Protein semi-synthesis in living cells

Area of Science:

• Biochemistry
• Molecular Biology
• Chemical Biology

Background:

• Attaching chemical probes to proteins is crucial for understanding biological pathways and engineering new protein functions.
• Existing methods for intracellular protein modification can be limited in scope or efficiency.

Purpose of the Study:

• To develop a novel intracellular protein ligation strategy for precise chemical probe incorporation.
• To enable the engineering of cellular proteins with diverse abiotic molecules.

Main Methods:

• Genetically tagging cellular proteins with one half of a split intein.
• Linking the complementary intein half to a synthetic probe in vitro.
• Delivering the probe-intein fusion into cells using a transduction peptide, triggering protein trans-splicing and probe ligation.

Main Results:

• Successfully demonstrated intracellular ligation of synthetic probes to target proteins via a peptide bond.
• The method is specific and effective for both cytosolic and integral membrane proteins.
• Validated the ability to elaborate cellular proteins with various abiotic probes.

Conclusions:

• The described split intein-based protein trans-splicing technology enables efficient and specific intracellular ligation of synthetic molecules to proteins.
• This approach offers a versatile platform for chemical biology research and protein engineering applications.