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Site-Specific Functionalization of Recombinant Spider Silk Janus Fibers.

ABSTRACT: Biotechnological production is a powerful tool to design materials with customized properties. The aim of this work was to apply designed spider silk proteins to produce Janus fibers with two different functional sides. First, functionalization was established through a cysteine-modified silk protein, ntag^Cys eADF4(κ16). After fiber spinning, gold nanoparticles (AuNPs) were coupled via thiol-ene click chemistry. Significantly reduced electrical resistivity indicated sufficient loading density of AuNPs on such fiber surfaces. Then, Janus fibers were electrospun in a side-by-side arrangement, with "non-functional" eADF4(C16) on the one and "functional" ntag^Cys eADF4(κ16) on the other side. Post-treatment was established to render silk fibers insoluble in water. Subsequent AuNP binding was highly selective on the ntag^Cys eADF4(κ16) side demonstrating the potential of such silk-based systems to realize complex bifunctional structures with spatial resolutions in the nano scale.

SUBMITTER: Lang G

PROVIDER: S-EPMC9303884 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Recombinant Spider Silk: Promises and Bottlenecks.

Project description:Spider silk threads have exceptional mechanical properties such as toughness, elasticity and low density, which reach maximum values compared to other fibre materials. They are superior even compared to Kevlar and steel. These extraordinary properties stem from long length and specific protein structures. Spider silk proteins can consist of more than 20,000 amino acids. Polypeptide stretches account for more than 90% of the whole protein, and these domains can be repeated more than a hundred times. Each repeat unit has a specific function resulting in the final properties of the silk. These properties make them attractive for innovative material development for medical or technical products as well as cosmetics. However, with livestock breeding of spiders it is not possible to reach high volumes of silk due to the cannibalistic behaviour of these animals. In order to obtain spider silk proteins (spidroins) on a large scale, recombinant production is attempted in various expression systems such as plants, bacteria, yeasts, insects, silkworms, mammalian cells and animals. For viable large-scale production, cost-effective and efficient production systems are needed. This review describes the different types of spider silk, their proteins and structures and discusses the production of these difficult-to-express proteins in different host organisms with an emphasis on plant systems.

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Assembly mechanism of recombinant spider silk proteins.

Project description:Spider silk threads are formed by the irreversible aggregation of silk proteins in a spinning duct with dimensions of only a few micrometers. Here, we present a microfluidic device in which engineered and recombinantly produced spider dragline silk proteins eADF3 (engineered Araneus diadematus fibroin) and eADF4 are assembled into fibers. Our approach allows the direct observation and identification of the essential parameters of dragline silk assembly. Changes in ionic conditions and pH result in aggregation of the two proteins. Assembly of eADF3 fibers was induced only in the presence of an elongational flow component. Strikingly, eADF4 formed fibers only in combination with eADF3. On the basis of these results, we propose a model for dragline silk aggregation and early steps of fiber assembly in the microscopic regime.

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Proteomic Evidence for Components of Spider Silk Synthesis from Black Widow Silk Glands and Fibers.

Project description:Spider silk research has largely focused on spidroins, proteins that are the primary components of spider silk fibers. Although a number of spidroins have been characterized, other types of proteins associated with silk synthesis are virtually unknown. Previous analyses of tissue-specific RNA-seq libraries identified 647 predicted genes that were differentially expressed in silk glands of the Western black widow, Latrodectus hesperus. Only ∼5% of these silk-gland specific transcripts (SSTs) encode spidroins; although the remaining predicted genes presumably encode other proteins associated with silk production, this is mostly unverified. Here, we used proteomic analysis of multiple silk glands and dragline silk fiber to investigate the translation of the differentially expressed genes. We find 48 proteins encoded by the differentially expressed transcripts in L. hesperus major ampullate, minor ampullate, and tubuliform silk glands and detect 17 SST encoded proteins in major ampullate silk fibers. The observed proteins include known silk-related proteins, but most are uncharacterized, with no annotation. These unannotated proteins likely include novel silk-associated proteins. Major and minor ampullate glands have the highest overlap of identified proteins, consistent with their shared, distinctive ampullate shape and the overlapping functions of major and minor ampullate silks. Our study substantiates and prioritizes predictions from differential expression analysis of spider silk gland transcriptomes.

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Silkworms transformed with chimeric silkworm/spider silk genes spin composite silk fibers with improved mechanical properties.

Project description:The development of a spider silk-manufacturing process is of great interest. However, there are serious problems with natural manufacturing through spider farming, and standard recombinant protein production platforms have provided limited progress due to their inability to assemble spider silk proteins into fibers. Thus, we used piggyBac vectors to create transgenic silkworms encoding chimeric silkworm/spider silk proteins. The silk fibers produced by these animals were composite materials that included chimeric silkworm/spider silk proteins integrated in an extremely stable manner. Furthermore, these composite fibers were, on average, tougher than the parental silkworm silk fibers and as tough as native dragline spider silk fibers. These results demonstrate that silkworms can be engineered to manufacture composite silk fibers containing stably integrated spider silk protein sequences, which significantly improve the overall mechanical properties of the parental silkworm silk fibers.

| S-EPMC3271896 | biostudies-literature

Properties of Biomimetic Artificial Spider Silk Fibers Tuned by PostSpin Bath Incubation.

