Norwegian College of Fishery Science, Faculty of Biosciences, Fisheries and Economics, University of Tromsø, Breivika, N-9037 Tromsø, Norway.
Dev Comp Immunol. 2010 Mar;34(3):286-92. doi: 10.1016/j.dci.2009.10.006. Epub 2009 Oct 30.
The cysteine-rich strongylocins were the first antimicrobial peptides (AMPs) discovered from the sea urchin species, Strongylocentrotus droebachiensis. Homologous putative proteins (called SpStrongylocin) were found in the sister species, S. purpuratus. To demonstrate that they exhibit the same antibacterial activity as strongylocins, cDNAs encoding the 'mature' peptides (SpStrongylocins 1 and 2) were cloned into a direct expression system fusing a protease cleavage site and two purification tags to the recombinant peptide. Both recombinant fusion peptides were expressed in a soluble form in an Escherichia coli strain tolerant to toxic proteins. Enterokinase was used to remove the fusion tags and purified recombinant SpStrongylocins 1 and 2 showed antimicrobial activity against both Gram-negative and Gram-positive bacteria. The results of membrane integrity assays against cytoplasmic membranes of E. coli suggest that both recombinant SpStrongylocins 1 and 2 conduct their antibacterial activity by intracellular killing mechanisms because no increase in membrane permeability was detected.
富含半胱氨酸的强肌肽是第一种从海胆物种 Strongylocentrotus droebachiensis 中发现的抗菌肽 (AMP)。在其姐妹种 S. purpuratus 中发现了同源的假定蛋白（称为 SpStrongylocin）。为了证明它们具有与强肌肽相同的抗菌活性，编码“成熟”肽（SpStrongylocins 1 和 2）的 cDNA 被克隆到直接表达系统中，该系统在重组肽上融合了蛋白酶切割位点和两个纯化标签。两种重组融合肽均以可溶形式在耐受毒性蛋白的大肠杆菌菌株中表达。肠激酶用于去除融合标签，纯化的重组 SpStrongylocins 1 和 2 对革兰氏阴性菌和革兰氏阳性菌均表现出抗菌活性。针对大肠杆菌细胞质膜的膜完整性测定结果表明，两种重组 SpStrongylocins 1 和 2 均通过细胞内杀伤机制发挥其抗菌活性，因为未检测到膜通透性增加。
