Research Mentor(s)
Jeanine Amacher
Description
Sortases are cysteine transpeptidase enzymes, found in gram positive bacteria, that are used in sortase-mediated ligation (SML) reactions in vitro for a variety of protein engineering applications. Historically, sortase A from Staphylococcus aureus (saSrtA) has been the protein of choice for SML reactions, but the stringent specificity of saSrtA for the sequence motif LPXTG limits its uses. We have explored the activity of ��4-��5 loop swapped sortases, while also working to crystallize these chimeras in order to provide a structural basis for any alterations in activity. Streptoccocus pyogenes was used as the scaffold, as we have had success with chimeric S. pyogenes enzyme activity and crystallization with ��7-��8 loop swaps in the past. Class B sortase enzymes recognize sequence motifs that differ markedly from that of saSrtA, making them attractive candidates for SML, however their slow reaction rates have limited their use. Here, we expressed and purified class B sortases from Bacillus anthracis, Clostridium difficile, and Staphylococcus aureus and explored their activity in vitro. In the future we hope to use engineered chimeric enzymes to research similar structural loops in SrtB to those we have characterized previously in SrtA, as well as develop a bioluminescent luciferase assay for measuring sortase enzyme activity.
Document Type
Event
Start Date
May 2022
End Date
May 2022
Location
Carver Gym (Bellingham, Wash.)
Department
CSE - Chemistry
Genre/Form
student projects; posters
Type
Image
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
Expanding on sortase characterization: B4-B5 loop swap chimera and Class B enzymatic activity
Carver Gym (Bellingham, Wash.)
Sortases are cysteine transpeptidase enzymes, found in gram positive bacteria, that are used in sortase-mediated ligation (SML) reactions in vitro for a variety of protein engineering applications. Historically, sortase A from Staphylococcus aureus (saSrtA) has been the protein of choice for SML reactions, but the stringent specificity of saSrtA for the sequence motif LPXTG limits its uses. We have explored the activity of ��4-��5 loop swapped sortases, while also working to crystallize these chimeras in order to provide a structural basis for any alterations in activity. Streptoccocus pyogenes was used as the scaffold, as we have had success with chimeric S. pyogenes enzyme activity and crystallization with ��7-��8 loop swaps in the past. Class B sortase enzymes recognize sequence motifs that differ markedly from that of saSrtA, making them attractive candidates for SML, however their slow reaction rates have limited their use. Here, we expressed and purified class B sortases from Bacillus anthracis, Clostridium difficile, and Staphylococcus aureus and explored their activity in vitro. In the future we hope to use engineered chimeric enzymes to research similar structural loops in SrtB to those we have characterized previously in SrtA, as well as develop a bioluminescent luciferase assay for measuring sortase enzyme activity.