Anderluh G, Pungercar J, Krizaj I, Strukelj B, Gubensek F, Macek P
Department of Biology, Biotechnical Faculty, University of Ljubljana, Slovenia.
Protein Eng. 1997 Jul;10(7):751-5. doi: 10.1093/protein/10.7.751.
The role of the N-terminal segment 1-33 of equinatoxin II, a 20 kDa pore-forming protein from the sea anemone Actinia equina, was studied by N-truncation mutagenesis. A part of this segment was classified as being amphiphilic and membrane seeking. Wild-type equinatoxin II and its mutants lacking 5, 10 and 33 amino acid residues, respectively, were produced in Escherichia coli using T7 RNA polymerase-based expression vector. Soluble recombinant proteins were isolated from bacterial lysates and assayed for their inhibition by sphingomyelin, binding to red blood cells and hemolytic activity. The N-terminal deletion of 33 amino acids resulted in an insoluble protein, while mutants lacking 5 and 10 residues expressed increased relative avidity for sphingomyelin and red blood cell membranes. Their specific hemolytic activity was decreased, however, with increasing truncation. The results suggest that the N-terminus, which has been found to be conserved in sea anemone pore-forming toxins, contributes to the solubility of the equinatoxin II, but it is not essential for binding to lipid membranes. It is very likely that the N-terminus play a role in the formation of functional pores.
通过N端截短诱变研究了来自海葵（Actinia equina）的20 kDa成孔蛋白海葵毒素II（equinatoxin II）N端1 - 33片段的作用。该片段的一部分被归类为具有两亲性且趋向于膜。分别缺失5、10和33个氨基酸残基的野生型海葵毒素II及其突变体，使用基于T7 RNA聚合酶的表达载体在大肠杆菌中产生。从细菌裂解物中分离出可溶性重组蛋白，并检测它们被鞘磷脂抑制的情况、与红细胞的结合以及溶血活性。N端缺失33个氨基酸导致产生不溶性蛋白，而缺失5和10个残基的突变体对鞘磷脂和红细胞膜表现出增加的相对亲和力。然而，随着截短程度的增加，它们的特异性溶血活性降低。结果表明，已发现海葵成孔毒素中保守的N端有助于海葵毒素II的溶解性，但对于与脂质膜的结合并非必需。N端很可能在功能性孔的形成中起作用。
