Proximity-Driven Protein Ligation Beyond the Concentration Limit

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.