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A promising method for recycling phosphate from wastewater is through precipitation of struvite (MgNH 4 PO 4 ·6H 2 O), a slow-release fertilizer. Peptides have been shown to increase the yield of struvite formation, but producing peptides via solid phase synthesis is cost prohibitive. This work investigates the effects of peptide-expressing bacteria on struvite precipitation to provide a sustainable and cost-efficient means to enhance struvite precipitation. A peptide known for increased struvite yield was expressed on a membrane protein in Escherichia coli ( E. coli ), and then 5 mL precipitation reactions were performed in 50 mL culture tubes for at least 15 min. The yield of struvite crystals was examined, with the presence of peptide-expressing E. coli inducing significantly higher yields than nonpeptide-expressing E. coli when normalized to the amount of bacteria. The precipitate was identified as struvite through Fourier transform infrared spectroscopy and energy dispersive spectroscopy, while the morphology and size of the crystals were analyzed through optical microscopy and scanning electron microscopy. Crystals were found to have a larger area when precipitated with the peptide-expressing bacteria. Additionally, bacteria-struvite samples were thermogravimetrically analyzed to quantify their purity and determine their thermal decomposition behavior. Overall, this study presents the benefits of a novel, microbe-driven method of struvite precipitation, offering a means for scalable implementation., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Rare earth elements (REEs) are a vital part of many technologies with particular importance to the renewable energy sector and there is a pressing need for environmentally friendly and sustainable processes to recover and recycle them from waste streams. Functionalized polymer scaffolds are a promising means to recover REEs due to the ability to engineer both transport properties of the porous material and specificity for target ions. In this work, REE adsorbing polymer scaffolds were synthesized by first introducing poly(glycidyl methacrylate) (GMA) brushes onto porous polyvinylidene fluoride (PVDF) surface through activator generated electron transfer atom transfer radical polymerization (AGET ATRP). Azide moieties were then introduced through a ring opening reaction of GMA. Subsequently, REE-binding peptides were conjugated to the polymer surface through copper catalyzed azide alkyne cycloaddition (CuAAC) click chemistry. The presence of GMA, azide, and peptide was confirmed through Fourier transform infrared spectroscopy. Polymer scaffolds functionalized with the REE-binding peptide bound cerium, while polymer scaffolds functionalized with a scrambled control peptide bound significantly less cerium. Importantly, this study shows that the REE binding peptide retains its functionality when bound to a polymer surface. The conjugation strategy employed in this work can be used to introduce peptides onto other polymeric surfaces and tailor surface specificity for a wide variety of ions and small molecules.

Surface-grafted elastin has found a wide range of uses such as sensing, tissue engineering and capture/release applications because of its ability to undergo stimuli-responsive phase transition. While various methods exist to control surface grafting in general, it is still difficult to control orientation as attachment occurs. This study investigates using an electric field as a new approach to control the surface-grafting of short elastin-like polypeptide (ELP). Characterization of ELP grafting to gold via quartz crystal microbalance with dissipation, atomic force microscopy and temperature ramping experiments revealed that the charge/hydrophobicity of the peptides, rearrangement kinetics and an applied electric field impacted the grafted morphology of ELP. Specifically, an ELP with a negative charge on the opposite end of the surface-binding moiety assembled in a more upright orientation, and a sufficient electric field pushed the charge away from the surface compared to when the same peptide was assembled in no electric field. In addition, this study demonstrated that assembling charged ELP in an applied electric field impacts transition behavior. Overall, this study reveals new strategies for achieving desirable and predictable surface properties of surface-bound ELP., (© 2022. The Author(s).)

Polyproline peptide sequences have gained popularity as anchors for peptide-based self-assembled monolayers (SAMs) due to their attractive properties. In this work, peptides containing the polyproline II helix (PPII) conformation were designed and assembled on gold (Au). A quartz crystal microbalance with dissipation was used to characterize SAM formation kinetics and related properties. Peptides were designed with the sequence (GPPPPPG) 2 C. It was discovered that a biexponential adsorption and rearrangement model describes the binding kinetics of the PPII-containing peptide on Au. In this model, an initial reversible binding step is followed by an irreversible rearrangement step, given by parameter k t . This study found k t to be approximately 0.00064 s -1 for the PPII-containing peptides. Similarly, we found that the adsorption of the PPII-containing peptide on Au, given by Δ G ads , was thermodynamically favorable (-7.8 kcal mol -1 ) and comparable to other common thiol terminated SAMs on Au. Furthermore, we characterized SAM properties via QCM-D, Fourier-transform infrared (FTIR) spectroscopy, and electrochemical techniques to reveal high molecular density SAMs consisting of PPII helices. In addition, these SAMs were found to have high antifouling properties. Overall, this study characterizes the fundamental assembly mechanisms, particularly, rearrangement of PPII-containing peptides for the first time, which will be useful in the designing of future peptide-based SAMs with high surface coverage and antifouling properties.

