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Sequence-definable polymers are seen as a prerequisite for design of future materials, with many polymer scientists regarding such polymers as the holy grail of polymer science. Recombinant proteins are sequence-defined polymers. Proteins are dictated by DNA templates and therefore the sequence of amino acids in a protein is defined, and molecular biology provides tools that allow redesign of the DNA as required. Despite this advantage, proteins are underrepresented in materials science. In this publication we investigate the advantages and limitations of using proteins as templates for rational design of new materials. [ABSTRACT FROM AUTHOR]

A focused ion beam has been used to mill both individual carbon fibres as well as fibres in an epoxy composite, with a view to preparing flat surfaces for nano-indentation. The milled surfaces have been assessed for damage using scanning probe microscopy nano-indentation and Raman micro-probe analysis, revealing that FIB milling damages the carbon fibre surface and covers surrounding areas with debris of disordered carbon. The debris is detected as far as 100 μm from the milling site. The energy of milling as well as the orientation of the beam was varied and shown to have an effect when assessed by Raman spectroscopy. [ABSTRACT FROM AUTHOR]

Abstract: A range of polyacrylonitrile (PAN) and pitch based carbon fibre types (high, standard and intermediate modulus fibres) have been characterised using both physical and chemical techniques, the results highlighting the heterogeneity of the fibre. Nano-indentation showed variation in stiffness between different fibres of the same type as well as variation along a 20μm length of a single fibre. Tensile tests showed variance of approximately 25% in tenacity for three different carbon fibre types but less variability in modulus with values from 8% to 19%. Raman spectroscopy showed variation in the graphitic content both between fibres of different origin as well as variation, with 0.5μm spatial resolution, along the length of a single fibre. Inverse gas chromatography surface energy measurements of larger samples of fibres were carried out using the novel approach of incremental surface coverage by varying the probe molecule concentration and revealed different levels of energetic heterogeneity for PAN based fibres collected at different stages of carbon fibre production. The heterogeneity of the unoxidised fibres (collected after carbonisation) was restricted to about 15% of the fibre surface whereas the surface oxidised fibre sample (collected after the electrolytic oxidation bath) was heterogeneous over more than 30% and the sized fibres were shown to be quite homogeneous. [Copyright &y& Elsevier]

Honeybee larvae produce silken cocoons that provide mechanical stability to the hive. The silk proteins are small and nonrepetitive and therefore can be produced at large scale by fermentation in E. coli. The recombinant proteins can be fabricated into a range of forms; however the resultant material is soluble in water and requires a post production stabilizing treatment. In this study, we describe the structural and mechanical properties of sponges fabricated from artificial honeybee silk proteins that have been stabilized in aqueous methanol baths or by dry heating. Aqueous methanol treatment induces formation of ß-sheets, with the amount of ß-sheet dictated by methanol concentration. Formation of ßsheets renders sponges insoluble in water and generates a reversibly compressible material. Dry heat treatments at 190°C produce a water insoluble material, that is stiffer than the methanol treated equivalent but without significant secondary structural changes. Honeybee silk proteins are particularly high in Lys, Ser, Thr, Glu and Asp. The properties of the heat treated material are attributed to generation of lysinoalanine, amide (isopeptide) and/or ester covalent cross-links. The unique ability to stabilize material by controlling secondary structure rearrangement and covalent cross-linking allows us to design recombinant silk materials with a wide range of properties. [ABSTRACT FROM AUTHOR]

We demonstrate the utilisation of an azomethine 1,3-dipolar cycloaddition reaction with carbon fibre to graft complex molecules onto the fibre surface. In an effort to enhance the interfacial interaction of the fibre to the matrix, the functionalised fibres possessed a pendant amine that is able to interact with epoxy resins. Functionalisation was supported by X-ray photoelectron spectroscopy and the grafting process had no detrimental effects on tensile strength compared with the control (untreated) fibres. Also, microscopic roughness (as determined by atomic force microscopy) and fibre topography were unchanged after the described treatment process. This methodology complements existing methodology aimed at enhancing the surface of carbon fibres for advanced material applications while not compromising the desirable strength profile. Single-fibre fragmentation tests show a statistically significant decrease in fragment length compared with the control fibres in addition to transverse cracking within the curing resin, both of which indicate an enhanced interaction between fibre and resin. [ABSTRACT FROM AUTHOR]

