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Updated: Jul 1, 2026

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

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Proximity-Driven Protein Ligation Beyond the Concentration Limit.

Jianhui Yin¹, Xinliang Liu², Zhe Meng¹

¹Shanghai Key Laboratory for Antibody-Drug Conjugates with Innovative Target, National Key Laboratory of Innovative Immunotherapy, School of Chemistry and Chemical Engineering, Shanghai Jiao Tong University, Shanghai 200240, China.

Journal of the American Chemical Society

|June 30, 2026

Summary

A new photocleavable picolyl linker enables mild, postsynthetic installation of bio-orthogonal reagents. This strategy enhances protein ligation efficiency at low concentrations, overcoming challenges with large or hydrophobic proteins.

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Published on: September 2, 2019

Area of Science:

Chemical Biology
Biochemistry
Synthetic Chemistry

Background:

Classic protein synthesis faces limitations in kinetics and concentration requirements, hindering the production of large or hydrophobic proteins.
Existing auxiliary strategies for protein modification often require harsh conditions and are incompatible with expressed protein ligation (EPL).

Purpose of the Study:

To develop a novel linker strategy for mild, postsynthetic installation of bio-orthogonal reagents onto peptides and proteins.
To overcome low-concentration synthesis bottlenecks and enable efficient ligation of challenging protein targets.

Main Methods:

Development of a photocleavable picolyl (Pic) linker for installing inverse-electron-demand Diels-Alder (IEDDA) reagents.
Application of the Pic linker strategy under mild conditions for postsynthetic modification of expressed proteins.
Demonstration of one-pot IEDDA ligation, EPL, and desulfurization/Pic linker cleavage.

Main Results:

Successful postsynthetic installation of IEDDA reagents onto synthetic peptides and expressed proteins under mild conditions.
Efficient ligation of expressed G-CSF and SARS-CoV-2 RBD fragments at micromolar concentrations using the IEDDA strategy.
Demonstration that the developed method overcomes limitations of conventional approaches for low-concentration protein ligation.

Conclusions:

The photocleavable picolyl linker strategy provides a powerful tool for accessing challenging hydrophobic or large proteins.
This method enhances effective peptide concentration through rapid IEDDA kinetics, enabling efficient ligation at low concentrations.
The one-pot procedure combining IEDDA ligation, EPL, and linker cleavage offers a versatile approach for complex protein synthesis.