THE CLONING OF A CDNA-ENCODING A PROTEIN (LATRODECTIN) WHICH COPURIFIES WITH THE ALPHA-LATROTOXIN FROM THE BLACK-WIDOW SPIDER LATRODECTUS-TREDECIMGUTTATUS (THERIDIIDAE)

A cDNA encoding a polypeptide of 88 amino acids was cloned following t he rapid amplification of cDNA ends (RACE) procedure using mRNA isolat ed from the venom glands of the Mediterranean black widow spider (Latr odectus tredecinguttatus) and oligonucleotides based on the sequence o f a tryptic fragment putatively from alpha-latrotoxin. Apart from a po tential signal peptide, the rest of this small protein, named latrodec tin, was highly hydrophilic, having a calculated molecular mass of 794 5 Da and a pI of 4.3. Northern-blot analysis showed that the mRNA was specifically expressed in the venom gland of L. tredecinguttatus and t hat it was well conserved between two geographically remote species (L . geometricus and L. indistinctus). A polyclonal serum raised in rabbi ts against the C-terminal sequence of latrodectin detected cross-react ive proteins in the venom fluid, venom gland extracts, and in purified alpha-latrotoxin, suggesting that latrodectin is intimately associate d with alpha-latrotoxin. Finally, we produced a recombinant protein in a cell system infected with baculovirus and developed an immunoaffini ty purification procedure for latrodectin to facilitate further struct ural and functional analyses of the molecule.