Engineering Proteins for PEDOT Dispersions: A New Horizon for Highly Mixed Ionic-Electronic Biocompatible Conducting Materials.

Poly (3,4-ethylenedioxythiophene) (PEDOT) doped with polystyrene sulfonate (PSS) is the most used conducting polymer from energy to biomedical applications. Despite its exceptional properties, there is a need for developing new materials that can improve some of its inherent limitations, e.g., biocompatibility. In this context, doping PEDOT is propose with a robust recombinant protein with tunable properties, the consensus tetratricopeptide repeated protein (CTPR). The doping consists of an oxidative polymerization, where the PEDOT chains are stabilized by the negative charges of the CTPR protein. CTPR proteins are evaluated with three different lengths (3, 10, and 20 identical CTPR units) and optimized varied synthetic conditions. These findings revealed higher doping rate and oxidized state of the PEDOT chains when doped with the smallest scaffold (CTPR3). These PEDOT:CTPR hybrids possess ionic and electronic conductivity. Notably, PEDOT:CTPR3 displayed an electronic conductivity of 0.016 S cm-1, higher than any other reported protein-doped PEDOT. This result places PEDOT:CTPR3 at the level of PEDOT-biopolymer hybrids, and brings it closer in performance to PEDOT:PSS gold standard. Furthermore, PEDOT:CTPR3 dispersion is successfully optimized for inkjet printing, preserving its electroactivity properties after printing. This approach opens the door to the use of these novel hybrids for bioelectronics.

An Electroactive and Self-Assembling Bio-Ink, based on Protein-Stabilized Nanoclusters and Graphene, for the Manufacture of Fully Inkjet-Printed Paper-Based Analytical Devices.

Hundreds of new electrochemical sensors are reported in literature every year. However, only a few of them makes it to the market. Manufacturability, or rather the lack of it, is the parameter that dictates if new sensing technologies will remain forever in the laboratory in which they are conceived. Inkjet printing is a low-cost and versatile technique that can facilitate the transfer of nanomaterial-based sensors to the market. Herein, an electroactive and self-assembling inkjet-printable ink based on protein-nanomaterial composites and exfoliated graphene is reported. The consensus tetratricopeptide proteins (CTPRs), used to formulate this ink, are engineered to template and coordinate electroactive metallic nanoclusters (NCs), and to self-assemble upon drying, forming stable films. The authors demonstrate that, by incorporating graphene in the ink formulation, it is possible to dramatically improve the electrocatalytic properties of the ink, obtaining an efficient hybrid material for hydrogen peroxide (H2 O2 ) detection. Using this bio-ink, the authors manufactured disposable and environmentally sustainable electrochemical paper-based analytical devices (ePADs) to detect H2 O2 , outperforming commercial screen-printed platforms. Furthermore, it is demonstrated that oxidoreductase enzymes can be included in the formulation, to fully inkjet-print enzymatic amperometric biosensors ready to use.

Generation of Thermally Stable Affinity Pairs for Sensitive, Specific Immunoassays.

Many point-of-care diagnostic tests rely on a pair of monoclonal antibodies that bind to two distinct epitopes of a molecule of interest. This protocol describes the identification and generation of such affinity pairs based on an easily produced small protein scaffold rcSso7d which can substitute monoclonal antibodies. These strong binding variants are identified from a large yeast display library. The approach described can be significantly faster than antibody generation and epitope binning, yielding affinity pairs synthesized in common bacterial protein synthesis strains, enabling the rapid generation of novel diagnostic tools.

Improving Bioavailability of Hydrophobic Prodrugs through Supramolecular Nanocarriers Based on Recombinant Proteins for Osteosarcoma Treatment.

Supramolecular nanodrug assembly driven by supramolecular chemistry is becoming a powerful strategy for medication. The potential of engineered proteins as building blocks for nanoformulations is rarely investigated. Here, we developed a new generation of recombinant protein-based nanodrug carriers, which is very efficient for loading and delivering the hydrophobic prodrug aldoxorubicin. Significantly enhanced anti-tumor effects in osteosarcoma (OS) models were observed. The half-life of the nanodrug reached almost two days and the corresponding bioavailability increased by 17-fold. This is significantly superior to other drug counterparts, empowering long-acting OS treatment scenarios. Importantly, off-target side effects of the prodrug, including cardiotoxicity and lung-metastasis, were greatly mitigated with our medication. Thus, our assembly strategy enables the customized design of advanced nanodelivery systems employing broader biomaterial building blocks for cancer therapy.

