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Proteins are involved in several cellular processes and biochemical reactions. Analyzing a specific protein of interest requires it to be isolated from the other proteins in the cell. This is achieved by overexpressing the specific gene in a suitable host to produce large quantities of the target protein. A tag or label is recombined with the gene to produce a fusion protein containing the target protein and the tag. The tags on these fusion proteins can then be used for easy detection and...
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Updated: Oct 26, 2025

OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy
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DogCatcher allows loop-friendly protein-protein ligation.

Anthony H Keeble1, Vikash K Yadav1, Matteo P Ferla1

  • 1Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.

Cell Chemical Biology
|July 29, 2021
PubMed
Summary
This summary is machine-generated.

We developed DogTag/DogCatcher, a novel protein ligation system that efficiently connects proteins within loops. This method enables precise protein engineering and labeling, even for challenging membrane proteins like TRPC5.

Keywords:
SpyTagTRPCbioconjugationchemical biologyepitope tagion channelprotein designprotein engineeringsplit proteinsynthetic biology

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Protein Engineering

Background:

  • Protein ligation is crucial for constructing complex protein architectures.
  • Connecting proteins at internal loops is challenging due to structural constraints.
  • Existing methods often struggle with efficiency and applicability to diverse protein environments.

Purpose of the Study:

  • To develop a novel protein ligation system, DogTag/DogCatcher, for efficient loop-based protein conjugation.
  • To optimize the DogTag/DogCatcher system for enhanced reaction kinetics and solubility.
  • To demonstrate the utility of DogTag/DogCatcher for labeling membrane proteins and enzymes within their functional loops.

Main Methods:

  • The DogTag/DogCatcher system was derived from a Streptococcus pneumoniae adhesin.
  • Rational design and directed evolution were employed to optimize the system's performance.
  • Kinetic assays, solubility measurements, and functional studies were conducted.
  • The system was tested in loop insertions within fluorescent proteins, enzymes, and the TRPC5 ion channel.

Main Results:

  • Optimized DogTag/DogCatcher exhibited a 250-fold increase in reaction rate and millimolar solubility.
  • Loop-inserted DogTag demonstrated significantly faster ligation kinetics compared to SpyTag003.
  • DogTag insertion into the TRPC5 extracellular loop enabled rapid, specific covalent cell labeling without affecting calcium flux.
  • The system achieved 98% conversion under diverse conditions and at nanomolar concentrations.

Conclusions:

  • DogTag/DogCatcher provides an efficient and versatile tool for loop-friendly protein ligation.
  • This technology expands the possibilities for creating novel protein architectures and functional protein conjugates.
  • The system's effectiveness with membrane proteins opens new avenues for studying and manipulating complex biological systems.