Mimicking reverse protein splicing by three-segment tandem peptide ligation

Area of Science:

• Chemical Biology
• Synthetic Chemistry
• Biochemistry

Background:

• Protein splicing is a natural process where inteins excise themselves from precursor proteins.
• This process is highly specific and efficient, offering potential for protein engineering.
• Current methods for peptide synthesis and ligation have limitations in efficiency and modularity.

Purpose of the Study:

• To develop a novel method for peptide ligation.
• To mimic the reverse process of protein splicing for peptide synthesis.
• To create a bi-directional and interchangeable three-segment peptide ligation system.

Main Methods:

• Utilized a synthetic intein to mediate peptide ligation.
• Employed a three-segment strategy involving N, M, and C-segments.
• Designed the M-segment with specific N-terminal (Ser/Thr) and C-terminal thioester functionalities.

Main Results:

• Successfully achieved bi-directional and interchangeable ligation of N, M, and C-segments.
• Demonstrated the formation of a tripartite NMC-peptide.
• Validated the role of the synthetic intein and the M-segment's specific features in the ligation process.

Conclusions:

• The developed method provides a novel approach for synthesizing complex peptides.
• This technique offers a modular and efficient way to assemble peptides, inspired by reverse protein splicing.
• The synthetic intein-mediated ligation system has potential applications in peptide synthesis and drug discovery.