# Improved Understanding of Bacterial Sortase Activity Towards Next-Generation Protein Engineering

> **NIH NIH R15** · WESTERN WASHINGTON UNIVERSITY · 2024 · $382,912

## Abstract

Project Summary. Sortase enzymes are cysteine transpeptidases located on the cell surface of Gram-positive
bacteria, which attach a variety of proteins to the peptidoglycan cell wall. The first sortase discovered, the Class
A sortase from Staphylococcus aureus (saSrtA), is widely used in sortase-mediated ligation (SML) or sortagging
protein engineering applications. This is a versatile protein with numerous human health and disease
implications; recent examples include the use of an engineered saSrtA to identify and disrupt amyloid-b
aggregates in a neurodegeneration model and to construct multi-arm SARS-CoV-2 neutralizing nanobodies.
SrtA is also a potential antibiotic target, as it is an essential protein in these pathogenic bacteria. However, recent
work from ourselves and others reveals that the stringent target selectivity of saSrtA is not shared amongst other
Class A sortases, e.g., those from the Streptococcus or Listeria genera. Furthermore, other classes of sortases
are extremely understudied with respect to SrtA proteins, and could provide useful tools for SML applications.
Class B sortases (SrtBs), for example, recognize a different motif sequence, which may be exploited to expand
the capabilities of SML. Finally, early evidence suggests that the transmembrane region of saSrtA may play an
important role in the catalytic efficiency of this enzyme, but this has not been thoroughly investigated.
 The overall objectives in this application are to (i) dissect selectivity and activity determinants in sortase
enzymes from multiple classes, including the transmembrane regions of sortases and/or their substrates, and
(ii) use bacterial display to create a high throughput assay to measure sortase selectivity, which would be
applicable broadly to all classes of sortases. The central hypothesis is that we can use protein biochemistry,
chemical biology, and computational biology to better understand these enzymes. The rationale of this project is
that a deep understanding of the general characteristics of sortase biology can be leveraged to develop new
SML strategies for protein engineering, and assist in targeted antibiotic design to improve human health. The
central hypothesis will be tested by pursuing three specific aims: 1) dissect selectivity and activity determinants
in SrtA enzymes by characterizing differences in protein dynamics and elucidating extended motif preferences,
as well as develop a high throughout bacterial display assay for sortase selectivity, 2) advance a molecular
understanding of SrtB activities in order to improve reaction rates, and 3) characterize the role of the
transmembrane regions of sortase and its substrates in catalysis. All aims will utilize protein biochemistry,
structural biology, chemical biology, and computational biology. The research proposed in this application is
innovative in that we will be developing new assays and applying techniques not previously used with sortase
enzymes, continuing our work as a ...

## Key facts

- **NIH application ID:** 10874337
- **Project number:** 1R15GM154315-01
- **Recipient organization:** WESTERN WASHINGTON UNIVERSITY
- **Principal Investigator:** Jeanine Faye Amacher
- **Activity code:** R15 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $382,912
- **Award type:** 1
- **Project period:** 2024-05-01 → 2027-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10874337

## Citation

> US National Institutes of Health, RePORTER application 10874337, Improved Understanding of Bacterial Sortase Activity Towards Next-Generation Protein Engineering (1R15GM154315-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10874337. Licensed CC0.

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