This work investigates the mechanisms involved in the improvement of flexural properties of a jute/polyester composite when the reinforcement material has been atmospherically plasma treated using helium gas. All composites were laid-up by hand and cured using a Quickstep™ cure cycle. Surface characterization techniques including scanning probe microscopy (SPM), and surface wettability combined with fabric tensile strength, composite flexural strength and composite Mode-I properties have been used to quantify the effects of plasma modification. Flexural strength and modulus increased with plasma treatment time, reaching a maximum at 25 passes before decreasing. SPM topographical analysis showed that roughness of the fibre decreased as the plasma treatment time increased until 25 passes after which the roughness was found to increase again. The coefficient of friction increased rapidly after only a short plasma treatment time (5 passes) whilst wettability continued to increase until 25 passes after which it remained constant. The fabric tensile strength followed the same trend as the flexural properties of the composites. Decreasing fibre surface roughness is postulated as a reason for decreasing Mode-I interlaminar fracture toughness properties of the composites. [ABSTRACT FROM AUTHOR]

Abstract: We recently reported the generation of a highly elastic, crosslinked protein biomaterial via a rapid photochemical process using visible light illumination. In light of these findings, we predicted that other unmodified, tyrosine-rich, self-associating proteins might also be susceptible to this covalent crosslinking method. Here we show that unmodified native fibrinogen can also be photochemically crosslinked into an elastic hydrogel biomaterial through the rapid formation of intermolecular dityrosine. Photochemically crosslinked fibrinogen forms tissue sealant bonds at least 5-fold stronger than commercial fibrin glue and is capable of producing maximum bond strength within 20s. In vitro studies showed that components of the photochemical crosslinking reaction are non-toxic to cells. This material will find useful application in various surgical procedures where rapid curing for high strength tissue sealing is required. [Copyright &y& Elsevier]

Resilin is a member of a family of elastic proteins that includes elastin, as well as gluten, gliadin, abductin and spider silks. Resilin is found in specialized regions of the cuticle of most insects, providing low stiffness, high strain and efficient energy storage; it is best known for its roles in insect flight and the remarkable jumping ability of fleas and spittle bugs. Previously, the Drosophila melanogaster CG15920 gene was tentatively identified as one encoding a resilin-like protein (pro-resilin). Here we report the cloning and expression of the first exon of the Drosophila CG15920 gene as a soluble protein in Escherichia coli. We show that this recombinant protein can be cast into a rubber-like biomaterial by rapid photochemical crosslinking. This observation validates the role of the putative elastic repeat motif in resilin function. The resilience (recovery after deformation) of crosslinked recombinant resilin was found to exceed that of unfilled synthetic polybutadiene, a high resilience rubber. We believe that our work will greatly facilitate structural investigations into the functional properties of resilin and shed light on more general aspects of the structure of elastomeric proteins. In addition, the ability to rapidly cast samples of this biomaterial may enable its use in situ for both industrial and biomedical applications. [ABSTRACT FROM AUTHOR]

Low interface strength is a common challenge in taking full advantage of excellent physical performances of carbon fibre (CF) reinforced polymer composites. Herein, for the first time, we have used amino-functionalized nanoclay as a linkage between CF surface and epoxy matrix, which by a cation exchange process was grafted on the CF surface. Amino-functionalized nanoclay significantly increased surface roughness, coefficient of friction, and BET surface area of CF. The results showed that nanoclay-based modification does not change the tensile strength and Weibull modulus of CF significantly however, both specific and dispersive surface energies, obtained by inverse gas chromatography technique, were increased. A high compatibility of nanoclay-based modified CF (clay@CF) with epoxy resin was also observed by analysing the contact angle between epoxy droplets and fibre surface. Moreover, single fibre fragmentation tests (SFFT) alongside fractographic observations showed that the length of fibre pull-out and the size of cracks between the fibre and matrix were outstandingly reduced in clay@CF in comparison to untreated CF, demonstrating that the stress transfer and interfacial shear strength (IFSS) have been significantly improved. [ABSTRACT FROM AUTHOR]

