Biology subjects

Stover, K.

Publications and source records attributed to Stover, K..

2 recordsLinked to original sources

bioRxiv2026

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 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.

biochemistry↗

bioRxiv2018

Alanine scanning mutagenesis of the Suvratoxumab epitope reduces alpha toxin lytic activity in vitro and S. aureus fitness in infection models

Alpha toxin (AT) is a cytolytic pore-forming toxin that plays a key role in Staphylococcus aureus pathogenesis, consequently extensive research was undertaken to understand the AT mechanism of action and its utility as a target for novel prophylaxis and treatment strategies against S. aureus infections. MEDI4893 (Suvratoxumab) is a human anti-AT IgG1 monoclonal antibody (mAb), which targets AT and is currently in Phase 2 clinical development. As shown previously, the MEDI4893-binding epitope on AT is comprised of the highly conserved amino acid regions 177-200 and 261-271, suggesting these amino acids are important for AT function. To test this hypothesis, and gain insight into the effect mutations in the epitope on AT neutralization by MEDI4893, nine MEDI4893 contact residues in AT were individually mutated to alanine. Consistent with our hypothesis, 8 out of 9 mutants exhibited >2-fold loss in lytic activity resulting from a defect in cell binding and pore formation. MEDI4893 binding affinity was reduced >2-fold (2 - 27-fold) for 7 out of 9 mutants and no binding detected for W187A mutant. MEDI4893 effectively neutralized all of the lytic mutants in vitro and in vivo. When the defective mutants were introduced into a S. aureus clinical isolate, the mutant-expressing strains exhibited less severe disease in mouse models and were effectively neutralized by MEDI4893. These results indicate the MEDI4893 epitope is highly conserved due, in part to its role in AT pore-formation and bacterial fitness, thus decreasing the likelihood for the emergence of mAb-resistant variants.

microbiology↗