The biochemical and biophysical properties of the extracellular matrix (ECM) play a pivotal role in regulating cellular behaviors such as proliferation, migration, and differentiation. Engineered protein-based hydrogels, with highly tunable multifunctional properties, have the potential to replicate key features of the native ECM. Formed by self-assembly or crosslinking, engineered protein-based hydrogels can induce a range of cell behaviors through bioactive and functional domains incorporated into the polymer backbone. Using recombinant techniques, the amino acid sequence of the protein backbone can be designed with precise control over the chain-length, folded structure, and cell-interaction sites. In this review, the modular design of engineered protein-based hydrogels from both a molecular- and network-level perspective are discussed, and summarize recent progress and case studies to highlight the diverse strategies used to construct biomimetic scaffolds. This review focuses on amino acid sequences that form structural blocks, bioactive blocks, and stimuli-responsive blocks designed into the protein backbone for highly precise and tunable control of scaffold properties. Both physical and chemical methods to stabilize dynamic protein networks with defined structure and bioactivity for cell culture applications are discussed. Finally, a discussion of future directions of engineered protein-based hydrogels as biomimetic cellular scaffolds is concluded.

OBJECTIVE: Conventional coating strategies and materials for bio-applications with protective, diagnostic, and therapeutic functions are commonly limited by their arduous preparation processes and lack of on-demand functionalities. Herein, inspired by the \'root-leaf\' structure of grass, a series of novel polyacrylate-conjugated proteins can be engineered with sticky bovine serum albumin (BSA) protein as a \'root\' anchoring layer and a multifunctional polyacrylate as a \'leaf\' functional layer for the facile coating procedure and versatile surface functionalities.
METHODS: The engineered proteins were synthesized based on click chemistry, where the \'root\' layer can universally anchor onto both organic and inorganic substrates through a facile dip/spraying method with excellent stability in harsh solution conditions, thanks to its multiple adaptive molecular interactions with substrates that further elucidated by molecular force measurements between the \'root\' BSA protein and substrates. The \'leaf\' conjugated-polyacrylates imparted coatings with versatile on-demand functionalities, such as resistance to over 99% biofouling in complex biofluids, pH-responsive performance, and robust adhesion with various nanomaterials.
RESULTS: By synergistically leveraging the universal anchoring capabilities of BSA with the versatile physicochemical properties of polyacrylates, this study introduces a promising and facile strategy for imparting novel functionalities to a myriad of surfaces through engineering natural proteins and biomaterials for biotechnical and nanotechnical applications.

Rheumatoid arthritis (RA) is a relatively common inflammatory disease that affects the synovial tissue, eventually results in joints destruction and even long-term disability. Although Janus kinase inhibitors (Jakinibs) show a rapid efficacy and are becoming the most successful agents in RA therapy, high dosing at frequent interval and severe toxicities cannot be avoided. Here, we developed a new type of fully compatible nanocarriers based on recombinant chimeric proteins with outstanding controlled release of upadacitinib. In addition, the fluorescent protein component of the nanocarriers enabled noninvasive fluorescence imaging of RA lesions, thus allowing real-time detection of RA therapy. Using rat models, the nanotherapeutic is shown to be superior to free upadacitinib, as indicated by extended circulation time and sustained bioefficacy. Strikingly, this nanosystem possesses an ultralong half-life of 45 h and a bioavailability of 4-times higher than pristine upadacitinib, thus extending the dosing interval from one day to 2 weeks. Side effects such as over-immunosuppression and leukocyte levels reduction were significantly mitigated. This smart strategy boosts efficacy, safety and visuality of Jakinibs in RA therapy, and potently enables customized designs of nanoplatforms for other therapeutics.
UNASSIGNED: Supplementary material (further details of DLS analysis, biocompatibility of PCP-UPA, CIA models construction, etc.) is available in the online version of this article at 10.1007/s12274-023-5838-0.

Many molecular targets for cancer therapy are located in the cytosol. Therapeutic macromolecules are generally not able to spontaneously translocate across membranes to reach these cytosolic targets. Therefore a strong need exists for tools that enhance cytosolic delivery. Shiga toxin B-subunit (STxB) is used to deliver therapeutic principles to disease-relevant cells that express its receptor, the glycolipid Gb3. Based on its naturally existing membrane translocation capacity, STxB delivers antigens to the cytosol of Gb3-positive dendritic cells, leading to the induction of CD8+ T cells. Here, we have explored the possibility of further increasing the membrane translocation of STxB to enable other therapeutic applications. For this, our capacity to synthesize STxB chemically was exploited to introduce unnatural amino acids at different positions of the protein. These were then functionalized with hydrophobic entities to locally destabilize endosomal membranes. Intracellular trafficking of these functionalized STxB was measured by confocal microscopy and their cytosolic arrival with a recently developed highly robust, sensitive, and quantitative translocation assay. From different types of hydrophobic moieties that were linked to STxB, the most efficient configuration was determined. STxB translocation was increased by a factor of 2.5, paving the path for new biomedical opportunities.

