Expression of recombinant alpha and beta tubulins from the yew Taxus cuspidata and analysis of the microtubule assembly in the presence of taxol
Expression of recombinant alpha and beta tubulins from the yew Taxus cuspidata and analysis of the microtubule assembly in the presence of taxol
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DOI：
10.1080/09168451.2014.940837
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发表时间：
2014-11-02
影响因子：
1.6
通讯作者：
Konoki, Keiichi
中科院分区：
文献类型：
--
作者：
Kudo, Yuma;Abe, Akihiro;Konoki, Keiichi
紫杉醇最初是从短叶红豆杉中分离出来的。由于紫杉醇抑制微管的解聚，红豆杉中存在的自我抗性机制。是假设的。从东北红豆杉和人胚肾细胞系HEK 293 T中克隆α和β微管蛋白的cDNA揭示，人β微管蛋白中对紫杉醇结合重要的（26）Asp、（359）Arg和（361）Leu残基被东北红豆杉β微管蛋白中的Glu、Trp和Met取代。cuspidata，分别。用比浊法对重组α和β微管蛋白的微管组装进行了监测，结果清楚地表明，来自T。虎杖对紫杉醇的敏感性低于HEK293T细胞。由野生型α微管蛋白和E26D突变的β微管蛋白组成的红豆杉微管恢复了对紫杉醇的敏感性。因此，我们推测T. cuspidata在该物种对紫杉醇的自身抗性中起作用。
Taxol was originally isolated from the yew Taxus brevifolia. Because taxol inhibits the depolymerization of microtubules, the presence of a self-resistance mechanism in Taxus spp. was hypothesized. The cloning of the cDNA for alpha and beta tubulins from Taxus cuspidata and those from the human embryonic kidney cell line HEK293T revealed that the (26)Asp, (359)Arg, and (361)Leu residues in the human beta tubulin, which are important for taxol binding, were replaced with Glu, Trp, and Met in the beta tubulin of T. cuspidata, respectively. The microtubule assembly of the recombinant alpha and beta tubulins was monitored turbidimetrically, and the results clearly demonstrated that the microtubule from T. cuspidata is less sensitive to taxol than that from HEK293T cells. The Taxus microtubule composed of the wild-type alpha tubulin and the beta tubulin with the E26D mutation restored the sensitivity to taxol. We thus postulated that the mutation identified in the beta tubulin of T. cuspidata plays a role in the self-resistance of this species against taxol.