The tobacco etch virus (TEV) protease has become a popular choice for cleaving fusion proteins because of its high stringency in sequence recognition. Procedures for isolating recombinant protein from the cytoplasm of E. coli require rupturing of the cell wall via enzymatic treatment combined with sonication or French press. Here we present an expedited method for producing laboratory-grade TEV protease in E. coli using a freeze-thaw method, followed by purification with immobilized metal affinity chromatography. Protease is obtained by expression from the pDZ2087 plasmid in BL21 (DE3) cells. Proteolysis resulting from this product, cleaves a maltose-binding protein fusion to completion at a fusion-to-protease molar ratio of 50:1.

中文翻译：

---

一种从大肠杆菌生产实验室级重组TEV蛋白酶的简化方法。

烟草蚀刻病毒（TEV）蛋白酶由于其在序列识别中的高度严格性，已成为切割融合蛋白的普遍选择。从大肠杆菌的细胞质中分离重组蛋白的程序需要通过酶处理结合超声处理或French press使细胞壁破裂。在这里，我们提出了一种使用冻融法在大肠杆菌中生产实验室级TEV蛋白酶的快速方法，然后用固定的金属亲和色谱法进行纯化。通过在BL21（DE3）细胞中从pDZ2087质粒表达获得蛋白酶。由该产物产生的蛋白水解以融合与蛋白酶的摩尔比为50：1的方式裂解麦芽糖结合蛋白融合体。