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Related Experiment Video

Updated: May 24, 2026

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells
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Protein ligation in living cells using sortase.

Karin Strijbis1, Eric Spooner, Hidde L Ploegh

  • 1Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, USA.

Traffic (Copenhagen, Denmark)
|February 22, 2012
PubMed
Summary

This study demonstrates intracellular sortagging using Streptococcus pyogenes sortase A (SrtA) in yeast and mammalian cells. This protein ligation method enables site-specific protein modification within living cells and the endoplasmic reticulum.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Background:

  • Sortagging is a powerful in vitro technique for site-specific protein modification.
  • Adapting sortagging for in vivo applications presents unique challenges and opportunities.

Purpose of the Study:

  • To investigate the feasibility of intracellular sortagging in living cells.
  • To adapt the sortase A (SrtA) method for protein ligation within eukaryotic cells.

Main Methods:

  • Utilized Ca²⁺-independent sortase A (SrtA) from Streptococcus pyogenes.
  • Engineered substrate proteins with a C-terminal LPXTG recognition motif.
  • Employed N-terminally (oligo)glycine-tagged proteins as nucleophiles.
  • Performed sortagging in Saccharomyces cerevisiae and HEK293T mammalian cells, including cytosol and ER lumen.

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Detection of Detergent-sensitive Interactions Between Membrane Proteins
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Published on: March 7, 2018

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Last Updated: May 24, 2026

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells
08:58

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

Published on: September 2, 2019

Detection of Detergent-sensitive Interactions Between Membrane Proteins
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Detection of Detergent-sensitive Interactions Between Membrane Proteins

Published on: March 7, 2018

Main Results:

  • Successfully achieved sortase-dependent protein ligation in both yeast and mammalian cells.
  • Demonstrated successful sortagging within the cytosol and the endoplasmic reticulum lumen.
  • Showcased ER luminal sortagging for secretion of ligation products, including circular polypeptides.
  • Observed protein ligation occurring rapidly, within 30 minutes of translation.
  • Illustrated the method's versatility through ligation with multiple substrates and fluorescent protein nucleophiles in various cellular compartments.

Conclusions:

  • Intracellular sortagging is a viable method for site-specific protein modification in living cells.
  • The SrtA-mediated ligation system functions effectively in diverse cellular compartments.
  • This technique offers new possibilities for protein engineering and functional studies in vivo.