Probing the potential of CnaB-type domains for the design of tag/catcher systems

Abstract

Building proteins into larger, post-translational assemblies in a defined and stable way is still a challenging task. A promising approach relies on so-called tag/catcher systems that are fused to the proteins of interest and allow a durable linkage via covalent intermolecular bonds. Tags and catchers are generated by splitting protein domains that contain intramolecular isopeptide or ester bonds that form autocatalytically under physiological conditions. There are already numerous biotechnological and medical applications that demonstrate the usefulness of covalent linkages mediated by these systems. Additional covalent tag/catcher systems would allow creating more complex and ultra-stable protein architectures and networks. Two of the presently available tag/catcher systems were derived from closely related CnaB-domains of Streptococcus pyogenes and Streptococcus dysgalactiae proteins. However, it is unclear whether domain splitting is generally tolerated within the CnaB-family or only by a small subset of these domains. To address this point, we have selected a set of four CnaB domains of low sequence similarity and characterized the resulting tag/catcher systems by computational and experimental methods. Experimental testing for intermolecular isopeptide bond formation demonstrated two of the four systems to be functional. For these two systems length and sequence variations of the peptide tags were investigated revealing only a relatively small effect on the efficiency of the reaction. Our study suggests that splitting into tag and catcher moieties is tolerated by a significant portion of the naturally occurring CnaB-domains, thus providing a large reservoir for the design of novel tag/catcher systems.

New Routes and Opportunities for Modular Construction of Particulate Vaccines: Stick, Click, and Glue

“…In other cases, adjustment of the spacer between SpyCatcher/coat protein and SpyTag/antigen may be needed for large antigens. Alternative Catcher/Tag pairs have also been engineered that can cater to different pH-dependent conjugation optima, which may be more suitable for a particular demanding antigen or expression system ( 74 , 85 , 104 – 106 ).…”

Advantages and Prospects of Tag/Catcher Mediated Antigen Display on Capsid-Like Particle-Based Vaccines

“…Split-protein (Tag/Catcher) conjugation systems have been developed [61][62][63][64][65][66][67][68][69][70] and used for covalent anchoring of vaccine antigens onto CLPs [39,51]. The split-protein technology is based on the separation of a bacterial pili protein, into a reactive peptide (Tag) and corresponding protein binding partner (Catcher).…”

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.…”

“…We have developed a SARS-CoV-2 vaccine based on a platform similar to the well-characterized Tag/Catcher-AP205 derived technology 26,27 . Accordingly, a split-protein Tag/catcher system 28,29,30 is used to conjugate and display the RBD of the SARS-CoV-2 spike protein on the protein surface of preassembled AP205 capsid-like particles (CLPs).…”