Screening and Engineering of Ancestral Sortase Es That Enable High-Fidelity Enzyme Design with Activity Trade-Off.

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Protein grafting enables new protein functions through conjugation of diverse compounds.
• Sortase-mediated ligation (SML) is a key technique utilizing sortases for protein conjugation.

Purpose of the Study:

• To identify novel sortase E variants for enhanced SML.
• To investigate the structural basis of sortase E specificity.

Main Methods:

• In silico enzyme screening and ancestral sequence reconstruction (ASR) to design ancestral sortase Es.
• Expression and characterization of ancestral sortase E variants (1AcSE-8AcSE).
• HPLC analysis to assess enzymatic activity and specificity; structural analysis of sortase variants.

Main Results:

• Four ancestral sortase E variants (1AcSE, 2AcSE, 3AcSE, 8AcSE) were successfully expressed with moderate thermal stability.
• Sortase 2AcSE demonstrated unique substrate specificity, with structural analysis pointing to Loop A differences.
• A designed variant, ΔAcSE5, showed improved specificity and reduced byproduct formation in antibody conjugation.

Conclusions:

• Database-driven screening and ASR are effective for designing novel sortase E variants.
• Structure-function analysis guides the optimization of sortase E for specific SML applications.
• Engineered sortase E variants hold potential for advanced protein conjugation strategies.