Partial purification and characterization of an antigen-specific helper factor synthesized by a T-cell continuous line.

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Abstract
Antigen-specific factors produced by the T-cell growth factor-dependent [mouse] T-cell continuous line E-9M(+) were partially purified. Gel analysis of the twice-affinity-purified eluate of a poly(Tyr,Glu)-poly(DL-Ala)--poly(LLys) [(T,G)--A--L] column revealed the existence of iodinated bands with MW of 17,000 and 15,000 [daltons], in addition to a diffuse band of high MW. The specific helper activity of the E-9M(+) supernatants was associated with a precipitate from 65-75% ammonium sulfate. Gel electrophoresis of either the eluate of a (T,G)-A--L column or of the 65-75% salt precipitate indicated that in both preparations 2 fractions contained the biological activity of the factor, 1 of a high (< 67,000) MW and the other of a low (15,000-17,000) MW. Culture supernatants of the internally [35S]methionine-labeled E-9M(+) line were subjected to a combined purification of sequential ammonium sulfate precipitations followed by affinity chromatography. Polyacrylamide gel patterns obtained of the eluates of the different salt precipitates demonstrated that the 65-75% ammonium sulfate precipitate contained 2 35S-labeled bands with apparent MW in the range of 60,000 and 15,000, similar to the activity patterns obtained by the gel electrophoresis fractionation experiments. Apparently, a fraction of low MW preserves the antigen specificity, and the helper activity of the factor produced by the T-cell line.