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Sortase-Catalyzed Protein Domain Inversion.

Yan Zhou1, Thomas Durek1, David J Craik1

  • 1ARC Centre of Excellence for Innovations in Peptide and Protein Science, Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, 4072, Australia.

Angewandte Chemie (International Ed. in English)
|February 17, 2024
PubMed
Summary
This summary is machine-generated.

Researchers developed a novel protein domain inversion technique, altering protein structure by reversing a C-terminal segment. This unnatural permutation, achieved using engineered sortase A, expands protein engineering possibilities.

Keywords:
NanobodyPermutationSortaseTopologyTranspeptidase

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Area of Science:

  • Biochemistry
  • Protein Engineering
  • Synthetic Biology

Background:

  • Protein topological transformations, including cyclization and circular permutation, are key to generating novel protein functions and stability.
  • Existing methods, inspired by natural modifications, typically preserve the polypeptide backbone's N-to-C orientation.
  • These transformations are achieved through genetic encoding or enzymatic processing.

Purpose of the Study:

  • To introduce a new type of unnatural protein permutation: protein domain inversion.
  • To enzymatically invert a C-terminal protein segment from N-to-C to a C-to-C configuration relative to the N-terminal portion.
  • To demonstrate the feasibility and efficiency of this novel protein engineering strategy.

Main Methods:

  • Utilized an engineered sortase A, a transpeptidase enzyme, to catalyze the protein domain inversion.
  • Performed reactions under mild conditions, ranging from 4 to 25°C.
  • Employed heterologously-produced protein substrates compatible with the enzymatic reaction.

Main Results:

  • Successfully achieved protein domain inversions, creating a C-to-C linkage.
  • Demonstrated efficient reaction kinetics under mild temperature conditions.
  • Confirmed compatibility with proteins produced via heterologous expression.

Conclusions:

  • Protein domain inversion represents a novel topological transformation for protein engineering.
  • Engineered sortase A provides an effective tool for catalyzing this unnatural permutation.
  • The method offers a new avenue for designing proteins with altered structures and potentially novel functionalities.