Humanization and directed evolution of the selenium-containing scFv phage abzyme.

Area of Science:

• Biochemistry and Molecular Biology
• Enzyme Engineering
• Drug Discovery

Background:

• Glutathione peroxidase (GPX) is crucial for cellular defense against oxidative stress.
• Developing artificial enzymes (abzymes) with GPX activity is a key goal in bioengineering.
• Synthetic antibody fragments (scFv) offer a versatile platform for abzyme development.

Purpose of the Study:

• To synthesize glutathione derivatives (haptens) for antibody selection.
• To create and select single-chain variable fragment (scFv) phage displaying abzymes with GPX activity.
• To enhance the catalytic activity and binding affinity of engineered abzymes.

Main Methods:

• Synthesis of three glutathione derivatives (haptens): GSH-S-DNP butyl ester, GSH-S-DNP hexyl ester, and GSH-S-DNP hexamethylene ester.
• Four-round panning of a human synthetic scFv phage library against synthesized haptens.
• Chemical mutation of serine residues to selenocysteine in selected scFv phage particles.
• Directed evolution using DNA shuffling to improve hapten binding affinity and catalytic activity.

Main Results:

• Successfully synthesized three hapten derivatives.
• Selected scFv phage particles with specific binding activity to each hapten.
• Engineered selenium-containing scFv abzymes exhibiting GPX activity up to 3000 U μmol⁻¹.
• Achieved a 17% increase in catalytic activity after directed evolution and DNA shuffling.

Conclusions:

• Demonstrated the successful creation of functional GPX-mimicking abzymes using scFv phage display and selenium incorporation.
• Highlighted the potential of these engineered abzymes for therapeutic applications and drug development.
• Provided a novel strategy for designing high-affinity and high-activity abzymes for specific targets.