Mutational analysis of human tumor necrosis factor-alpha.

To understand the structure-function relationship of human tumor necrosis factor-alpha (TNF-alpha), mutational analysis was carried out on the lower regions (regions 1-6) of the molecule. The muteins were prepared as a soluble form by using a chaperonin co-expression system and the cytotoxic activities of the purified muteins were evaluated on TNF-sensitive murine fibrosarcoma L929 cells. Three regions (regions 1, 2 & 4) were found where mutations significantly influenced the bioactivity. In region 1 (residues 1-10), the number of deleted residues and the positioning of positive charges are important to achieve a maximum activity and in region 4 (residues 84-88), introduction of charged residues in one of the positions 86-88 significantly increased the cytotoxic activity. On the other hand, any mutation introduced in region 2 (residues 37-41) had a deleterious effect. The present study provides a structural basis for the design of highly potent TNF-alpha as a therapeutic agent.