q000032:analysis*; D008297:Male; D051379:Mice; D008809:Mice, Inbred C3H; D011506:Proteins / Q000302:isolation & purification; D013462:Sulfur Radioisotopes; D013922:Thrombocytosis / Q000097:blood; D013926:Thrombopoietin / Q000302:isolation & purification*
D003470:Culture Media; D006023:Glycoproteins; D011506:Proteins; D013462:Sulfur Radioisotopes; D013926:Thrombopoietin; D000645:Ammonium Sulfate