Directed evolution of APEX2 for electron microscopy and proximity labeling

Area of Science:

• Biochemistry
• Cell Biology
• Proteomics

Background:

• Engineered peroxidase APEX is used as an electron microscopy tag and labeling enzyme for live-cell proteomics.
• Limited sensitivity of APEX restricts its use in applications requiring low expression levels.

Purpose of the Study:

• To improve the catalytic efficiency and sensitivity of APEX for broader applications in live-cell research.
• To develop a more active peroxidase enzyme for enhanced electron microscopy and proteomics.

Main Methods:

• Yeast-display evolution was employed to enhance the catalytic efficiency of the APEX enzyme.
• The engineered enzyme, APEX2, was tested for activity and sensitivity in cellular environments.

Main Results:

• APEX2 exhibits significantly higher activity in cells compared to the original APEX.
• The enhanced sensitivity of APEX2 enables electron microscopy to resolve submitochondrial localization of proteins like MICU1.
• APEX2 facilitates superior enrichment of endogenous mitochondrial and endoplasmic reticulum membrane proteins.

Conclusions:

• APEX2 represents a substantial improvement over APEX, offering enhanced catalytic efficiency and sensitivity.
• The improved enzyme expands the utility of APEX technology for high-resolution cellular imaging and deep proteomics.
• APEX2 is a valuable tool for studying protein localization and interactions within organelles.