Spider Silk Proteins Research Articles

Aqueous electrospinning of recombinant spider silk proteins.

There has been a significant increase in the use of sensitive biological components, e.g., growth factors or enzymes, in implanted scaffolds/devices. To prevent diffusion away from the targeted area and to maximize access of the biological agent to the desired target, it is necessary to provide a supportive substrate to immobilize and protect biological agents from the environment. For this purpose, nanofiber fabrics are highly promising due to their high porosity, capacity for solution flow-through and high surface-to-volume ratio. However, electrospinning often requires harsh processing conditions, such as the use of volatile solutions, which can result in loss of activity of the incorporated biological components. In this study we developed a mild process for electrospinning of eADF4(C16), a recombinant spider silk protein. eADF4(C16) is non-cytotoxic, displays excellent stability against hydrolytic and enzymatic degradation and opens the opportunity for genetic addition of bioactive factors. Therefore, an aqueous spinning dope of eADF4(C16) was loaded with either green fluorescence protein (GFP) or the recombinant fusion protein GFP-eADF4(C16). The fluorescence activity of GFP is dependent on its proper folding, which does not occur in organic solvents, making it an attractive model protein. We were able to demonstrate the usability as well as the significance of the all-aqueous processing conditions for the activity of GFP in electrospun spider silk scaffolds.

Microwave Assisted Sol-Gel Synthesis of Silica-Spider Silk Composites.

This study introduces a simple and environmentally friendly method to synthesize silica-protein nanocomposite materials using microwave energy to solubilize hydrophobic protein in an aqueous solution of pre-hydrolyzed organo- or fluoro-silane. Sol-gel functionality can be enhanced through biomacromolecule incorporation to tune mechanical properties, surface energy, and biocompatibility. Here, synthetic spider silk protein and organo- and fluoro-silane precursors were dissolved and mixed in weakly acidic aqueous solution using microwave technology. Scanning electron microscopy (SEM) and Atomic force microscopy (AFM) images revealed the formation of spherical nanoparticles with sizes ranging from 100 to 500 nm depending, in part, on silane fluoro- or organo-side chain chemistry. The silane-protein interaction in the nanocomposite was assessed through infrared spectroscopy. Deconvoluted ATR-FTIR (Attenuated total reflectance Fourier-transform infrared spectroscopy) spectra revealed silane chemistry-specific conformational changes in the protein-silane nanocomposites. Relative to microwave-solubilized spider silk protein, the β structure content increased by 14% in the spider silk-organo-silica nanocomposites, but decreased by a net 20% in the spider silk-fluoro-silica nanocomposites. Methods of tuning the secondary structures, and in particular β-sheets that are the cross-linking moieties in spider silks and other self-assembling fibrillar proteins, may provide a unique means to promote protein interactions, favor subsequent epitaxial growth process, and enhance the properties of the protein-silane nanocomposites.

Bioactive Silk Coatings Reduce the Adhesion of Staphylococcus aureus while Supporting Growth of Osteoblast-like Cells.

Orthopedic and dental implants are associated with a substantial risk of failure due to biomaterial-associated infections and poor osseointegration. To prevent such outcomes, a coating can be applied on the implant to ideally both reduce the risk of bacterial adhesion and support establishment of osteoblasts. We present a strategy to construct dual-functional silk coatings with such properties. Silk coatings were made from a recombinant partial spider silk protein either alone (silkwt) or fused with a cell-binding motif derived from fibronectin (FN-silk). The biofilm-dispersal enzyme Dispersin B (DspB) and two peptidoglycan degrading endolysins, PlySs2 and SAL-1, were produced recombinantly. A sortase recognition tag (SrtTag) was included to allow site-specific conjugation of each enzyme onto silkwt and FN-silk coatings using an engineered variant of the transpeptidase Sortase A (SrtA*). To evaluate bacterial adhesion on the samples, Staphylococcus aureus was incubated on the coatings and subsequently subjected to live/dead staining. Fluorescence microscopy revealed a reduced number of bacteria on all silk coatings containing enzymes. Moreover, the bacteria were mobile to a higher degree, indicating a negative influence on the bacterial adhesion. The capability to support mammalian cell interactions was assessed by cultivation of the osteosarcoma cell line U-2 OS on dual-functional surfaces, prepared by conjugating the enzymes onto FN-silk coatings. U-2 OS cells could adhere to silk coatings with enzymes and showed high spreading and viability, demonstrating good cell compatibility.

