A FRET Ligation Assay using Fluorescent Proteins for Bacterial Sortase Enzymes
Bacterial sortases are widely used in sortase-mediated ligation (SML) experiments for various protein engineering applications. The power of these enzymes to bind and cleave a specific recognition motif, followed by ligation to another substrate using a ping-pong reaction mechanism has numerous applications in vaccine and antibody/nanobody drug conjugate development, as a diagnostic and…
A FRET-based assay for monitoring ligation products generated through sortase-mediated ligation (SML) has been optimized using fluorescent proteins, GFP derivatives mTurquoise2 and SYFP2. Bacterial sortases, particularly class A sortase (SrtA) from Staphylococcus aureus (saSrtA) and its engineered derivatives, are widely employed in SML experiments for various protein engineering applications.
However, saSrtA and other endogenous sortases are relatively inefficient enzymes and require specific recognition of the Cell Wall Sorting Signal (CWSS) for optimal performance. To address this limitation, the researchers aimed to develop a screening assay for SML. The presented optimization procedures involve a FRET-based assay utilizing the GFP derivatives mTurquoise2 and SYFP2 to directly monitor the formation of ligation products during SML.
The recombinant substrates used in the assay can be easily manipulated to screen various parameters such as the substrate recognition motif, second substrate nucleophile, and sortase variants. The researchers believe that further optimization of this assay could prove beneficial for high-throughput sortase screening strategies, providing a proof-of-concept approach for continued development of SML reagents.
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