Scalable Geometrically Designed Protein Cages Assembled via Genetically Encoded Split Inteins.

Engineering proteins to assemble into user-defined structures is key in their development for biotechnological applications. However, designing generic rather than bespoke solutions is challenging. Here we describe an expandable recombinant assembly system that produces scalable protein cages via split intein-mediated native chemical ligation. Three types of component are used: two complementary oligomeric "half-cage" protein fusions and an extendable monomeric "linker" fusion. All are composed of modular protein domains chosen to fulfill the required geometries, with two orthogonal pairs of split intein halves to drive assembly when mixed. This combination enables both one-pot construction of two-component cages and stepwise assembly of larger three-component scalable cages. To illustrate the system's versatility, trimeric half-cages and linker constructs comprising consensus-designed repeat proteins were ligated in one-pot and stepwise reactions. Under mild conditions, rapid high-yielding ligations were obtained, from which discrete proteins cages were easily purified and shown to form the desired trigonal bipyramidal structures.

Programmed Protein Self-Assembly Driven by Genetically Encoded Intein-Mediated Native Chemical Ligation.

Harnessing and controlling self-assembly is an important step in developing proteins as novel biomaterials. With this goal, here we report the design of a general genetically programmed system that covalently concatenates multiple distinct protein domains into specific assembled arrays. It is driven by iterative intein-mediated native chemical ligation (NCL) under mild native conditions. The system uses a series of initially inert recombinant protein fusions that sandwich the protein modules to be ligated between one of a number of different affinity tags and an intein protein domain. Orthogonal activation at opposite termini of compatible protein fusions, via protease and intein cleavage, coupled with sequential mixing directs an irreversible and traceless stepwise assembly process. This gives total control over the composition and arrangement of component proteins within the final product, enabled the limits of the system-reaction efficiency and yield-to be investigated, and led to the production of "functional" assemblies.