Intron-tagged epitope:: a tool for facile detection and purification of proteins expressed in Agrobacterium-transformed plant cells
Intron-tagged epitope:: a tool for facile detection and purification of proteins expressed in Agrobacterium-transformed plant cells
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DOI：
10.1046/j.1365-313x.2000.00763.x
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发表时间：
2000-06-01
期刊：
影响因子：
7.2
通讯作者：
Koncz, C
中科院分区：
文献类型：
--
作者：
Ferrando, A;Farràs, R;Koncz, C
表位标记为体内蛋白质的免疫学检测和细胞定位提供了有用的工具。利用T-DNA介导的转化，目前在植物中检测表位标记蛋白是可行的，因为在细菌中由植物启动子驱动的cDNA和基因结构的人工表达掩盖了在农杆菌感染的植物细胞中早期检测表位标记蛋白的过程。我们已经开发了一种方法来标记带有内含子标记的表位编码域的植物编码序列，这些结构域在农杆菌中没有被处理。在这里，我们发现HA表位标记的编码β-葡萄糖醛酸酶和S相蛋白的构建体可以在培养的拟南芥细胞中特异和唯一地表达，早在农杆菌感染后5天就可以检测到。这种表位标记方法为在农杆菌转化的植物细胞中单独或同时表达的蛋白质的纯化和定位提供了无限的转化材料来源。
Epitope tagging provides a useful tool for immunological detection and cellular localization of proteins in vivo. Using T-DNA-mediated transformation, the detection of epitope-tagged proteins in planta is currently feasible only in transgenic plants, because an artificial expression of cDNA and gene constructs driven by plant promoters in bacteria obscures an early detection of epitope-tagged proteins in Agrobacterium-infected plant cells. We have developed a method for labelling plant coding sequences with intron-tagged epitope-coding domains that are not processed in Agrobacterium. Here we show that the expression of HA-epitope-tagged constructs encoding beta-glucuronidase and S-phase kinase-associated (AtSKP1/ASK1) proteins can be specifically and exclusively detected in cultured Arabidopsis cells as early as five days after Agrobacterium infection. This epitope-tagging approach offers an unlimited source of transformed material for purification and localization of proteins expressed individually or simultaneously in Agrobacterium-transformed plant cells.