A Disintegrin and Metalloprotease 10, also known as ADAM10, is a cell surface protease ubiquitously expressed in mammalian cells where it cuts several membrane proteins implicated in multiple physiological processes. The dysregulation of ADAM10 expression and function has been implicated in pathological conditions, including Alzheimer\'s disease (AD). Although it has been suggested that ADAM10 is expressed as a zymogen and the removal of the prodomain results in its activation, other potential mechanisms for the ADAM10 proteolytic function and activation remain unclear. Another suggested mechanism is post-translational modification of the cytoplasmic domain, which regulates ADAM10-dependent protein ectodomain shedding. Therefore, the precise and temporal activation of ADAM10 is highly desirable to reveal the fine details of ADAM10-mediated cleavage mechanisms and protease-dependent therapeutic applications. Here, we present a strategy to control prodomain and cytosolic tail cleavage to regulate ADAM10 shedding activity without the intervention of small endogenous molecule signaling pathways. We generated a series of engineered ADAM10 analogs containing Tobacco Etch Virus protease (TEV) cleavage site (TEVcs), rendering ADAM10 cleavable by TEV. This strategy revealed that, in the absence of other stimuli, the TEV-mediated removal of the prodomain could not activate ADAM10. However, the TEV-mediated cleavage of the cytosolic domain significantly increased ADAM10 activity. Then, we generated ADAM10 with a minimal constitutively catalytic activity that increased significantly in the presence of TEV or after activating a chemically activatable TEV. Our results revealed a bioengineering strategy for controlling the ADAM10 activity in living cells, paving the way to obtain spatiotemporal control of ADAM10. Finally, we proved that our approach of controlling ADAM10 promoted α-secretase activity and the non-amyloidogenic cleavage of amyloid-β precursor protein (APP), thereby increasing the production of the neuroprotective soluble ectodomain (sAPPα). Our bioengineering strategy has the potential to be exploited as a next-generation gene therapy for AD.

In multicellular organisms, cell-adhesion molecules connect cells into tissues and mediate intercellular signaling between these cells. In vertebrate brains, synaptic cell-adhesion molecules (SAMs) guide the formation, specification, and plasticity of synapses. Some SAMs, when overexpressed in cultured neurons or in heterologous cells co-cultured with neurons, drive formation of synaptic specializations onto the overexpressing cells. However, genetic deletion of the same SAMs from neurons often has no effect on synapse numbers, but frequently severely impairs synaptic transmission, suggesting that most SAMs control the function and plasticity of synapses (i.e., organize synapses) instead of driving their initial establishment (i.e., make synapses). Since few SAMs were identified that mediate initial synapse formation, it is difficult to develop methods that enable experimental control of synaptic connections by targeted expression of these SAMs. To overcome this difficulty, we engineered novel SAMs from bacterial proteins with no eukaryotic homologues that drive synapse formation. We named these engineered adhesion proteins \"Barnoligin\" and \"Starexin\" because they were assembled from parts of Barnase and Neuroligin-1 or of Barstar and Neurexin3β, respectively. Barnoligin and Starexin robustly induce the formation of synaptic specializations in a specific and directional manner in cultured neurons. Synapse formation by Barnoligin and Starexin requires both their extracellular Barnase- and Barstar-derived interaction domains and their Neuroligin- and Neurexin-derived intracellular signaling domains. Our findings support a model of synapse formation whereby trans-synaptic interactions by SAMs drive synapse organization via adhesive interactions that activate signaling cascades.

The development of cancer vaccines based on tumor-associated antigens is hurdled by lack of an efficient adjuvant and insufficient efficacy. To improve the efficacy of vaccines, a genetically-engineered method was employed in this work to achieve the codelivery of antigen and adjuvant to enhance immune responses. Trichosanthin is a plant-derived protein that possesses cancer immune stimulation function. A genetically engineered protein vaccine composed of trichosanthin (adjuvant) and legumain domain (a peptidic antigen) was constructed, which was further chemically modified with mannose for targeting dendritic cells (DCs). The method is facile and ready for scaling up for massive production. Such a \"two-in-one\" vaccine is advantageous for codelivery for augmenting the immune responses. The vaccine inhibited the tumors by triggering a robust cytotoxic T lymphocyte response in the orthotopic-breast-tumor mice. Furthermore, the vaccine was loaded into the temperature-sensitive hydrogel based on Pluronic F127 for implanting use in the post-surgical site. The sustained-released vaccine from the hydrogel inhibited not only the tumor recurrence but also the lung metastases of breast cancer. These findings demonstrated that it was a safe and effective vaccination for breast cancer immunotherapy in a prophylactical and therapeutical manner for remodeling the tumor immune microenvironment and arresting tumor growth.

