Tumor necrosis factor (TNF) is a pleiotropic cytokine with a wide range of biological activities including cytotoxicity, immune-cell proliferation, and mediation of inflammatory responses. Mutational analysis of mature TNF has been facilitated by the high expression levels that were obtained inEscherichia colicells. Furthermore, the fact that mature TNF does not form inclusion bodies, but remains soluble in bacterial extracts, allows a fast and easy characterization. We describe an efficient method for the introduction of a specific mutation in mature murine TNF making use of double-stranded plasmid DNA and two oligonucleotides. Two in vitro protocols are given that allow assessment of the binding of wild-type TNF and/or TNF muteins to TNF receptors (TNFR) (radioligand competition binding and Biacore). The biological activity of wild-type TNF and/or TNF muteins can be assessed in cellular assays. TNF-induced cytotoxicity toward murine L929s cells and human Kym39A6 cells is mediated by interaction with cellular TNFR-I, whereas TNF-induced proliferation of murine CT6 cells is mediated by triggering of cellular TNFR-II.
