2025 Theses Doctoral

Characterization and engineering of calcium-binding proteins for recovery and separation of rare earth elements

Khoury, Farid Farid

Rare earth elements (REEs), which include the 15 lanthanides plus scandium and yttrium, are essential components in permanent magnets, electronics, and green energy technologies. However, the separation of REEs remains a major industrial and environmental challenge due to their highly similar physicochemical properties. Conventional separation processes are chemically and energy intensive, generating significant waste. Proteins that natively bind calcium ions offer a promising bio-based platform for selective REE recognition due to the shared ionic radii and coordination geometries between REEs and calcium. This dissertation explores protein-based strategies to selectively bind and separate REEs using rational protein engineering, bioinformatics, and directed evolution.

In Chapter One, we investigated the REE-binding potential of the Block V of the RTX domain from the adenylate cyclase protein of Bordetella pertussis. This domain is intrinsically disordered and folds into a β-roll structure upon binding eight calcium ions. Using a FRET-based assay, we showed that the RTX domain binds trivalent lanthanide ions with significantly higher affinities than calcium, exhibiting apparent dissociation constants ranging from 20–75 µM. Notably, the domain demonstrates higher selectivity for heavy REEs over light REEs. Circular dichroism spectroscopy revealed that the domain undergoes pH-induced folding even in the absence of metal ions, suggesting protonation of key residues enables structure formation in acidic environments. Equilibrium ultrafiltration confirmed that the domain can coordinate up to four REE ions under extreme acidic conditions (pH < 1). Furthermore, we demonstrated practical application of the RTX domain by selectively recovering Nd and Dy from Fe and Co in a simulated NdFeB magnet leachate at pH 6.

In Chapter Two, we expanded our search for REE-binding proteins by conducting a bioinformatic analysis of calcium-binding peptides and domains. Seven unique candidates with distinct calcium-binding loop geometries were selected for experimental evaluation. This study revealed that the loop charge strongly correlates with REE affinity: highly charged, aspartic acid-rich loops demonstrated enhanced ion binding due to electrostatic repulsion and stabilization effects. Affinity trends across the lanthanide series favored ions with radii closest to calcium (~1 Å), consistent with evolutionary pressure for calcium selectivity. In solution, binding selectivity varied between proteins; however, immobilized proteins showed enhanced selectivity for intermediate REEs. One top-performing candidate, HEW5 from Nocardioides zeae, was immobilized in a 7 mL column and successfully achieved chelator-free, single-step separation of an equimolar La/Nd mixture. The separation yielded over 90% purity and 90% recovery. Moreover, immobilized HEW5 effectively removed non-REE ions from a simulated leachate stream and enabled selective separation of lanthanum from other REEs in a single chromatographic stage.

In Chapter Three, we addressed the need for rapid and efficient engineering of REE-selective proteins by developing a phage-assisted continuous evolution (PACE) system tailored for lanthanide binding. We constructed a selection circuit based on a lanthanide-mediated protein protein interaction, enabling high-throughput evolution of a calmodulin-derived peptide library. Within days, the system yielded a dominant evolved sequence with a restructured hydrogen bond network that enhanced second-shell ion coordination and overall protein packing. The evolved protein exhibited improved binding affinity and thermal stability, enabling high-purity, singlestage, chelator-free separations of individual REE ions. This work establishes a platform that can be extended to evolve binding domains for other critical metals, significantly expanding the scope of protein-based separation technologies.

In Chapter Four, we applied rational protein engineering to improve the selectivity and performance of the RTX domain. A truncated variant, termed RTX(2), was designed to reduce the heterogeneity of ion-binding sites. This engineered scaffold displayed enhanced REE binding affinity, greater structural stability, and pH-tunable elution characteristics. RTX(2) achieved over 200-fold selectivity between REE species during chromatographic separation, demonstrating improved resolution within the lanthanide series. This construct offers a robust, tunable platform for future protein-based separation technologies with direct applications in REE recycling and purification.

Together, these four studies demonstrate the power of combining natural protein scaffolds, bioinformatic screening, directed evolution, and rational design to address one of the most pressing materials challenges of the 21st century. This dissertation aimed to advance the development of protein-based REE separation platforms by revealing fundamental structure–function relationships and providing novel tools for scalable, sustainable metal recovery.