Research Mentor(s)
Amacher, Jeanine
Description
Bacterial sortase enzymes are a beneficial tool in innovative mechanisms of protein engineering. However, important limitations to utilization of sortases for engineered purposes exist; namely, that sortase A (SrtA) is a relatively poor enzyme and very specific for the substrate containing LPATXG motif. Exciting previous work from our collaborators reveals that sortases from different species recognize different sequences and that activity can vary. Therefore, we wanted to create and investigate hybrid sortase enzymes between SrtA from S. aureus and Streptococcus pneumoniae, wherein we swapped a substrate-interacting loop between the beta-E and beta-F strands. Our hypothesis is that these residues are responsible for the differing specificities of these two enzymes and that our loop-swapped S. aureus enzyme will show S. pneumoniae sequence selectivity, and vice versa. In addition to the creation of loop swapped complexes, we have also used ancestral sequence reconstruction (ASR) to investigate the specificity and activity of ancestral sortase sequences in gram negative bacteria. We have engineered two ancestral sortase complexes, ancestral SrtAstaph and SrtAstrep using ASR of sequences obtained from NCBI. Our hypothesis is that SrtAstaph and SrtAstrep will be more active, promiscuous enzymes than their extant relatives. Here, we present activity and selectivity data for our loop swapped variants, as well as our ancestral enzymes, in comparison to the two native S. aureus and S. pneumoniae SrtA enzymes. The discovery or development of a more promiscuous sortase enzyme could lead to more efficient mechanisms of protein engineering then are currently available to researchers.
Document Type
Event
Start Date
15-5-2019 9:00 AM
End Date
15-5-2019 5:00 PM
Location
Carver Gym (Bellingham, Wash.)
Department
Chemistry
Genre/Form
student projects, posters
Subjects – Topical (LCSH)
Staphylococcal protein A--Research; Streptococcus pneumoniae; Structural bioinformatics
Type
Image
Keywords
Sortase, Sortase A, Ancestral Sequence Reconstruction, Structural Biology, Enzymology
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.
Language
English
Format
application/pdf
Included in
Engineering Sortase; Activity and Selectivity of New Hybrid and Ancestral Variants of Sortase A
Carver Gym (Bellingham, Wash.)
Bacterial sortase enzymes are a beneficial tool in innovative mechanisms of protein engineering. However, important limitations to utilization of sortases for engineered purposes exist; namely, that sortase A (SrtA) is a relatively poor enzyme and very specific for the substrate containing LPATXG motif. Exciting previous work from our collaborators reveals that sortases from different species recognize different sequences and that activity can vary. Therefore, we wanted to create and investigate hybrid sortase enzymes between SrtA from S. aureus and Streptococcus pneumoniae, wherein we swapped a substrate-interacting loop between the beta-E and beta-F strands. Our hypothesis is that these residues are responsible for the differing specificities of these two enzymes and that our loop-swapped S. aureus enzyme will show S. pneumoniae sequence selectivity, and vice versa. In addition to the creation of loop swapped complexes, we have also used ancestral sequence reconstruction (ASR) to investigate the specificity and activity of ancestral sortase sequences in gram negative bacteria. We have engineered two ancestral sortase complexes, ancestral SrtAstaph and SrtAstrep using ASR of sequences obtained from NCBI. Our hypothesis is that SrtAstaph and SrtAstrep will be more active, promiscuous enzymes than their extant relatives. Here, we present activity and selectivity data for our loop swapped variants, as well as our ancestral enzymes, in comparison to the two native S. aureus and S. pneumoniae SrtA enzymes. The discovery or development of a more promiscuous sortase enzyme could lead to more efficient mechanisms of protein engineering then are currently available to researchers.