Spider Silk Proteins Research Articles

Characteristic Evaluation of Recombinant MiSp/Poly(lactic-co-glycolic) Acid (PLGA) Nanofiber Scaffolds as Potential Scaffolds for Bone Tissue Engineering.

Biomaterial-based nanofibrous scaffolds are the most effective alternative to bone transplantation therapy. Here, two recombinant minor ampullate spidroins (spider silk proteins), R1SR2 and NR1SR2C, were blended with Poly(lactic-co-glycolic) Acid (PLGA), respectively, to generate nanofiber scaffolds by electrospinning. The N-terminal (N), C-terminal (C), repeating (R1 and R2) and spacer (S) modules were all derived from the minor ampullate spidroins (MiSp). The physical properties and structures of the blended scaffolds were measured by scanning electron microscopy (SEM), water contact angle measurement, Fourier transform infrared spectroscopy (FTIR), Differential scanning calorimetry (DSC), and Tensile mechanical testing. The results showed that blending of MiSp (R1SR2 and NR1SR2C) reduced the diameter of nanofibers, increased the porosity and glass transition temperatures of nanofibrous scaffolds, and effectively improved the hydrophilicity and ultimate strain of scaffolds. It is worth noting that the above changes were more significant in the presence of the N- and C-termini of MiSp. In cell culture assays, human bone mesenchymal stem cells (HBMSCs) grown on NR1SR2C/PLGA (20/80) scaffolds displayed markedly enhanced proliferative and adhesive abilities compared with counterparts grown on pure PLGA scaffolds. Jointly, these findings indicated recombinant MiSp/PLGA, particularly NR1SR2C/PLGA (20/80) blend nanofibrous scaffolds, is promising for bone tissue engineering.

Development of an Injection Method for the Genetic Engineering of Diapause Silkworm Egg Using Dimethyl Sulfoxide

Genetic engineering of the domesticated silkworm Bombyx mori is a useful tool for developing sericulture. New races that produce recombinant silk with fluorescent color and spider silk protein have been constructed. Strains resistant to B. mori nuclear polyhedrosis virus have also been constructed and started to rear in the field. In this genetic engineering method, the use of nondiapause eggs that do not arrest embryonic development is indispensable as it includes the injection process of vector DNA into the eggs. However, most silkworm strains are bivoltine or univoltine and lay diapause eggs that arrest their embryonic development. Thus, applying this method to these silkworm strains is impossible. To enable the application, whether dimethyl sulfoxide (DMSO), which can prevent the diapause of eggs, can be used to develop an injection method for the genetic engineering of diapause strains was investigated. The DMSO treatment worked well for this purpose, and an injection method for the genetic engineering of diapause eggs was successfully developed. This method gave high hatchability in the injection of diapause eggs. Genetically modified silkworms were efficiently created in three different diapause silkworms: hybrid race and Japanese and Chinese strains. Thus, DMSO treatment is useful for creating genetically modified silkworms.

Advancing large-scale production of TEV protease through an innovative NT* tag-based fusion construct

Tobacco etch virus Protease (TEVp), a cysteine protease, is renowned for its remarkable specific proteolysis, making it an invaluable tool for removing fusion tags from recombinant proteins. However, TEV protease's inherent insolubility limits its broad application. Fusion constructs like an N-terminal MBP fusion, known for its improved solubility, have been employed for TEVp production to address this issue. In this study, we fused the TEVp with the N-terminal domain of the spider silk protein, specifically utilizing a charge-reversed mutant (D40K/K65D) of the N-terminal domain of major ampullate spidroin-1 protein from Euprosthenops australis, referred to as NT*. This fusion construct contains a TEVp cleavage site, enabling intracellular self-processing and the release of a His7-tagged protease. The significant increase in soluble protein expression allowed us to purify approximately 90–100 mg of TEVp from a 1-L E. coli culture, surpassing previous findings by a considerable margin. The enzyme remained stable and catalytically active even after several months of storage in a deep freezer (−80 °C).