Precipitation of struvite (MgNH 4 PO 4 ·6H 2 O), a slow-release fertilizer, provides a means of recycling phosphate from wastewater streams. In this work, a high-throughput struvite precipitation method is developed to investigate the effects of a peptide additive. The reactions occurred in small volumes (300 μL or less) in a 96-well plate for 45 minutes. The formation of struvite was monitored by fitting absorbance at 600 nm over time to a first order model with induction time, with the addition of peptide inducing significant changes to the yield parameter and formation constant in that model. The impact of struvite seed dosing was also investigated, highlighting the importance of optimization when peptide is present. The composition of the precipitate was confirmed through Fourier-transform infrared spectroscopy, while morphology and crystal size were analyzed through optical microscopy. Crystals had a higher aspect ratio when precipitated with the peptide. Finally, the utility of the high-throughput platform was demonstrated with a 2 5 full factorial design to capture the effects and interactions of: magnesium dose, mixing time, seed dose, pH, and temperature. Overall, this study quantifies novel effects of a sequence-defined peptide on struvite formation and morphology via a newly developed high throughput platform., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Rare-earth elements (which include all lanthanides, scandium, and yttrium) play a key role in many fields including oil refining, metallurgy, electronics manufacturing, and other high-technology applications. Although the available lanthanide resources are enough for current levels of manufacturing, increased future demand for lanthanides will require new, efficient recovery methods to provide a sustainable supply. Membrane adsorbers are promising separation materials to recover lanthanides from high volumes of wastewater due to their tailorable surface chemistry, high binding capacity and high throughput. In this work, membrane adsorbers were synthesized by first using ultraviolet-initiated free radical polymerization to graft a poly(glycidyl methacrylate) (p-GMA) layer to the surface of polyethersulfone membranes. Then, the reactive epoxy groups of the grafted p-GMA were used for the covalent attachment of lysine molecules via a zinc perchlorate-catalyzed, epoxide ring-opening reaction at 35°C. Changes in membrane surface chemistry throughout the functionalization process were monitored with Fourier Transform Infrared Spectroscopy. The degree of grafting for the p-GMA film was quantified gravimetrically and increased with increasing polymerization time. Equilibrium adsorption experiments were performed for single specie solutions of La 3+ , Ce 3+ , Nd 3+ , Na + , Ca 2+ , and Mg 2+ at pH 5.25 ± 0.25. Lysine-modified membranes showed negligible uptake of Na + , Ca 2+ , and Mg 2+ . The maximum capacities modeled by the Langmuir isotherm for La 3+ and Ce 3+ were 6.11 ± 0.58 and 6.45 ± 1.29 mg/g adsorbent, respectively. Nd 3+ adsorbed to the membrane; however, the fit of the Langmuir model was not significant and it adsorbed to a lower extent than La 3+ and Ce 3+ . Lower adsorption of the higher charge density species indicates that the primary binding mode is through the amine moieties of lysine and not the carboxylic acid. Dynamic adsorption experiments were conducted with 1 ppm La 3+ feed solutions at different flow rates using either a single membrane or three membranes in series. The dynamic binding capacity at 50% breakthrough was independent of flowrate within the tested range. The low-temperature membrane functionalization methodology presented in this work can be used to immobilize biomolecules with even higher specificity, like engineered peptides or proteins, on membrane surfaces., (Copyright © 2020 Yu, Renner and Duval.)

Proton-exchange-membrane (PEM)-based devices are promising technologies for hydrogen production and electricity generation. Currently, the amount of expensive platinum catalyst used in these devices must be reduced to be cost-competitive with other technologies. These devices typically contain Nafion ionomer thin films in the catalyst layers, which are responsible for transporting protons and gaseous species to and from electrochemically active sites. The morphology of the Nafion ionomer thin films in the catalyst layers with reduced platinum loading is impacted by interactions with the catalyst and the confinement to nanometer thicknesses, which leads to performance losses in PEM-based devices. In this study, an elastin-like polypeptide (ELP) is designed to modulate the morphology of Nafion ionomer on platinum surfaces. The ELP shows an ability to assemble into a monolayer on platinum and change the ionomer interaction with platinum, thereby modifying its thin-film structure and improving the Nafion ionomer coverage. As a proof of concept, an ELP-modified catalyst ink was prepared and morphological differences were observed. Overall, we discovered an engineered ELP that can modulate the ionomer-catalyst interface in the electrodes of PEM-based devices.

