Rat tumour necrosis factor-alpha: expression in recombinant Pichia pastoris, purification, characterization and development of a novel ELISA

Tumour necrosis factor-alpha is a pro-inflammatory cytokine involved in man y aspects of acute phase and immune responses, Species specificity in the b iological action and receptor binding of TNF-alpha make it desirable to use homologous reagents in experimental models, both in vivo and irt vitro. As the rat is the model of choice in many investigations on fever, trauma and pathology, there is a need for specific rat reagents, In this paper, we de scribe the production of recombinant rat TNF-alpha in milligram quantities, using a methylotrophic yeast expression system, Pichia pastoris, Recombina nt TNF-alpha was produced intracellularly in a soluble form, cells were lys ed and the protein purified by ammonium sulphate precipitation, Sephadex G7 5 fractionation and finally, ion-exchange chromatography, The purified reco mbinant rat TNF-alpha had a molecular mass of 17401.38 +/- 0.38 Da, which i s within 1 Da of the value predicted by the sequence data, taking into acco unt N-acetylation of the initial methionine residue and a single disulphide bridge between amino acids 70 and 101, Recombinant rat TNF-alpha was shown to be 20 x fold more biologically active in the WEHI cytotoxicity assay, t han the human standard preparation. Polyclonal antibodies were raised again st purified recombinant rat TNF-alpha, these reagents were used to develop a novel enzyme-linked immunosorbant assay (ELISA), The ELISA was sensitive to 10 pg.ml-(1) rat TNF-alpha and was specific for TNF-alpha, showing no cr oss-reactivity with rat IL-1 alpha, rat IL-1 alpha, rat IL-1Ra or rat IL-6, The ELISA was used to measure TNF-alpha in the plasma of rats injected wit h bacterial endotoxin and in cultures of rat white blood cells. The ELISA w as shown to be a robust method suitable for use in assaying samples generat ed in both in vivo or in vitro experiments.