Laboratory evolution of a sortase enzyme that modifies amyloid-β protein

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Site-specific protein modification is crucial for research and therapeutics.
• Current methods often require genetic manipulation of target proteins.
• Amyloid-beta (Aβ) is a key protein implicated in neurodegenerative diseases.

Purpose of the Study:

• To evolve an epitope-specific enzyme for recognizing endogenous amyloid-beta (Aβ) without genetic manipulation.
• To develop a novel method for labeling and detecting Aβ in biological samples.
• To investigate the potential of sortase-mediated modification in preventing Aβ aggregation.

Main Methods:

• Laboratory evolution of bacterial sortase A using yeast display selection.
• Directed evolution to alter substrate specificity from LPESG to LMVGG.
• Application of the evolved sortase for labeling endogenous Aβ in human cerebrospinal fluid.

Main Results:

• Achieved a >1,400-fold increase in substrate preference for the LMVGG sequence.
• Successfully labeled endogenous Aβ in cerebrospinal fluid with high sensitivity.
• Demonstrated that conjugation of a peptide to Aβ42 impedes its aggregation.

Conclusions:

• Laboratory evolution can create epitope-specific enzymes for endogenous targets without genetic manipulation.
• Evolved sortase A provides a sensitive tool for Aβ detection and modification.
• Sortase-mediated labeling offers a potential strategy to inhibit Aβ aggregation.