The Properties and Application of Natural Spider Silk: a Literature Review

Drag silk, which is produced by the main ampullate silk gland, is a kind of natural insect silk which outperforms some other kinds of silk. Given its exceptional mechanical characteristics and excellent biocompatibility, drag silk, a kind of spider silk, does have the potential to be utilized by humans. The excellent properties of natural spider silk make it useful in many fields. But due to the cannibalistic nature of spiders, spiders cannot be reared on a large scale to meet the huge demand for spider silk. The development of recombinant spider silk protein and artificial spider silk through research and development is vital for the commercialization of spider silk and its applications. This research outlines the possible applications of natural spider silk by examining its physical, biological, and characteristic features. Due to its extraordinary physical and biological properties, drag silk and its corresponding silk proteins have been found to have the potential for the applications in the aerospace industry and biomedical treatments.

Recombinant Spider Silk Fiber with High Dimensional Stability in Water and Its NMR Characterization

Spider dragline silk has unique characteristics of strength and extensibility, including supercontraction. When we use it as a biomaterial or material for textiles, it is important to suppress the effect of water on the fiber by as much as possible in order to maintain dimensional stability. In order to produce spider silk with a highly hydrophobic character, based on the sequence of ADF-3 silk, we produced recombinant silk (RSSP(VLI)) where all QQ sequences were replaced by VL, while single Q was replaced by I. The artificial RSSP(VLI) fiber was prepared using formic acid as the spinning solvent and methanol as the coagulant solvent. The dimensional stability and water absorption experiments of the fiber were performed for eight kinds of silk fiber. RSSP(VLI) fiber showed high dimensional stability, which is suitable for textiles. A remarkable decrease in the motion of the fiber in water was made evident by 13C solid-state NMR. This study using 13C solid-state NMR is the first trial to put spider silk to practical use and provide information regarding the molecular design of new recombinant spider silk materials with high dimensional stability in water, allowing recombinant spider silk proteins to be used in next-generation biomaterials and materials for textiles.

Recombinant protein condensation inside E.coli enables the development of building blocks for bioinspired materials engineering - Biomimetic spider silk protein as a case study.

Recombinant expression of proteins destined to form biological materials often results in poor production yields or loss of their function due to premature aggregation. Recently, liquid-liquid phase separation has been proposed as a mechanism to control protein solubility during expression and accumulation in the cytoplasm. Here, we investigate this process in vivo during the recombinant overexpression of the mimetic spider silk mini-spidroin NT2RepCT in Escherichia coli. The protein forms intracellular liquid-like condensates that shift to a solid-like state triggered by a decrease in their microenvironmental pH. These features are also maintained in the purified sample in vitro both in the presence of a molecular crowding agent mimicking the bacterial intracellular environment, and during a biomimetic extrusion process leading to fiber formation. Overall, we demonstrate that characterization of protein condensates inside E.coli could be used as a basis for selecting proteins for both materials applications and their fundamental structure-function studies.

Patterned Duplex Fabric Based on Genetically Modified Spidroin for Smart Wound Management.

The treatment of diabetic wounds remains a great challenge for the medical community. Here, a smart patterned DNA double helix (duplex)-like fabric based on genetically modified spider silk protein (PDF-S) which is inspired by soft plant tendrils, is proposed for diabetic wound treatment. Benefiting from spider silk protein (spidroin); PDF-S is equipped with high strength; high toughness, and excellent biocompatibility. Notably, the fabric crimped through the biomimetic DNA double-helix-like structure can effectively adapt to tensile impact and the maximum stretch rate reaches 1500%. A pattern-based microfluidic channel of PDF-S allowed wound secretion to flow spontaneously through the channel. Meanwhile; due to the optical properties of the introduced photonic crystal structure; PDF-S is equipped with fluorescence enhancement properties; enabling PDF-S to display color-sensitive behavior suitable for wound monitoring and guiding clinical treatment. In addition, to enable sensitive motion monitoring, microelectronic circuits are integrated on the surface of the PDF-S. These unique material features suggest that this study will lead to a new generation of biomimetic artificial spider silk materials for design and application in the biomedical field.

Preparation and Characterization of Nanofibrous Membranes Electro-Spun from Blended Poly(l-lactide-co-ε-caprolactone) and Recombinant Spider Silk Protein as Potential Skin Regeneration Scaffold.

