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

• Published in: Veterinary immunology and immunopathology 1995-02, Vol.44 (3), p.279-291
• Main Authors: Seow, H.-F., Rothel, J.S., Pepin, M., David, M.-J., Wood, P.R.
• Format: Article
• Language: English
• Subjects: Animals; ARN; Base Sequence; Cells, Cultured; Cloning, Molecular; Cytotoxicity, Immunologic - immunology; DNA Primers - chemistry; DNA, Complementary - analysis; Escherichia coli - genetics; Gene Expression; Lymphocyte Activation - immunology; MACROFAGOS; MACROPHAGE; Macrophage Activation - immunology; MACROPHAGES; Macrophages, Alveolar - immunology; Macrophages, Alveolar - metabolism; Molecular Sequence Data; NEOPLASMAS; NEOPLASME; NEOPLASMS; OVIN; OVINOS; Polymerase Chain Reaction; Recombinant Fusion Proteins - genetics; RNA; RNA - isolation & purification; RNA, Messenger - biosynthesis; SHEEP; Sheep - genetics; Sheep - immunology; T-Lymphocytes - immunology; Transfection; Tumor Necrosis Factor-alpha - biosynthesis; Tumor Necrosis Factor-alpha - genetics; Tumor Necrosis Factor-alpha - immunology
• ISSN: 0165-2427; 1873-2534
• DOI: 10.1016/0165-2427(94)05305-C

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.