Wu Xudong, Wu Di, Lu Zhisheng, Chen Wentao, Hu Xiaojian, Ding Yu
Department of Physiology and Biophysics, School of Life Sciences, Fudan University, Shanghai 200433, China.
J Biomed Biotechnol. 2009;2009:591923. doi: 10.1155/2009/591923. Epub 2010 Feb 23.
Because of its stringent sequence specificity, tobacco etch virus (TEV) protease is widely used to remove fusion tags from recombinant proteins. Due to the poor solubility of TEV protease, many strategies have been employed to increase the expression level of this enzyme. In our work, we introduced a novel method to produce TEV protease by using visible superfolder green fluorescent protein (sfGFP) as the fusion tag. The soluble production and catalytic activity of six variants of sfGFP-TEV was examined, and then the best variant was selected for large-scale production. After purified by Ni-NTA affinity chromatography and Q anion exchange chromatography, the best variant of sfGFP-TEV fusion protease was obtained with purity of over 98% and yield of over 320 mg per liter culture. The sfGFP-TEV had a similar catalytic activity to that of the original TEV protease. Our research showed a novel method of large-scale production of visible and functional TEV protease for structural genomics research and other applications.
由于烟草蚀纹病毒（TEV）蛋白酶具有严格的序列特异性，它被广泛用于从重组蛋白中去除融合标签。由于TEV蛋白酶的溶解性较差，人们采用了许多策略来提高这种酶的表达水平。在我们的工作中，我们引入了一种新方法，利用可见超折叠绿色荧光蛋白（sfGFP）作为融合标签来生产TEV蛋白酶。检测了sfGFP-TEV六种变体的可溶性表达和催化活性，然后选择最佳变体进行大规模生产。通过镍-氮三乙酸（Ni-NTA）亲和层析和Q阴离子交换层析纯化后，获得了纯度超过98%、每升培养物产量超过320毫克的sfGFP-TEV融合蛋白酶最佳变体。sfGFP-TEV具有与原始TEV蛋白酶相似的催化活性。我们的研究展示了一种用于结构基因组学研究和其他应用的大规模生产可见且具有功能的TEV蛋白酶的新方法。
