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Date Permissions Signed

8-4-2020

Date of Award

Summer 2020

Document Type

Masters Thesis

Department or Program Affiliation

Chemistry

Degree Name

Master of Science (MS)

Department

Chemistry

First Advisor

Amacher, Jeanine

Second Advisor

Antos, John M.

Third Advisor

Spiegel, P. Clint

Abstract

Bacterial sortases are cysteine transpeptidases that anchor virulence factors to the surface of bacterial cells. Sortases are a powerful tool utilized for protein engineering that allow researchers to modify proteins at the protein level, not the DNA level. However, important limitations to utilization of sortases for engineering purposes exist; namely, SrtA from S. aureus is a relatively modest enzyme compared to other SrtA enzymes and is very specific for the LPXTG motif. Previous work from our collaborators and others revealed that sortases from different species can recognize alternative sequences and that activities can vary widely. We were curious about how natural sequence variation in class A sortases affects activity and selectivity. To that end, a principle component analysis revealed that the structurally conserved beta7-beta8 substrate-interacting loop region may be a key component in substrate recognition and activity. We investigated this in two ways, by engineering eight S. pneumoniae beta7-beta8 loop variants with loop sequences from different bacterial species and by performing ancestral sequence reconstruction on extant class A sortase sequences. We then assayed all of our variants and found a SrtA construct, SPSfaec (S. pneumoniae core with a beta7-beta8 substrate-interacting loop from E. faecalis) which not only possessed an enhanced substrate promiscuity profile, recognizing seven 5th position substrates LPATGG, LPATSG, LPATAG, LPATVG, LPATTG, LPATNG, and LPATFG, but also displayed improved catalytic efficiency for all six of these substrates compared to the WT enzymes SrtA from S. aureus and SrtA from S. pneumoniae. Overall our engineered constructs provide further insight into the role of this beta7-beta8 substrate-interacting loop in class A sortases and provide additional framework for the design of sortases for future engineering purposes.

Type

Text

Keywords

Sortase A, Sortase, Protein Engineering, Ancestral Protein Reconstruction, Enzymology, Sortase Mediated Ligation

Publisher

Western Washington University

OCLC Number

1182637684

Subject – LCSH

Enzymes; Protein engineering; Virulence (Microbiology); Proteomics

Format

application/pdf

Genre/Form

masters theses

Language

English

Rights

Copying of this document in whole or in part is allowable only for scholarly purposes. It is understood, however, that any copying or publication of this document for commercial purposes, or for financial gain, shall not be allowed without the author’s written permission.

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