Biotechnologically produced tumor necrosis factor alpha (TNF-α) neutralizing agents have proven efficient in patients suffering from disparate autoimmune diseases. The rhesus monkey (Macaca mulatta) could be developed as a model for human autoimmune disease. Consequently, a large amount of M. mulatta TNF-α (mmTNFα) is required to further understand TNF-α-related pathogenesis and evaluate novel human TNF-α (hTNFα) neutralizing agents. We therefore attempted to express mmTNFα by using a small ubiquitin-like modifier (SUMO) fusion system. The synthetic gene, encoding the fusion protein SUMO-mmTNFα, was inserted into a pQE30 plasmid and was transformed into Escherichi... More
Biotechnologically produced tumor necrosis factor alpha (TNF-α) neutralizing agents have proven efficient in patients suffering from disparate autoimmune diseases. The rhesus monkey (Macaca mulatta) could be developed as a model for human autoimmune disease. Consequently, a large amount of M. mulatta TNF-α (mmTNFα) is required to further understand TNF-α-related pathogenesis and evaluate novel human TNF-α (hTNFα) neutralizing agents. We therefore attempted to express mmTNFα by using a small ubiquitin-like modifier (SUMO) fusion system. The synthetic gene, encoding the fusion protein SUMO-mmTNFα, was inserted into a pQE30 plasmid and was transformed into Escherichia coli M15. The fusion protein was expressed as both soluble and insoluble protein in E. coli. Approximately 10-12 mg of SUMO-mmTNFα was obtained from the soluble fraction of 1 L of bacterial culture. Cleavage of the fusion protein with SUMO protease produced native-like mmTNFα. Both native-like and SUMO-modified mmTNFα formed functional trimers and showed excellent cytotoxicity (ED(50), 0.05-0.1 ng/ml) in standard L929 cells. In addition, SUMO-mmTNFα and mmTNFα also exhibited cytotoxicity in human cancer cell types, such as, breast, lung, and liver cancer cells. The hTNFα neutralizing agents, including soluble receptors of hTNFα and antibodies against hTNFα, interacted with the mmTNFα. These results demonstrate that the bioactive mmTNFα produced with the SUMO fusion system is useful for further research, especially for the in vitro preclinical evaluation of biological hTNFα neutralizing agents.