Structure of the N-terminal domain of Euprosthenops australis dragline silk suggests that conversion of spidroin dope to spider silk involves a conserved asymmetric dimer intermediate.

Spider silk is a biomaterial with exceptional mechanical toughness, and there is great interest in developing biomimetic methods to produce engineered spider silk-based materials. However, the mechanisms that regulate the conversion of spider silk proteins (spidroins) from highly soluble dope into silk are not completely understood. The N-terminal domain (NT) of Euprosthenops australis dragline silk protein undergoes conformational and quaternary-structure changes from a monomer at a pH above 7 to a homodimer at lower pH values. Conversion from the monomer to the dimer requires the protonation of three conserved glutamic acid residues, resulting in a low-pH `locked' dimer stabilized by symmetric electrostatic interactions at the poles of the dimer. The detailed molecular events during this transition are still unresolved. Here, a 2.1 Å resolution crystal structure of an NT T61A mutant in an alternative, asymmetric, dimer form in which the electrostatic interactions at one of the poles are dramatically different from those in symmetrical dimers is presented. A similar asymmetric dimer structure from dragline silk of Nephila clavipes has previously been described. It is suggested that asymmetric dimers represent a conserved intermediate state in spider silk formation, and a revised `lock-and-trigger' mechanism for spider silk formation is presented.

CRISPR/Cas9 Initiated Transgenic Silkworms as a Natural Spinner of Spider Silk.

Using transgenic silkworms with their natural spinning apparatus has proven to be a promising way to spin spider silk-like fibers. The challenges are incorporating native-size spider silk proteins and achieving an inheritable transgenic silkworm strain. In this study, a CRISPR/Cas9 initiated fixed-point strategy was used to successfully incorporate spider silk protein genes into the Bombyx mori genome. Native-size spider silk genes (up to 10 kb) were inserted into an intron of the fibroin heavy or light chain (FibH or FibL) ensuring that any sequence changes induced by the CRISPR/Cas9 would not impact protein production. The resulting fibers are as strong as native spider silks (1.2 GPa tensile strength). The transgenic silkworms have been tracked for several generations with normal inheritance of the transgenes. This strategy demonstrates the feasibility of using silkworms as a natural spider silk spinner for industrial production of high-performance fibers.

Material Formation of Recombinant Spider Silks through Aqueous Solvation using Heat and Pressure.

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order to reproduce the remarkable mechanical properties of spider silks, they must be produced in heterologous hosts as spiders are both territorial and cannibalistic. The synthetic analogs of spider silk also tend to be insoluble in aqueous solutions. Thus, a large percentage of research in recombinant spider silks rely upon organic solvents that are detrimental to large scale production of materials. Our group's method forces the solvation of these recombinant spider silks into water. Remarkably, when these proteins are prepared using this method of heat and pressure, a wide range of material forms can be prepared from the same solution of recombinant spider silk proteins (rSSp) including: films, fibers, sponge, hydrogel, lyogel, and adhesives. This article demonstrates the production of the solvated rSSp and material forms in a manner that is more easily understood than from written materials and methods alone.

Intrinsic Vascularization of Recombinant eADF4(C16) Spider Silk Matrices in the Arteriovenous Loop Model.

The surgically induced angiogenesis by means of arteriovenous (AV) loops represents a powerful method to significantly enhance vascularization of biomaterials. Regarding tissue engineering applications, spider silk is a promising biomaterial with a good biocompatibility and slow biodegradation. This study aims at investigating vascularization as well as de novo tissue formation of fibrous matrices made of electro-spun (ES) or wet-spun (WS) engineered ADF4(C16) spider silks in the rat AV loop model. Either ES or WS spider silk fibrous matrices were filled into Teflon chambers. Intrinsic vascularization was induced by means of an AV loop. After 4 weeks of vascularization, de novo tissue formation and biocompatibility were analyzed. Regardless of their significantly differing fiber diameters, both ES and WS eADF4(C16) fiber matrices displayed a good biocompatibility and initiated de novo tissue formation as well as vessel formation. Both matrices demonstrated partial vascularization originating from the AV loop, with more vessels in spider silk matrices with lower fiber diameters. We were able to demonstrate intrinsic vascularization of spider silk fibrous matrices by means of the AV loop. Moreover, our study indicates that the adjustment of the fiber diameter of engineered spider silks enables new possibilities to optimize vascularization. Impact Statement Spider silk is a promising biomaterial demonstrating excellent biocompatibility and biodegradation. Biotechnology allows the high-volume production of recombinant spider silk proteins, such as eADF4(C16), with the required purity for biomedical applications. In this study, eADF4(C16) fibrous matrices were produced by either electro- or wet-spinning, resulting in different fiber diameters. Forming an arteriovenous fistula, surgical vascularization of the scaffolds was induced. After 4 weeks, both silks demonstrated a good biocompatibility and tissue formation. The thinner electro-spun fibers displayed a faster biodegradation and vascularization, indicating that the adjustment of the fiber diameter is a valuable tool to fine-tune vascularization and biodegradation.

