Novel muteins of human tumor necrosis factor α |
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Authors: | Ryoji Ito Hiroshi Matsumoto Kumiko Uchida Toshiyuki Kubo Yuji Tsukii Tohru Endo Akira Kaji |
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Institution: | 1. Research Institute for Molecular Genetics, Tsumura and Company, Ibaraki, Japan;2. Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia, PA, U.S.A. |
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Abstract: | For chemical synthesis of a gene coding for human necrosis factor α (TNF-α), DNA sequence predicted by the amino acid sequence of human TNF molecule was prepared. Codons were chosen according to the codon usage in Escherichia coli (E. coli). The 490 hp gene was assembled by enzymic ligation of 42 oligonucleotides and was cloned into vector (]KK223-3) for high expression of active TNF-α in E. coli. With use of site-directed mutagenesis on this DNA, five different muteins of TNF-α were synthesized. TNF-M1 and TNF-M4 have deletions of His-73 and Gln-102, respectively. These deletions didn't cause loss of the cytotoxic activity against L929 cells. TNF-M5, which has a substitution of Asp-10 to Arg, had the similar cytotoxic activity to that of TNF-α. The cytotoxic spectra against several tumor cells were not changed by this substitution. TNF-M3 has an amino acid substitution of Glu-116 to His which occupies this position in human TNF-β. This substitution didn't change the cytotoxicity. In addition, evidence was presented that the change of the carboxyl terminal residue doesn't always influence the cytotoxic activity of TNF-α. Many different muteins were also isolated by random mutagenesis with hydroxylamine-HCl. One of the muteins, which carries a mutation of His-15 to Tyr, lost the cytotoxic activity almost completely. |
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Keywords: | Tumor necrosis factor Synthetic gene Gene expression Site-directed mutagenesis Random mutagenesis Mutein (Human) cDNA complementary deoxynucleic acid DEAE diethylaminoethyl ELISA enzyme-linked immunosorbent assay HIC hydrophobic interaction chromatography HPLC high-performance liquid chromatography IPTG MHC major histocompatibility complex MTT 3-(4 5-dimethylthiazol-2-yl)-2 5-diphenyltetrazolium bromide SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis |
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