Mutational analysis of structure—activity relationships in human tumor necrosis factor-alpha
| Autor: | Toshikazu Fukui, Noriyuki Matsuo, Ryuji Furuta, Mayumi Ohue, Katsuji Nakano, Masaaki Yamada, Hirotada Kotani, Junichi Yamagishi, Hitoshi Kawashima, Michiko Yamayoshi |
|---|---|
| Rok vydání: | 1990 |
| Předmět: |
Cell Survival
Molecular Sequence Data Mutant Mutagenesis (molecular biology technique) Bioengineering Biology Protein Engineering medicine.disease_cause Biochemistry Cell Line Mice Structure-Activity Relationship Sequence Homology Nucleic Acid Aspartic acid Escherichia coli medicine Animals Humans Amino Acid Sequence Site-directed mutagenesis Molecular Biology Peptide sequence Genetics Expression vector Base Sequence Tumor Necrosis Factor-alpha Biological activity Gene Expression Regulation Bacterial Hydrogen-Ion Concentration Molecular biology Genes Mutation Mutagenesis Site-Directed Biotechnology |
| Zdroj: | "Protein Engineering, Design and Selection". 3:713-719 |
| ISSN: | 1741-0134 1741-0126 |
| DOI: | 10.1093/protein/3.8.713 |
| Popis: | To determine the region of human tumor necrosis factor-alpha (TNF-alpha), essential for cytotoxic activity against mouse L-M cells, single amino-acid-substituted TNF-alpha mutant proteins (muteins) were produced in Escherichia coli by protein engineering techniques. An expression plasmid for TNF-alpha was mutagenized by passage through an E. coli mutD5 mutator strain and by oligonucleotide-directed mutagenesis. Approximately 100 single amino-acid-substituted TNF-alpha muteins were produced and assayed for cytotoxic activity. The cytotoxic activities of purified TNF-alpha muteins, e.g. TNF-31T, -32Y, -82D, -85H, -115L, -141Y, -144K and -146E, were less than 1% of that of parent TNF-alpha. These results indicate that the integrity of at least four distinct regions of the TNF-alpha molecule is required for full biological activity. These regions are designated as follows: region I, from position 30 to 32; region II, from position 82 to 89; region III, from position 115 to 117; region IV, from position 141 to 146. In addition, TNF-141Y could not completely compete with parent TNF-alpha for binding to the receptor. This demonstrates that region IV, and at least aspartic acid at position 141, must be involved in the TNF receptor binding site. |
| Databáze: | OpenAIRE |
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