Morgan, HE, Arnott, ZLP, Kamiński, TP et al. (2 more authors) (2022) Combined Application of Orthogonal Sortases and Depsipeptide Substrates for Dual Protein Labeling. Bioconjugate Chemistry, 33 (12). pp. 2341-2347. ISSN 1043-1802
Abstract
Staphylococcus aureus sortase A is a transpeptidase that has been extensively exploited for site-specific modification of proteins and was originally used to attach a labeling reagent containing an LPXTG recognition sequence to a protein or peptide with an N-terminal glycine. Sortase mutants with other recognition sequences have also been reported, but in all cases, the reversibility of the transpeptidation reaction limits the efficiency of sortase-mediated labeling reactions. For the wildtype sortase, depsipeptide substrates, in which the scissile peptide bond is replaced with an ester, allow effectively irreversible sortase-mediated labeling as the alcohol byproduct is a poor competing nucleophile. In this paper, the use of depsipeptide substrates for evolved sortase variants is reported. Substrate specificities of three sortases have been investigated allowing identification of an orthogonal pair of enzymes accepting LPEToG and LPESoG depsipeptides, which have been applied to dual N-terminal labeling of a model protein mutant containing a second, latent N-terminal glycine residue. The method provides an efficient orthogonal site-specific labeling technique that further expands the biochemical protein labeling toolkit.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2022 The Authors. Published by American Chemical Society.This is an open access article, under the terms of the Creative Commons Attribution License (CC-BY 4.0), which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited. |
Dates: |
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Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Engineering & Physical Sciences (Leeds) > School of Chemistry (Leeds) > Organic Chemistry (Leeds) |
Funding Information: | Funder Grant number BBSRC (Biotechnology & Biological Sciences Research Council) BB/R005540/1 |
Depositing User: | Symplectic Publications |
Date Deposited: | 16 Nov 2022 11:49 |
Last Modified: | 25 Jun 2023 23:09 |
Status: | Published |
Publisher: | American Chemical Society |
Identification Number: | 10.1021/acs.bioconjchem.2c00411 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:193300 |