Fibers Made of Recombinant Spidroins—A Brief Review

Spider silk is a promising biomaterial, with a wide range of potential applications in the medical, military, and commercial sectors. In the past decade, researchers have been developing recombinant spider silk (RSS) proteins with the help of advanced genetic engineering techniques, including gene design, cloning, and protein expression in different foreign hosts. The extraordinary strength and extensibility of natural spider silk fiber are constituted from the tandemly repeated amino acid sequences and antiparallel crystalline β-sheet segments formed by polyalanine motifs. Additionally, mechanical properties are also influenced by extrusion and drawing of fibers in the natural spinning process. To date, the wet spinning process was found to be a commercially successful method for spinning recombinant silks with properties comparable to their natural counterparts.

Insight into spider silk proteins

Insight into spider silk proteins

SpiderMAEn: recombinant spider silk-based hybrid materials for advanced energy technology

A growing energy demand requires new and preferably renewable energy sources. The infinite availability of solar radiation makes its conversion into storable and transportable energy forms attractive for research as well as for the industry. One promising example of a transportable fuel is hydrogen (H2), making research into eco-friendly hydrogen production meaningful. Here, a hybrid system was developed using newly designed recombinant spider silk protein variants as a template for mineralization with inorganic titanium dioxide and gold. These bioinspired organic/inorganic hybrid materials allow for hydrogen production upon light irradiation. To begin with, recombinant spider silk proteins bearing titanium dioxide and gold-binding moieties were created and processed into structured films. These films were modified with gold and titanium dioxide in order to produce a photocatalyst. Subsequent testing revealed hydrogen production as a result of light-induced hydrolysis of water. Therefore, the novel setup presented here provides access to a new principle of generating advanced hybrid materials for sustainable hydrogen production and depicts a promising platform for further studies on photocatalytic production of hydrogen, the most promising future fuel.

Recombinant Silk Fiber Properties Correlate to Prefibrillar Self-Assembly.

Spider silks are desirable materials with mechanical properties superior to most synthetic materials coupled with biodegradability and biocompatibility. In order to replicate natural silk properties using recombinant spider silk proteins (spidroins) and wet-spinning methods, the focus to date has typically been on modifying protein sequence, protein size, and spinning conditions. Here, an alternative approach is demonstrated. Namely, using the same ≈57 kDa recombinant aciniform silk protein with a consistent wet-spinning protocol, fiber mechanical properties are shown to significantly differ as a function of the solvent used to dissolve the protein at high concentration (the "spinning dope" solution). A fluorinated acid/alcohol/water dope leads to drastic improvement in fibrillar extensibility and, correspondingly, toughness compared to fibers produced using a previously developed fluorinated alcohol/water dope. To understand the underlying cause for these mechanical differences, morphology and structure of the two classes of silk fiber are compared, with features tracing back to dope-state protein structuring and preassembly. Specifically, distinct classes of spidroin nanoparticles appear to form in each dope prior to fiber spinning and these preassembled states are, in turn, linked to fiber morphology, structure, and mechanical properties. Tailoring of dope-state spidroin nanoparticle assembly, thus, appears a promising strategy to modulate fibrillar silk properties.

