Mutational analysis of structure--activity relationships in human tumor necrosis factor-alpha |
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Authors: | Yamagishi, Jun-ichi Kawashima, Hitoshi Matsuo, Noriyuki Ohue, Mayumi Yamayoshi, Michiko Fukui, Toshikazu Kotani, Hirotada Furuta, Ryuji Nakano, Katsuji Yamada, Masaaki |
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Affiliation: | Research Laboratories, Dainippon Pharmaceutical Co. Ltd 33-94, Enoki, Suita, Osaka 564, Japan |
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Abstract: | To determine the region of human tumor necrosis factor-alpha(TNF-), essential for cytotoxic activity against mouse L-M cells,single amino-acid-substituted TNF- mutant proteins (muteins)were produced in Escherichia coli by protein engineering techniques.An expression plasmid for TNF- was mutagenized by passage throughan E.coli mutD5 mutator strain and by oligonucleotide-directedmutagenesis. Approximately 100 single amino-acid-substitutedTNF- muteins were produced and assayed for cytotoxic activity.The cytotoxic activities of purified TNF- muteins, e.g. TNF-31T,-32Y, -82D, -85H, -115L, -141Y, -144K and -146E, were < 1%of that of parent TNF-. These results indicate that the integrityof at least four distinct regions of the TNF- molecule is requiredfor full biological activity. These regions are designated asfollows: region I, from position 30 to 32; region II, from position82 to 89; region III, from position 115 to 117; region FV, fromposition 141 to 146. In addition, TNF-141Y could not completelycompete with parent TNF- for binding to the receptor. This demonstratesthat region IV, and at least aspartk acid at position 141, mustbe involved in the TNF receptor binding site. |
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Keywords: | cytotoxic activity/ human tumor necrosis factor-alpha/ mutD mutagenesis/ oligonucleotide-directed mutagenesis/ receptor binding activity |
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