Enzyme-Cleavable Linkers for Protein Chemical Synthesis through Solid-Phase Ligations

Area of Science:

• Biochemistry
• Organic Chemistry
• Synthetic Biology

Background:

• Total protein synthesis relies on fragment assembly via ligation, but intermediate purification limits yield.
• Solid-supported chemical ligation (SPCL) offers a solution by immobilizing peptide segments on a solid support.
• Existing SPCL methods often use chemical linkers with cleavage conditions incompatible with sensitive proteins.

Purpose of the Study:

• To develop an alternative enzymatic approach for cleaving SPCL linkers under mild and selective conditions.
• To overcome the limitations of chemical cleavage in SPCL for sensitive protein targets.
• To demonstrate the utility of this enzymatic cleavage strategy in assembling long proteins.

Main Methods:

• Immobilization of a peptide segment on a solid support via a cleavable linker.
• Optimization of linker structure for compatibility with enzymatic cleavage.
• Utilizing a small enzyme capable of diffusing into the solid support for linker cleavage.
• Assembly of three peptide segments using native chemical ligation.

Main Results:

• Successful development of an enzymatic cleavage strategy for SPCL linkers.
• Demonstrated extremely mild and selective cleavage conditions.
• Optimization of linker structure and enzyme selection were critical for success.
• Assembly of a 15 kDa polypeptide from three peptide segments was achieved.

Conclusions:

• Enzymatic cleavage of SPCL linkers offers a significant advancement over traditional chemical methods.
• This strategy enables the synthesis of long proteins with improved yields and compatibility with sensitive targets.
• The developed method provides a mild and selective approach for protein assembly on solid supports.