DogCatcher allows loop-friendly protein-protein ligation. Issue 2 (17th February 2022) - Keeble, Anthony H.
Summary: 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. Graphical abstract: Highlights: Spontaneous transamidation at internal sites harnessing a DogTag/DogCatcher pair DogCatcher is designed and bred for high solubility and rapid reaction Within protein loops DogTag can clamp on its partner faster than SpyTag003 Fast and faithful fluorescent labeling of an ion channel at the cell surface via DogTag Abstract : Loopophilic protein coupling is achieved here by re-engineering a pathogenic adhesion protein. Many conjugation approaches only function at exposed termini. Keeble et al. create DogTag/DogCatcher for high-yield covalent labeling. Reactivity is robust to conditions and maintains protein function for catalysis, fluorescence and ion conductance after DogTag loop insertion.