Biomaterial scaffolding serves as an important strategy in skin tissue engineering. In this research, recombinant spider silk protein (RSSP) and poly(L-lactide-co-ε-caprolactone) (PLCL) were blended in different ratios to fabricate nanofibrous membranes as potential skin regeneration scaffolds with an electro-spinning process. Scanning electron microscopy (SEM), water contact angles measurement, Fourier transform infrared (FTIR) spectroscopy, wide angle X-ray diffraction (WAXD), tensile mechanical tests and thermo-gravimetric analysis (TGA) were carried out to characterize the nanofibrous membranes. The results showed that the blending of RSSP greatly decreased the nanofibers' average diameter, enhanced the hydrophilicity, changed the microstructure and thermal properties, and could enable tailored mechanical properties of the nanofibrous membranes. Among the blended membranes, the PLCL/RSSP (75/25) membrane was chosen for further investigation on biocompatibility. The results of hemolysis assays and for proliferation of human foreskin fibroblast cells (hFFCs) confirmed the membranes potential use as skin-regeneration scaffolds. Subsequent culture of mouse embryonic fibroblast cells (NIH-3T3) demonstrated the feasibility of the blended membranes as a human epidermal growth factor (hEGF) delivery matrix. The PLCL/RSSP (75/25) membrane possessed good properties comparable to those of human skin with high biocompatibility and the ability of hEGF delivery. Further studies can be carried out on such membranes with chemical or genetic modifications to make better scaffolds for skin regeneration.

Characterization of two full-length tubuliform silk gene sequences from Neoscona theisi reveals intragenic concerted evolution and multiple copies in genome

Orb-web weaving spiders use a variety of silk types for particular tasks, and each silk type is composed of at least two spider silk proteins (spidroins). In the early stage of divergence, however, the molecular evolutionary processes act on spidroin variants are still unclear because of a lack of knowledge for full-length paralogous and orthologous gene sequences among closely related species. Here, we present two complete gene sequences encoding the tubuliform spidroin TuSp1 variants (TuSp1-v2 and TuSp1-v3) from orb-weaving spider Neoscona theisi. Both N. theisi TuSp1-v2 and TuSp1-v3 genes contain a single enormous exon (14,139 bp for TuSp1-v2 and 13,152 bp for TuSp1-v3) and dozens of tandemly arrayed repeats (25 repeats for TuSp1-v2 and 23 repeats for TuSp1-v3) with extreme intragenic homogenization. The pattern of expression for these two spidroins revealed that the level of TuSp1-v3 mRNA is ~3-fold higher than that of TuSp1-v2 in tubuliform gland. Phylogenetic analyses of spidroins not only show the occurrence of a gene duplication event for TuSp1-v2 and TuSp1-v3 in the common ancestor of the Neoscona and Araneus lineage but reinforce the role of concerted evolution for the extreme homogenization of TuSp1 repeats.

Spatially Directed Biosynthesis of Quantum Dots via Spidroin Templating in Escherichia coli

AbstractSpatially directed synthesis of quantum dots (QDs) is intriguing yet challenging in organisms, due to the dispersed feature of templating biomolecules and precursors. Whether this task could be accomplished by biomolecular condensates, an emerging type of membraneless compartments in cells remains unknown. Here we report synthetic protein condensates for templated synthesis of QDs in bacterium Escherichia coli. This was realized by overexpression of spider silk protein to bind precursor ions and recruit other necessary components, which induced the spidroin to form more β‐sheet structures for assembly and maturation of the protein condensates. This in turn enabled formation and co‐localization of the fluorescent QDs to “light up” the condensates, and alleviated cytotoxicity of the precursor heavy metal ions and resulting QDs. Thus, our results suggest a new strategy for nanostructure synthesis and deposition in subcellular compartments with great potential for in situ applications.

Spatially Directed Biosynthesis of Quantum Dots via Spidroin Templating in Escherichia coli.

Spatially directed synthesis of quantum dots (QDs) is intriguing yet challenging in organisms, due to the dispersed feature of templating biomolecules and precursors. Whether this task could be accomplished by biomolecular condensates, an emerging type of membraneless compartments in cells remains unknown. Here we report synthetic protein condensates for templated synthesis of QDs in bacterium Escherichia coli. This was realized by overexpression of spider silk protein to bind precursor ions and recruit other necessary components, which induced the spidroin to form more β-sheet structures for assembly and maturation of the protein condensates. This in turn enabled formation and co-localization of the fluorescent QDs to "light up" the condensates, and alleviated cytotoxicity of the precursor heavy metal ions and resulting QDs. Thus, our results suggest a new strategy for nanostructure synthesis and deposition in subcellular compartments with great potential for in situ applications.

