EXPRESSION AND CHARACTERIZATION OF BIOACTIVE RECOMBINANT OVINE TNF-ALPHA - SOME SPECIES-SPECIFICITY IN CYTOTOXIC RESPONSE TO TNF

We have expressed and partially purified recombinant ovine tumour necrosis factor α (rovTNF-α) using a yeast Ty, virus like particle, expression system. RovTNF-α is at least as active as recombinant human TNF-α (rhTNF-α) in two different bio-assays performed on ovine material, whilst approximately 1000-fold more rovrTNF-α than rhTNF-α is required to induce the same level of cytotoxicity in TNF-sensitive murine cell lines L929 and WEHI 164 clone 13. When cytotoxic assays are performed on the porcine TNF sensitive cell line PK(15)-1512, rovTNF-α shows about 2 logs greater activity than on murine cells, whilst rhTNF-α is about 1 log more active. A monoclonal antibody, raised against rovTNF-α, has been used to demonstrate the presence of nanogram amounts of an appropriately sized glycoprotein to be native ovine TNF-α in supernatants of LPS stimulated ovine alveolar macrophages. These samples show no detectable cytotoxicity to L929 cells, although they show activity attributable to TNF-α (through neutralization by a polyclonal antiserum raised to rovTNF-α) in an assay on ovine material. The relative lack of activity on murine cells helps to explain previous reports of inability to assay native ovine TNF-α using these cells, in spite of their routine use to assay TNF-α from several other species. The sequence features in ovine TNF-α which might reduce its affinity for the murine TNF type 1 receptor are discussed. © 1993 Academic Press Limited.