Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin

Abstract

Protein interactions with peptides generally have low thermodynamic and mechanical stability. Streptococcus pyogenes fibronectin-binding protein FbaB contains a domain with a spontaneous isopeptide bond between Lys and Asp. By splitting this domain and rational engineering of the fragments, we obtained a peptide (SpyTag) which formed an amide bond to its protein partner (SpyCatcher) in minutes. Reaction occurred in high yield simply upon mixing and amidst diverse conditions of pH, temperature, and buffer. SpyTag could be fused at either terminus or internally and reacted specifically at the mammalian cell surface. Peptide binding was not reversed by boiling or competing peptide. Single-molecule dynamic force spectroscopy showed that SpyTag did not separate from SpyCatcher until the force exceeded 1 nN, where covalent bonds snap. The robust reaction conditions and irreversible linkage of SpyTag shed light on spontaneous isopeptide bond formation and should provide a targetable lock in cells and a stable module for new protein architectures. agging with peptides (e.g., HA, myc, FLAG, His 6 ) is one of the most common ways to detect, purify, or immobilize proteins (1-4). Peptides are very useful minimally disruptive probes (5) but they are also "slippery"-antibodies or other proteins typically bind peptides with low affinity and poor mechanical strength (6-9). We sought to form a rapid covalent bond to a peptide tag without the use of chemical modification, artificial amino acids, or cysteines (disulfide bond formation is reversible and restricted to particular cellular locations).It has recently been found that Streptococcus pyogenes, like many other Gram-positive bacteria, contains extracellular proteins stabilized by spontaneous intramolecular isopeptide bonds (10). Here we explored the second immunoglobulin-like collagen adhesin domain (CnaB2) from the fibronectin binding protein FbaB, found in invasive strains of S. pyogenes (11,12) and essential for phagocytosis-like uptake of the bacteria by endothelial cells (13). CnaB2 contains a single isopeptide bond conferring exceptional stability: CnaB2 remains folded even at pH 2 or up to 100°C (12). By splitting CnaB2 into peptide and protein fragments, followed by rational modification of the parts, we developed a peptide tag of 13 amino acids that rapidly formed a covalent bond with its protein partner (138 amino acids, 15 kDa) and characterized the conditions for reaction, cellular specificity of bond formation, and resilience of the reacted product.

Programmable biofilm-based materials from engineered curli nanofibres

“…The library members, detailed in Table 1, range in length from 7-59 amino acids and encode a wide variety of functions such as binding to various inorganic substrates (GBP, CNBP, QBP, MBD and AFP8), nucleation of mineral and metallic nanostructures (A3, CLP12, CT43 and Mms6), and a highly specific catalytic interaction with a protein (SpyTag) 15,[21][22][23][24][25][26][27][28][29][30][31] . Each peptide domain was cloned as C-terminal fusions to CsgA with an intervening six-amino-acid flexible linker (Table 1) and these plasmids were expressed in LSR10 cells to produce 12 different BIND biofilms.…”

“…5b-g). We recombinantly produced GFP-SpyCatcher and a non-functional mutant 28 (GFP-SpyCatcher E77Q ) and applied cell lysates containing these proteins to SpyTag-BIND or wild-type CsgA biofilms. Analysis by epifluorescence microscopy revealed, as expected, that only the combination of biofilms composed of CsgA-SpyTag incubated with GFP-SpyCatcher resulted in covalent attachment (Fig.…”

B 12 -dependent photoresponsive protein hydrogels for controlled stem cell/protein release

“…The gelation rate of the current CarH C hydrogel system mainly depends on two events: the SpyTag-SpyCatcher reaction and AdoB 12 -dependent CarH C self-assembly. The SpyTag-SpyCatcher reaction is likely to be the rate-limiting step, because its secondorder rate constant is ∼1.4 × 10 3 M −1 s −1 , which is far from the diffusion limit and substantially slower than some other biomolecular interactions, such as the widely used streptavidin-biotin interaction (26). The reaction kinetics of SpyTag-SpyCatcher can be optimized through directed evolution to accelerate the gelation.…”

“…Genetically encoded SpyTag (A)-SpyCatcher (B) chemistry, inspired by an isopeptide bond-containing bacterial adhesin, consists of a peptide/protein pair that can spontaneously form an isopeptide bond on binding under mild physiological conditions (26). Because of its high efficiency and modularity, this chemistry has led to a number of applications, including control of biomacromolecular topology, synthesis of bioactive and "living" materials, and biomolecular imaging (16,18,(27)(28)(29)(30)(31)(32)(33)(34)(35)(36).…”

Virus Like Nanoparticles: Display Matters

“…CnaB2 domain locks itself together by forming a spontaneous isopeptide bond between Lys and Asp and this reaction is irreversible. Zakeri and colleagues earlier showed that CnaB2 domain could be split and engineered into two parts, a peptide (SpyTag) and a protein (SpyCatcher) and that these two could form amide bond irreversibly just by mixing the two components together (Zakeri et al, 2012). VLPs expressing SpyTag or Spy Catcher can thus be linked potentially to any antigen that is fused to either SpyCatcher or SpyTag respectively.…”