Biotechnologically produced tumor necrosis factor alpha (TNF-alpha) 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-alpha (mmTNF alpha) is required to further understand TNF-alpha-related pathogenesis and evaluate novel human TNF-alpha (hTNF alpha) neutralizing agents. We therefore attempted to express mmTNF alpha by using a small ubiquitin-like modifier (SUMO) fusion system. The synthetic gene, encoding the fusion protein SUMO-mmTNF alpha, 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-mmTNF alpha was obtained from the soluble fraction of 1 L of bacterial culture. Cleavage of the fusion protein with SUMO protease produced native-like mmTNF alpha. Both native-like and SUMO-modified mmTNF alpha formed functional trimers and showed excellent cytotoxicity (ED50, 0.05-0.1 ng/ml) in standard L929 cells. In addition, SUMO-mmTNF alpha and mmTNF alpha also exhibited cytotoxicity in human cancer cell types, such as, breast, lung, and liver cancer cells. The hTNF alpha neutralizing agents, including soluble receptors of hTNF alpha and antibodies against hTNF alpha, interacted with the mmTNF alpha. These results demonstrate that the bioactive mmTNF alpha produced with the SUMO fusion system is useful for further research, especially for the in vitro preclinical evaluation of biological hTNF alpha neutralizing agents.