Dataset Information

Catching a SPY: Using the SpyCatcher-SpyTag and Related Systems for Labeling and Localizing Bacterial Proteins.

ABSTRACT: The SpyCatcher-SpyTag system was developed seven years ago as a method for protein ligation. It is based on a modified domain from a Streptococcus pyogenes surface protein (SpyCatcher), which recognizes a cognate 13-amino-acid peptide (SpyTag). Upon recognition, the two form a covalent isopeptide bond between the side chains of a lysine in SpyCatcher and an aspartate in SpyTag. This technology has been used, among other applications, to create covalently stabilized multi-protein complexes, for modular vaccine production, and to label proteins (e.g., for microscopy). The SpyTag system is versatile as the tag is a short, unfolded peptide that can be genetically fused to exposed positions in target proteins; similarly, SpyCatcher can be fused to reporter proteins such as GFP, and to epitope or purification tags. Additionally, an orthogonal system called SnoopTag-SnoopCatcher has been developed from an S. pneumoniae pilin that can be combined with SpyCatcher-SpyTag to produce protein fusions with multiple components. Furthermore, tripartite applications have been produced from both systems allowing the fusion of two peptides by a separate, catalytically active protein unit, SpyLigase or SnoopLigase. Here, we review the current state of the SpyCatcher-SpyTag and related technologies, with a particular emphasis on their use in vaccine development and in determining outer membrane protein localization and topology of surface proteins in bacteria.

SUBMITTER: Hatlem D

PROVIDER: S-EPMC6539128 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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Publications

Catching a SPY: Using the SpyCatcher-SpyTag and Related Systems for Labeling and Localizing Bacterial Proteins.

Hatlem Daniel D Trunk Thomas T Linke Dirk D Leo Jack C JC

International journal of molecular sciences 20190430 9

The SpyCatcher-SpyTag system was developed seven years ago as a method for protein ligation. It is based on a modified domain from a <i>Streptococcus pyogenes</i> surface protein (SpyCatcher), which recognizes a cognate 13-amino-acid peptide (SpyTag). Upon recognition, the two form a covalent isopeptide bond between the side chains of a lysine in SpyCatcher and an aspartate in SpyTag. This technology has been used, among other applications, to create covalently stabilized multi-protein complexes ...[more]

PMID: 31052154

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Project description:BackgroundUp-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.ResultsWe 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.ConclusionThe 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.

Dataset Information

Catching a SPY: Using the SpyCatcher-SpyTag and Related Systems for Labeling and Localizing Bacterial Proteins.

Publications

Catching a SPY: Using the SpyCatcher-SpyTag and Related Systems for Labeling and Localizing Bacterial Proteins.

Similar Datasets

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