Implantable medical devices have been widely applied in diagnostics, therapeutics, organ restoration, and other biomedical areas, but often suffer from dysfunction and infections due to irreversible biofouling. Inspired by the self-defensive \"vine-thorn\" structure of climbing thorny plants, a zwitterion-conjugated protein is engineered via grafting sulfobetaine methacrylate (SBMA) segments on native bovine serum albumin (BSA) protein molecules for surface coating and antifouling applications in complex biological fluids. Unlike traditional synthetic polymers of which the coating operation requires arduous surface pretreatments, the engineered protein BSA@PSBMA (PolySBMA conjugated BSA) can achieve facile and surface-independent coating on various substrates through a simple dipping/spraying method. Interfacial molecular force measurements and adsorption tests demonstrate that the substrate-foulant attraction is significantly suppressed due to strong interfacial hydration and steric repulsion of the bionic structure of BSA@PSBMA, enabling coating surfaces to exhibit superior resistance to biofouling for a broad spectrum of species including proteins, metabolites, cells, and biofluids under various biological conditions. This work provides an innovative paradigm of using native proteins to generate engineered proteins with extraordinary antifouling capability and desired surface properties for bioengineering applications.

Vaccination is critical for population protection from pathogenic infections. However, its efficiency is frequently compromised by a failure of antigen retention and presentation. Herein, we designed a dextran-binding protein DexBP, which is composed of the carbohydrate-binding domains of Trichoderma reesei cellobiohydrolases Cel6A and Cel7A, together with the sequence of the fluorescent protein mCherry. DexBP was further prepared by engineered Escherichia coli cells and grafted to magnetic nanoparticles. The magnetic nanoparticles were integrated with a dextran/poly(vinyl alcohol) framework and a reactive oxygen species-responsive linker, obtaining magnetic polymeric microgels for carrying pathogen antigen. Similar to amoeba aggregation, the microgels self-assembled to form aggregates and further induced dendritic cell aggregation. This step-by-step assembly retained antigens at lymph nodes, promoted antigen presentation, stimulated humoral immunity, and protected the mice from life-threatening systemic infections. This study developed a magnetic microgel-assembling platform for dynamically regulating immune response during protection of the body from dangerous infections.
UNASSIGNED: Supplementary material (AFM image and zeta potential of MG; TEM, FT-IR, DLS, and zeta potential of MNP-DexBP; zeta potential of MG+CaAg and MG+MNP-DexBP+CaAg; antigen release profile of MG+CaAg and MG+MNP-DexBP+CaAg; aggregation and dispersion of dendritic cells induced by MG+MNP-DexBP+CaAg; uptake of FITC-labeled CaAg (fCaAg) and intracellular distribution of fCaAg in the dendritic cells; antigen retention and dendritic cell activation in lymph nodes; and serum anti-CaAg antibody levels on day 3 after C. albicans infection in the mice pre-immunized by PBS (control), CaAg, MG+CaAg, and MG+MNP-DexBP+CaAg) is available in the online version of this article at 10.1007/s12274-022-4809-1.

Genetic code expansion (GCE) enables directed incorporation of noncoded amino acids (NCAAs) and unnatural amino acids (UNAAs) into the active core that confers dedicated structure and function to engineered proteins. Many protein biomaterials are tandem repeats that intrinsically include NCAAs generated through post-translational modifications (PTMs) to execute assigned functions. Conventional genetic engineering approaches using prokaryotic systems have limited ability to biosynthesize functionally active biomaterials with NCAAs/UNAAs. Codon suppression and reassignment introduce NCAAs/UNAAs globally, allowing engineered proteins to be redesigned to mimic natural matrix-cell interactions for tissue engineering. Expanding the genetic code enables the engineering of biomaterials with catechols - growth factor mimetics that modulate cell-matrix interactions - thereby facilitating tissue-specific expression of genes and proteins. This method of protein engineering shows promise in achieving tissue-informed, tissue-compliant tunable biomaterials.