A combined approach to improving large-scale production of tobacco etch virus protease
A combined approach to improving large-scale production of tobacco etch virus protease
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DOI：
10.1016/j.pep.2007.04.013
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发表时间：
2007-09-01
影响因子：
1.6
通讯作者：
Fox, Brian G.
中科院分区：
文献类型：
--
作者：
Blommel, Paul G.;Fox, Brian G.
烟草蚀纹病毒NIa蛋白酶（TEV蛋白酶）是从重组蛋白中去除融合标签的重要酶。大肠杆菌中TEV蛋白酶的生产受到不溶性的阻碍，并通过许多不同的策略来解决。然而，以前最好的结果和蛋白质表达的新方法还没有结合起来，以测试是否有可能进一步改进。在这里，我们使用定量，高通量测定TEV蛋白酶的活性在细胞裂解物中，以评估结合新的表达宿主和诱导方法的几个以前的修改的功效。小规模的筛选，纯化和质谱分析表明，TEV蛋白酶与C-末端聚精氨酸标签在细胞中的蛋白水解，以消除四个精氨酸残基。截短的形式是活性的和可溶的，但相比之下，标记的形式也是活性的，但可溶性大大降低。然后将缺乏C-末端残基238-242的工程化TEV蛋白酶用于进一步的表达优化。由此，通过使用37 ℃的自诱导培养基，获得了高水平和高溶解度的TEV蛋白酶表达。结合表达工作，开发了自动化两步纯化方案，其产生具有>99%纯度、高催化活性和类似于400 mg/L表达培养物的纯化产率（类似于15 mg纯TEV蛋白酶/克E. coli细胞糊）。以类似产率（类似于每克大肠杆菌12 mg纯蛋白酶融合物）生产谷胱甘肽-S-转移酶标记的TEV的方法。大肠杆菌细胞糊）。(C)2007爱思唯尔公司All rights reserved.
Tobacco etch virus NIa proteinase (TEV protease) is an important too] for the removal of fusion tags from recombinant proteins. Production of TEV protease in Escherichia coli has been hampered by insolubility and addressed by many different strategies. However, the best previous results and newer approaches for protein expression have not been combined to test whether further improvements are possible. Here, we use a quantitative, high-throughput assay for TEV protease activity in cell lysates to evaluate the efficacy of combining several previous modifications with new expression hosts and induction methods. Small-scale screening, purification and mass spectral analysis showed that TEV protease with a C-terminal poly-Arg tag was proteolysed in the cell to remove four of the five arginine residues. The truncated form was active and soluble but in contrast, the tagged version was also active but considerably less soluble. An engineered TEV protease lacking the C-terminal residues 238-242 was then used for further expression optimization. From this work, expression of TEV protease at high levels and with high solubility was obtained by using auto-induction medium at 37 degrees C. In combination with the expression work, an automated two-step purification protocol was developed that yielded His-tagged TEV protease with >99% purity, high catalytic activity and purified yields of similar to 400 mg/L of expression culture (similar to 15 mg pure TEV protease per gram of E. coli cell paste). Methods for producing glutathione-S-transferase-tagged TEV with similar yields (similar to 12 mg pure protease fusion per gram of E. coli cell paste) are also reported. (C) 2007 Elsevier Inc. All rights reserved.