The aim of our study was to test the applicability of the SpySystem for the development of a programmable and specific organelle labeling and isolation toolbox. Sp圜atcher and SpyTag are known to efficiently work in vitro and in vivo, extra- and intracellular, but so far little is known to which extend they are also functional in planta. The SpySystem has been used for a broad range of applications, including the production of vaccines, hydrogels, the functionalization of surfaces and the construction and spatial organization of multiprotein complexes, among others. The connection is stable under a wide range of conditions, including heat, pH, detergents and mechanical forces.
The most efficient, best characterized and therefore widely used posttranslational protein coupling reagents, Sp圜atcher and SpyTag, can be used for stable, rapid, irreversible and specific linkage of proteins. Simultaneous labeling of different organelles with specific Tag/Catcher combinations will enable simultaneous isolation of different organelles from one plant extract in future experiments.Īn elegant and innovative way to covalently link proteins and create artificial multiprotein assemblies or team works, is the use of molecular superglue systems based on engineered Ig-like domains. 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. The beauty of the system is that it works as a covalent toolbox. The SpySystem can be used to in planta label subcellular structures, which enables the one-step purification of organelles from crude plant extracts. The isolated organelles were intact, showed high yield and hardly contaminants and can be subsequently used for further molecular or biochemical analysis. To isolate tagged organelles, crude plant filtrates were mixed with Sp圜atcher-coated beads which allowed isolation of SpyTag-labelled chloroplasts and mitochondria. For one-step organelle purification, recombinantly expressed Sp圜atcher protein was immobilized on magnetic microbeads via covalent thiol-etherification. By co-expression of a cytosolic, soluble eGFP-Sp圜atcher fusion protein, we could demonstrate intermolecular isopeptide formation in planta and proper organelle targeting of the SpyTag peptides to the respective organelles. 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. The functionality of the SpySystem in planta, combined with downstream applications, was proven. This was achieved by expressing the covalent split-isopeptide interaction system, consisting of SpyTag and Sp圜atcher, in Nicotiana benthamiana leaves.
We developed a simple and specific method to in vivo tag and visualize, as well as isolate organelles of interest from crude plant extracts. Here we describe the use of this system to tag and purify plant organelles. The recently published SpySystem enables the in vitro and in vivo covalent linkage between proteins and protein complexes.
Moreover, barely a protocol allows rapid and flexible isolation of different subcellular compartments. These procedures are often time consuming, require substantial amounts of plant material, have low yield or do not result in pure organelle fractions. Up-to-now, several biochemical methods have been developed to allow specific organelle isolation from plant tissues.
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