ALT Recombinant protein-based biomaterials offer exciting opportunities in materials design and controlled therapeutic delivery. Though their precursors can be readily synthesized with near-perfect monodispersity and sequence specificity through scalable fermentation processes, recombinant protein materials have yet to achieve the same level of multi-stimuli-responsiveness as their synthetic counterparts. Integrating cutting-edge tools from chemical biology, we autonomously compile topologically specified protein crosslinkers that can be degraded following user-programmable Boolean logic. Covalent step polymerization of these linkers into protein hydrogels yields smart materials whose cargo (e.g., bioactive proteins, cellular therapeutics) can be liberated following bulk degradation in response to user-specified input combinations.

ALT Protein-based biomaterials are growing in popularity for biomedical applications, in part owing to their innate ability to interface with biological systems. These materials, in the form of fibres, nanoparticles and hydrogels, have shown promise as drug delivery vehicles, tissue scaffolds and vaccines. Moreover, the explosion of protein engineering tools and the inception of de novo protein design have transformed our ability to explore new protein structures, enabling the creation of novel materials with diverse properties and furthering their customization for various applications. Here we explore the coming of age of protein engineering technologies and their impact on biomaterials. Starting with naturally sourced materials, we highlight common protein building blocks and fabrication methods, as well as recent applications of each. We subsequently explore rationally designed materials and conclude by discussing the potential impacts that de novo design will have on biomaterial dev.

ALT Stimuli-responsive biomaterials hold great promise in controlled therapeutic delivery, tissue engineering, and biosensing applications. Recently, molecular assembly via autonomous compilation has been employed to create topologically specified protein cargos that can be site-specifically tethered to and conditionally released from biomaterials following user-programmable Boolean logic. Prior implementation has been confined to simple fluorescent protein outputs and model protease inputs. Here, we extend the applicability of this framework by assembling all 7 unique logical operations emanating from a YES/OR/AND 3-input operator set to deliver bioactive proteins spanning diverse categories: growth factors, enzymes, therapeutic nanobodies, de novo-engineered cytokines, and fluorescent proteins. Through inclusion of a photocleavable protein motif, we further establish that visible light can be employed as an additional input in specifying logic-based protein release.

ALT In this work, we employ de novo protein design methodologies to generate a suite of self-assembling multimeric proteins, whose step-growth heteropolymerization into bulk hydrogels and condensates can be exogenously triggered through small-molecule addition. Our results highlight how changes in programmed multimer valency and their triggered assembly yield materials with varying structures and viscoelasticity. We anticipate that these approaches will prove useful in rapidly generating large libraries of stimuli-responsive biomaterials that are precisely tailored to specific applications in the biosciences and beyond.

ALT Harnessing the power of recombinant expression, we integrate emerging chemical biology tools to create topologically specified protein cargos that can be site-specifically tethered to and conditionally released from biomaterials following user-programmable Boolean logic. Critically, construct topology is autonomously compiled during expression through spontaneous intramolecular ligations, enabling direct and scalable synthesis of advanced operators. Using this framework, we specify protein release from biomaterials following all 17 possible YES/OR/AND logic outputs from input combinations of three orthogonal protease actuators, multiplexed delivery of three distinct biomacromolecules from hydrogels, five-input-based conditional cargo liberation and logically defined protein localization on or within living mammalian cells.