Hasegawa, H.; Wang, S.; Pelegri-O'Day, E.

Crystalline inclusion proteins CipA and CipB from Photorhabdus luminescens serve as versatile scaffolds for clustering genetically fused heterologous enzymes into crystalline inclusion bodies. Although engineered Cip crystals are known to function as solid biocatalysts for improving metabolite production in bacterial cells, the phase separation behavior of Cip proteins in non-bacterial cellular environments, as well as their biochemical attributes in a soluble, non-crystalline state, remain poorly understood. This study demonstrates that CipA and CipB efficiently undergo crystallization in the cytosol of human embryonic kidney cells both at normal and hypothermic cell culture conditions. Within 72 hours post-transfection, CipA and CipB become the most abundant proteins in transfected cells and produce distinctive cytosolic crystals often exceeding 10 m at least in one of the dimensions. Co-expression of CipA and CipB drives spontaneous demixing into two distinct crystal populations, and the orthogonality is maintained even when an unrelated third protein crystallizes in the same cytosol, permitting three crystal types to coexist simultaneously. Intracellular crystals are readily isolable from cells, and once purified, these crystals are stable under physiological pH conditions. However, CipA and CipB show notable differences in their crystal dissolution kinetics and protein oligomerization states when solubilized under acidic or alkaline conditions. These findings suggest that CipA/CipB forms a robust orthogonal self-assembly pair and establish CipA/CipB crystals as an efficient platform for producing biochemically programmable intracellular crystals. These properties should extend the Cip-based scaffolding approach to mammalian cell systems for synthetic biology applications.