Scalable Geometrically Designed Protein Cages Assembled via Genetically Encoded Split Inteins

Abstract

Engineering proteins to assembly into user-defined structures is key in their development for biotechnological applications. However, designing generic rather than bespoke solutions is a challenging task. Here, we describe an expandable recombinant assembly system that produces scalable protein cages via split intein-mediated native chemical ligation. Three types of construct are used: a pair of oligomeric mirror-image “half-cages” and an extendable “linker. All constructs are composed of modular protein domains chosen to fulfil the required geometries, fused to 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, tetrahedral 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.