A simple method for expression and purification of Shiga toxin 1 (Stx1) with biological activities by using a single-promoter vector and native signal peptide.
A simple method for expression and purification of Shiga toxin 1 (Stx1) with biological activities by using a single-promoter vector and native signal peptide.
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一种利用单启动子载体和天然信号肽表达和纯化具有生物活性的志贺毒素 1 (Stx1) 的简单方法。
DOI：
10.1002/bab.1398
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发表时间：
2016
影响因子：
2.8
通讯作者：
Wang Hui
中科院分区：
文献类型：
--
作者：
Tu Wei;Li Tao;Wang Qin;Cai Kun;Gao Xiang;Wang Hui
将大肠杆菌O 157：H7的restx 1区（含两个开放阅读框stx 1a和stx 1b）克隆到含有单一启动子的pET-32 a中。将该区域转化为E. coliTransB（DE 3）为atrx突变株。在E.在完全可溶形式的大肠杆菌周质中，纯化rStx 1，并通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）、ELISA和Western印迹分析进行验证。我们的rStx 1具有Vero细胞中位细胞毒性剂量（CD 50）和中位致死剂量（LD 50）值，分别约为30 ng和1.5 µg。通过一步镍亲和凝胶柱层析，纯化的rStx 1的最终产量为2至3 mg/L。该方法是以合理成本大规模制备Stx 1的简单方法。
The entirestx1region fromEscherichia coliO157:H7, containing two open reading frames (stx1aandstx1b), was cloned into pET‐32a with a single promoter. This region was transformed intoE. coliTransB (DE3), which is atrxBandgormutation strain. After expression in theE. coliperiplasm in a completely soluble form, the rStx1 was purified and verified by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE), ELISA, and Western blot analysis. Our rStx1 have Vero cell median cytotoxic dose (CD50) and median lethal dose (LD50) values of approximately 30 ng and 1.5 µg, respectively. The final yield of the purified rStx1 ranged from 2 to 3 mg/L by one‐step nickel affinity gel column chromatography. This method is an easy approach to the large‐scale preparation of Stx1 at a reasonable cost.