A recently established means of surface functionalization of unsized carbon fibres for enhanced compatibility with epoxy resins was optimised and evaluated using interfacial shear stress measurements. Interfacial adhesion has a strong influence on the bulk mechanical properties of composite materials. In this work we report on the optimisation of our aryl diazo-grafting methodology via a series of reagent concentration studies. The fibres functionalised at each concentration are characterised physically (tensile strength, modulus, coefficient of friction, and via AFM), and chemically (XPS). The interfacial shear strength (IFSS) of all treated fibres was determined via the single fibre fragmentation test, using the Kelly–Tyson model. Large increases in IFSS for all concentrations (28–47%) relative to control fibres were observed. We show that halving the reagent concentration increased the coefficient of friction of the fibre and the interfacial shear strength of the composite while resulting in no loss of the key performance characteristics in the treated fibre. [ABSTRACT FROM AUTHOR]

Abstract: The use of coiled coil proteins as the basis of silk materials is an engineering solution that has evolved convergently in at least five insect lineages—the stinging hymenopterans (ants, bees, hornets), argid sawflies, fleas, lacewings, and praying mantises—and persisted throughout large radiations of these insect families. These coiled coil silk proteins share a characteristic distinct from other coiled coil proteins, in that they are fabricated into solid materials after accumulating as highly concentrated solutions within dedicated glands. Here, we relate the amino acid sequences of these proteins to the secondary and tertiary structural information available from biophysical methods such as X-ray scattering, nuclear magnetic resonance and Raman spectroscopy. We investigate conserved and convergently evolved features within these proteins and compare these to the features of classic coiled coil proteins including tropomyosin and leucine zippers. Our analysis finds that the coiled coil domains of insect silk proteins have several common structural anomalies including a high prevalence of alanine residues in core positions. These atypical features of the coiled coil fibrous proteins – which likely produce deviations from canonical coiled-coil structure – likely exist due to selection pressures related to the process of silk fabrication and the final function of the proteins. [Copyright &y& Elsevier]

Collagen is ubiquitous throughout the animal kingdom, where it comprises some 28 diverse molecules that form the extracellular matrix within organisms. In the 1960s, an extracorporeal animal collagen that forms the cocoon of a small group of hymenopteran insects was postulated. Here we categorically demonstrate that the larvae of a sawfly species produce silk from three small collagen proteins. The native proteins do not contain hydroxyproline, a post translational modification normally considered characteristic of animal collagens. The function of the proteins as silks explains their unusual collagen features. Recombinant proteins could be produced in standard bacterial expression systems and assembled into stable collagen molecules, opening the door to manufacture a new class of artificial collagen materials. [ABSTRACT FROM AUTHOR]

Abstract: The surface of both oxidized and unoxidized unsized carbon fiber was functionalized using an aziridine linking group derived from reactive nitrenes. The aziridine functionality arose from the cyclization of a reactive nitrene species onto the highly electron rich graphitic surface of the carbon fibers; the nitrene species evolved from thermal N2 elimination from the corresponding (room temperature stable) azide. Surface functionalization using the nitrene approach was supported by X-ray Photoelectron Spectroscopy, in both oxidized and unoxidized carbon fiber. Attempts were also made to functionalize using amide chemistry, the two-step acid chloride coupling being successful for oxidized fibers by utilizing the carboxylic acid rich defect sites on the carbon fiber. None of the chemical treatment pathways had a significant impact on the tensile strength of the individual fibers, and atomic force microscopy revealed that fibers undergoing these treatment methodologies remained intact, without creating additional surface defects. [Copyright &y& Elsevier]

Abstract: Resilin is an important elastomeric protein of insects, with roles in the storage and release of energy during a variety of different functional categories including flight and jumping. To date, resilin genes and protein function have been characterised only in a small number of flying insects, despite their importance in fleas and other jumping insects. Microscopy and immunostaining studies of resilin in flea demonstrate the presence of resilin pads in the pleural arch at the top of the hind legs, a region responsible for the flea’s jumping ability. A degenerate primer approach was used to amplify resilin gene transcripts from total RNA isolated from flea (Ctenocephalides felis), buffalo fly (Haematobia irritans exigua) and dragonfly (Aeshna sp.) pharate adults, and full-length transcripts were successfully isolated. Two isoforms (A and B) were amplified from each of flea and buffalo fly, and isoform B only in dragonfly. Flea and buffalo fly isoform B transcripts were expressed in an Escherichia coli expression system, yielding soluble recombinant proteins Cf-resB and Hi-resB respectively. Protein structure and mechanical properties of each protein before and after crosslinking were assessed. This study shows that resilin gene and protein sequences are broadly conserved and that crosslinked recombinant resilin proteins share similar mechanical properties from flying to jumping insects. A combined use of degenerate primers and polyclonal sera will likely facilitate characterisation of resilin genes from other insect and invertebrate orders. [Copyright &y& Elsevier]

