Up-to-now, several biochemical methods have been developed to allow specific organelle isolation from plant tissues. These procedures are often time consuming, require substantial amounts of plant material, have low yield or do not result in pure organelle fractions. Moreover, barely a protocol allows rapid and flexible isolation of different subcellular compartments. The recently published SpySystem enables the in vitro and in vivo covalent linkage between proteins and protein complexes. Here we describe the use of this system to tag and purify plant organelles. We developed a simple and specific method to in vivo tag and visualize, as well as isolate organelles of interest from crude plant extracts. This was achieved by expressing the covalent split-isopeptide interaction system, consisting of SpyTag and SpyCatcher, in Nicotiana benthamiana leaves. The functionality of the SpySystem in planta, combined with downstream applications, was proven. Using organelle-specific membrane anchor sequences to program the sub-cellular localization of the SpyTag peptide, we could tag the outer envelope of chloroplasts and mitochondria. By co-expression of a cytosolic, soluble eGFP-SpyCatcher fusion protein, we could demonstrate intermolecular isopeptide formation in planta and proper organelle targeting of the SpyTag peptides to the respective organelles. For one-step organelle purification, recombinantly expressed SpyCatcher protein was immobilized on magnetic microbeads via covalent thiol-etherification. To isolate tagged organelles, crude plant filtrates were mixed with SpyCatcher-coated beads which allowed isolation of SpyTag-labelled chloroplasts and mitochondria. The isolated organelles were intact, showed high yield and hardly contaminants and can be subsequently used for further molecular or biochemical analysis. The SpySystem can be used to in planta label subcellular structures, which enables the one-step purification of organelles from crude plant extracts. The beauty of the system is that it works as a covalent toolbox. Labeling of different organelles with individual tags under control of cell-specific and/or inducible promoter sequences will allow the rapid organelle and cell-type specific purification. Simultaneous labeling of different organelles with specific Tag/Catcher combinations will enable simultaneous isolation of different organelles from one plant extract in future experiments.

中文翻译：

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标记和捕获：使用植物中的共价间谍系统快速分离和有效标记细胞器。


到目前为止，已经开发了几种生化方法来从植物组织中分离特定的细胞器。这些程序通常很耗时，需要大量的植物材料，产量低或不能产生纯的细胞器级分。此外，几乎没有一种方案可以快速灵活地分离不同的亚细胞区室。最近发布的 SpySystem 能够在体外和体内实现蛋白质和蛋白质复合物之间的共价连接。在这里，我们描述了使用该系统来标记和纯化植物细胞器。我们开发了一种简单而具体的方法来进行体内标记和可视化，以及从粗植物提取物中分离感兴趣的细胞器。这是通过在本塞姆氏烟草叶子中表达由 SpyTag 和 SpyCatcher 组成的共价分裂异肽相互作用系统来实现的。 SpySystem 在植物中的功能与下游应用相结合已得到验证。使用细胞器特异性膜锚定序列对 SpyTag 肽的亚细胞定位进行编程，我们可以标记叶绿体和线粒体的外膜。通过胞质、可溶性 eGFP-SpyCatcher 融合蛋白的共表达，我们可以证明植物中分子间异肽的形成以及 SpyTag 肽对相应细胞器的正确细胞器靶向。为了进行一步细胞器纯化，重组表达的 SpyCatcher 蛋白通过共价硫醇醚化固定在磁性微珠上。为了分离标记的细胞器，将粗植物滤液与 SpyCatcher 包被的珠子混合，从而分离出 SpyTag 标记的叶绿体和线粒体。 分离的细胞器完整，产量高，几乎没有污染物，可用于后续的进一步分子或生化分析。 SpySystem 可用于在植物中标记亚细胞结构，从而能够从粗植物提取物中一步纯化细胞器。该系统的美妙之处在于它可以作为一个共价工具箱。在细胞特异性和/或诱导型启动子序列的控制下用单独的标签标记不同的细胞器将允许快速细胞器和细胞类型特异性纯化。使用特定标签/捕手组合同时标记不同的细胞器将能够在未来的实验中从一种植物提取物中同时分离不同的细胞器。