Programmable adhesion and morphing of protein hydrogels for underwater robots.

Soft robots capable of efficiently implementing tasks in fluid-immersed environments hold great promise for diverse applications. However, it remains challenging to achieve robotization that relies on dynamic underwater adhesion and morphing capability. Here we propose the construction of such robots with designer protein materials. Firstly, a resilin-like protein is complexed with polyoxometalate anions to form hydrogels that can rapidly switch between soft adhesive and stiff non-adhesive states in aqueous environments in response to small temperature variation. To realize remote control over dynamic adhesion and morphing, Fe3O4 nanoparticles are then integrated into the hydrogels to form soft robots with photothermal and magnetic responsiveness. These robots are demonstrated to undertake complex tasks including repairing artificial blood vessel, capturing and delivering multiple cargoes in water under cooperative control of infrared light and magnetic field. These findings pave an avenue for the creation of protein-based underwater robots with on-demand functionalities.

Direct and Efficient Incorporation of DOPA into Resilin-Like Proteins Enables Cross-Linking into Tunable Hydrogels.

3,4-Dihydroxyphenylalanine (DOPA), a naturally occurring yet noncanonical amino acid, endows protein polymers with diverse chemical reactivities and novel functionalities. Although many efforts have been made to incorporate DOPA into proteins, the incorporation efficiency and production titer remain low and severely hinder the exploration of these peculiar proteins for biomaterial fabrication. Here, we report an efficient biosynthetic strategy to produce large amounts of DOPA-incorporated structural proteins for the fabrication of hydrogels with tunable mechanical properties. First, synthetic genes were constructed that encode repetitive resilin-like proteins (RLPs) with varying proportions of tyrosine residues and molecular weights (Mw). Decoding of these genes into RLPs incorporated with DOPA was achieved via mis-aminoacylation of DOPA by endogenous tyrosyl-tRNA synthetase (TyrRS) in recombinant Escherichia coli cells. By developing a stoichiometry-guided two-phase culture strategy, we achieved independent control of the bacterial growth and protein synthesis phases. This enabled hyperproduction of the DOPA-incorporated RLPs at gram-per-liter levels and with a high DOPA incorporation yield of 76-85%. The purified DOPA-containing RLPs were then successfully cross-linked into bulk hydrogels via facile DOPA-Fe3+ complexations. Interestingly, these hydrogels exhibited viscoelastic and self-healing properties that are highly dependent on the catechol content and Mw of the RLPs. Finally, exploration of the molecular cross-linking mechanisms revealed that higher DOPA contents of the proteins would result in the concomitant occurrence of metal coordination and oxidative covalent cross-linking. In summary, our results suggest a useful platform to generate DOPA-functionalized protein materials and provide deeper insights into the gelation systems based on DOPA chemistry.

Synthetic protein condensates for cellular and metabolic engineering.

Protein condensates are distinct structures assembled in living cells that concentrate molecules via phase separation in a confined subcellular compartment. In the past decade, remarkable advances have been made to discover the fundamental roles of the condensates in spatiotemporal control of cellular metabolism and physiology and to reveal the molecular principles, components and driving forces that underlie their formation. Here we review the unique properties of the condensates, the promise and hurdles for harnessing them toward purposeful design and manipulation of biological functions in living cells. In particular, we highlight recent advances in mining and understanding the proteinaceous components for creating designer condensates, along with the engineering approaches to manipulate their material properties and biological functions. With these advances, a greater variety of complex organelle-like structures can be built for diverse applications, with unprecedented effects on synthetic biology.

Fibrous Structure and Stiffness of Designer Protein Hydrogels Synergize to Regulate Endothelial Differentiation of Bone Marrow Mesenchymal Stem Cells.

Matrix stiffness and fibrous structure provided by the native extracellular matrix have been increasingly appreciated as important cues in regulating cell behaviors. Recapitulating these physical cues for cell fate regulation remains a challenge due to the inherent difficulties in making mimetic hydrogels with well-defined compositions, tunable stiffness, and structures. Here, we present two series of fibrous and porous hydrogels with tunable stiffness based on genetically engineered resilin-silk-like and resilin-like protein polymers. Using these hydrogels as substrates, the mechanoresponses of bone marrow mesenchymal stem cells to stiffness and fibrous structure were systematically studied. For both hydrogel series, increasing compression modulus from 8.5 to 14.5 and 23 kPa consistently promoted cell proliferation and differentiation. Nonetheless, the promoting effects were more pronounced on the fibrous gels than their porous counterparts at all three stiffness levels. More interestingly, even the softest fibrous gel (8.5 kPa) allowed the stem cells to exhibit higher endothelial differentiation capability than the toughest porous gel (23 kPa). The predominant role of fibrous structure on the synergistic regulation of endothelial differentiation was further explored. It was found that the stiffness signal activated Yes-associated protein (YAP), the main regulator of endothelial differentiation, via spreading of focal adhesions, whereas fibrous structure reinforced YAP activation by promoting the maturation of focal adhesions and associated F-actin alignment. Therefore, our results shed light on the interplay of physical cues in regulating stem cells and may guide the fabrication of designer proteinaceous matrices toward regenerative medicine.

