A dual-drive strategy for enhanced protein crystallization with sodium alginate/hyaluronic acid film: Protein adsorption and supersaturation regulation.

Protein crystallization is essential for determining the three-dimensional structures of biomacromolecules and advancing biopharmaceutical development, yet it remains a major challenge in structural biology due to common issues like slow nucleation rates and inconsistent crystal quality. Herein, a dual-drive crystallization (DDC) strategy, relying on a composite film of sodium alginate (SA) and hyaluronic acid (HA), is reported to synergistically regulate both protein adsorption and solution supersaturation. Driven by the electrostatic interactions of SA and the water absorption properties of HA, the SA/HA film achieves enhanced crystallization efficiency and controlled crystal quality mainly. It significantly reduces lysozyme nucleation time by over 66.0 % and better controls crystal size distribution. Molecular simulations further reveal a strong electrostatic interaction energy of -17.0 kcal·mol between protein and SA, which enhances protein adsorption and then promotes cluster formation, nucleation, and crystal growth. Additionally, the DDC strategy efficiently promotes the crystallization of both thaumatin and proteinase K, enhancing the crystallization success rate for proteins with opposite charges. These results highlight the advantages and promising potential of SA/HA film-assisted protein crystallization for effectively producing protein crystals suitable for diverse applications.