![]() Tag-MBP are shown alone or mixed for the indicated times (triplicate samples), before SDS-PAGE with Coomassie staining.Ī MBP A 312 I 317 172 -173 175 -176 Bound sugar - + - 1 1 2 2 3 3 4 4 Spy. (c) Sample gel to test for quantitative reaction of Snoop. Catcher are shown alone or mixed for 32 min (triplicate samples), before SDS-PAGE with Coomassie staining. (b) Sample gel to test for quantitative reaction of Snoop. The Lys, Asn and Glu involved in isopeptide bond formation are shown in stick-format in yellow and the rest of Snoop. A in cartoon format (based on PDB 2 WW 8), residues mutated are shown in space-fill with carbons in cyan (G 842 was changed to T and D 848 to G). Tag-MBP Reactive triad c Time (min) Tag Catcher 0 0 Tag and Catcher (4: 1) 32 64 128 Snoop. Tag b Tag 0 G 842 Tag and Catcher (1: 2) 0 32 min Snoop. Up-to-now, several biochemical methods have been developed to allow specific organelle isolation from plant tissues.A Residues mutated to make Snoop. 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 Sp圜atcher, 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. For one-step organelle purification, recombinantly expressed Sp圜atcher protein was immobilized on magnetic microbeads via covalent thiol-etherification.īy 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. To isolate tagged organelles, crude plant filtrates were mixed with Sp圜atcher-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.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. ![]()
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