BIOMATERIAL APPLICATIONS OF CROSS-LINKED POROUS PROTEIN MICROCRYSTALS

Abstract

Porous protein crystals represent a promising but underexplored class of biomaterials for therapeutic delivery, molecular capture, and biofunctional scaffolding. This dissertation investigates the synthesis, stabilization, and biomedical applications of cross-linked porous protein microcrystals derived from a putative polyisoprenoid-binding protein from Campylobacter jejuni (“CJ”). These crystals possess a highly ordered P622 lattice with large solvent channels and can be reproducibly generated as microcrystals through batch crystallization and stabilized via chemical cross-linking.Chapter 1 provides a review of the broader landscape of large-pore protein crystals, including methods of synthesis, stabilization, validation, and emerging applications in catalysis, delivery, and functional materials. In chapter 2, I demonstrate that CJ microcrystals can adsorb and retain diverse nucleic acid cargos, including single-stranded DNA, RNA, and plasmid DNA. Guest loading is rapid, dependent on pH and guest size, and can be partially reversed by exposure to high ATP concentrations, indicating specific but heterogeneous interactions between nucleic acids and the crystal scaffold. Because unmodified pores remain accessible to nucleases, I further show that a hexameric protein complex derived from the D2 domain of N-ethylmaleimide sensitive factor can function as a physical pore cap, significantly reducing nuclease-mediated degradation of guest nucleic acids. In chapter 3, I extend the CJ platform toward selective capture of virus-sized particles by engineering SpyTag-bearing “SpyCrystals” that covalently host SpyCatcher fusion proteins. Functionalized crystals displaying SARS-CoV-2 spike binders selectively adsorb virions in vitro and can be integrated into a pump-driven filtration workflow for capture from wastewater. In chapter 4, I also evaluate ex vivo biocompatibility in precision-cut lung slices, showing that unloaded CJ crystals do not measurably impair tissue health or alter B-cell abundance, while lipopolysaccharide-loaded crystals elicit a moderated biological response consistent with guest release. Finally, in chapter 5, I investigate in vivo biodistribution using bioluminescent CJ microcrystals and find evidence consistent with rapid pulmonary exposure followed by partial retention in skeletal muscle microvasculature. Collectively, this dissertation establishes cross-linked porous protein microcrystals as structurally precise, modular biomaterials with potential applications in nucleic acid delivery, pathogen capture, localized depot formation, and engineered therapeutic scaffolding.