Keeble, AH, Yadav, VK, Ferla, MP et al. (5 more authors) (2022) DogCatcher allows loop-friendly protein-protein ligation. Cell chemical biology, 29 (2). pp. 339-350. ISSN 2451-9448
Abstract
There are many efficient ways to connect proteins at termini. However, connecting at a loop is difficult because of lower flexibility and variable environment. Here, we have developed DogCatcher, a protein that forms a spontaneous isopeptide bond with DogTag peptide. DogTag/DogCatcher was generated initially by splitting a Streptococcus pneumoniae adhesin. We optimized DogTag/DogCatcher through rational design and evolution, increasing reaction rate by 250-fold and establishing millimolar solubility of DogCatcher. When fused to a protein terminus, DogTag/DogCatcher reacts slower than SpyTag003/SpyCatcher003. However, inserted in loops of a fluorescent protein or enzyme, DogTag reacts much faster than SpyTag003. Like many membrane proteins, the ion channel TRPC5 has no surface-exposed termini. DogTag in a TRPC5 extracellular loop allowed normal calcium flux and specific covalent labeling on cells in 1 min. DogTag/DogCatcher reacts under diverse conditions, at nanomolar concentrations, and to 98% conversion. Loop-friendly ligation should expand the toolbox for creating protein architectures.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2021 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). |
Keywords: | bioconjugation, protein engineering, protein design, split protein, synthetic biology, SpyTag, chemical biology, TRPC, epitope tag, ion channel |
Dates: |
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Institution: | The University of Leeds |
Academic Units: | The University of Leeds > Faculty of Medicine and Health (Leeds) > School of Medicine (Leeds) > Leeds Institute of Cardiovascular and Metabolic Medicine (LICAMM) > Discovery & Translational Science Dept (Leeds) |
Funding Information: | Funder Grant number British Heart Foundation PG/19/2/34084 |
Depositing User: | Symplectic Publications |
Date Deposited: | 03 Aug 2021 13:01 |
Last Modified: | 25 Jun 2023 22:43 |
Status: | Published |
Publisher: | Elsevier |
Identification Number: | 10.1016/j.chembiol.2021.07.005 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:176727 |