Anderluh G, Pungercar J, Strukelj B, Macek P, Gubensek F
Department of Biology, Biotechnical Faculty, University of Ljubljana, Slovenia.
Biochem Biophys Res Commun. 1996 Mar 18;220(2):437-42. doi: 10.1006/bbrc.1996.0391.
Equinatoxin II (EqtII), a basic protein of 179 amino acids lacking cysteine residues, is the most abundant cytolysin isolated from the sea anemone Actinia equina. Its mode of action is still poorly understood. In order to initiate further structure-function studies by protein engineering, cDNA library was prepared from the whole animal and hybridized with a PCR-derived probe, deduced from the EqtII primary structure. The longest positive clone of 899 bp was shown to encode a 214 residue precursor of EqtII. The mature protein region was amplified by PCR, cloned into a T7 RNA polymerase-based expression vector and expressed in Escherichia coli. Recombinant toxin was isolated by a simple, two-step isolation procedure including separation on CM-cellulose and gel filtration using an FPLC system. Its biochemical properties and hemolytic activity were practically indistinguishable from those of native toxin.
海葵毒素II（EqtII）是一种由179个氨基酸组成的碱性蛋白，不含半胱氨酸残基，是从海葵（Actinia equina）中分离出的最丰富的细胞溶素。其作用方式仍知之甚少。为了通过蛋白质工程开展进一步的结构-功能研究，从整个动物体制备了cDNA文库，并与根据EqtII一级结构推导的PCR衍生探针进行杂交。最长的899 bp阳性克隆被证明编码EqtII的214个残基前体。通过PCR扩增成熟蛋白区域，克隆到基于T7 RNA聚合酶的表达载体中，并在大肠杆菌中表达。重组毒素通过简单的两步分离程序进行分离，包括在CM-纤维素上分离和使用FPLC系统进行凝胶过滤。其生化特性和溶血活性与天然毒素几乎没有区别。
