DogCatcher allows loop-friendly protein-protein ligation

Journal article


Heeble, A. H., Yadav, VK, Ferla, M. P., Bauer, C. C., Chuntharpursat-Bon, C., Huang, J., Bon, R. S. and Howarth, M. 2022. DogCatcher allows loop-friendly protein-protein ligation. Cell Chemical Biology. 29 (2), p. 339–350. https://doi.org/10.1016/j.chembiol.2021.07.005
AuthorsHeeble, A. H., Yadav, VK, Ferla, M. P., Bauer, C. C., Chuntharpursat-Bon, C., Huang, J., Bon, R. S. and Howarth, M.
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.

KeywordsProtein engineering; isopeptide bond; covalent labeling; Ligation
Year2022
JournalCell Chemical Biology
Journal citation29 (2), p. 339–350
PublisherElsevier
ISSN2451-9448
Digital Object Identifier (DOI)https://doi.org/10.1016/j.chembiol.2021.07.005
Web address (URL)https://www.cell.com/cell-chemical-biology/fulltext/S2451-9456(21)00315-9?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS2451945621003159%3Fshowall%3Dtrue
https://pubmed.ncbi.nlm.nih.gov/34324879/
Accepted author manuscript
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Open
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File Access Level
Open
Output statusPublished
Publication dates
Online28 Jul 2021
Publication process dates
Deposited16 Dec 2022
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