High order assembly of multiple protein cages with homogeneous sizes and shapes via limited cage surface engineering

Protein cages are attractive building blocks to build high order materials such as 3D cage lattices, which offer accurately ordered bio-templates. However, controlling the size or valency of these cage-to-cage assemblies is extremely difficult due to highly multivalent and symmetric cage structures. Here, various high order cage assemblies with homogeneous sizes and geometries are constructed by developing an anisotropic ferritin cage with limitedly exposed binding modules, leucine zipper. The anisotropic ferritin is produced as expressed in cells without the need of complex in vitro cage fabrication by careful subunit manipulation. Ferritin cages with limitedly exposed zippers are assembled around a core ferritin with fully exposed opposing zippers, generating homogeneous high order structures, whereas two fully exposed ferritins are assembled into heterogeneous cage aggregates. Diverse fully exposed core cages are prepared by varying the zipper-ferritin fusion geometries and even by using larger cage structures. With these core cages and the anisotropic ferritin, a range of high order cage assemblies with diverse ferritin valencies (3 to over 12) and sizes (over 40 nm) are created. Cell surface binding and internalization of cage structures are greatly varied by assembly sizes, where high order ferritins are clearly more effective than monomeric ferritin.