Design and Evolution of Enhanced Peptide-Peptide Ligation for Modular Transglutaminase Assembly

Area of Science:

• Synthetic Biology
• Biochemistry
• Protein Engineering

Background:

• Synthetic nanoarchitectures require robust tools for enhanced functionality and resilience.
• Existing protein conjugation methods often lack speed, efficiency, or compatibility with biological systems.

Purpose of the Study:

• To engineer a fast-acting molecular superglue for precise protein functionalization.
• To develop a genetically encoded system for efficient transamidation in biological environments.

Main Methods:

• Directed evolution and rational design of bacterial adhesion proteins.
• Phage display screening to optimize SnoopTag2 and DogTag2 peptides for rapid reaction.
• Engineering of transglutaminase 2 (TG2) for enhanced stability and minimal self-reactivity.
• Application of the SnoopLigase2 system for covalent display and functionalization in mammalian cells.

Main Results:

• Generation of the SnoopLigase2 coupling system, achieving over 99% completion in transamidation.
• Over 1000-fold acceleration of the reaction rate, with broad compatibility across buffers, pH, and temperatures.
• Successful covalent display of TG2:TGFα conjugates on the plasma membrane via the mammalian secretory pathway.
• Demonstration of retained TG2 activity and stable anchoring of TGFα for signal activation, leading to reprogrammed cell behavior.

Conclusions:

• The SnoopLigase2 system provides a powerful, genetically encoded tool for rapid and specific protein conjugation.
• This modular toolbox enables novel applications in biomaterials and the engineering of complex cellular environments.
• The developed system overcomes limitations of genetic fusion for protein functionalization in vivo.