Axon regeneration constitutes a fundamental challenge for regenerative neurobiology, which necessitates the use of tailor-made biomaterials for controllable delivery of cells and biomolecules. An increasingly popular approach for creating these materials is to directly assemble engineered proteins into high-order structures, a process that often relies on sophisticated protein chemistry. Here, we present a simple approach for creating injectable, photoresponsive hydrogels via metal-directed assembly of His6-tagged proteins. The B12-dependent photoreceptor protein CarHC can complex with transition metal ions through an amino-terminal His6-tag, which can further undergo a sol-gel transition upon addition of AdoB12, leading to the formation of hydrogels with marked injectability and photodegradability. The inducible phase transitions further enabled facile encapsulation and release of cells and proteins. Injecting the Zn2+-coordinated gels decorated with leukemia inhibitory factor into injured mouse optic nerves led to prolonged cellular signaling and enhanced axon regeneration. This study illustrates a powerful strategy for designing injectable biomaterials.
human life. [1][2][3] To achieve such a dream, choosing natural biomaterials such as nucleic acids, [4][5][6] peptides, [7] proteins, [8,9] and polysaccharides [10] as the substitutes of fossil source-based synthetic chemicals is a promising way. Correspondingly, micro/ nanoscale fabrication on natural biomaterials receives great of importance, [11] and a spatially definable property with high precision to exactly position biomaterials on a surface is highly desirable [12][13][14][15][16][17][18] toward biodegradable and biocompatible green electronic, [19][20][21][22] optical, [23][24][25][26][27][28][29] sensor, [30] energy, [31] and environmental devices. [32] Typical examples down this road include silk fibroin, [12][13][14] polysaccharides, [10] and DNA origami-mediated lithography. [5] These approaches are part of great efforts to develop "green" lithographic processing in semiconductor industry that is eligible to lessen the exposure of workers and environment toward noxious chemical and reduce waste. [13] The adoption of these strategies allows the replacement of organic solvent with water to cast and develop a resist, accompanied with the exclusion of environmentally harmful components from material processing and fabrication. However, so far the lithographic processes based on existing natural biomaterials are difficult to be exploited in scale-up industrial application, because their limited quantities, high material, and processing cost as well as small deposition area impede the practical engineering of these biomaterials. [9] Moreover, the less-controlled variation on polymorph and polydispersity of biomaterials often influences the repeatability of results. [33] Recently, our group reported the use of phase-transited lysozyme nanofilm as the resist for photolithography and electron-beam lithography (EBL), with the cost being decreased and practical applicability being potentially demonstrated. [17] Continuous efforts along this way further require much cheaper material and simpler fabrication process for large-scale industrial uses. In this respect, we pay attention to egg white due to the following reasons. First, egg white is a very common natural biomaterial just taken from egg as our daily food, which makes it has great natural abundance with a low cost. Second, in contrast to other natural competitors usually requiring strict demands on the purity, polydispersity, and polymorph stability, egg white is a combination of multiple functional nutrients, [34] which could be directly utilized without further purification and Complex lithographic steps and the use of toxic chemicals in these processes are in conflict with a sustainable human society. Development of new inexpensive and green resist, simple alternative procedures, and nontoxic solvents is the key to move recyclable micro/nanofabrication from laboratory level to industrial application in large scale. Herein, precise control on protein fragmentation/aggregation upon photo/electron irradiation is conceived into egg white-based green resist...
Design of proteins with nonlinear topologies has emerged as a nascent branch of protein engineering, but significant applications remain to be seen. Here, we demonstrate the cellular synthesis of (SpyCatcher) 4 GFP, a 4-arm star-like protein enabled by spontaneous split GFP reconstitution, which further led to the creation of various protein networks exhibiting tunable mechanics and suitability for cell encapsulation. A derivative 4-arm star-like protein, (CarH C ) 4 GFP, resulting from the conjugation of (SpyCatcher) 4 GFP with the SpyTag-fusion CarH C photoreceptors, can undergo rapid sol-gel and gel-sol transitions in response to AdoB 12 and light, respectively. The chemo-and photo-induced phase transitions enabled encapsulation and controlled release of protein molecules such as the biofilm-degrading glycosyl hydrolase PslG, a potential agent for combatting multidrug-resistant bacterial species in chronic infections. The creation of those uncommon protein architectures promises great opportunities for materials biology and ''smart'' therapeutic delivery.
