SnoopLigase Catalyzes Peptide-Peptide Locking and Enables Solid-Phase Conjugate Isolation

Area of Science:

• Protein engineering
• Bioconjugation chemistry
• Enzyme technology

Background:

• Efficient methods for linking biological molecules are crucial for advancing biotechnology.
• Protein engineering offers a powerful approach to create novel catalysts with specific functions.

Purpose of the Study:

• To design and characterize a protein ligase (SnoopLigase) for efficient and site-specific peptide bond formation.
• To engineer peptide tags (SnoopTagJr and DogTag) for use with SnoopLigase.
• To demonstrate the application of SnoopLigase in enhancing enzyme stability and creating reusable catalysts.

Main Methods:

• Protein engineering guided by structural, bioinformatic, and computational analyses.
• Site-specific transamidation reaction catalyzed by SnoopLigase.
• Solid-phase immobilization of SnoopLigase for catalyst reuse.
• Enzyme cyclization to enhance thermoresilience.

Main Results:

• SnoopLigase achieved >95% efficiency in forming isopeptide bonds between SnoopTagJr and DogTag.
• SnoopTagJr and DogTag were functional at various protein positions, including internal sites.
• Solid-phase immobilized SnoopLigase allowed for high-purity product elution and catalyst reuse.
• Enzyme cyclization with SnoopLigase conferred exceptional thermoresilience (up to 100 °C) to model enzymes.

Conclusions:

• SnoopLigase is a highly efficient tool for protein conjugation and nanoassembly.
• The developed system enables the creation of robust, reusable biocatalysts.
• This approach offers a generic strategy for improving enzyme stability and utility.