Design of a Genetically Programmable and Customizable\nProtein Scaffolding System for the Hierarchical Assembly of Robust,\nFunctional Macroscale Materials

Inspired\nby the properties of natural protein-based biomaterials,\nprotein nanomaterials are increasingly designed with natural or engineered\npeptides or with protein building blocks. Few examples describe the\ndesign of functional protein-based materials for biotechnological\napplications that can be readily manufactured, are amenable to functionalization,\nand exhibit robust assembly properties for macroscale material formation.\nHere, we designed a protein-scaffolding system that self-assembles\ninto robust, macroscale materials suitable for in vitro cell-free\napplications. By controlling the coexpression in Escherichia\ncoli of self-assembling scaffold building blocks with\nand without modifications for covalent attachment of cross-linking\ncargo proteins, hybrid scaffolds with spatially organized conjugation\nsites are overproduced that can be readily isolated. Cargo proteins,\nincluding enzymes, are rapidly cross-linked onto scaffolds for the\nformation of functional materials. We show that these materials can\nbe used for the in vitro operation of a coimmobilized two-enzyme reaction\nand that the protein material can be recovered and reused. We believe\nthat this work will provide a versatile platform for the design and\nscalable production of functional materials with customizable properties\nand the robustness required for biotechnological applications.