Control of ionomer thin films on metal surfaces is important for a range of electrodes used in electrochemical applications. Engineered peptides have emerged as powerful tools in electrode assembly because binding sites and peptide structures can be modulated by changing the amino acid sequence. However, no studies have been conducted showing peptides can be engineered to interact with ionomers and metals simultaneously. In this study, we design a single-repeat elastin-like peptide to bind to gold using a cysteine residue, and bind to a perfluorinated sulfonic-acid ionomer called Nafion® using a lysine guest residue. Quartz crystal microbalance with dissipation monitoring and atomic force microscopy are used to show that an elastin-like peptide monolayer attached to gold facilitates the formation of a thin, phase-separated ionomer layer. Dynamic light scattering confirms that the interaction between the peptide with the lysine residue and the ionomer also happens in solution, and circular dichroism shows that the peptides maintain their secondary structures in the presence of ionomer. These results demonstrate that elastin-like peptides are promising tools for ionomer control in electrode engineering.

Bioelectrochemical technologies have an important and growing role in healthcare, with applications in sensing and diagnostics, as well as the potential to be used as implantable power sources and be integrated with automated drug delivery systems. Challenges associated with enzyme-based electrodes include low current density and short functional lifetimes. Protein engineering is emerging as a powerful tool to overcome these issues. By taking advantage of the ability to precisely define protein sequences, electrodes can be organized into high performing structures, and enable the next generation of medical devices., (© 2016 by the Society for Experimental Biology and Medicine.)

Protein-based biomaterials have emerged as powerful tools for tissue engineering applications. Recombinant DNA techniques can be used to precisely tune material properties at the molecular level, and multiple peptide modules can be incorporated into a single material. Here, we genetically engineered biomaterials that incorporate a peptide derived from bone morphogenetic protein-2 (BMP-2) and investigated whether the BMP-2 peptide, within the context of these materials, can promote stem cells to produce bone matrix and synergize with a cell-binding sequence (RGD). Our study is the first to demonstrate that when the BMP-2 peptide is incorporated within the backbone of protein-based biomaterials, it is active and accelerates osteogenic differentiation of mesenchymal stem cells. In particular, cells seeded on proteins containing the BMP-2 peptide had increased levels of alkaline phosphatase (AP) activity, calcium deposition, and expression of bone-related genes. However, the BMP-2 peptide did not synergize with the RGD cell-binding domain within the context of these protein-based materials. Overall, these results suggest that incorporation of the BMP-2 peptide within the context of modular proteins is a successful strategy for engineering biomaterials for applications in bone tissue engineering.

Recombinant proteins are promising tools for tissue engineering and drug delivery applications. Protein-based biomaterials have several advantages over natural and synthetic polymers, including precise control over amino acid composition and molecular weight, modular swapping of functional domains, and tunable mechanical and physical properties. In this work, we describe recombinant proteins based on abductin, an elastomeric protein that is found in the inner hinge of bivalves and functions as a coil spring to keep shells open. We illustrate, for the first time, the design, cloning, expression, and purification of a recombinant protein based on consensus abductin sequences derived from Argopecten irradians . The molecular weight of the protein was confirmed by mass spectrometry, and the protein was 94% pure. Circular dichroism studies showed that the dominant structures of abductin-based proteins were polyproline II helix structures in aqueous solution and type II β-turns in trifluoroethanol. Dynamic light scattering studies illustrated that the abductin-based proteins exhibit reversible upper critical solution temperature behavior and irreversible aggregation behavior at high temperatures. A LIVE/DEAD assay revealed that human umbilical vein endothelial cells had a viability of 98 ± 4% after being cultured for two days on the abductin-based protein. Initial cell spreading on the abductin-based protein was similar to that on bovine serum albumin. These studies thus demonstrate the potential of abductin-based proteins in tissue engineering and drug delivery applications due to the cytocompatibility and its response to temperature.