Impact of physio-chemical spinning conditions on the mechanical properties of biomimetic spider silk fibers

Artificial spider silk has emerged as a biobased fiber that could replace some petroleum-based materials that are on the market today. Recent progress made it possible to produce the recombinant spider silk protein NT2RepCT at levels that would make the commercialization of fibers spun from this protein economically feasible. However, for most applications, the mechanical properties of the artificial silk fibers need to be improved. This could potentially be achieved by redesigning the spidroin, and/or by changing spinning conditions. Here, we show that several spinning parameters have a significant impact on the fibers’ mechanical properties by tensile testing more than 1000 fibers produced under 92 different conditions. The most important factors that contribute to increasing the tensile strength are fast reeling speeds and/or employing post-spin stretching. Stretching in combination with optimized spinning conditions results in fibers with a strength of >250 MPa, which is the highest reported value for fibers spun using natively folded recombinant spidroins that polymerize in response to shear forces and lowered pH.

Untangling the biological and inflammatory behavior of silk-like sutures In vivo

Recombinant spider silk materials with antimicrobial peptides are a promising new class of drug-free antimicrobial materials capable of preventing surgical site infections (SSI), but their potential to impede infections is unclear. Herein, we aimed to unravel the biological and inflammatory potential of bioengineered spider silk materials to prevent SSI using an infection animal model. Silk-like fibers made of silk fibroin and spider silk proteins with antimicrobial peptides (6mer-HNP1) held improved stiffness (2.9 GPa) and had a slow biodegradation profile while inhibiting bacterial adherence in vitro by 5-log and 6-log reduction on Methicillin-Resistant Staphylococcus aureus (MRSA) and Escherichia coli (E. coli), respectively. In vivo studies showed that fibers with 6mer-HNP1 elicited a short-term low to mild local inflammatory response, similar to implanted commercial sutures. In the presence of a bacterial infection, the mediators related to infection and inflammation were downregulated suggesting that the fibers maintained a low but active response to bacterial infection. Thus, the presence of 6mer-HNP1 helped the host maintain an active response to bacterial infection, impairing the development of an acute infection. Our findings further support the use of bioengineered spider silk proteins to develop drug-free antimicrobial sutures capable to impair SSI.

Construction of spider silk protein small-caliber tissue engineering vascular grafts based on dynamic culture and its performance evaluation.

Tissue engineering is an alternative method for preparing small-caliber (<6 mm) vascular grafts. Dynamic mechanical conditioning is being researched as a method to improve mechanical properties of tissue engineered blood vessels. This method attempts to induce unique reaction in implanted cells that regenerate the matrix around them, thereby improving the overall mechanical stability of the grafts. In this study, we used a bioreactor to seed endothelial cells and smooth muscle cells into the inner and outer layers of the electrospun spider silk protein scaffold respectively to construct vascular grafts. The cell proliferation, mechanical properties, blood compatibility and other indicators of the vascular grafts were characterized in vitro. Furthermore, the vascular grafts were implanted in Sprague Dawley rats, and the vascular grafts' patency, extracellular matrix formation, and inflammatory response were evaluated in vivo. We aimed to construct spider silk protein vascular grafts with the potential for in vivo implantation by using a pulsating flow bioreactor. The results showed that, when compared with the static culture condition, the dynamic culture condition improved cell proliferation on vascular scaffolds and enhanced mechanical function of vascular scaffolds. In vivo experiments also showed that the dynamic culture of vascular grafts was more beneficial for the extracellular matrix deposition and anti-thrombogenesis, as well as reducing the inflammatory response of vascular grafts. In conclusion, dynamic mechanical conditioning aid in the resolution of challenges impeding the application of electrospun scaffolds and have the potential to construct small-caliber blood vessels with regenerative function for cardiovascular tissue repair.

Spider silk proteins form hydrogels

Spider silk proteins form hydrogels

Spider silk proteins form hydrogels at body temperature

Spider silk proteins form hydrogels at body temperature

Dual Patterning of Self‐Assembling Spider Silk Protein Nanofibrillar Networks

AbstractSelf‐assembly of a recombinant spider silk protein into nanofibrillar networks in combination with photolithography is used to produce diversely functionalized micropattern. Amino‐modified substrates coated with a positive tone photoresist are processed into 1 µm deep arbitrarily shaped microwells, at the bottom of which spider silk proteins are covalently coupled to the deprotected aminated surface. The protein layer serves to seed the self‐assembly of nanofibrils from the same protein in the microwells, forming immobilized few nanometers thin networks after the stripping of the photoresist. The nanofibrous micropattern can be functionalized by employing fluorescently modified spider silk variants during the self‐assembly or by later covalent modification with nucleic acids. By repeating the photolithography and fibril assembly procedures, two functionally different and spatially defined pattern are created.

