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.

The inverse autotransporters of Yersinia ruckeri, YrInv and YrIlm, contribute to biofilm formation and virulence

“…Therefore, only SpyTags exposed on the extracellular surface of the cell can react with SpyCatcher. For easy readout, SpyCatcher can be fused to fluorescent proteins such as sfGFP (Chauhan et al ., 2019; Hatlem et al ., 2019). Escherichia coli BL21‐Gold (DE3) was transformed with the plasmid pASK_IBA3_ yrInv _SpyTag, which contains an intact yrInv gene and its own promoter, followed by a DNA sequence encoding for a SpyTag before the stop codon.…”

Greatest Hits—Innovative Technologies for High Throughput Identification of Bispecific Antibodies

“…The system is applied in several modular "plug-and-display" approaches for the generation of enhanced immunostimulants against cancer or infectious diseases such as influenza or MERS-CoV [72][73][74][75][76]. Further applications include optimized protein purification procedures (in parts, in combination with inteins) [77,78], specific immobilization of targets for enhanced phage display antibody discovery campaigns [79], site-specific fluorescence labeling of antibodies for in vivo optical imaging [80], up to in vivo assembling of proteins or enzymes [81,82], and versatile further applications, in depth reviewed by Hatlem et al [27].…”

“…In order to overcome this bottleneck, technologies such as controlled Fab-arm exchange used for DuoBodies [22], paired light chain single cell production approaches [23], microbial transglutaminase [24] or Sortase A [25] mediated bioconjugation, the SpyTag/SpyCatcher system [26,27], or split inteins [28] were adapted to enable broad bispecific antibody screening ( Figure 1). These methodologies harbor the benefit that bispecific entities can be generated from a pre-existing set of antibody fragments in a mix-and-match manner, significantly reducing hands-on time as well as overall efforts for bispecific screening.…”

Capsid-like particles decorated with the SARS2-CoV-2 receptor-binding domain elicit strong virus neutralization activity

“…1C). Mixing of Catcher-RBD and Tag-CLPs result in the formation of a covalent isopeptide bond between the Catcher and Tag [46][47][48][49][50][51] . Covalent coupling of the RBD antigens to the CLPs was confirmed by SDS-PAGE analysis, by the appearance of a protein band of 60kDa, corresponding to the added size of the RBD antigen (43 kDa) and Tag-CLP subunit (16.5 kDa) (Fig.…”