Cytotoxic Cyplasin of the Sea Hare, Aplysia punctata, cDNA Cloning, and Expression of Bioactive Recombinants in Insect Cells
A 56-kDa protein isolated from the mucus of the European sea hare Aplysia punctata shows a preferential toxicity to autonomously growing transformed mammalian cells. Cell death induced by this protein differs from both apoptosis and necrosis. The cytotoxic effects are irreversible and become apparen...
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| Format: | Article |
| Language: | English |
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Elsevier
2002-01-01
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| Series: | Neoplasia: An International Journal for Oncology Research |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S1476558602800471 |
| _version_ | 1819136982877470720 |
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| author | Christian Petzelt Gaby Joswig Hermann Stammer Dieter Werner |
| author_facet | Christian Petzelt Gaby Joswig Hermann Stammer Dieter Werner |
| author_sort | Christian Petzelt |
| collection | DOAJ |
| description | A 56-kDa protein isolated from the mucus of the European sea hare Aplysia punctata shows a preferential toxicity to autonomously growing transformed mammalian cells. Cell death induced by this protein differs from both apoptosis and necrosis. The cytotoxic effects are irreversible and become apparent at nanomolar concentrations in a cell type-dependent manner. In contrast, injection of micromolar concentrations into mice is tolerated without apparent negative consequences. Microsequencing of the 56-kDa protein released a peptide sequence whose corresponding nucleotide sequence was used as probe to screen A. punctata RNA-based cDNA and to select cDNA clones encoding polypeptides comprising the target peptide. Two closely related cDNA were detected. The cDNA encoding a polypeptide 558 as in length was considered to reflect a bona fide clone encoding the cytotoxic protein. Its protein-coding section was recloned in vectors suitable for expression in Escherichia coli, in mammalian cells, and in insect cells, respectively. The E. coli-expressed polypeptide was biologically inactive. Transfected mammalian cells expressed a cytotoxic factor and died thereof as if treated with the genuine cytotoxic protein. In contrast, transfected insect cells, which proved to be much less sensitive when treated with the genuine protein, expressed the cytotoxic factor and continued to proliferate, allowing to establish stable insect cell lines expressing sufficient amounts of the cytotoxic factor for further characterization. |
| first_indexed | 2024-12-22T10:43:38Z |
| format | Article |
| id | doaj.art-b6379978cf51488ca3c590950cb81821 |
| institution | Directory Open Access Journal |
| issn | 1476-5586 1522-8002 |
| language | English |
| last_indexed | 2024-12-22T10:43:38Z |
| publishDate | 2002-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Neoplasia: An International Journal for Oncology Research |
| spelling | doaj.art-b6379978cf51488ca3c590950cb818212022-12-21T18:28:58ZengElsevierNeoplasia: An International Journal for Oncology Research1476-55861522-80022002-01-0141495910.1038/sj.neo.7900202Cytotoxic Cyplasin of the Sea Hare, Aplysia punctata, cDNA Cloning, and Expression of Bioactive Recombinants in Insect CellsChristian Petzelt0Gaby Joswig1Hermann Stammer2Dieter Werner3Laboratoire International de Biologie Marine, LIBM, F-85350 Ile d'Yeu, FranceGerman Cancer Research Center, Division of Biochemistry of the Cell, D-69120 Heidelberg, GermanyGerman Cancer Research Center, Division of Biochemistry of the Cell, D-69120 Heidelberg, GermanyGerman Cancer Research Center, Division of Biochemistry of the Cell, D-69120 Heidelberg, GermanyA 56-kDa protein isolated from the mucus of the European sea hare Aplysia punctata shows a preferential toxicity to autonomously growing transformed mammalian cells. Cell death induced by this protein differs from both apoptosis and necrosis. The cytotoxic effects are irreversible and become apparent at nanomolar concentrations in a cell type-dependent manner. In contrast, injection of micromolar concentrations into mice is tolerated without apparent negative consequences. Microsequencing of the 56-kDa protein released a peptide sequence whose corresponding nucleotide sequence was used as probe to screen A. punctata RNA-based cDNA and to select cDNA clones encoding polypeptides comprising the target peptide. Two closely related cDNA were detected. The cDNA encoding a polypeptide 558 as in length was considered to reflect a bona fide clone encoding the cytotoxic protein. Its protein-coding section was recloned in vectors suitable for expression in Escherichia coli, in mammalian cells, and in insect cells, respectively. The E. coli-expressed polypeptide was biologically inactive. Transfected mammalian cells expressed a cytotoxic factor and died thereof as if treated with the genuine cytotoxic protein. In contrast, transfected insect cells, which proved to be much less sensitive when treated with the genuine protein, expressed the cytotoxic factor and continued to proliferate, allowing to establish stable insect cell lines expressing sufficient amounts of the cytotoxic factor for further characterization.http://www.sciencedirect.com/science/article/pii/S1476558602800471antitumorrecombinantmelanomasecretion signalGFP |
| spellingShingle | Christian Petzelt Gaby Joswig Hermann Stammer Dieter Werner Cytotoxic Cyplasin of the Sea Hare, Aplysia punctata, cDNA Cloning, and Expression of Bioactive Recombinants in Insect Cells Neoplasia: An International Journal for Oncology Research antitumor recombinant melanoma secretion signal GFP |
| title | Cytotoxic Cyplasin of the Sea Hare, Aplysia punctata, cDNA Cloning, and Expression of Bioactive Recombinants in Insect Cells |
| title_full | Cytotoxic Cyplasin of the Sea Hare, Aplysia punctata, cDNA Cloning, and Expression of Bioactive Recombinants in Insect Cells |
| title_fullStr | Cytotoxic Cyplasin of the Sea Hare, Aplysia punctata, cDNA Cloning, and Expression of Bioactive Recombinants in Insect Cells |
| title_full_unstemmed | Cytotoxic Cyplasin of the Sea Hare, Aplysia punctata, cDNA Cloning, and Expression of Bioactive Recombinants in Insect Cells |
| title_short | Cytotoxic Cyplasin of the Sea Hare, Aplysia punctata, cDNA Cloning, and Expression of Bioactive Recombinants in Insect Cells |
| title_sort | cytotoxic cyplasin of the sea hare aplysia punctata cdna cloning and expression of bioactive recombinants in insect cells |
| topic | antitumor recombinant melanoma secretion signal GFP |
| url | http://www.sciencedirect.com/science/article/pii/S1476558602800471 |
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