The cage-forming protein lumazine synthase is readily modified, evolved and assembled with other components.

Significance: Viruses consist of a protective proteinaceous shell that packages a DNA or RNA genome. The critical step in the evolution of primitive viruses was presumably the emergence of protein cages that could load, protect, and transfer their own genetic information. Here, we show that a nonviral protein cage formed by Aquifex aeolicus lumazine synthase and its encoding mRNA can be engineered and evolved into virus-like nucleocapsids. The optimized proteins specifically recognize designed motifs on cognate mRNAs, and these RNA tags can also be employed for the encapsulation of other RNA molecules. These model systems have great potential for the investigation of nucleocapsid assembly and evolution and as tailored protein compartments for protective packaging and delivery of nucleic acids.

Compartmentalization of proteases enables spatially and temporally controlled protein degradation in cells. Here we show that an engineered lumazine synthase protein cage, which possesses a negatively supercharged lumen, can exploit electrostatic effects to sort substrates for an encapsulated protease. This proteasome-like nanoreactor preferentially cleaves positively charged polypeptides over both anionic and zwitterionic substrates, inverting the inherent substrate specificity of the guest enzyme approximately 480 fold. Our results suggest that supercharged nanochambers could provide a simple and potentially general means of conferring substrate specificity to diverse encapsulated catalysts.