Pescatori M, Bradbury A, Bouet F, Gargano N, Mastrogiacomo A, Grasso A
Istituto di Biologia Cellulare, CNR, Roma, Italy.
Eur J Biochem. 1995 May 15;230(1):322-8. doi: 10.1111/j.1432-1033.1995.tb20566.x.
A cDNA encoding a polypeptide of 88 amino acids was cloned following the rapid amplification of cDNA ends (RACE) procedure using mRNA isolated from the venom glands of the Mediterranean black widow spider (Latrodectus tredecimguttatus) and oligonucleotides based on the sequence of a tryptic fragment putatively from alpha-latrotoxin. Apart from a potential signal peptide, the rest of this small protein, named latrodectin, was highly hydrophilic, having a calculated molecular mass of 7945 Da and a pI of 4.3. Northern-blot analysis showed that the mRNA was specifically expressed in the venom gland of L. tredecimguttatus and that it was well conserved between two geographically remote species (L. geometricus and L. indistinctus). A polyclonal serum raised in rabbits against the C-terminal sequence of latrodectin detected cross-reactive proteins in the venom fluid, venom gland extracts, and in purified alpha-latrotoxin, suggesting that latrodectin is intimately associated with alpha-latrotoxin. Finally, we produced a recombinant protein in a cell system infected with baculovirus and developed an immunoaffinity purification procedure for latrodectin to facilitate further structural and functional analyses of the molecule.
利用从地中海黑寡妇蜘蛛（间斑寇蛛）毒腺中分离的mRNA和基于推定来自α- latrotoxin的胰蛋白酶片段序列的寡核苷酸，通过cDNA末端快速扩增（RACE）程序克隆了一个编码88个氨基酸多肽的cDNA。除了一个潜在的信号肽外，这个名为latrodectin的小蛋白的其余部分具有高度亲水性，计算分子量为7945 Da，pI为4.3。Northern印迹分析表明，该mRNA在地中海黑寡妇蜘蛛的毒腺中特异性表达，并且在两个地理上相距遥远的物种（华丽寇蛛和隐匿寇蛛）之间高度保守。用针对latrodectin C末端序列的兔多克隆血清检测毒液、毒腺提取物和纯化的α- latrotoxin中的交叉反应蛋白，表明latrodectin与α- latrotoxin密切相关。最后，我们在感染杆状病毒的细胞系统中产生了重组蛋白，并开发了一种latrodectin的免疫亲和纯化程序，以促进对该分子的进一步结构和功能分析。