Bioselectivity of silk protein-based materials and their bio-inspired applications.

Adhesion to material surfaces is crucial for almost all organisms regarding subsequent biological responses. Mammalian cell attachment to a surrounding biological matrix is essential for maintaining their survival and function concerning tissue formation. Conversely, the adhesion and presence of microbes interferes with important multicellular processes of tissue development. Therefore, tailoring bioselective, biologically active, and multifunctional materials for biomedical applications is a modern focus of biomaterial research. Engineering biomaterials that stimulate and interact with cell receptors to support binding and subsequent physiological responses of multicellular systems attracted much interest in the last years. Further to this, the increasing threat of multidrug resistance of pathogens against antibiotics to human health urgently requires new material concepts for preventing microbial infestation and biofilm formation. Thus, materials exhibiting microbial repellence or antimicrobial behaviour to reduce inflammation, while selectively enhancing regeneration in host tissues are of utmost interest. In this context, protein-based materials are interesting candidates due to their natural origin, biological activity, and structural properties. Silk materials, in particular those made of spider silk proteins and their recombinant counterparts, are characterized by extraordinary properties including excellent biocompatibility, slow biodegradation, low immunogenicity, and non-toxicity, making them ideally suited for tissue engineering and biomedical applications. Furthermore, recombinant production technologies allow for application-specific modification to develop adjustable, bioactive materials. The present review focusses on biological processes and surface interactions involved in the bioselective adhesion of mammalian cells and repellence of microbes on protein-based material surfaces. In addition, it highlights the importance of materials made of recombinant spider silk proteins, focussing on the progress regarding bioselectivity.

Recombinant Spider Silk Bioinks for Continuous Protein Release by Encapsulated Producer Cells.

Targeted therapies using biopharmaceuticals are of growing clinical importance in disease treatment. Currently, there are several limitations of protein-based therapeutics (biologicals), including suboptimal biodistribution, lack of stability, and systemic side effects. A promising approach to overcoming these limitations could be a therapeutic cell-loaded 3D construct consisting of a suitable matrix component that harbors producer cells continuously secreting the biological of interest. Here, the recombinant spider silk proteins eADF4(C16), eADF4(C16)-RGD, and eADF4(C16)-RGE have been processed together with HEK293 producer cells stably secreting the highly traceable reporter biological TNFR2-Fc-GpL, a fusion protein consisting of the extracellular domain of TNFR2, the Fc domain of human IgG1, and the luciferase of Gaussia princeps as a reporter domain. eADF4(C16) and eADF4(C16)-RGD hydrogels provide structural and mechanical support, promote HEK293 cell growth, and allow fusion protein production by the latter. Bioink-captured HEK293 producer cells continuously release functional TNFR2-Fc-GpL over 14 days. Thus, the combination of biocompatible, printable spider silk bioinks with drug-producing cells is promising for generating implantable 3D constructs for continuous targeted therapy.

Nerve Growth Factor‐Binding Engineered Silk Films Promote Neuronal Attachment and Neurite Outgrowth

AbstractSpider silks have outstanding potential as biomaterials due to their sought‐after mechanical properties and low immunogenicity. The toughest spider silk is aciniform silk, which is used by spiders to wrap prey and produce egg sacs. A variety of recombinant aciniform silk constructs are now been developed, including hybrid silks with domains from multiple spider silk proteins fused together. In this study, an engineered aciniform silk construct, termed NBSilk, fused both N‐ and C‐terminally to heptapeptide motifs that bind the neurotrophic factor and nerve growth factor‐β (NGF) is introduced. NBSilk is shown to be amenable to casting into robust films that remain intact while sequestering and maintaining bioactive NGF at the film surface for at least 7 days. These films support cell survival while enhancing differentiation and neurite density and outgrowth in neuron‐like PC12 cells, with elevation of signaling through both the mitogen‐activated protein kinase (MAPK) and protein kinase B (AKT) signaling pathways. Strikingly, preloading of NBSilk films with NGF enhances neuritogenesis even over conditions where cells are grown with NGF supplementation in the culture medium. NBSilk scaffolds, thus, warrant future development and evaluation as biomaterials for nerve regeneration.