Nam-Kyu Shin

High resolution crystal structure of a human tumor necrosis factor-alpha mutant with low systemic toxicity.

A human tumor necrosis factor-α (TNF-α) mutant (M3S) with low systemic toxicity in vivo was designed, and its structures in two different crystal packings were determined crystallographically at 1.8 and 2.15-Å resolution, respectively, to explain altered biological activities of the mutant. M3S contains four changes: a hydrophilic substitution of L29S, two hydrophobic substitutions of S52I and Y56F, and a deletion of the N-terminal seven amino acids that is disordered in the structure of wild-type TNF-α. Compared with wild-type TNF-α, it exhibits 11- and 71-fold lower binding affinities for the human TNF-R55 and TNF-R75 receptors, respectively, and in vitro cytotoxic effect andin vivo systemic toxicity of M3S are 20 and 10 times lower, respectively. However, in a transplanted solid tumor mouse model, M3S suppresses tumor growth more efficiently than wild-type TNF-α. M3S is highly resistant to proteolysis by trypsin, and it exhibits increased thermal stability and a prolonged half-life in vivo. The L29S mutation causes substantial restructuring of the loop containing residues 29–36 into a rigid segment as a consequence of induced formation of intra- and intersubunit interactions, explaining the altered receptor binding affinity and thermal stability. A mass spectrometric analysis identified major proteolytic cleavage sites located on this loop, and thus the increased resistance of M3S to the proteolysis is consistent with the increased rigidity of the loop. The S52I and Y56F mutations do not induce a noticeable conformational change. The side chain of Phe56 projects into a hydrophobic cavity, while Ile52 is exposed to the bulk solvent. Ile52 should be involved in hydrophobic interactions with the receptors, since a mutant containing the same mutations as in M3S except for the L29S mutation exhibits an increased receptor binding affinity. The low systemic toxicity of M3S appears to be the effect of the reduced and selective binding affinities for the TNF receptors, and the superior tumor-suppression of M3S appears to be the effect of its weak but longer antitumoral activity in vivo compared with wild-type TNF-α. It is also expected that the 1.8-Å resolution structure will serve as an accurate model for explaining the structure-function relationship of wild-type TNF-α and many TNF-α mutants reported previously and for the design of new TNF-α mutants.

Production of recombinant human glucagon in Escherichia coli by a novel fusion protein approach

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-α). 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‐α mutant with significantly enhanced cytotoxicity and receptor binding affinity

A novel tumor necrosis factor‐α 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 wild‐type 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‐α 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‐α mutant utilizing increase of hydrophobicity in the region 52‐56 may provide a structural basis for a design of optimized TNF‐α as a therapeutic purpose.