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Sortase-Mediated Multi-Fragment Assemblies by Ligation Site Switching.

Jan Bierlmeier1, Miguel Álvaro-Benito2, Maren Scheffler1

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Summary

This study introduces a novel multi-fragment sortase-mediated ligation (SML) technique using switchable substrates. This method enables the assembly of complex peptide structures, overcoming previous limitations in protein chemistry.

Keywords:
Chemical BiologyPeptide ligationProtein bioconjugationProtein semisynthesisSortase-mediated ligation

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Area of Science:

  • Biochemistry
  • Chemical Biology
  • Molecular Biology

Background:

  • Sortase-mediated ligation (SML) is a key protein chemistry technique for joining peptides with LPxTG motifs and glycine nucleophiles.
  • Current SML methods are limited in assembling multiple peptide fragments due to the irreversible nature of the sorting motif in the product.

Purpose of the Study:

  • To develop a novel multi-fragment SML strategy enabling the assembly of more complex peptide structures.
  • To overcome the limitations of existing SML techniques for sequential peptide assembly.

Main Methods:

  • Developed switchable sortase substrates by replacing Leu with disulfide-containing Cys(StBu) in LPxTG motifs.
  • Utilized a photo-protected N-Gly nucleophile to enable sequential ligation and site switching.
  • Demonstrated feasibility through a four-fragment ligation and assembly of peptide probes.

Main Results:

  • Successfully demonstrated multi-fragment SML by creating switchable sortase substrates.
  • Assembled peptide probes for bivalent chromatin binding proteins and oligomerized peptide antigens.
  • Confirmed the functionality of the assembled probes using biochemical and immuno-assays.

Conclusions:

  • The developed switchable SML approach facilitates complex peptide assembly, overcoming previous limitations.
  • The method is versatile, enabling the creation of functional peptide probes for applications in immunology and chromatin biochemistry.