Isolation and stability of histidine-tagged proteins produced in plants via potyvirus gene vectors.
Isolation and stability of histidine-tagged proteins produced in plants via potyvirus gene vectors.
复制标题
通过马铃薯Y病毒基因载体在植物中产生的组氨酸标签蛋白的分离和稳定性。
DOI：
10.1006/viro.1998.9458
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Hong,J
中科院分区：
文献类型：
--
作者：
Dolja,VV;Peremyslov,VV;Keller,KE;Martin,RR;Hong,J
使用烟草蚀刻病毒 (TEV) 衍生的基因载体开发了一种从植物中表达和纯化组氨酸标签蛋白的系统。该载体提供了方便的多接头以及重组蛋白 N 或 C 末端组氨酸标签的选择。这些载体用于表达甜菜黄线状病毒 (BYV) 编码的蛋白质。从被杂交 TEV 系统感染的植物中很容易分离出约 4 μg/g 的 20-kDa BYV 蛋白。相比之下，只能纯化极少量 N 或 C 末端带有组氨酸标签的 22-kDa BYV 衣壳蛋白 (CP)。重组 CP 的快速降解与其在受感染植物中积累的失败有关。与 TEV HC-Pro 的融合稳定了组氨酸标记的 BYV CP，并促进了从受感染植物中纯化融合产物。同样的融合方法已成功用于 24-kDa 小 BYV CP。在免疫印迹分析中，重组蛋白被组氨酸标签特异性单克隆抗体识别。这些结果证明了一系列设计的 TEV 载体在重组蛋白表达、检测和纯化方面的实用性，并表明内在蛋白稳定性是从植物中回收重组蛋白的主要因素。
A system for the expression and purification of histidine-tagged proteins from plants has been developed using a tobacco etch potyvirus (TEV)-derived gene vectors. The vectors offered a convenient polylinker and a choice of histidine tagging at the recombinant proteins' N or C termini. These vectors were utilized for expression of proteins encoded by beet yellows closterovirus (BYV). Approximately 4 μg/g of 20-kDa BYV protein was readily isolated from plants systemically infected by hybrid TEV. In contrast, only minute quantities of 22-kDa BYV capsid protein (CP) histidine-tagged at its N or C terminus could be purified. Rapid degradation of the recombinant CP has been implicated in its failure to accumulate in infected plants. Fusion with TEV HC-Pro stabilized the histidine-tagged BYV CP and facilitated purification of the fusion product from infected plants. This same fusion approach was successfully used with the 24-kDa minor BYV CP. The recombinant proteins were recognized by histidine-tag-specific monoclonal antibody in immunoblot analysis. These results demonstrate the utility of a designed series of TEV vectors for expression, detection, and purification of the recombinant proteins and suggest that intrinsic protein stability is a major factor in a recovery of recombinant proteins from plants.