Many modern pursuits of molecular biology and biochemistry are built on precise and selective protein labeling techniques. Traditional bioconjugation methods such as N-hydroxysuccinimide or maleimide-based chemistry often involve the modification of lysine or cysteine residues in the protein. These approaches are simple and quick; however, they lack control over the site and frequency of conjugation reactions, resulting in heterogeneous and poorly defined material. In the context of complex biological systems, off-target labeling can have unpredictable effects. Site-specifically modified conjugates often exhibit improved behavior compared to their counterparts synthesized using traditional methods [1].

Substantial effort has been invested in applying the enzyme sortase A (SrtA), initially discovered operating in the Gram-positive bacterium S. aureus as part of its cell wall assembly mechanism, to facilitate site-specific protein modifications, also known as “sortagging” [1]. SrtA enzymatic activity has been honed through directed evolution campaigns that provided variants with significantly improved transpeptidation activity [1], which serve as versatile tools in protein engineering applications. Sortagging has been applied in various contexts, including site-specific ligation reactions to link protein domains, cyclization chemistry to constrain peptides into bioactive conformations, and transformations that result in the attachment of proteins to material surfaces [1, 2, 3, 4]. Sortase-mediated ligation (SML) enables the attachment of a diverse array of molecules to targets of interest with high specificity and in mild reaction conditions (aqueous buffers at room temperature or cooler) allowing for modification of biomolecules that are sensitive to harsher labeling chemistries. Bioconjugates produced using sortagging allow scientists to interrogate and modulate biological systems that would not be possible using conventional chemical and biological approaches [1].

Here, we survey recent strides made in SML strategies in three key areas: (1) new SrtA-based labeling chemistries, 2) the development of specialized bioconjugates for advanced imaging techniques through SML and 3) applications of sortagged reagents for interrogating and controlling complex biological processes.