Artificial Spider Silk Research Articles

Control of silicification by genetically engineered fusion proteins: Silk–silica binding peptides

In the present study, an artificial spider silk gene, 6mer, derived from the consensus sequence of Nephila clavipes dragline silk gene, was fused with different silica-binding peptides (SiBPs), A1, A3 and R5, to study the impact of the fusion protein sequence chemistry on silica formation and the ability to generate a silk–silica composite in two different bioinspired silicification systems: solution–solution and solution–solid. Condensed silica nanoscale particles (600–800nm) were formed in the presence of the recombinant silk and chimeras, which were smaller than those formed by 15mer-SiBP chimeras [1], revealing that the molecular weight of the silk domain correlated to the sizes of the condensed silica particles in the solution system. In addition, the chimeras (6mer-A1/A3/R5) produced smaller condensed silica particles than the control (6mer), revealing that the silica particle size formed in the solution system is controlled by the size of protein assemblies in solution. In the solution–solid interface system, silicification reactions were performed on the surface of films fabricated from the recombinant silk proteins and chimeras and then treated to induce β-sheet formation. A higher density of condensed silica formed on the films containing the lowest β-sheet content while the films with the highest β-sheet content precipitated the lowest density of silica, revealing an inverse correlation between the β-sheet secondary structure and the silica content formed on the films. Intriguingly, the 6mer-A3 showed the highest rate of silica condensation but the lowest density of silica deposition on the films, compared with 6mer-A1 and -R5, revealing antagonistic crosstalk between the silk and the SiBP domains in terms of protein assembly. These findings offer a path forward in the tailoring of biopolymer–silica composites for biomaterial related needs.

Bioinspired one-dimensional materials for directional liquid transport.

One-dimensional materials (1D) capable of transporting liquid droplets directionally, such as spider silks and cactus spines, have recently been gathering scientists' attention due to their potential applications in microfluidics, textile dyeing, filtration, and smog removal. This remarkable property comes from the arrangement of the micro- and nanostructures on these organisms' surfaces, which have inspired chemists to develop methods to prepare surfaces with similar directional liquid transport ability. In this Account, we report our recent progress in understanding how this directional transport works, as well our advances in the design and fabrication of bioinspired 1D materials capable of transporting liquid droplets directionally. To begin, we first discuss some basic theories on droplet directional movement. Then, we discuss the mechanism of directional transport of water droplets on natural spider silks. Upon contact with water droplets, the spider silk undergoes what is known as a wet-rebuilt, which forms periodic spindle-knots and joints. We found that the resulting gradient of Laplace pressure and surface free energy between the spindle-knots and joints account for the cooperative driving forces to transport water droplets directionally. Next, we discuss the directional transport of water droplets on desert cactus. The integration of multilevel structures of the cactus and the resulting integration of multiple functions together allow the cactus spine to transport water droplets continuously from tip to base. Based on our studies of natural spider silks and cactus spines, we have prepared a series of artificial spider silks (A-SSs) and artificial cactus spines (A-CSs) with various methods. By changing the surface roughness and chemical compositions of the artificial spider silks' spindle-knots, or by introducing stimulus-responsive molecules, such as thermal-responsive and photoresponsive molecules, onto the spindle-knots, we can reversibly manipulate the direction of water droplet's movement on the prepared A-SSs. In addition, the A-SSs with nonuniform spindle-knots, such as multilevel sized spindle-knots and gradient spindle-knots, further demonstrate integrated directional transport ability for water droplets. Through mimicking the main principle of cactus spines in transporting water droplets, we were able to fabricate both single and array A-CSs, which are able to transport liquid droplets directionally both in air and under water. Lastly, we demonstrated some applications of this directional liquid transport, from aspects of efficient fog collection to oil/water separation. In addition, we showed some potential applications in smart catalysis, tracer substance enrichment, smog removal, and drug delivery.

