POMPOMS: Crosslinked biomolecular condensates as a versatile platform for multifunctional protein microparticles.

Abstract

Protein-based microparticles are promising materials for applications such as biocatalysis and biomolecular capture, yet their fabrication by existing techniques remains challenging due to protein denaturation or lack of spatial control. Here, we present a method for synthesizing microscale protein-based materials by chemically crosslinking biomolecular condensates. Leveraging the liquid-liquid phase separation behavior of intrinsically disordered RGG domains, we sequestered RGG-tagged fusion proteins into droplets, then we solidified them into porous microparticles using the homobifunctional, amine-reactive crosslinker BS3. By modulating protein concentration and condensate coalescence, we controlled microparticle size from 40 {micro}m. We then demonstrated three encodable functionalities: We used the SpyCatcher/SpyTag system to capture cargo proteins, we crosslinked core-shell condensates to generate microparticles with controlled spatial organization, and we immobilized a thermostable alcohol dehydrogenase with 31% retained enzymatic activity. These POMPOMS (protein-based, self-organized microparticles of multifunctional significance) represent a sustainable, tunable platform for versatile protein-based materials.