Mickey G. Huson

Synthesis and properties of crosslinked recombinant pro-resilin

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.

Environmentally Sustainable Fibers from Regenerated Protein

Concerns for the environment and consumer demand are driving research into environmentally friendly fibers as replacements for part of the 38 million tonnes of synthetic fiber produced annually. While much current research focuses on cellulosic fibers, we highlight that protein fibers regenerated from waste or byproduct sources should also be considered. Feather keratin and wheat gluten may both be suitable. They are annually renewable, commercially abundant, of consistent quality, and have guaranteed supply. They contain useful amino acids for fiber making, with interchain cross-linking possible via cysteine residues or through the metal-catalyzed photocrosslinking of tyrosine residues. Previous commercially produced fibers suffered from poor wet strength. Contemporary nanoparticle and cross-linking technology has the potential to overcome this, allowing commercial production to resume. This would bring together two existing large production and processing pipelines, agricultural protein production and textile processing, to divert potential waste streams into useful products.

A highly elastic tissue sealant based on photopolymerised gelatin

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 >100 kPa) 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.

The development of photochemically crosslinked native fibrinogen as a rapidly formed and mechanically strong surgical tissue sealant.

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.

Honeybee silk: Recombinant protein production, assembly and fiber spinning

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.

Comparisons of Recombinant Resilin-like Proteins: Repetitive Domains Are Sufficient to Confer Resilin-like Properties

Two novel recombinant proteins An16 and Dros16 have recently been generated. These recombinant proteins contain, respectively, sixteen copies of an 11 amino acid repetitive domain (AQTPSSQYGAP) observed in a resilin-like gene from Anopheles gambiae and sixteen copies of a 15 amino acid repetitive domain (GGRPSDSYGAPGGGN) observed in the first exon of the Drosophila melanogaster CG15920 gene. We compare structural characteristics of the proteins and material properties of resulting biopolymers relative to Rec1-resilin, a previously characterized resilin-like protein encoded by the first exon of the Drosophila melanogaster CG15920 gene. While the repetitive domains of natural resilins display significant variation both in terms of amino acid sequence and length, our synthetic polypeptides have been designed as perfect repeats. Using techniques including circular dichroism, atomic force microscopy, and tensile testing, we demonstrate that both An16 and Dros16 have similar material properties to those previously observed in insect and recombinant resilins. Modulus, elasticity, resilience, and dityrosine content in the cross-linked biomaterials were assessed. Despite the reduced complexity of the An16 and Dros16 proteins compared to natural resilins, we have been able to produce elastic and resilient biomaterials with similar properties to resilin.

Single Honeybee Silk Protein Mimics Properties of Multi-Protein Silk

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.

New insights into the nature of the wool fibre surface.

Wool fibres have been treated to remove the covalently bound lipid and characterised using lipid analysis, wettability and scanning probe microscopy. A model substrate consisting of alternating stripes of hydrophobic (predominantly CH(3) terminated molecules) and hydrophilic (COOH terminated molecules) surfaces, micro-printed onto a gold-coated mica surface was assessed using the SPM techniques of adhesion, friction and phase imaging and showed that SPM can easily distinguish these surfaces. When KOH/methanol treated wool fibres were examined, SPM showed an increase in coefficient of friction and a decrease in adhesion as the lipid is removed. The increased friction is consistent with studies on the model surface and confirms the hypothesis that the lipid layer decreases the surface friction of fibres. The decreased adhesion is consistent with results in the literature on hair, but is at odds with the results on the model surface. The strong contrast shown between the methyl and carboxylic acid surfaces in the friction image of the micro-patterned surface, and the complete absence of any such contrast developing with time of treatment of the wool fibres strongly suggests that the surface lipid is not present as a discrete outer layer on the fibre. A new model is proposed in which the lipid is intimately associated with the surface proteins and allows for changes in lipid concentration at the surface in response to changes in environmental conditions.

Molecular and functional characterisation of resilin across three insect orders

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 fleas 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.

Fifty years later: The sequence, structure and function of lacewing cross-beta silk

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 67 kDa 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.