Zhang, R.; Kang, S.; Gaascht, F. J.; Pena, E. L.; Schmidt-Dannert, C.

Inspired by the properties of natural protein-based biomaterials, protein nanomaterials are increasingly designed with natural or engineered peptides, or with protein building blocks. Very few examples describe the design of functional protein-based materials for biotechnological applications that can be readily manufactured, are amenable to functionalization, and exhibit robust assembly properties for macroscale material formation. Here, we designed a protein-scaffolding system that self-assembles into robust, macroscale materials suitable for cell-free applications. By controlling the co-expression in E. coli of self-assembling scaffold building blocks with and without modifications for covalent attachment of cross-linking cargo proteins, hybrid scaffolds with spatially organized conjugation sites are overproduced that can be readily isolated. Cargo proteins, including enzymes, are rapidly cross-linked onto scaffolds for the formation of functional materials. We show that these materials can be used for the cell-free operation of a co-immobilized two-enzyme reaction and that the protein material can be recovered and reused. We believe that this work will provide a versatile platform for the design and scalable production of functional materials with customizable properties and the robustness required for biotechnological applications.