Nanoparticle self-assembly by a highly stable recombinant spider wrapping silk protein subunit

Artificial spider silk proteins may form fibers with exceptional strength and elasticity. Wrapping silk, or aciniform silk, is the toughest of the spider silks, and has a very different protein composition than other spider silks. Here, we present the characterization of an aciniform protein (AcSp1) subunit named W1, consisting of one AcSp1 199 residue repeat unit from Argiope trifasciata. The structural integrity of recombinant W1 is demonstrated in a variety of buffer conditions and time points. Furthermore, we show that W1 has a high thermal stability with reversible denaturation at ∼71°C and forms self-assembled nanoparticle in near-physiological conditions. W1 therefore represents a highly stable and structurally robust module for protein-based nanoparticle formation.

Bioinspired Materials: Controlled Fabrication and Water Collection Ability of Bioinspired Artificial Spider Silks (Adv. Mater. 32/2011)

Bioinspired Materials: Controlled Fabrication and Water Collection Ability of Bioinspired Artificial Spider Silks (Adv. Mater. 32/2011)

Controlled Fabrication and Water Collection Ability of Bioinspired Artificial Spider Silks

Spider silks with periodic spindle-knots are skillful at collecting water from the foggy air. Under controlled fabrication conditions, a series of bioinspired artificial spider silks (BAS) with different sized spindle-knots are realized. Furthermore, the water collection ability of these BASs is investigated. The study is helpful for the practical application of BASs as water collecting materials.

Microdissection of Black Widow Spider Silk-producing Glands

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of developing offspring. Spiders accomplish these tasks by spinning several distinct fiber types that have diverse mechanical properties. Such specialization of fiber types has occurred through the evolution of different silk-producing glands, which function as small biofactories. These biofactories manufacture and store large quantities of silk proteins for fiber production. Through a complex series of biochemical events, these silk proteins are converted from a liquid into a solid material upon extrusion. Mechanical studies have demonstrated that spider silks are stronger than high-tensile steel. Analyses to understand the relationship between the structure and function of spider silk threads have revealed that spider silk consists largely of proteins, or fibroins, that have block repeats within their protein sequences. Common molecular signatures that contribute to the incredible tensile strength and extensibility of spider silks are being unraveled through the analyses of translated silk cDNAs. Given the extraordinary material properties of spider silks, research labs across the globe are racing to understand and mimic the spinning process to produce synthetic silk fibers for commercial, military and industrial applications. One of the main challenges to spinning artificial spider silk in the research lab involves a complete understanding of the biochemical processes that occur during extrusion of the fibers from the silk-producing glands. Here we present a method for the isolation of the seven different silk-producing glands from the cobweaving black widow spider, which includes the major and minor ampullate glands [manufactures dragline and scaffolding silk], tubuliform [synthesizes egg case silk], flagelliform [unknown function in cob-weavers], aggregate [makes glue silk], aciniform [synthesizes prey wrapping and egg case threads] and pyriform [produces attachment disc silk]. This approach is based upon anesthetizing the spider with carbon dioxide gas, subsequent separation of the cephalothorax from the abdomen, and microdissection of the abdomen to obtain the silk-producing glands. Following the separation of the different silk-producing glands, these tissues can be used to retrieve different macromolecules for distinct biochemical analyses, including quantitative real-time PCR, northern- and western blotting, mass spectrometry (MS or MS/MS) analyses to identify new silk protein sequences, search for proteins that participate in the silk assembly pathway, or use the intact tissue for cell culture or histological experiments.

