A novel approach to the production of a human glucagon in E. coli is described. The 29 amino acids of human glucagon and pentapeptide linker containing enzyme processing site were fused at the amino terminus to a 57 residue N-terminal portion of the human tumor necrosis factor-alpha (hTNF-a). The fusion protein was expressed in the E. coli cytoplasm at levels up to 30% of the total cell protein. Precipitation of the fusion protein near its isoelectric point, specific enterokinase cleavage at the linker site and subsequent HPLC purification makes this approach suitable for the production of glucagon as well as other relatively small peptides with therapeutic interests.
A novel tumor necrosis factor-u, mutant (mutant M3), in which Ser and Tyr at positions 52 and 56 were substituted by Ile and Phe, respectively, along with deletion of 7 N-terminal amino acids, was prepared and its biological activities were investigated. The mutant exhibited a 14-to 24-fold increase in the cytotoxicity relative to the wildtype TNF on various cancer cell lines. The binding affinity of the mutant to TNF-R55 and TNF-R75 receptors was over 10-fold higher than that of the wild-type. TNF-c~ and the mutant show similar CD spectra in the far-UV region, indicating that the overall structure was not influenced by the mutations. The production of highly potent TNF-c~ mutant utilizing increase of hydrophobicity in the region 52-56 may provide a structural basis for a design of optimized TNF-ct as a therapeutic purpose.
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