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 alpha (rovTNF-alpha) using a yeast Ty, virus like particle, expression system. RovTNF-alpha is at least as active as recombinant human TNF-alpha (rhTNF-alpha) in two different bio-assays performed on ovine material, whilst approximately 1000-fold more rovrTNF-alpha than rhTNF-alpha 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-alpha shows about 2 logs greater activity than on murine cells, whilst rhTNF-alpha is about 1 log more active. A monoclonal antibody, raised against rovTNF-alpha, has been used to demonstrate the presence of nanogram amounts of an appropriately sized glycoprotein to be native ovine TNF-alpha in supernants of LPS stimulated ovine alveolar macrophages. These samples show no detectable cytotoxicity to L929 cells, although they show activity attributable to TNF-alpha (through neutralization by a polyclonal antiserum raised to rovTNF-alpha) 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-alpha using these cells, in spite of their routine use to assay TNF-alpha from several other species. The sequence features in ovine TNF-alpha which might reduce its affinity for the murine TNF type 1 receptor are discussed.
Green
I R
IR
Department of Veterinary Pathology, University of Edinburgh, UK.
Fiskerstrand
C
C
Bertoni
G
G
Roy
D J
DJ
Peterhans
E
E
Sargan
D R
DR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002358:Cartilage, Articular / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D017931:DNA Primers; D005354:Fibrosarcoma; D006801:Humans; D008213:Lymphocyte Activation / Q000187:drug effects; D008262:Macrophage Activation; D008264:Macrophages / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000633:toxicity; D017386:Sequence Homology, Amino Acid; D012756:Sheep; D013045:Species Specificity; D013552:Swine; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis* / Q000302:isolation & purification / Q000633:toxicity*
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha