Williamson, DJ, Webb, ME orcid.org/0000-0003-3574-4686 and Turnbull, WB orcid.org/0000-0002-7352-0360 (2014) Depsipeptide substrates for sortase-mediated N-terminal protein ligation. Nature Protocols, 9 (2). pp. 253-262. ISSN 1754-2189
Abstract
Technologies that allow the efficient chemical modification of proteins under mild conditions are widely sought after. Sortase-mediated peptide ligation provides a strategy for modifying the N or C terminus of proteins. This protocol describes the use of depsipeptide substrates (containing an ester linkage) with sortase A (SrtA) to completely modify proteins carrying a single N-terminal glycine residue under mild conditions in 4–6 h. The SrtA-mediated ligation reaction is reversible, so most labeling protocols that use this enzyme require a large excess of both substrate and sortase to produce high yields of ligation product. In contrast, switching to depsipeptide substrates effectively renders the reaction irreversible, allowing complete labeling of proteins with a small excess of substrate and catalytic quantities of sortase. Herein we describe the synthesis of depsipeptide substrates that contain an ester linkage between a threonine and glycolic acid residue and an N-terminal FITC fluorophore appended via a thiourea linkage. The synthesis of the depsipeptide substrate typically takes 2–3 d.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | (c) 2014 Nature America, Inc. All rights reserved. This is an author produced version of a paper published in Nature Protocols Uploaded in accordance with the publisher's self-archiving policy. Available online here; https://doi.org/10.1038/nprot.2014.003. |
Keywords: | Biosynthesis; Chemical Modification; Peptides; Sensors and probes |
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) |
Depositing User: | Symplectic Publications |
Date Deposited: | 19 Mar 2018 14:13 |
Last Modified: | 20 Mar 2018 04:54 |
Status: | Published |
Publisher: | Springer Nature |
Identification Number: | 10.1038/nprot.2014.003 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:121578 |