Kinetic Controlled Tag-Catcher Interactions for Directed Covalent Protein Assembly

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Protein assembly strategies are crucial for controlling macromolecular structures.
• The SpyTag/SpyCatcher system from Streptococcus pyogenes offers a rapid protein ligation approach.
• Expanding protein ligation tools is essential for advanced molecular engineering.

Purpose of the Study:

• To engineer and characterize a novel Tag-Catcher protein pair from Streptococcus dysgalactiae.
• To assess the specificity and utility of the new SdyTag/SdyCatcher system.
• To demonstrate sequential protein assembly and in vivo protein circularization using the new system.

Main Methods:

• Engineering of SdyTag based on the CnaB domain and SdyCatcher.
• Specificity assays comparing SdyTag/SdyCatcher with SpyTag/SpyCatcher.
• In vitro demonstration of sequential assembly of tagged proteins.
• In vivo demonstration of Tag-Catcher specific protein circularization.

Main Results:

• SdyTag exhibits high specificity, with SpyCatcher showing 320-fold lower reactivity.
• SdyTag demonstrates 75-fold specificity for SdyCatcher over SpyCatcher.
• Successful sequential assembly of tagged proteins in vitro was achieved.
• Tag-Catcher specific in vivo protein circularization was demonstrated.

Conclusions:

• The novel SdyTag/SdyCatcher system provides enhanced specificity for protein ligation.
• This system enables precise, sequential control over protein assembly.
• The engineered system holds potential for constructing novel protein architectures and multiplexed assembly.