Directed Evolution of a Protein Container

An engineered protein container protects its bacterial host by efficient and selective encapsulation of a toxic protease. Confinement of enzymes in protein nanocompartments represents a potentially powerful strategy for controlling catalytic activity in cells. By using a simple electrostatically based tagging system for protein encapsulation, we successfully sequestered HIV protease, a toxic enzyme when produced cytoplasmically, within an engineered lumazine synthase capsid. The growth advantage resulting from protecting the Escherichia coli host from the protease enabled directed evolution of improved capsids. After four rounds of mutagenesis and selection, we obtained a variant with a 5- to 10-fold higher loading capacity than the starting capsid, which permitted efficient growth even at high intracellular concentrations of HIV protease. The superior properties of the evolved capsid can be ascribed to multiple mutations that increase the net negative charge on its luminal surface and thereby enhance engineered Coulombic interactions between host and guest. Such structures could be used for diverse biotechnological applications in living cells.