Engineered Near-Infrared Fluorescent Protein Assemblies for Robust Bioimaging and Therapeutic Applications.

Fluorescent proteins are investigated extensively as markers for the imaging of cells and tissues that are treated by gene transfection. However, limited transfection efficiency and lack of targeting restrict the clinical application of this method rooted in the challenging development of robust fluorescent proteins for in vivo bioimaging. To address this, a new type of near-infrared (NIR) fluorescent protein assemblies manufactured by genetic engineering is presented. Due to the formation of well-defined nanoparticles and spectral operation within the phototherapeutic window, the NIR protein aggregates allow stable and specific tumor imaging via simple exogenous injection. Importantly, in vivo tumor metastases are tracked and this overcomes the limitations of in vivo imaging that can only be implemented relying on the gene transfection of fluorescent proteins. Concomitantly, the efficient loading of hydrophobic drugs into the protein nanoparticles is demonstrated facilitating the therapy of tumors in a mouse model. It is believed that these theranostic NIR fluorescent protein assemblies, hence, show great potential for the in vivo detection and therapy of cancer.

Solvent-Free Plasticity and Programmable Mechanical Behaviors of Engineered Proteins.

Biopolymeric networks with plasticity show great competences in diverse fields owing to the combined biocompatible and mechanical characteristics. However, to realize such plasticity external complicated treatments, e.g., UV or organic solvent have to be applied, which in turn impair the biological nature and even mechanical properties of those systems. To address this challenge, one new type of anhydrous protein liquid crystalline (LC) gels, which exhibit flexible morphological plasticity and mechanical programmability is demonstrated. Supramolecular interactions in the smectic biogels play an important role for their high plasticity. Remarkably, the samples exhibit outstanding mechanical behaviors. The tensile strength and Young's modulus at MPa levels are comparable or even higher than chemically cross-linked hydrogels and LC elastomers. More importantly, mechanical programmability of the LC gels is achieved by genetically tuning the charge density of protein backbones. Consequently, the mechanical performance is manipulated in the range of one order of magnitude. Thus, this type of anhydrous protein LC gels offers great opportunities for load-bearing high-tech applications.

Protein-Engineered Functional Materials.

Proteins are versatile macromolecules that can perform a variety of functions. In the past three decades, they have been commonly used as building blocks to generate a range of biomaterials. Owing to their flexibility, proteins can either be used alone or in combination with other functional molecules. Advances in synthetic and chemical biology have enabled new protein fusions as well as the integration of new functional groups leading to biomaterials with emergent properties. This review discusses protein-engineered materials from the perspectives of domain-based designs as well as physical and chemical approaches for crosslinked materials, with special emphasis on the creation of hydrogels. Engineered proteins that organize or template metal ions, bear noncanonical amino acids (NCAAs), and their potential applications, are also reviewed.

X-ray-Mediated Release of Molecules and Engineered Proteins from Nanostructure Surfaces.

Many applications call for initiation of chemical reactions with highly penetrating X-rays with nanometer precision and little damage to the surroundings, which is difficult to realize because of low interaction cross-sections between hard X-rays and organic matters. Here, we demonstrate that a combination of computational protein design of single conjugation site green fluorescent proteins and nanomaterial engineering of silica-covered gold nanoparticles can enhance the release efficiencies of proteins from the surface of nanoparticles. The nanoparticles, to which the proteins are attached through DNA linkers, provide increased X-ray absorption without scavenging radicals, and single conjugation sites allow efficient release of proteins.

Boosting Antitumor Drug Efficacy with Chemically Engineered Multidomain Proteins.

A facile chemical approach integrating supramolecular chemistry, site-selective protein chemistry, and molecular biology is described to engineer synthetic multidomain protein therapeutics that sensitize cancer cells selectively to significantly enhance antitumor efficacy of existing chemotherapeutics. The desired bioactive entities are assembled via supramolecular interactions at the nanoscale into structurally ordered multiprotein complexes comprising a) multiple copies of the chemically modified cyclic peptide hormone somatostatin for selective targeting and internalization into human A549 lung cancer cells expressing SST-2 receptors and b) a new cysteine mutant of the C3bot1 (C3) enzyme from Clostridium botulinum, a Rho protein inhibitor that affects and influences intracellular Rho-mediated processes like endothelial cell migration and blood vessel formation. The multidomain protein complex, SST3-Avi-C3, retargets C3 enzyme into non-small cell lung A549 cancer cells and exhibits exceptional tumor inhibition at a concentration ≈100-fold lower than the clinically approved antibody bevacizumab (Avastin) in vivo. Notably, SST3-Avi-C3 increases tumor sensitivity to a conventional chemotherapeutic (doxorubicin) in vivo. These findings show that the integrated approach holds vast promise to expand the current repertoire of multidomain protein complexes and can pave the way to important new developments in the area of targeted and combination cancer therapy.

