Biocrystallization: Harnessing natural scaffolds for stable protein crystal formulations

There is a growing interest in developing precisely engineered and reliable crystalline formulations of proteins for medical and biotechnological applications. However, protein crystallization remains a complex and often unpredictable process, influenced by numerous factors governing nucleation and crystal growth. Furthermore, industrial-scale crystallization and purification techniques are still underdeveloped, presenting a significant bottleneck for broad implementation. Consequently, innovative strategies are required to address the challenges associated with large-scale crystal particle formulation, particularly for fragile therapeutic and industrial relevant proteins. Certain natural protein scaffolds possess intrinsic sequence features that promote spontaneous crystal lattice formation, often serving functional roles in their native organisms. These scaffolds can drive crystal formation either in native stage or with foreign cargo proteins through heterologous expression in recombinant host cells. By leveraging these properties, target proteins can be packed into stable crystalline matrices that support encapsulation, protection, and sustained release of functional cargo proteins. Such scaffold-based in cellulo systems offer advantages in terms of reproducibility, manipulability, and potential for nanoscale engineering. They are emerging as powerful platforms for drug delivery, protein immobilisation, and functional biomaterials. This review provides a comprehensive overview of scaffold-assisted in-cell biocrystallization of cargo proteins. We discussed naturally occurring crystalline scaffolds, their fusion design strategies, potential applications, and highlighted critical challenges and future directions. Together, these insights illustrate how harnessing natural crystallization mechanisms can enable the next generation of protein-based technologies for healthcare, catalysis, and synthetic biology. • Spontaneous self-assembly events are central to highly ordered protein architectures • In-cell biocrystallization offers an efficient route for stable protein formulation. • It is a genetically encoded and scalable way to produce synthetic crystalline particles. • Such cargo encapsulated particles are suitable for drug delivery and biomaterial engineering.