Scalable Production of Genetically Engineered Nanofibrous Macroscopic Materials via Filtration

As interest in using proteins to assemble functional, biocompatible, and environmentally friendly materials is growing, developing scalable protocols for producing recombinant proteins with customized functions coupled to straightforward fabrication processes is becoming crucial. Here, we use E. coli bacteria to produce amyloid protein nanofibers that are key constituents of the biofilm extracellular matrix and show that protein nanofiber aggregates can be purified using a fast and easily accessible vacuum filtration procedure. With their extreme resistance to heat, detergents, solvents, and denaturing agents, engineered curli nanofibers remain functional throughout the rigorous processing and can be used to assemble macroscopic materials directly from broth culture. As a demonstration, we show that engineered curli nanofibers can be fabricated into self-standing films while maintaining the functionality of various fused domains that confer new specific binding activity to the material. We also demonstrate that purified curli fibers can be disassembled, reassembled into thin films, and recycled for further materials processing. Our scalable approach, which combines established purification techniques for amyloid fibers, is applicable to a new class of recombinant amyloid proteins whose sequence can be easily tailored for diverse applications through genetic