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bioRxiv · 10.64898/2026.08.21.746329

A FRET Ligation Assay using Fluorescent Proteins for Bacterial Sortase Enzymes

Wachsman, A.·Walkenhauer, E. G.·Stover, K.·Richardson, B. C.·Jackson, S. N.·Amacher, J.·Antos, J. M.
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Abstract

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 therapeutic tool, in creating novel insulin derivatives, etc. The most widely used sortase for SML is the class A sortase (SrtA) from Staphylococcus aureus (saSrtA), and its engineered derivatives. Despite its utility, saSrtA and other endogenous sortases are relatively inefficient enzymes and use can be limited by the need for specific recognition of the Cell Wall Sorting Signal (CWSS), sequence Leu-Pro-X-Thr-Gly, where X=any amino acid. Therefore, there is a need to continue to identify new tools for SML and to develop screening assays towards these endeavors. Here, we present optimization procedures for a FRET-based assay utilizing the GFP derivatives mTurquoise2 and SYFP2 to directly monitor formation of ligation products generated via SML. Similar to related assays, our recombinant substrates can be easily manipulated to screen either the substrate recognition motif, second substrate nucleophile, and/or sortase variants themselves. We believe continued optimization of this assay for a variety of high throughput uses in sortase screening strategies is possible, providing a proof-of-concept approach for continued SML reagent development.

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Wachsman, A., Walkenhauer, E. G., Stover, K., Richardson, B. C., Jackson, S. N., Amacher, J., Antos, J. M.. 2026-08-24. A FRET Ligation Assay using Fluorescent Proteins for Bacterial Sortase Enzymes. https://doi.org/10.64898/2026.08.21.746329

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