New Generation of Tunable Bioactive Shape Memory Mats Integrated with Genetically Engineered Proteins.

Aligned poly(l-lactide)/poly(methyl methacrylate) binary blend fibers and mats loaded with a chimeric green fluorescence protein having a bioactive peptide with hydroxyapatite binding and mineralization property are prepared by pressurized gyration. The effect of processing parameters on the product morphologies, and the shape memory properties of these samples are investigated. Integration of hydroxyapatite nanoparticles into the fiber assembly is self-directed using the hydroxyapatite-binding property of the peptide genetically engineered to green fluorescence protein. Fluorescence microscopy analysis corroborated with Fourier transform infrared spectroscopy (FTIR) data confirms the integration of the chimeric protein with the fibers. An enzyme based remineralization assay is conducted to study the effects of peptide-mediated mineralization within the fiber mats. Raman and FTIR spectral changes observed following the peptide-mediated mineralization provides an initial step toward a soft-hard material transition. These results show that programmable shape memory properties can be obtained by incorporating genetically engineered bioactive peptide domains into polymer fibers.

Engineered protein coatings to improve the osseointegration of dental and orthopaedic implants.

Here we present the design of an engineered, elastin-like protein (ELP) that is chemically modified to enable stable coatings on the surfaces of titanium-based dental and orthopaedic implants by novel photocrosslinking and solution processing steps. The ELP includes an extended RGD sequence to confer bio-signaling and an elastin-like sequence for mechanical stability. ELP thin films were fabricated on cp-Ti and Ti6Al4V surfaces using scalable spin and dip coating processes with photoactive covalent crosslinking through a carbene insertion mechanism. The coatings withstood procedures mimicking dental screw and hip replacement stem implantations, a key metric for clinical translation. They promoted rapid adhesion of MG63 osteoblast-like cells, with over 80% adhesion after 24 h, compared to 38% adhesion on uncoated Ti6Al4V. MG63 cells produced significantly more mineralization on ELP coatings compared to uncoated Ti6Al4V. Human bone marrow mesenchymal stem cells (hMSCs) had an earlier increase in alkaline phosphatase activity, indicating more rapid osteogenic differentiation and mineral deposition on adhesive ELP coatings. Rat tibia and femur in vivo studies demonstrated that cell-adhesive ELP-coated implants increased bone-implant contact area and interfacial strength after one week. These results suggest that ELP coatings withstand surgical implantation and promote rapid osseointegration, enabling earlier implant loading and potentially preventing micromotion that leads to aseptic loosening and premature implant failure.

Coupling Infusion and Gyration for the Nanoscale Assembly of Functional Polymer Nanofibers Integrated with Genetically Engineered Proteins.

Nanofibers featuring functional nanoassemblies show great promise as enabling constituents for a diverse range of applications in areas such as tissue engineering, sensing, optoelectronics, and nanophotonics due to their controlled organization and architecture. An infusion gyration method is reported that enables the production of nanofibers with inherent biological functions by simply adjusting the flow rate of a polymer solution. Sufficient polymer chain entanglement is obtained at Berry number > 1.6 to make bead-free fibers integrated with gold nanoparticles and proteins, in the diameter range of 117-216 nm. Integration of gold nanoparticles into the nanofiber assembly is followed using a gold-binding peptide tag genetically conjugated to red fluorescence protein (DsRed). Fluorescence microscopy analysis corroborated with Fourier transform infrared spectroscopy (FTIR) data confirms the integration of the engineered red fluorescence protein with the nanofibers. The gold nanoparticle decorated nanofibers having red fluorescence protein as an integral part keep their biological functionality including copper-induced fluorescence quenching of the DsRed protein due to its selective Cu(+2) binding. Thus, coupling the infusion gyration method in this way offers a simple nanoscale assembly approach to integrate a diverse repertoire of protein functionalities into nanofibers to generate biohybrid materials for imaging, sensing, and biomaterial applications.

Presentation of fibronectin fragments using affinity protein interactions for enhanced retention and function.

We present a protein immobilization system, based on the Src Homology 3 (SH3) affinity domain, allowing for a transient interaction between a fibronectin ligand and a biomaterial surface. This strategy leads to enhanced retention of the fibronectin fragment over adsorbed fibronectin, and increased cellular proliferation and motility over either covalently immobilized or adsorbed fibronectin. The results indicate that intermediate affinity protein immobilization could provide benefits for tissue engineering beyond the traditional immobilization techniques, adsorption or covalent attachment.