Microdissection of Black Widow Spider Silk-producing Glands

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of developing offspring 1,2. Spiders accomplish these tasks by spinning several distinct fiber types that have diverse mechanical properties. Such specialization of fiber types has occurred through the evolution of different silk-producing glands, which function as small biofactories. These biofactories manufacture and store large quantities of silk proteins for fiber production. Through a complex series of biochemical events, these silk proteins are converted from a liquid into a solid material upon extrusion. Mechanical studies have demonstrated that spider silks are stronger than high-tensile steel 3. Analyses to understand the relationship between the structure and function of spider silk threads have revealed that spider silk consists largely of proteins, or fibroins, that have block repeats within their protein sequences 4. Common molecular signatures that contribute to the incredible tensile strength and extensibility of spider silks are being unraveled through the analyses of translated silk cDNAs. Given the extraordinary material properties of spider silks, research labs across the globe are racing to understand and mimic the spinning process to produce synthetic silk fibers for commercial, military and industrial applications. One of the main challenges to spinning artificial spider silk in the research lab involves a complete understanding of the biochemical processes that occur during extrusion of the fibers from the silk-producing glands. Here we present a method for the isolation of the seven different silk-producing glands from the cobweaving black widow spider, which includes the major and minor ampullate glands [manufactures dragline and scaffolding silk] 5,6, tubuliform [synthesizes egg case silk] 7,8, flagelliform [unknown function in cob-weavers], aggregate [makes glue silk], aciniform [synthesizes prey wrapping and egg case threads] 9 and pyriform [produces attachment disc silk] 10. This approach is based upon anesthetizing the spider with carbon dioxide gas, subsequent separation of the cephalothorax from the abdomen, and microdissection of the abdomen to obtain the silk-producing glands. Following the separation of the different silk-producing glands, these tissues can be used to retrieve different macromolecules for distinct biochemical analyses, including quantitative real-time PCR, northern- and western blotting, mass spectrometry (MS or MS/MS) analyses to identify new silk protein sequences, search for proteins that participate in the silk assembly pathway, or use the intact tissue for cell culture or histological experiments.

Artifi cal Spider Silk: Direction Controlled Driving of Tiny Water Drops on Bioinspired Artificial Spider Silks (Adv. Mater. 48/2010)

Artifi cal Spider Silk: Direction Controlled Driving of Tiny Water Drops on Bioinspired Artificial Spider Silks (Adv. Mater. 48/2010)

Direction Controlled Driving of Tiny Water Drops on Bioinspired Artificial Spider Silks

www.MaterialsViews.com C O M M Direction Controlled Driving of Tiny Water Drops on Bioinspired Artifi cial Spider Silks U N IC A By Hao Bai , Xuelin Tian , Yongmei Zheng ,* Jie Ju , Yong Zhao , and Lei Jiang * IO N Directional driving of liquid drops is of signifi cant interest in many applications, such as microfl uidic devices, [ 1–6 ] fog harvesting, [ 7 ] fi ltration, [ 8 ] and condensers. [ 9 ] For this purpose, great progress has been made in driving drops larger than hundreds of micrometers [ 9–23 ] by introducing chemical, [ 1 , 10 , 12–15 , 23 ] thermal, [ 16–19 ] or shape [ 20–22 ] gradients on surfaces. However, driving micrometer-sized drops is much more diffi cult because they encounter a larger contact angle (CA) hysteresis effect. [ 7 , 24 ] In nature, the wetted silk of cribellate spider offers new insights into solving this problem by combining different gradients together. [ 7 ] Here, inspired by the spider silk, we fabricated a series of artifi cial spider silks with spindle-knots in which the chemical compositions and surface nanostructures were subtly designed. Our investigations demonstrated that tiny water drops (tens of picoliters) could be driven with controllable direction (“toward” or “away from” the knot) by optimizing the cooperation of curvature, chemical, and roughness gradients on the fi ber surfaces. The study will pave the way for designing smart materials and devices to drive tiny water drops in a controllable manner. When a nylon fi ber was immersed into polymer solution and drawn out horizontally, a string of polymer drops, which became spindle-knots after being dried, formed on the fi ber due to the Rayleigh instability [ 25 ] of the polymer solution. The surface energy of the spindle-knots was tailored by choosing different polymers including poly(vinyl acetate) (PVAc), poly(methyl methacrylate) (PMMA), polystyrene (PS), and poly(vinylidene fl uoride) (PVDF), which have intrinsic water contact angles of 56.7 ° , 68.4 ° , 93.3 ° , and 92.7 ° , respectively (see Supporting Information, Figure S1). On the other hand, the surface roughness (porous nanostructures) of the spindle-knots was also designed through phase separation