Microbially-synthesized protein-based materials are attractive replacements for petroleum-derived synthetic polymers. However, the high molecular weight, high repetitiveness, and highly-biased amino acid composition of high-performance protein-based materials have restricted their production and widespread use. Here we present a general strategy for enhancing both strength and toughness of low-molecular-weight protein-based materials by fusing intrinsically-disordered mussel foot protein fragments to their termini, thereby promoting end-to-end protein-protein interactions. We demonstrate that fibers of a ~60 kDa bi-terminally fused amyloid-silk protein exhibit ultimate tensile strength up to 481 ± 31 MPa and toughness of 179 ± 39 MJ*m−3, while achieving a high titer of 8.0 ± 0.70 g/L by bioreactor production. We show that bi-terminal fusion of Mfp5 fragments significantly enhances the alignment of β-nanocrystals, and intermolecular interactions are promoted by cation-π and π-π interactions between terminal fragments. Our approach highlights the advantage of self-interacting intrinsically-disordered proteins in enhancing material mechanical properties and can be applied to a wide range of protein-based materials.
Hydrogels made from proteins are attractive materials for diverse medical applications, as they are biocompatible, biodegradable, and amenable to chemical and biological modifications. Recent advances in protein engineering, synthetic biology, and material science have enabled the fine-tuning of protein sequences, hydrogel structures, and hydrogel mechanical properties, allowing for a broad range of biomedical applications using protein hydrogels. This article reviews recent progresses on protein hydrogels with special focus on those made of microbially produced proteins. We discuss different hydrogel formation strategies and their associated hydrogel properties. We also review various biomedical applications, categorized by the origin of protein sequences. Lastly, current challenges and future opportunities in engineering protein-based hydrogels are discussed. We hope this review will inspire new ideas in material innovation, leading to advanced protein hydrogels with desirable properties for a wide range of biomedical applications.
High molecular weight (MW), highly repetitive protein polymers are attractive candidates to replace petroleum-derived materials as these protein-based materials (PBMs) are renewable, biodegradable, and have outstanding mechanical properties. However, their high MW and highly repetitive sequence features make them difficult to synthesize in fast-growing microbial cells in sufficient amounts for real applications. To overcome this challenge, various methods were developed to synthesize repetitive PBMs. Here, we review recent strategies in the construction of repetitive genes, expression of repetitive proteins from circular mRNAs, and synthesis of repetitive proteins by ligation and protein polymerization. We discuss the advantages and limitations of each method and highlight future directions that will lead to scalable production of highly repetitive PBMs for a wide range of applications.
Spider silk is recognized for its exceptional mechanical properties and biocompatibility, making it a versatile platform for developing functional materials. In this study, a modular functionalization strategy for recombinant spider silk is presented using SpyTag/SpyCatcher chemistry, a prototype of genetically encoded click chemistry. The approach involves AlphaFold2‐aided design of SpyTagged spider silk coupled with bacterial expression and biomimetic spinning, enabling the decoration of silk with various SpyCatcher‐fusion motifs, such as fluorescent proteins, enzymes, and cell‐binding ligands. The silk threads can be coated with a silica layer using silicatein, an enzyme for silicification, resulting in a hybrid inorganic–organic 1D material. The threads installed with RGD or laminin cell‐binding ligands lead to enhanced endothelial cell attachment and proliferation. These findings demonstrate a straightforward yet powerful approach to 1D protein materials.
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