Enhancing Sortase mediated transpeptidation reaction conversion via metal-peptide complexation
Research Mentor(s)
Antos, John M.
Description
Sortase A from Staphylococcus aureus has grown in popularity as a robust and reliable enzyme for site-specifically modifying proteins. Recognizing and attacking the five amino acid recognition sequence (LPXTG), Sortase A forms a thioester intermediate which is susceptible to nucleophilic attack by the N-terminal amine of a glycine. During formation of the intermediate, sortase A excises the region of the peptide C-terminal to threonine. The excised fragment thus has an N-terminal glycine, and can attack the enzyme-protein intermediate, leading to reversal of the reaction. To attenuate reaction reversal, the recognition motif has been extended to LPETGGH, as GGH peptides are known to be strong chelators of metal ions. Sortase-catalyzed reactions performed using this modified recognition sequence in the presence of Ni2+ have been found to have a higher conversion to product; although little to no effect has been seen in the presence of other metal ions. To demonstrate the increase of efficiency and the potential for decreased cost, here we present the results of applying this metal chelation strategy to a range of model proteins and peptides.
Document Type
Event
Start Date
18-5-2017 9:00 AM
End Date
18-5-2017 12:00 PM
Department
Chemistry
Genre/Form
student projects; posters
Subjects – Topical (LCSH)
Proteins--Biotechnology; Catalysis; Streptococcus pneumoniae--Research
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 documentation for commercial purposes, or for financial gain, shall not be allowed without the author's written permission.
Language
English
Format
application/pdf
Enhancing Sortase mediated transpeptidation reaction conversion via metal-peptide complexation
Sortase A from Staphylococcus aureus has grown in popularity as a robust and reliable enzyme for site-specifically modifying proteins. Recognizing and attacking the five amino acid recognition sequence (LPXTG), Sortase A forms a thioester intermediate which is susceptible to nucleophilic attack by the N-terminal amine of a glycine. During formation of the intermediate, sortase A excises the region of the peptide C-terminal to threonine. The excised fragment thus has an N-terminal glycine, and can attack the enzyme-protein intermediate, leading to reversal of the reaction. To attenuate reaction reversal, the recognition motif has been extended to LPETGGH, as GGH peptides are known to be strong chelators of metal ions. Sortase-catalyzed reactions performed using this modified recognition sequence in the presence of Ni2+ have been found to have a higher conversion to product; although little to no effect has been seen in the presence of other metal ions. To demonstrate the increase of efficiency and the potential for decreased cost, here we present the results of applying this metal chelation strategy to a range of model proteins and peptides.
Comments
Outstanding Poster Award Recipient