Continuous Evolution of Calmodulin for High-Purity Separation of Rare Earth Elements

Area of Science:

• Biochemistry
• Materials Science
• Chemical Engineering

Background:

• Separating rare earth elements (REEs) is challenging due to their similar properties, necessitating complex industrial processes.
• Protein-based methods present a sustainable and selective alternative for REE separation.
• Current protein engineering approaches for REE separation are limited by low throughput, restricting optimization.

Purpose of the Study:

• To develop a high-throughput protein engineering method for enhanced REE separation.
• To evolve a calmodulin-derived peptide for selective binding and separation of individual REEs.
• To demonstrate the utility of phage-assisted continuous evolution (PACE) for metal-binding protein engineering.

Main Methods:

• Developed a lanthanide-mediated protein-protein interaction selection circuit for PACE.
• Utilized PACE to rapidly evolve a calmodulin-derived peptide library against REEs.
• Employed molecular dynamics simulations to analyze structural changes in the evolved protein.

Main Results:

• Identified a dominant evolved peptide sequence within days using the PACE system.
• Molecular dynamics simulations revealed enhanced second-shell ion coordination and protein packing.
• The evolved protein demonstrated improved binding affinity and thermal stability.
• Achieved single-stage, chelator-free, high-purity separation of individual REEs.

Conclusions:

• The developed PACE system significantly accelerates the engineering of metal-binding proteins.
• The evolved peptide enables efficient and selective separation of critical rare earth elements.
• This high-throughput strategy is applicable for engineering other metal-binding proteins for various applications.