Investigating Recombinant Aciniform Silk Nanoparticles as Potential Drug Carriers and as Intermediates in Silk Fibrillogenesis

Spider silks form biomaterials including fibres, films, and nanospheres with excellent mechanical properties and biocompatibility. As such, silk materials have been investigated as a component of biomedical textiles for grafts and implants, as a scaffold for tissue regeneration, and as a drug delivery vehicle. Silk fibres generally form in vivo in a process where soluble proteins are forced through a narrowing spinning duct, giving rise to shear force. One theory of fibre formation is that silk proteins are stored in micellar form, with these micelles elongating while being forced together in the spinning duct, undergoing morphological and conformational transitions as shear forces are applied. Silk nanoparticles can be self-assembled, with concomitant or post-assembly incorporation of a variety of drug-like molecules into their interior. Here, we focus on the mechanism of formation of previously uncharacterized nanoparticles by recombinant aciniform spider silk proteins, their potential as drug carriers, and the role of these self-assembled species in the initial stages of fibre formation. The aciniform spidroin protein (AcSp1) from Argiope trifasciata consists of ≥14 repeat units of 200 amino acids (“W”, from wrapping silk) flanked by a non-repetitive N- and C-terminal domains. Recombinant proteins consisting of a single repeat (W1) spontaneously self-assemble into spherical micelle-like nanoparticles in potassium phosphate buffer. Insoluble W1 nanoparticles also form in highly concentrated phosphate buffer. Features of conformational transitions associated with nanoparticle formation from monomeric W1 are delineated here using tryptophan mutants of W1 through intrinsic tryptophan fluorescence and 19F NMR spectroscopy. Nanoparticle properties are correlated to these features using electron microscopy, dynamic light scattering, and Raman spectroscopy. Ultimately, reproducible and specific production of these nanoparticles will assist in the design and optimization of aciniform silk-based materials for biomedical and other materials applications.

Nanostructured, Self-Assembled Spider Silk Materials for Biomedical Applications.

The extraordinary mechanical properties of spider silk fibers result from the interplay of composition, structure and self-assembly of spider silk proteins (spidroins). Genetic approaches enabled the biotechnological production of recombinant spidroins which have been employed to unravel the self-assembly and spinning process. Various processing conditions allowed to explore non-natural morphologies including nanofibrils, particles, capsules, hydrogels, films or foams. Recombinant spider silk proteins and materials made thereof can be utilized for biomedical applications, such as drug delivery, tissue engineering or 3D-biomanufacturing.

A proteotranscriptomic study of silk-producing glands from the orb-weaving spiders.

Orb-weaving spiders can produce different silk fibers, which constitute outstanding materials characterized by their high strength and elasticity. Researchers have tried to reproduce the fibers of these proteins synthetically and/or by using recombinant DNA technology, but only a few of the natural physicochemical and biophysical properties have been obtained to date. Female orb-web-spiders present seven silk-glands, which synthesize the spidroins and a series of other proteins, which interact with the spidroins, resulting in silk fibers with notable physicochemical properties. Despite the recognized importance of the silk-glands for understanding how the fibers are produced and processed, the investigation of these glands is at a nascent stage. In the current study we present the assembled transcriptome of silk-producing glands from the orb-weaving spider Nephila clavipes, as well as develop a large-scale proteomic approach for in-depth analyses of silk-producing glands. The present investigation revealed an extensive repertoire of hitherto undescribed proteins involved in silk secretion and processing, such as prevention of degradation during the silk spinning process, transportation, protection against proteolytic autolysis and against oxidative stress, molecular folding and stabilization, and post-translational modifications. Comparative phylogenomic-level evolutionary analyses revealed orthologous genes among three groups of silk-producing organisms - (i) Araneomorphae spiders, (ii) Mygalomorphae spiders, and (iii) silk-producing insects. A common orthologous gene, which was annotated as silk gland factor-3 is present among all species analysed. This protein belongs to a transcription factor family, that is important and related to the development of the silk apparatus synthesis in the silk glands of silk-producing arthropods.

Ion effects on the conformation and dynamics of repetitive domains of a spider silk protein: implications for solubility and β-sheet formation.

The effect of ions on the structure and dynamics of a spider silk protein is elucidated. Chaotropic ions prevent intra- and inter-molecular interactions on the repetitive domain, which are required to maintain the solubility, while kosmotropic ions promote hydrogen bond interactions in the glycine-rich region, which are a prerequisite for β-sheet formation.

Triboelectric Nanogenerators: “Genetically Engineered” Biofunctional Triboelectric Nanogenerators Using Recombinant Spider Silk (Adv. Mater. 50/2018)

In article number 1805722, Tiger H. Tao and co-workers report a new strategy for developing a set of “genetic-engineered” bioelectronics with programmable triboelectric property, multiple functionalization, large-scale-fabrication capability, and transcendent output performance using recombinant spider silk proteins.