Dissection of the BCR-ABL signaling network using highly specific monobody inhibitors to the SHP2 SH2 domains.

The dysregulated tyrosine kinase BCR-ABL causes chronic myelogenous leukemia in humans and forms a large multiprotein complex that includes the Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2). The expression of SHP2 is necessary for BCR-ABL-dependent oncogenic transformation, but the precise signaling mechanisms of SHP2 are not well understood. We have developed binding proteins, termed monobodies, for the N- and C-terminal SH2 domains of SHP2. Intracellular expression followed by interactome analysis showed that the monobodies are essentially monospecific to SHP2. Two crystal structures revealed that the monobodies occupy the phosphopeptide-binding sites of the SH2 domains and thus can serve as competitors of SH2-phosphotyrosine interactions. Surprisingly, the segments of both monobodies that bind to the peptide-binding grooves run in the opposite direction to that of canonical phosphotyrosine peptides, which may contribute to their exquisite specificity. When expressed in cells, monobodies targeting the N-SH2 domain disrupted the interaction of SHP2 with its upstream activator, the Grb2-associated binder 2 adaptor protein, suggesting decoupling of SHP2 from the BCR-ABL protein complex. Inhibition of either N-SH2 or C-SH2 was sufficient to inhibit two tyrosine phosphorylation events that are critical for SHP2 catalytic activity and to block ERK activation. In contrast, targeting the N-SH2 or C-SH2 revealed distinct roles of the two SH2 domains in downstream signaling, such as the phosphorylation of paxillin and signal transducer and activator of transcription 5. Our results delineate a hierarchy of function for the SH2 domains of SHP2 and validate monobodies as potent and specific antagonists of protein-protein interactions in cancer cells.

Dynamic dual-tracer MRI-guided fluorescence tomography to quantify receptor density in vivo.

The up-regulation of cell surface receptors has become a central focus in personalized cancer treatment; however, because of the complex nature of contrast agent pharmacokinetics in tumor tissue, methods to quantify receptor binding in vivo remain elusive. Here, we present a dual-tracer optical technique for noninvasive estimation of specific receptor binding in cancer. A multispectral MRI-coupled fluorescence molecular tomography system was used to image the uptake kinetics of two fluorescent tracers injected simultaneously, one tracer targeted to the receptor of interest and the other tracer a nontargeted reference. These dynamic tracer data were then fit to a dual-tracer compartmental model to estimate the density of receptors available for binding in the tissue. Applying this approach to mice with deep-seated gliomas that overexpress the EGF receptor produced an estimate of available receptor density of 2.3 ± 0.5 nM (n = 5), consistent with values estimated in comparative invasive imaging and ex vivo studies.

Biomarker discovery and applications for foods and beverages: proteomics to nanoproteomics.

Foods and beverages have been at the heart of our society for centuries, sustaining humankind - health, life, and the pleasures that go with it. The more we grow and develop as a civilization, the more we feel the need to know about the food we eat and beverages we drink. Moreover, with an ever increasing demand for food due to the growing human population food security remains a major concern. Food safety is another growing concern as the consumers prefer varied foods and beverages that are not only traded nationally but also globally. The 21st century science and technology is at a new high, especially in the field of biological sciences. The availability of genome sequences and associated high-throughput sensitive technologies means that foods are being analyzed at various levels. For example and in particular, high-throughput omics approaches are being applied to develop suitable biomarkers for foods and beverages and their applications in addressing quality, technology, authenticity, and safety issues. Proteomics are one of those technologies that are increasingly being utilized to profile expressed proteins in different foods and beverages. Acquired knowledge and protein information have now been translated to address safety of foods and beverages. Very recently, the power of proteomic technology has been integrated with another highly sensitive and miniaturized technology called nanotechnology, yielding a new term nanoproteomics. Nanoproteomics offer a real-time multiplexed analysis performed in a miniaturized assay, with low-sample consumption and high sensitivity. To name a few, nanomaterials - quantum dots, gold nanoparticles, carbon nanotubes, and nanowires - have demonstrated potential to overcome the challenges of sensitivity faced by proteomics for biomarker detection, discovery, and application. In this review, we will discuss the importance of biomarker discovery and applications for foods and beverages, the contribution of proteomic technology in this process, and a shift towards nanoproteomics to suitably address associated issues. This article is part of a Special Issue entitled: Translational plant proteomics.