An improved strategy for high-level production of TEV protease in Escherichia coli and its purification and characterization
An improved strategy for high-level production of TEV protease in Escherichia coli and its purification and characterization
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DOI：
10.1016/j.pep.2006.07.003
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发表时间：
2007-01-01
影响因子：
1.6
通讯作者：
Hua, Zi-Chun
中科院分区：
文献类型：
--
作者：
Fang, Lei;Jia, Kun-Zhi;Hua, Zi-Chun
烟草蚀纹病毒（TEV）蛋白酶由于其严格的序列特异性，在重组融合蛋白的切割中有着广泛的应用。然而，在大肠杆菌中表达的TEV蛋白酶的溶解性极低。在本研究中，我们引入了一个更有效的系统，以提高和促进TEV蛋白酶在E。杆菌可溶性His 6-TEV的最佳表达通过检查伴侣共表达和较低温度发酵的贡献来实现。当通过Ni 2+亲和色谱进一步纯化时，从IL培养物中分离出65 mg His 6-TEV，纯度超过95%。His 6-TEV的酶活性通常通过使用GST-EGFP和His 6-L-TNF融合蛋白作为底物来表征，其在两个部分之间含有TEV切割位点。(c)2006年爱思唯尔公司All rights reserved.
Because of its stringent sequence specificity, tobacco etch virus (TEV) protease emerges as a useful reagent with wide application in the cleavage of recombinant fusion proteins. However, the Solubility of TEV protease expressed in Escherichia coli is extremely low. In the present study, we introduced a more efficient system to improve and facilitate the soluble production of TEV protease in E. Coli. Optimal expression of soluble His6-TEV was achieved by examining the contribution of chaperone co-expression and lower temperature fermentation. When further purified by Ni2+ affinity chromatography, 65 mg of His6-TEV was isolated with purity over 95% from I L of culture. The enzyme activity of His6-TEV was generally characterized by using GST-EGFP and His6-L-TNF fusion protein as substrates, which contained a TEV cleavage site between two moieties. (c) 2006 Elsevier Inc. All rights reserved.