Nanoscale Patterning of Surfaces via DNA Directed Spider Silk Assembly

Oligonucleotide-spider silk conjugates can be placed on silicon wafers by complementary DNA strands, which are coupled chemically to the surface. Such specific immobilization of spider silk proteins allows the nucleation and guided growth of β-sheet-rich nanofibrils in the presence of phosphate ions on the surface. Adjustment of the concentration of the immobilized conjugate, phosphate concentration and time of the assembly reaction enables control over fibril surface density and length. Furthermore, soft lithography was used to direct the conjugates on predetermined spots with a submicron resolution yielding high contrast surface patterns. This approach, which combines bottom-up and top-down surface structuring, opens up new possibilities in protein fibril based bionanotechnology.

"Genetically Engineered" Biofunctional Triboelectric Nanogenerators Using Recombinant Spider Silk.

Self-powered electronics using triboelectric nanogenerators (TENGs) is drawing increasing efforts and rapid advancements in eco/biocompatible energy harvesting, intelligent sensing, and biomedical applications. Currently, the triboelectric performances are mainly determined by the pair materials' inherent electron affinity difference, and merely tuned by chemical or physical methods, which significantly limit the optional variety and output capability, especially for natural-biomaterial-based TENGs. Herein, a biocompatible triboelectric material with a programmable triboelectric property, multiple functionalization, large-scale-fabrication capability, and transcendent output performance is designed, by genetically engineering recombinant spider silk proteins (RSSP). Featuring totally "green" large-scale manufacturing, the water lithography technique is introduced to the RSSP-TENG with facilely adjustable surface morphology, chemically modifiable surface properties, and controllable protein conformation. By virtue of the high electrical power, a proof-of-principle drug-free RSSP-patch is built, showing outstanding antibacterial performances both in vitro and in vivo. This work provides a novel high-performance biomaterial-based TENG and extends its potential for multifunctional applications.

Silk genes and silk gene expression in the spider Tengella perfuga (Zoropsidae), including a potential cribellar spidroin (CrSp).

Most spiders spin multiple types of silk, including silks for reproduction, prey capture, and draglines. Spiders are a megadiverse group and the majority of spider silks remain uncharacterized. For example, nothing is known about the silk molecules of Tengella perfuga, a spider that spins sheet webs lined with cribellar silk. Cribellar silk is a type of adhesive capture thread composed of numerous fibrils that originate from a specialized plate-like spinning organ called the cribellum. The predominant components of spider silks are spidroins, members of a protein family synthesized in silk glands. Here, we use silk gland RNA-Seq and cDNA libraries to infer T. perfuga silks at the protein level. We show that T. perfuga spiders express 13 silk transcripts representing at least five categories of spider silk proteins (spidroins). One category is a candidate for cribellar silk and is thus named cribellar spidroin (CrSp). Studies of ontogenetic changes in web construction and spigot morphology in T. perfuga have documented that after sexual maturation, T. perfuga females continue to make capture webs but males halt web maintenance and cease spinning cribellar silk. Consistent with these observations, our candidate CrSp was expressed only in females. The other four spidroin categories correspond to paralogs of aciniform, ampullate, pyriform, and tubuliform spidroins. These spidroins are associated with egg sac and web construction. Except for the tubuliform spidroin, the spidroins from T. perfuga contain novel combinations of amino acid sequence motifs that have not been observed before in these spidroin types. Characterization of T. perfuga silk genes, particularly CrSp, expand the diversity of the spidroin family and inspire new structure/function hypotheses.

Facile Photochemical Modification of Silk Protein-Based Biomaterials.

Silk protein-based materials show promise for application as biomaterials for tissue engineering. The simple and rapid photochemical modification of silk protein-based materials composed of either Bombyx mori silkworm silk or engineered spider silk proteins (eADF4(C16)) is reported. Radicals formed on the silk-based materials initiate the polymerization of monomers (acrylic acid, methacrylic acid, or allylamine) which functionalize the surface of the silk materials with poly(acrylic acid) (PAA), poly(methacrylic acid) (PMAA), or poly(allylamine) (PAAm). To demonstrate potential applications of this type of modification, the polymer-modified silks are mineralized. The PAA- and PMAA-functionalized silks are mineralized with calcium carbonate, whereas the PAAm-functionalized silks are mineralized with silica, both of which provide a coating on the materials that may be useful for bone tissue engineering, which will be the subject of future investigations.