Human mesenchymal stem cells (MSCs) are attractive for use in cartilage tissue engineering. Cells are often seeded in a structural scaffold containing growth factors. Peptide mimics of full-length growth factors are a promising alternative because they are less expensive and easier to manufacture. We investigated four short peptides for their effect on the chondrogenesis of human MSCs. The peptides were originally designed to mimic bone morphogenetic protein-2 (BMP-2), transforming growth factor-beta 1 (TGF-β1), and insulin, all of which have been shown to affect MSC chondrogenesis. Previous studies demonstrated that the peptides elicited bioactivity in other cell types, but the peptides have not been investigated for their effect on chondrogenesis in human MSCs. In a preliminary investigation, peptides were added to a pellet culture of human MSCs and assayed for their effect on glycosaminoglycan (GAG) production. These experiments determined peptide concentrations used in a full-factorial experiment to investigate any interactions. The experiment revealed the BMP peptide as a robust stimulant for GAG production. ., (© 2013 American Institute of Chemical Engineers.)

Directing chondrogenic differentiation of human mesenchymal stem cells (MSCs) is currently a challenging problem in tissue engineering of cartilage. Short-peptide motifs are promising new tools to aid in controlling chondrogenesis. The aim of this study was to investigate whether a short bone morphogenetic protein-2 (BMP-2)-derived peptide (BMP peptide) stimulates chondrogenesis of human MSCs in the absence of other growth factors. A high-throughput pellet culture system was used to rapidly collect biochemical data such as glycosaminoglycan (GAG), total collagen, and DNA content, as well as alkaline phosphatase (AP) activity. Cells cultured with ≥100 μg/mL of the peptide produced 74% of the GAG content that cells cultured with BMP-2 produced. Comparable levels of GAG production were promoted by the peptide and BMP-2 over 4 weeks of culture. However, histology revealed that the peptide promoted a more homogenous distribution of GAG than BMP-2. The BMP peptide directed human MSCs to increase collagen production after 3 weeks, but at significantly lower levels compared to BMP-2. Treatment with BMP-2 resulted in a large increase in hypertrophic markers such as AP activity and gene expression of type X collagen, whereas treatment with the peptide resulted in little-to-no increase in these markers. These results suggest that the BMP peptide could be an effective new tool for cartilage tissue engineering.

Modular proteins have emerged as powerful tools in tissue engineering because both the mechanical and biochemical properties can be precisely controlled through amino acid sequence. Resilin is an attractive candidate for use in modular proteins because it is well-known for having low stiffness, high fatigue lifetime, and high resilience. However, no studies have been conducted to assess resilin's compressive properties, cytocompatibility with clinically relevant cells, or effect on cell spreading. We designed a modular protein containing repeating sequences of a motif derived from Anopheles gambiae and cell-binding domains derived from fibronectin. Rapid cross-linking with tris(hydroxymethyl)phosphine was observed. The hydrogels had a complex modulus of 22 ± 1 kPa and yield strain of 63%. The elastic modulus in compression, or unconfined compressive modulus, was 2.4 ± 0.2 MPa, which is on the same order as human cartilage. A LIVE/DEAD assay demonstrated that human mesenchymal stem cells cultured on the resilin-based protein had a viability of 95% after three days. A cell-spreading assay revealed that the cells interacted with the fibronectin-derived domain in a sequence-specific manner and resulted in a mean cell area ~1.4-fold larger than when cells were seeded on a sequence-scrambled negative control protein. These results demonstrate that our resilin-based biomaterial is a promising biomaterial for cartilage tissue engineering.

Resilin has emerged as a promising new biomaterial possessing attractive properties for tissue engineering applications. To date, proteins with repeating resilin motifs have been expressed with molecular weights less than 30 kDa. This work describes the development of resilin-based proteins (repeating motif derived from Anopheles gambiae) 50 kDa in size. A modular cloning scheme was utilized and features a recursive cloning technique that can seamlessly and precisely tune the number of resilin repeats. Previously-established resilin expression protocols (based on the Studier auto-induction method) were employed to express the proteins in Escherichia coli BL21(DE3)pLysS. Western blot and densitometry results demonstrated that only ~50% of expressed proteins were the desired molecular weight. This finding suggested that either protein truncation or degradation occurred during protein expression. Preventing leaky expression, lowering the culture temperature, and harvesting during exponential phase resulted in up to 94% of the expressed proteins having the desired molecular weight. These expression conditions differ from previously-published resilin expression methods and are recommended when expressing proteins with a larger number of repetitive resilin sequences., (Copyright © 2011 Elsevier Inc. All rights reserved.)