Honeybee silk is composed of four fibrous proteins that, unlike other silks, are readily synthesized at full-length and high yield. The four silk genes have been conserved for over 150 million years in all investigated bee, ant and hornet species, implying a distinct functional role for each protein. However, the amino acid composition and molecular architecture of the proteins are similar, suggesting functional redundancy. In this study we compare materials generated from a single honeybee silk protein to materials containing all four recombinant proteins or to natural honeybee silk. We analyse solution conformation by dynamic light scattering and circular dichroism, solid state structure by Fourier Transform Infrared spectroscopy and Raman spectroscopy, and fiber tensile properties by stress-strain analysis. The results demonstrate that fibers artificially generated from a single recombinant silk protein can reproduce the structural and mechanical properties of the natural silk. The importance of the four protein complex found in natural silk may lie in biological silk storage or hierarchical self-assembly. The finding that the functional properties of the mature material can be achieved with a single protein greatly simplifies the route to production for artificial honeybee silk. [ABSTRACT FROM AUTHOR]

Abstract: Gelatin is widely used as a medical biomaterial because it is readily available, cheap, biodegradable and demonstrates favourable biocompatibility. Many applications require stabilisation of the biomaterial by chemical crosslinking, and this often involves derivatisation of the protein or treatment with cytotoxic crosslinking agents. We have previously shown that a facile photochemical method, using blue light, a ruthenium catalyst and a persulphate oxidant, produces covalent di-tyrosine crosslinks in resilin and fibrinogen to form stable hydrogel biomaterials. Here we show that various gelatins can also be rapidly crosslinked to form highly elastic (extension to break >650%) and adhesive (stress at break >100kPa) biomaterials. Although the method does not require derivatisation of the protein, we show that when the phenolic (tyrosine-like) content of gelatin is increased, the crosslinked material becomes resistant to swelling, yet retains considerable elasticity and high adhesive strength. The reagents are not cytotoxic at the concentration used in the photopolymerisation reaction. When tested in vivo in sheep lung, the photopolymerised gelatin effectively sealed a wound in lung tissue from blood and air leakage, was not cytotoxic and did not produce an inflammatory response. The elastic properties, thermal stability, speed of curing and high tissue adhesive strength of this photopolymerised gelatin, offer considerable improvement over current surgical tissue sealants. [ABSTRACT FROM AUTHOR]

Abstract: Transgenic production of silkworm and spider silks as biomaterials has posed intrinsic problems due to the large size and repetitive nature of the silk proteins. In contrast the silk of honeybees (Apis mellifera) is composed of a family of four small and non-repetitive fibrous proteins. We report recombinant production and purification of the four full-length unmodified honeybee silk proteins in Escherichia coli at substantial yields of 0.2–2.5 g/L. Under the correct conditions the recombinant proteins self-assembled to reproduce the native coiled coil structure. Using a simple biomimetic spinning system we could fabricate recombinant silk fibers that replicated the tensile strength of the native material. [Copyright &y& Elsevier]

Abstract: Classic studies of protein structure in the 1950s and 1960s demonstrated that green lacewing egg stalk silk possesses a rare native cross-beta sheet conformation. We have identified and sequenced the silk genes expressed by adult females of a green lacewing species. The two encoded silk proteins are 109 and 67kDa in size and rich in serine, glycine and alanine. Over 70% of each protein sequence consists of highly repetitive regions with 16-residue periodicity. The repetitive sequences can be fitted to an elegant cross-beta sheet structural model with protein chains folded into regular 8-residue long beta strands. This model is supported by wide-angle X-ray scattering data and tensile testing from both our work and the original papers. We suggest that the silk proteins assemble into stacked beta sheet crystallites bound together by a network of cystine cross-links. This hierarchical structure gives the lacewing silk high lateral stiffness nearly threefold that of silkworm silk, enabling the egg stalks to effectively suspend eggs and protect them from predators. [Copyright &y& Elsevier]