Hagfish slime threads as a biomimetic model for high performance protein fibres

Textile manufacturing is one of the largest industries in the world, and synthetic fibres represent two-thirds of the global textile market. Synthetic fibres are manufactured from petroleum-based feedstocks, which are becoming increasingly expensive as demand for finite petroleum reserves continues to rise. For the last three decades, spider silks have been held up as a model that could inspire the production of protein fibres exhibiting high performance and ecological sustainability, but unfortunately, artificial spider silks have yet to fulfil this promise. Previous work on the biomechanics of protein fibres from the slime of hagfishes suggests that these fibres might be a superior biomimetic model to spider silks. Based on the fact that the proteins within these ‘slime threads’ adopt conformations that are similar to those in spider silks when they are stretched, we hypothesized that draw processing of slime threads should yield fibres that are comparable to spider dragline silk in their mechanical performance. Here we show that draw-processed slime threads are indeed exceptionally strong and tough. We also show that post-drawing steps such as annealing, dehydration and covalent cross-linking can dramatically improve the long-term dimensional stability of the threads. The data presented here suggest that hagfish slime threads are a model that should be pursued in the quest to produce fibres that are ecologically sustainable and economically viable.

Biomimetic calcium phosphate coatings on recombinant spider silk fibres

Calcium phosphate ceramic coatings, applied on surfaces of metallic and polymeric biomaterials, can improve their performance in bone repair and regeneration. Spider silk is biocompatible, strong and elastic, and hence an attractive biomaterial for applications in connective tissue repair. Recently, artificial spider silk, with mechanical and structural characteristics similar to those of native spider silk, has been produced from recombinant minispidroins. In the present study, supersaturated simulated body fluid was used to deposit calcium phosphate coatings on recombinant spider silk fibres. The mineralization process was followed in time using scanning electron microscopy equipped with an energy dispersive x-ray (EDX) detector and Raman spectroscope. Focused ion beam technology was used to produce a cross section of a coated fibre, which was further analysed by EDX. Preliminary in vitro experiments using a culture of bone marrow-derived human mesenchymal stem cells (hMSCs) on coated fibres were also performed. This study showed that recombinant spider silk fibres were successfully coated with a homogeneous and thick crystalline calcium phosphate layer. In the course of the mineralization process from modified simulated body fluid, sodium chloride crystals were first deposited on the silk surface, followed by the deposition of a calcium phosphate layer. The coated silk fibres supported the attachment and growth of hMSCs.

Blueprint for a high-performance biomaterial: full-length spider dragline silk genes.

Spider dragline (major ampullate) silk outperforms virtually all other natural and manmade materials in terms of tensile strength and toughness. For this reason, the mass-production of artificial spider silks through transgenic technologies has been a major goal of biomimetics research. Although all known arthropod silk proteins are extremely large (>200 kiloDaltons), recombinant spider silks have been designed from short and incomplete cDNAs, the only available sequences. Here we describe the first full-length spider silk gene sequences and their flanking regions. These genes encode the MaSp1 and MaSp2 proteins that compose the black widow's high-performance dragline silk. Each gene includes a single enormous exon (>9000 base pairs) that translates into a highly repetitive polypeptide. Patterns of variation among sequence repeats at the amino acid and nucleotide levels indicate that the interaction of selection, intergenic recombination, and intragenic recombination governs the evolution of these highly unusual, modular proteins. Phylogenetic footprinting revealed putative regulatory elements in non-coding flanking sequences. Conservation of both upstream and downstream flanking sequences was especially striking between the two paralogous black widow major ampullate silk genes. Because these genes are co-expressed within the same silk gland, there may have been selection for similarity in regulatory regions. Our new data provide complete templates for synthesis of recombinant silk proteins that significantly improve the degree to which artificial silks mimic natural spider dragline fibers.

