Abstract
Background Tumor necrosis factor-α (TNF-α) is a multifunctional cytokine that has cytotoxic, cytostatic and immunomodulatory effects on malignant tumors. However, clinical trials have revealed high systemic toxicity and this has hampered its utilization as an anti-cancer agent. In this study, a human TNF-α mutant was created and tested for its anti-tumor effects.
Methods The TNF mutant (recombinant mutated human TNF; rmhTNF) was prepared by protein engineering in which amino acids Pro, Ser and Asp at positions 8, 9 and 10 of TNF-α were substituted by Arg, Lys and Arg, and C terminal Leu157 was substituted by Phe, along with deletion of the first seven N-terminal amino acids. Prokaryotic expression recombinant vector pBV-mhTNF containing the PLPR promotor was constructed and transformed into E. coli DH5α. The rmhTNF was expressed in a partially soluble form in DH5α, purified from the supernatant of cell lysate by ammonia sulfate precipitation and two sequential chromatographic steps.
Results The purified rmhTNF was >95% pure by SDS–PAGE stained with silver and high-pressure size exclusion chromatography (SEC-HPLC). Its yield was about 1.22 mg/g wet cell paste. The mutant rmhTNF exhibited an approximately 50-fold increase in cytotoxicity relative to the wild-type rhTNF on the mouse fibroblast cell line L929 in a standard cytotoxicity test, and at least and at least 50 times higher LD50 as wild type rhTNF in mice. In vivo biological activity studies carried out on tumor cell transplanted mice and nude mice also showed a more effective cytotoxicity of rmhTNF than rhTNF.
Discussion These results suggest that rmhTNF has potential for developing an effective anti-tumor reagent for some tumors.