Controllable Fibrillization Reinforces Genetically Engineered Rubberlike Protein Hydrogels.

Rubberlike protein hydrogels are unique in their remarkable stretchability and resilience but are usually low in strength due to the largely unstructured nature of the constitutive protein chains, which limits their applications. Thus, reinforcing protein hydrogels while retaining their rubberlike properties is of great interest and has remained difficult to achieve. Here, we propose a fibrillization strategy to reinforce hydrogels from engineered protein copolymers with photo-cross-linkable resilin-like blocks and fibrillizable silklike blocks. First, the designer copolymers with an increased ratio of the silk to resilin blocks were photochemically cross-linked into rubberlike hydrogels with reinforced mechanical properties. The increased silk-to-resilin ratio also enabled self-assembly of the resulting copolymers into fibrils in a time-dependent manner. This allowed controllable fibrillization of the copolymer solutions at the supramolecular level for subsequent photo-cross-linking into reinforced hydrogels. Alternatively, the as-prepared chemically cross-linked hydrogels could be reinforced at the material level by inducing fibrillization of the constitutive protein chains. Finally, we demonstrated the advantage of reinforcing these hydrogels for use as piezoresistive sensors to achieve an expanded pressure detection range. We anticipate that this strategy may provide intriguing opportunities to generate robust rubberlike biomaterials for broad applications.

Development of Adhesive and Conductive Resilin-Based Hydrogels for Wearable Sensors.

Integrating multifunctionality such as stretchability, adhesiveness, and electroconductivity on a single protein hydrogel is highly desirable for various applications, and remains a challenge. Here we present the development of such multifunctional hydrogels based on resilin, a natural rubber-like material with remarkable extensibility and resilience. First, genetically engineered reslin-like proteins (RLPs) with varying molecular weight were biosynthesized to tune mechanical strength and stiffness of the cross-linked RLP hydrogels. Second, glycerol was incorporated into the hydrogels to endow adhesive properties. Next, a graphene-RLP conjugate was synthesized for cross-linking with the unmodified, pristine RLP to form an integrated network. The obtained hybrid hydrogel could be stretched to over four times of its original length, and self-adhered to diverse substrate surfaces due to its high adhesion strength of ∼24 kPa. Furthermore, the hybrid hydrogel showed high sensitivity, with a gauge factor of 3.4 at 200% strain, and was capable of real-time monitoring human activities such as finger bending, swallowing, and phonating. Due to these favorable attributes, the graphene/resilin hybrid hydrogel was a promising material for use in wearable sensors. In addition, the above material design and functionalization strategy may provide intriguing opportunities to generate innovative materials for broad applications.

Rational Design and Hierarchical Assembly of a Genetically Engineered Resilin-Silk Copolymer Results in Stiff Hydrogels.

Genetically engineered protein polymers, which can combine different unique peptide sequences from natural protein materials, offer great opportunities for making advanced materials with well-defined structures and properties. Here we report for the first time biosynthesis and self-assembly of a recombinant resilin-silk (RS) copolymer consisting of repeating units of silk and resilin blocks. The copolymer in aqueous solution self-assembled into nanoparticles, and the assembled nanoparticles further form nano- to microscale fibers in a time-dependent manner at body temperature, whereas such fibers were not formed upon incubation of the copolymer at either low or high temperatures. In contrast, a resilin-like polypeptide without the silk blocks exhibited a typical thermoresponsive dual-phase transition behavior and was incapable of self-assembling into fibers. More interestingly, the microscale fibers self-assembled from a moderately concentrated RS solution (20 wt %) could interact to give a self-supporting, semitransparent hydrogel with elastic modulus at approximately 195 Pa. Furthermore, photo-cross-linking of either freshly prepared or annealed RS copolymer led to the formation of stiff hydrogels and the material mechanical property was superior upon annealing of the RS solution for a longer time up to 4 h, with elastic modulus ranging from 2.9 to 7.0 kPa. These results not only shed light on the fundamental hierarchical assembly mechanism of a new family of genetically engineered RS copolymer but also suggest future opportunities for these thermoresponsive polymers in fabrication of hydrogel materials with tunable mechanical properties for diverse applications.