Macroscopic Fibers Self-Assembled from Recombinant Miniature Spider Silk Proteins

Strength, elasticity, and biocompatibility make spider silk an attractive resource for the production of artificial biomaterials. Spider silk proteins, spidroins, contain hundreds of repeated poly alanine/glycine-rich blocks and are difficult to produce recombinantly in soluble form. Most previous attempts to produce artificial spider silk fibers have included solubilization steps in nonphysiological solvents. It is here demonstrated that a miniature spidroin from a protein in dragline silk of Euprosthenops australis can be produced in a soluble form in Escherichia coli when fused to a highly soluble protein partner. Although this miniature spidroin contains only four poly alanine/glycine-rich blocks followed by a C-terminal non-repetitive domain, meter-long fibers are spontaneously formed after proteolytic release of the fusion partner. The structure of the fibers is similar to that of dragline silks, and although self-assembled from recombinant proteins they are as strong as fibers spun from redissolved silk. Moreover, the fibers appear to be biocompatible because human tissue culture cells can grow on and attach to the fibers. These findings enable controlled production of high-performance biofibers at large scale under physiological conditions.

AMINO ACID COMPOSITION-DEPENDENT ELASTICITY OF SPIDER SILK

Spider silk exhibits excellent mechanical features in both toughness and extensibility. In recent years considerable investigations have focused on it. The understanding of spider silk protein is important for the development bionic silk. In this paper, we study by Monte Carlo simulation the force-of-extension property of spider silk proteins as a function of the residue composition for major and minor ampullate glands of typical Araneid orb weaver as well as that for one artificial spider silk. The results are also compared with those from a designed protein whose amino acid composition is uniform. The results clearly show that the major and minor ampullate gland proteins are much tougher than the designed protein, whereas the artificial protein, as a model of a nature spider silk, does have good mechanical properties. Our simulation reveals that mechanical property of a spider silk protein is dependent on its amino acid composition and that an excellent result of natural evolution is manifest in the composition of the spider silk protein.

Elastic theory of single spider silk protein molecule

Spider silk exhibits two excellent mechanical features, toughness and extensibility. In recent years, considerable investigations have been focused on it. The understanding of spider silk protein is significant for the bionic silk. In this article, we study the force extension property of spider silk proteins as the function of the amino acid compositions for major and minor ampullate glands of typical Araneid orb weaver, as well as one artificial spider silk by Monte Carlo simulation. The results are also compared with a designed protein whose amino acid composition is uniform. It shows clearly that the major and minor ampullate gland proteins are much tougher than the designed protein, and the artificial protein as a model of a nature spider silk do have good mechanical properties. Our simulation reveals that the mechanical property of a spider silk protein is dependent on amino acid composition and the composition of spider silk protein is an excellent result of natural evolution.

Supercontraction and Backbone Dynamics in Spider Silk: 13C and 2H NMR Studies

The high-performance mechanical properties of certain spider silks can be radically altered by the addition of water. For example, unconstrained silk fibers from the major ampullate gland of the golden orb-weaving spider, Nephila clavipes, contract to about half of their original length when immersed in water. In this paper we use solid-state 13C and 2H NMR to study N. clavipes silk fibers, so as to address the molecular origins of supercontraction in the wet silk. Using 13C NMR, we study backbone dynamics and demonstrate that, when in contact with water, a substantial fraction of the glycine, glutamine, tyrosine, serine, and leucine residues in the protein backbone show dramatic increases in the rate of large-amplitude reorientation. 2H NMR of silk samples that incorporate leucine deuterated at one terminal methyl group provides a probe for dynamics at specific side chains along the fiber. Only a subset of these leucine residues is strongly affected by water. We suggest that the highly conserved YGGLGS(N)QGAGR blocks found in the silk protein play a major role in the supercontraction process. Amino acid sequences are proposed to produce artificial spider silk with similar mechanical properties, but without the undesired phenomenon of supercontraction. A possible use of the “supercontracting sequence” is also suggested.