EXPRESSION, BIOLOGICAL-ACTIVITY AND KINETICS OF PRODUCTION OF RECOMBINANT OVINE TNF-ALPHA

Ovine tumour necrosis factor-alpha (OvTNF-alpha) was cloned by reverse transcription-polymerase reaction using RNA isolated from lipopolysac charide (LPS)-stimulated alveolar macrophages and primers based on the human TNF-alpha cDNA sequence. An expression vector carrying the codi ng sequence of the mature form of ovine TNF was constructed. The recom binant Ov-TNF alpha (rOvTNF-alpha) was expressed as a glutathione-S-tr ansferase (GST) fusion protein. It was cleaved with thrombin to yield rOvTNF free of the GST moiety. Growth at a lower temperature of 30 deg rees C and use of Escherichia coil strains AM207, AM305, E392 and NM52 2 did not improve the recovery of rOvTNF-alpha from the soluble fracti on to a significant extent. Purification of recombinant proteins was a chieved rapidly and easily by affinity chromatography using glutathion e-Sepharose. Yields of pure rOvTNF-alpha achieved in E. coli JM109 and AM207 were approximately 1 mg L(-1). Both rOvTNF-alpha and recombinan t human TNF-alpha (rhTNF-alpha) exerted cytotoxicity on L929 cells. Ho wever, rOvTNF-alpha but not rhTNF-alpha stimulated proliferation of ov ine thymocytes. Maximum levels of TNF-alpha mRNA expression by LPS-sti mulated ovine alveolar macrophages were detected at approximately 4 h post-stimulation.