The Sainsbury Laboratory, Norwich NR4 7UH, UK.
Plant J. 2009 Nov;60(4):744-54. doi: 10.1111/j.1365-313X.2009.03982.x. Epub 2009 Jul 22.
A key challenge in cell biology is to directly link protein localization to function. The green fluorescent protein (GFP)-binding protein, GBP, is a 13-kDa soluble protein derived from a llama heavy chain antibody that binds with high affinity to GFP as well as to some GFP variants such as yellow fluorescent protein (YFP). A GBP fusion to the red fluorescent protein (RFP), a molecule termed a chromobody, was previously used to trace in vivo the localization of various animal antigens. In this study, we extend the use of chromobody technology to plant cells and develop several applications for the in vivo study of GFP-tagged plant proteins. We took advantage of Agrobacterium tumefaciens-mediated transient expression assays (agroinfiltration) and virus expression vectors (agroinfection) to express functional GBP:RFP fusion (chromobody) in the model plant Nicotiana benthamiana. We showed that the chromobody is effective in binding GFP- and YFP-tagged proteins in planta. Most interestingly, GBP:RFP can be applied to interfere with the function of GFP fusion protein and to mislocalize (trap) GFP fusions to the plant cytoplasm in order to alter the phenotype mediated by the targeted proteins. Chromobody technology, therefore, represents a new alternative technique for protein interference that can directly link localization of plant proteins to in vivo function.
在细胞生物学中，一个关键的挑战是将蛋白质定位与功能直接联系起来。绿色荧光蛋白（GFP）结合蛋白（GBP）是一种 13kDa 的可溶性蛋白，来源于骆驼重链抗体，它与 GFP 以及一些 GFP 变体（如黄色荧光蛋白（YFP））具有高亲和力。将 GFP 融合到红色荧光蛋白（RFP）上，形成一种称为 chromobody 的融合蛋白，以前曾用于追踪各种动物抗原在体内的定位。在这项研究中，我们将 chromobody 技术扩展到植物细胞，并开发了几种应用来研究 GFP 标记的植物蛋白在体内的功能。我们利用农杆菌介导的瞬时表达测定（农杆菌浸润）和病毒表达载体（农杆菌感染）在模式植物烟草中表达功能性 GBP:RFP 融合（chromobody）。我们表明，chromobody 能够有效地结合 GFP 和 YFP 标记的蛋白。最有趣的是，GBP:RFP 可以用于干扰 GFP 融合蛋白的功能，并将 GFP 融合蛋白错误定位（捕获）到植物细胞质中，从而改变由靶向蛋白介导的表型。因此，chromobody 技术代表了一种新的蛋白质干扰替代技术，它可以将植物蛋白的定位与体内功能直接联系起来。
