背景:电泳迁移率变化测定(EMSA)是检测DNA-蛋白质相互作用的常用技术。然而,在大多数情况下,EMSA中使用的蛋白质是通过原核表达获得的,很少来自植物。同时,原核系统表达的蛋白质通常不能自然折叠,也没有翻译后修饰,这可能会影响蛋白质与DNA的结合。
结果:这里,我们开发了一种技术,从寄主植物中快速分离感兴趣的蛋白质,然后使用荧光EMSA对其进行分析。这种技术系统被称为:蛋白质从植物荧光EMSA方法(PPF-EMSA)。在PPF-EMSA中,一种特殊的瞬时转化方法被用来将基因瞬时传递到植物中,能够有效合成编码的蛋白质。然后,使用免疫沉淀分离目标蛋白,DNA探针用花青3(Cy3)标记。荧光EMSA和超移位荧光EMSA都可以使用来自植物的蛋白质进行。三种植物,白桦,杨树.davidiana×P.bolleana和拟南芥,在这项研究中使用。从植物中分离的蛋白质处于自然状态,可以自然折叠并经过翻译后修改,实现与其同源DNA的真正绑定。
结论:由于瞬时转化可以快速进行,而不依赖于稳定转化是否可用,我们相信这种方法将有广泛的应用,能够方便地从宿主植物中分离蛋白质。
BACKGROUND: The electrophoretic mobility shift assay (
EMSA) is a common technology to detect DNA-protein interactions. However, in most cases, the protein used in
EMSA is obtained via prokaryotic expression, and rarely from plants. At the same time, the proteins expressed from prokaryotic systems usually cannot fold naturally and have no post translationally modification, which may affect the binding of proteins to DNA.
RESULTS: Here, we develop a technique to quickly isolate proteins of interest from host plants and then analyze them using fluorescent EMSA. This technology system is called: protein from plants fluorescent
EMSA method (PPF-
EMSA). In PPF-
EMSA, a special transient transformation method is employed to transiently deliver genes into the plant, enabling efficient synthesis the encoded proteins. Then, the target protein is isolated using immunoprecipitation, and the DNA probes were labeled with cyanine 3 (Cy3). Both fluorescent
EMSA and super-shift fluorescent
EMSA can be performed using the proteins from plants. Three kinds of plants, Betula platyphylla, Populus. davidiana×P. bolleana and Arabidopsis thaliana, are used in this study. The proteins isolated from plants are in a natural state, can fold naturally and are posttranslationally modified, enabling true binding to their cognate DNAs.
CONCLUSIONS: As transient transformation can be performed quickly and not depended on whether stable transformation is available or not, we believe this method will have a wide application, enabling isolation of proteins from host plant conveniently.