Purification of human interleukin 2 to apparent homogeneity and its molecular heterogeneity.

Abstract

Interleukin 2 (IL-2), produced with and without co-stimulation by the Burkitt's lymphoma line Daudi, was purified 37,000-fold to apparent homogeneity from lymphocyte conditioned medium by (NH4)2SO4 precipitation, DEAE-cellulose ion-exchange chromatography, gel filtration, and chromatography on blue agarose and on Procion-red agarose. The purified IL-2 showed a 10(6) U/mg protein sp act. IL-2 produced in the absence of Daudi cells had a mol wt of 26,000 as measured by gel filtration and an isoelectric point of 6.7. This IL-2 showed a 16,000 and 17,000 mol wt in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). IL-2, produced in the presence of Daudi cells (10(6)/ml), showed a mol wt of approximately 14,000, as measured by both gel filtration and SDS-PAGE, and an isoelectric point of 8.1. The purified IL-2 lacked detectable interferon (alpha and gamma), granulocyte-macrophage colony-stimulating factor, B cell growth factor, T cell-replacing factor, and thymocyte-differentiating activity and was free of any contaminating proteins as judged by silver staining in SDS-PAGE. All three molecular forms of IL-2 were biologically active at concentrations of 10(-11) - 10(-10) M, supporting the growth of human and murine cytotoxic T cell lines.

Production of and in vitro response to interleukin 2 in the acquired immunodeficiency syndrome.

“…After 48 h, the culture supernatants were removed, sterilized by filtration, and stored at 4°C. As described elsewhere (32), supernatant IL-2 activity was determined using IL-2-dependent CTLL-l cells (33) In separate experiments (not shown) using the five antigens listed above with cells from healthy controls who either (a) were seronegative to T. gondii (7), CMV, or hISV, (b) had nonreactive Candida skin tests, or (c) had never received tetanus toxoid, the culture supernatants did not contain detectable IL-2 (<0.1 U/ml) or IFN-'y (<10 U/ml). These results indicated the specificity of the antigen-induced responses observed in the "positive" controls shown in the tables.…”

Cytokines with Possible Clinical Utility

“…The IL2 was purified from human PBL-conditioned medium in our laboratory [34]. The s.c. route of administration was chosen in order to achieve maximal lymphatic drainage.…”

Purification to Apparent Homogeneity and Biochemical Characterization of Human Pluripotent Hematopoietic Colony-Stimulating Factor

“…The first three steps of purification involved ammonium sulfate precipitation (80% saturation), anion-exchange chromatography [diethylaminoethanol (DEAE)-cellulose, DE 52, Whatman, Clifton, NJ], and gel filtration (AcA 54 Ultrogel, LKB Products, Inc., Rockland, MD) ( Table 1). These steps were used because they were highly effective for other cytokines, notably Interleukin 2 [18] and B-cell-differentiating factor [19] and have been described in detail elsewhere [18]. Pluripotent CSF eluted from the DE 52 cellulose column between 0.075 and 0.1 M NaCI in 0.05 M Tris/HCI, pH 7.8, and from the AcA 54 column with a single peak at around 32 000 molecular weight.…”

“…After 18 h eluted proteins were assayed for pluripotent activity (lower panel; GM-CSF activity, black columns). The following marker proteins (arrows) were used: ovalbumin (molecular weight, 43 000), chymotrypsinogen (molecular weight, 25 700), lactoglobulin (molecular weight, 18400), lysozyme (molecular weight, 14300), and cytochrome C (molecular weight, 12300) No an IEF column (LKB 8100) [18] using ampholines with a pH range of 3.5-10. Pluripotent CSF was localized in one fraction with a pH of 5.5 ( Table 2).…”