Preparative two-step purification of human IL-2 by HPLC and hydrophobic affinity chromatography.
Interleukin 2 (IL-2) has been purified by a protocol using gel filtration high performance liquid chromatography (HPLC) and hydrophobic affinity chromatography with blue-trisacryl M. Peripheral blood lymphocytes or tonsillar lymphocytes were stimulated with phytohemagglutinin (PHA). Serum free conditioned medium (CM) containing IL-2, other lymphokines and residual PHA molecules was analyzed after 3 variations of ammonium sulfate (AS) precipitation: (1) precipitation of CM with 50% AS yielded a precipitate containing most of the residual PHA but also a fraction of IL-2. (2) Precipitation with direct 80% AS of crude CM yielded both IL-2 and residual PHA. (3) A double step procedure (50% AS followed by 80% AS) yielded a precipitate containing IL-2 but free of residual lectin. HPLC purification of these various AS-precipitated materials or of lyophilized crude CM yielded 2 peaks with mitogenic activity as assayed with the CTLL2 murine clone or IL-2-dependent human Con A-stimulated lymphoblasts. IFN was easily separated from IL-2 and PHA, but BCGF still copurified with IL-2. Peak I (25 kDa) was enriched 400-fold for IL-2 while peak II (68 kDa) contained the residual PHA. The IL-2-containing fractions eluted from HPLC were further purified by blue-trisacryl M chromatography. The IL-2 eluted with 0.4 M NaCl. The entire protocol (HPLC followed by blue-trisacryl) led routinely to 8000-fold IL-2 enrichment. Preparative HPLC directly applied to lyophilized crude (CM) enriched IL-2 activity 400-fold with yield averaging 60% of the IL-2 input. The final material was free from interferon and IL-1, but BCGF still copurified with IL-2. The 2-step purified material (HPLC and blue-trisacryl) gave 2 bands in SDS-PAGE both of which contained IL-2.
D000645:Ammonium Sulfate / Q000494:pharmacology; D002471:Cell Transformation, Neoplastic / Q000276:immunology; D011232:Chemical Precipitation; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D007376:Interleukin-2 / Q000302:isolation & purification* / Q000502:physiology; D007945:Leukemia, Lymphoid / Q000276:immunology; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000032:analysis* / Q000276:immunology; D010835:Phytohemagglutinins / Q000494:pharmacology
D007376:Interleukin-2; D010835:Phytohemagglutinins; D000645:Ammonium Sulfate