Construction of novel tumor necrosis factor-alpha mutants with reduced toxicity and higher cytotoxicity on human tumor cells.
Two tumor necrosis factor-alpha mutants MT1 (32Trp157Phe) and MT2 (2Lys30Ser-32Trp 157Phe) were constructed by site-directed mutagenesis. These mutants were soluble and over-expressed inE. coli. The purity of purified mutants was above 95% by serial chromatography. The results of Western blot indicated that these mutants could be cross-reactive with monoclonal antibody against native hTNF-alpha. Compared to parent hTNF-alpha, the cytotoxicity of these mutants on murine fibrosarcoma L929 cell lines reduced 4-5 orders of magnitude but was equivalent to that of native hTNF-alpha on human tumor cell lines. The LD50 of mutant MT1 was reduced to 0.34% of wild type and the dose of MT2 that resulted in 30% death of mice reduced to less than 1/700 that of parent hTNF-alpha.
Liu
Hui
H
Shanghai Research Center of Biotechnology, Chinese Academy of Sciences, Shanghai 200233, China.
Lu
Fang
F
Chen
Jianjun
J
Ren
Hongyu
H
Chen
Changqing
C
D019943:Amino Acid Substitution; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology / Q000633:toxicity; D001483:Base Sequence; D045744:Cell Line, Tumor; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D006801:Humans; D007928:Lethal Dose 50; D008297:Male; D051379:Mice; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering; D018124:Receptors, Tumor Necrosis Factor / Q000187:drug effects; D011994:Recombinant Proteins / Q000235:genetics / Q000494:pharmacology / Q000633:toxicity; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics* / Q000494:pharmacology / Q000633:toxicity*
D000970:Antineoplastic Agents; D017931:DNA Primers; D018124:Receptors, Tumor Necrosis Factor; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha