Cytotoxic cyplasin of the sea hare, Aaplysia 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 prefer ential 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 aa in length was considered to reflect a bonafide 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.
Petzelt
Christian
C
Laboratoire International de Biologie Marine, Iie d'Yeu, France. petzelt@libmyeu.com
Joswig
Gaby
G
Stammer
Hermann
H
Werner
Dieter
D
D000595:Amino Acid Sequence; D000818:Animals; D000970:Antineoplastic Agents / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D001048:Aplysia / Q000737:chemistry*; D017209:Apoptosis / Q000187:drug effects; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D051379:Mice; D008811:Mice, Inbred DBA; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D019311:Staurosporine / Q000494:pharmacology; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000473:pathology
D000970:Antineoplastic Agents; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D008164:Luminescent Proteins; D009842:Oligopeptides; D011994:Recombinant Proteins; C461060:cyplasin; D049452:Green Fluorescent Proteins; D019311:Staurosporine