Christopher M. Elvin

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.

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.

Isolation of a trypsin‐like serine protease gene family from the sheep blowfly Lucilia cuprina

Various protease inhibitors active against both trypsin‐ and chymotrypsin‐like serine proteases were used to characterize gut proteases from Lucilia cuprina by in vitro feeding assays. Significant larval growth retardation was observed on feeding first‐instar larvae with trypsin inhibitors, particularly soybean trypsin inhibitor. Feeding of chymostatin, a specific chymotrypsin inhibitor, resulted in no significant growth retardation. This information suggests that trypsin‐like serine proteases are probably the major gut digestive enzymes. A DNA fragment obtained by PCR which coded for part of a putative trypsin gene from L. cuprina was used to isolate a four‐member multigene family of trypsins. The full nucleotide sequence of one of the genes and partial sequence from the other three genes were determined. Transcription of at least one of the genes has been confirmed. All four of the genes appear to have arisen by two separate gene duplication events.

A Synthetic Resilin Is Largely Unstructured

Proresilin is the precursor protein for resilin, an extremely elastic, hydrated, cross-linked insoluble protein found in insects. We investigated the secondary-structure distribution in solution of a synthetic proresilin (AN16), based on 16 units of the consensus proresilin repeat from Anopheles gambiae. Raman spectroscopy was used to verify that the secondary-structure distributions in cross-linked AN16 resilin and in AN16 proresilin are similar, and hence that solution techniques (such as NMR and circular dichroism) may be used to gain information about the structure of the cross-linked solid. The synthetic proresilin AN16 is an intrinsically unstructured protein, displaying under native conditions many of the characteristics normally observed in denatured proteins. There are no apparent alpha-helical or beta-sheet features in the NMR spectra, and the majority of backbone protons and carbons exhibit chemical shifts characteristic of random-coil configurations. Relatively few peaks are observed in the nuclear Overhauser effect spectra, indicating that overall the protein is dynamic and unstructured. The radius of gyration of AN16 corresponds to the value expected for a denatured protein of similar chain length. This high degree of disorder is also consistent with observed circular dichroism and Raman spectra. The temperature dependences of the NH proton chemical shifts were also measured. Most values were indicative of protons exposed to water, although smaller dependences were observed for glycine and alanine within the Tyr-Gly-Ala-Pro sequence conserved in all resilins found to date, which is the site of dityrosine cross-link formation. This result implies that these residues are involved in hydrogen bonds, possibly to enable efficient self-association and subsequent cross-linking. The beta-spiral model for elastic proteins, where the protein is itself shaped like a spring, is not supported by the results for AN16. Both the random-network elastomer model and the sliding beta-turn model are consistent with the data. The results indicate a flat energy landscape for AN16, with very little energy required to switch between conformations. This ease of switching is likely to lead to the extremely low energy loss on deformation of resilin.

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.

Localization of a Brain Sulfotransferase, SULT4A1, in the Human and Rat Brain: An Immunohistochemical Study

Cytosolic sulfotransferases are believed to play a role in the neuromodulation of certain neurotransmitters and drugs. To date, four cytosolic sulfotransferases have been shown to be expressed in human brain. Recently, a novel human brain sulfotransferase has been identified and characterized, although its role and localization in the brain are unknown. Here we present the first immunohistochemical (IHC) localization of SULT4A1 in human brain using an affinity-purified polyclonal antibody raised against recombinant human SULT4A1. These results are supported and supplemented by the IHC localization of SULT4A1 in rat brain. In both human and rat brains, strong reactivity was found in several brain regions, including cerebral cortex, cerebellum, pituitary, and brainstem. Specific signal was entirely absent on sections for which preimmune serum from the corresponding animal, processed in the same way as the postimmune serum, was used in the primary screen. The findings from this study may assist in determining the physiological role of this SULT isoform.

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.

A Genetically Engineered Protein Responsive to Multiple Stimuli

Smart protein: Careful design can yield novel biologically inspired materials that display advanced responsive behavior. A genetically engineered elastic protein displays both a lower and an upper critical solution temperature (LCST and UCST, see picture), and its photophysical behavior depends on solution pH value.

The effect of hydration on molecular chain mobility and the viscoelastic behavior of resilin-mimetic protein-based hydrogels

The outstanding rubber-like elasticity of resilin and resilin-mimetic proteins depends critically on the level of hydration. In this investigation, water vapor sorption and the role of hydration on the molecular chain dynamics and viscoelastic properties of resilin-mimetic protein, rec1-resilin is investigated in detail. The dynamic and equilibrium swelling behavior of the crosslinked protein hydrogels with different crosslink density are reported under various controlled environments. We propose three different stages of hydration; involving non-crystallizable water, followed by condensation or clustering of water around the already hydrated sites, and finally crystallizable water. The kinetics of water sorption for this engineering protein is observed to be comparable to hydrophilic polymers with a diffusion coefficient in the range of 10(-7) cm(2) s(-1). From the comparison between the absorption and desorption isotherms at a constant water activity, it has been observed that rec1-resilin exhibits sorption hysteresis only for the tightly bound water. Investigation of molecular mobility using differential scanning calorimetry, indicates that dehydrated crosslinked rec1-resilin is brittle with a glass transition temperature (T(g)) of >180 °C, which dramatically decreases with increasing hydration; and above a critical level of hydration rec1-resilin exhibits rubber-like elasticity. Nanoindentation studies show that even with little hydration (<10%), the mechanical properties of rec1-resilin gels change dramatically. Rheological investigations confirm that the equilibrium-swollen crosslinked rec1-resilin hydrogel exhibits outstanding elasticity and resilience of ∼ 92%, which exceeds that of any other synthetic polymer and biopolymer hydrogels.

Physical approaches for fabrication of organized nanostructure of resilin-mimetic elastic protein rec1-resilin

Protein adsorption on surfaces is a fundamental step in many applications. While various methods such as lithography, self assembly using nanoparticles, layer-by-layer attachment, etc. have been employed, here we report fabrication of controlled nanostructure of a new resilin-mimetic elastic protein rec1-resilin using physical approaches. We investigate the assembly, morphology and tunability of the nanostructure of adsorbed rec1-resilin architectures by atomic force microscopy (AFM) and scanning thermal microscopy (SThm) demonstrating that the protein conformation and structure during assembly can be controlled by tuning the physical conditions at the surface. Our findings show distinct morphology and height of monomolecular rec1-resilin film, dependent on substrate surface energy. We also show that these heights, a function of molecular orientation, can be maintained on swelling and drying.