*4.6. SpyCatcher and SpyTag for Nanoparticle Formation*

The SpyTag/SpyCatcher system (referred as Spy-System) is based on the internal isopeptide bond of the CnaB2 domain of FbaB protein from *Streptococcus pyogenes*. An internal isopeptide bond forms spontaneously in this domain between the reactive amine of lysine K31 and the side chain carboxyl of aspartate D117. This reaction is catalyzed by the spatially adjacent glutamate E77. The resulting isopeptide bond confers high stability to the CnaB2 domain. The CnaB2 domain is cleaved into two subcomponents, the large and incomplete immunoglobulin-like domain, named SpyCatcher, composed of 138 amino acids, and the short beta strand, named SpyTag, composed of 13 amino acids [116]. While the SpyCatcher contains the reactive lysine (K31) and catalytic glutamate (E77), the SpyTag includes the reactive aspartate (D117). The two components can still recognize each other with high affinity and the isopeptide is formed within minutes. Despite the fact that this system is not able to generate nanoparticles on its own, the two components of the Spy-System can be genetically fused to a number of viral or designed self-assembling proteins leading to the generation of a stable nanoparticle displaying a large variety of antigens. Moreover, the Spy-System offers a great flexibility since components can be inserted at the N- or C-termini of the target proteins. Its simplicity is of particular interest to quickly screen if a nanoparticle formulation will be of benefit for vaccine design, before generating genetic fusion on the nanoparticle of interest. The first nanoparticle assembly with Spy-System was performed by genetically fusing the SpyCatcher to the N-terminus of the viral coat protein (CP3) of the RNA bacteriophage AP205 [117]. AP205 VLPs have been then further investigated as vaccine candidates against a number of targets, including malaria and cancer [117–119].
