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. You need to first deactivate this app from the Device Administrator.Up-to-now, several biochemical methods have been developed to allow specific organelle isolation from plant tissues. * Activate the device admin and that's it.
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