Expression, biological activity and kinetics of production of recombinant ovine TNF-α

Ovine tumour necrosis factor-alpha (OvTNF-α) was cloned by reverse transcription-polymerase reaction using RNA isolated from lipopolysaccharide (LPS)-stimulated alveolar macrophages and primers based on the human TNF-α cDNA sequence. An expression vector carrying the coding sequence of the mature form of ovine TNF was constructed. The recombinant Ov-TNFα (rOvTNF-α) was expressed as a glutathione-S-transferase (GST) fusion protein. It was cleaved with thrombin to yield rOvTNF free of the GST moiety. Growth at a lower temperature of 30°C and use of Escherichia coli strains AM207, AM305, E392 and NM522 did not improve the recovery of rOvTNF-α from the soluble fraction to a significant extent. Purification of recombinant proteins was achieved rapidly and easily by affinity chromatography using glutathione-Sepharose. Yields of pure rOvTNF-α achieved in E. coli JM109 and AM207 were approximately 1 mg L−1. Both rOvTNF-α and recombinant human TNF-α (rhTNF-α) exerted cytotoxicity on L929 cells. However, rOvTNF-α but not rhTNF-α stimulated proliferation of ovine thymocytes. Maximum levels of TNF-α mRNA expression by LPS-stimulated ovine alveolar macrophages were detected at approximately 4 h post-stimulation.