Human tumor necrosis factor. Production, purification, and characterization.

作者：Bharat Bhushan Aggarwal, William J. Kohr, P E Hass, B Moffat, Steven A. Spencer, William J. Henzel, Timothy S. Bringman, Glenn E. Nedwin, David V. Goeddel, Richard N. Harkins · 发表于：Journal of Biological Chemistry · 年份：1985 · DOI：10.1016/s0021-9258(18)89560-6 · 被引用次数：924 · 研究领域：Monoclonal and Polyclonal Antibodies Research、Immune Response and Inflammation、Antimicrobial Peptides and Activities

Human tumor necrosis factor (TNF) was purified to homogeneity from serum-free tissue culture supernatants of the HL-60 promyelocytic leukemia cell line induced by 4 beta-phorbol 12-myristate 13-acetate. The purification scheme consisted of controlled-pore glass and DEAE-cellulose chromatography, Mono Q-fast-protein liquid chromatography, and reverse-phase high performance liquid chromatography. The purified protein was homogeneous by the criteria of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and NH2-terminal sequence analysis. The specific activity of purified tumor necrosis factor is approximately 10(8) units/mg. The protein has a molecular weight of approximately 17,000, an isoelectric point of 5.3, and contains two cysteines involved in a disulfide bridge. Approximately 50% homology between TNF and another cytolytic lymphokine, lymphotoxin, exists when the NH2-terminal 34 residues of TNF and internal sequence generated by tryptic, Staphylococcus aureus V8 protease, and chymotryptic digests of TNF are aligned with the complete amino acid sequence of lymphotoxin.