Biomolecular Click Reactions Using a Minimal pH-Activated Catcher/Tag Pair for Producing Native-Sized Spider-Silk...

Area of Science:

• Biochemistry
• Protein Engineering
• Biotechnology

Background:

• Catcher/Tag protein pairs spontaneously form isopeptide bonds for biomolecular click reactions.
• Covalent protein conjugation is vital in biotechnology and biomedicine.
• Expanding the library of orthogonal Catcher/Tag pairs is crucial for advanced applications like multiple-fragment ligation.

Purpose of the Study:

• To engineer novel Catcher/Tag pairs for protein ligation.
• To enhance the toolbox of orthogonal Catcher/Tag pairs for broader applications.
• To develop a new system for producing highly repetitive spider-silk-like proteins.

Main Methods:

• Engineering of novel Catcher/Tag pairs using a crystal structure of a minimal CnaB domain.
• Demonstration of pH-inducible protein ligation with the engineered SilkCatcher/Tag pair.
• Compatibility testing of SilkCatcher/Tag with the established SpyCatcher/Tag pair.

Main Results:

• Successful engineering of the SilkCatcher/Tag pair for efficient protein ligation.
• Demonstration of pH-inducible ligation capabilities.
• Compatibility of SilkCatcher/Tag with SpyCatcher/Tag, enabling orthogonal applications.
• Production of native-sized, highly repetitive spider-silk-like proteins with >90% purity.

Conclusions:

• The engineered SilkCatcher/Tag pair expands the available orthogonal Catcher/Tag systems.
• This new system facilitates efficient, pH-controllable protein conjugation.
• SilkCatcher/Tag enables novel recombinant production strategies for complex protein structures like spider silk.