An efficient method for the purification of tumor necrosis factor from rabbit serum

Tumor necrosis factor (TNF) has been purified to homogeneity from rabbit serum. TNF was isolated by a purification scheme consisting of chromatography on DEAE-Trisacryl M, concentration on an Amicon YM10 membrane filter, molecular exclusion on TSK-3000 SWG (HPLC), and then either by Reversed phase HPLC on a Vydac C 4 column or by hydrophobic interaction chromatography. This procedure gives a recovery of 50% of pure TNF. The purified material has a specific activity of 2 X 10(8) U/mg in vitro and is active in vivo causing necrosis in Meth A sarcoma bearing mice. It gives a single band at a molecular weight of 17.000 on 15% polyacrylamide gel electrophoresis and 40.000 on gel exclusion chromatography at pH 7.0. The protein has a pI of 5.0 and is stable when heated to 70 degrees C for 10 minutes.