Uechi Gen-ichiro, Toma Hiromu, Arakawa Takeshi, Sato Yoshiya
Division of Tropical Parasitology, Unit of Social and Environmental Medicine, School of Medicine, University of the Ryukyus, Nishihara, Okinawa 903-0215, Japan.
Protein Expr Purif. 2005 Apr;40(2):379-84. doi: 10.1016/j.pep.2004.12.003.
The full-length cDNA that encodes the hemolytic toxin Avt-I, with 226 amino acids, from the venomous sea anemone Actineria villosa has been cloned using the oligo-capping method. The cDNA contains 681bp open reading frame and its predicted amino acid sequences revealed that Avt-I was basic polypeptides without cysteine residues and Arg-Gly-Asp (RGD) motif sequence. The mature Avt-I has a predicted molecular weight of 19.6 kDa and its theoretical isoelectric point is 9.3. The Avt-I revealed 99, 61, 57, and 57% amino acid similarity with hemolytic toxins Pstx20, EqtII, StII, and HmT from Phyllodiscus semoni, Actinia Equina, Stichodactyla helianthus, and Heteractis magnifica, respectively. The characteristic amphiphilic alpha-helix structure was found at the N-terminal region of the mature Avt-I. Recombinant Avt-I (rAvt-I) was expressed in Escherichia coli BL21 (DE3) strain as a biologically active form and purified rAvt-I caused 50% hemolytic activity against 1% sheep erythrocytes at a concentration of 6.3 ng/ml (0.32 nM). M9Y medium led to more than 2-fold increase in rAvt-I yield than cultivation in Luria-Bertani medium.
利用寡核苷酸帽法克隆了来自有毒海葵绒毛海葵（Actineria villosa）的编码溶血毒素Avt-I的全长cDNA，该毒素含有226个氨基酸。该cDNA包含681bp的开放阅读框，其预测的氨基酸序列显示Avt-I是不含半胱氨酸残基和精氨酸-甘氨酸-天冬氨酸（RGD）基序序列的碱性多肽。成熟的Avt-I预测分子量为19.6 kDa，其理论等电点为9.3。Avt-I与来自半盘海葵（Phyllodiscus semoni）的溶血毒素Pstx20、马氏海葵（Actinia Equina）的EqtII、太阳海葵（Stichodactyla helianthus）的StII和巨大海葵（Heteractis magnifica）的HmT的氨基酸相似性分别为99%、61%、57%和57%。在成熟Avt-I的N端区域发现了特征性的两亲性α-螺旋结构。重组Avt-I（rAvt-I）在大肠杆菌BL21（DE3）菌株中以生物活性形式表达，纯化的rAvt-I在浓度为6.3 ng/ml（0.32 nM）时对1%的绵羊红细胞具有50%的溶血活性。与在Luria-Bertani培养基中培养相比，M9Y培养基使rAvt-I的产量提高了2倍以上。
