The solubility and stability of recombinant proteins are increased by their fusion to NusA
The solubility and stability of recombinant proteins are increased by their fusion to NusA
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DOI：
10.1016/j.bbrc.2004.07.189
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发表时间：
2004-09-24
影响因子：
3.1
通讯作者：
de Marco, A
中科院分区：
文献类型：
--
作者：
De Marco, V;Stier, G;de Marco, A
新的细菌载体pETM 60能够通过TEV蛋白酶识别序列表达与NusA的C-末端融合的His-标记的重组蛋白。编码两个蛋白结构域（Xklp 3A和Tep 3Ag）和一个膜结合病毒蛋白（E8 R）的三个序列不能在细菌中以可溶性形式表达。它们的GST融合蛋白大多可溶，但在纯化过程中迅速降解。克隆到pETM 60中的相同序列通过金属亲和有效纯化，并在去除融合伴侣后恢复可溶性。NusA融合的构建体能够产生13-20 mg融合蛋白/升培养物和2.5-5 mg纯蛋白/升培养物。结构分析表明，纯化的蛋白质是单分散的，并正确折叠。NusA已用于产生抗体，这些抗体已成功用于NusA融合蛋白的Western印迹和免疫沉淀。(C)2004年爱思唯尔公司All rights reserved.
The new bacterial vector pETM60 enables the expression of His-tagged recombinant proteins fused to the C-terminus of NusA through a TEV protease recognition sequence. Three sequences coding for two protein domains (Xklp3A and Tep3Ag) and one membrane-bound viral protein (E8R) could not be expressed in a soluble form in bacteria. Their GST-fusions were mostly soluble but quickly degraded during purification. The same sequences cloned in pETM60 were efficiently purified by metal affinity and recovered soluble after the removal of the fusion partner. The NusA-fused constructs enabled to yield 13-20 mg of fusion protein per litre of culture and 2.5-5 mg of pure protein per litre of culture. Structural analysis indicated that the purified proteins were monodispersed and correctly folded. NusA has been used to raise antibodies that have been successfully used for Western blot and immunoprecipitation of NusA fusion proteins. (C) 2004 Elsevier Inc. All rights reserved.