Project description:Efficient production of artificial spider silk fibers with properties that match its natural counterpart has still not been achieved. Recently, a biomimetic process for spinning recombinant spider silk proteins (spidroins) was presented, in which important molecular mechanisms involved in native spider silk spinning were recapitulated. However, drawbacks of these fibers included inferior mechanical properties and problems with low resistance to aqueous environments. In this work, we show that ≥5 h incubation of the fibers, in a collection bath of 500 mM NaAc and 200 mM NaCl, at pH 5 results in fibers that do not dissolve in water or phosphate buffered saline, which implies that the fibers can be used for applications that involve wet/humid conditions. Furthermore, incubation in the collection bath improved the strain at break and was associated with increased β-sheet content, but did not affect the fiber morphology. In summary, we present a simple way to improve artificial spider silk fiber strain at break and resistance to aqueous solvents.

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Production and secretion of recombinant spider silk in Bacillus megaterium

Project description: Not available

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Comprehensive Proteomic Analysis of Spider Dragline Silk from Black Widows: A Recipe to Build Synthetic Silk Fibers.

Project description:The outstanding material properties of spider dragline silk fibers have been attributed to two spidroins, major ampullate spidroins 1 and 2 (MaSp1 and MaSp2). Although dragline silk fibers have been treated with different chemical solvents to elucidate the relationship between protein structure and fiber mechanics, there has not been a comprehensive proteomic analysis of the major ampullate (MA) gland, its spinning dope, and dragline silk using a wide range of chaotropic agents, inorganic salts, and fluorinated alcohols to elucidate their complete molecular constituents. In these studies, we perform in-solution tryptic digestions of solubilized MA glands, spinning dope and dragline silk fibers using five different solvents, followed by nano liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis with an Orbitrap Fusion™ Tribrid™. To improve protein identification, we employed three different tryptic peptide fragmentation modes, which included collision-induced dissociation (CID), electron transfer dissociation (ETD), and high energy collision dissociation (HCD) to discover proteins involved in the silk assembly pathway and silk fiber. In addition to MaSp1 and MaSp2, we confirmed the presence of a third spidroin, aciniform spidroin 1 (AcSp1), widely recognized as the major constituent of wrapping silk, as a product of dragline silk. Our findings also reveal that MA glands, spinning dope, and dragline silk contain at least seven common proteins: three members of the Cysteine-Rich Protein Family (CRP1, CRP2 and CRP4), cysteine-rich secretory protein 3 (CRISP3), fasciclin and two uncharacterized proteins. In summary, this study provides a proteomic blueprint to construct synthetic silk fibers that most closely mimic natural fibers.

| S-EPMC5037812 | biostudies-literature

Recombinant spider silk coatings functionalized with enzymes targeting bacteria and biofilms.

Project description:Bacteria forming biofilms on surgical implants is a problem that might be alleviated by the use of antibacterial coatings. In this article, recombinant spider silk was functionalized with the peptidoglycan degrading endolysin SAL-1 from the staphylococcal bacteriophage SAP-1 and the biofilm-matrix-degrading enzyme Dispersin B from Aggregatibacter actinomycetemcomitans using direct genetic fusion and/or covalent protein-protein fusion catalyzed by Sortase A. Spider silk assembly and enzyme immobilization was monitored using quartz crystal microbalance analysis. Enzyme activity was investigated both with a biochemical assay using cleavage of fluorescent substrate analogues and bacterial assays for biofilm degradation and turbidity reduction. Spider silk coatings functionalized with SAL-1 and Disperin B were found to exhibit bacteriolytic effect and inhibit biofilm formation, respectively. The strategy to immobilize antibacterial enzymes to spider silk presented herein show potential to be used as surface coatings of surgical implants and other medical equipment to avoid bacterial colonization.

| S-EPMC7142364 | biostudies-literature

Silk fibroin fibers-based shape memory membrane with Janus wettability for multitiered wearable protection.

Project description:Realizing breathable shape memory fiber-based material with antibacterial and waterproof performances is important for multitiered wearable protection to address the increasing concerns of air pollution. Herein, using an alternating electrospinning-electrospraying technology, we develop a fiber-based membrane with Janus wettability based on a silk fibroin nanofibers-substrate (SFNFs), a polyurethane nanospheres-top layer (PUNSs), and a middle layer of PU nanofibers-mat with in-situ grown silver nanoparticles (PUNFs-AgNPs), which serves separately for skin contact, a self-cleaning physical barrier to resist external aerosol/bacteria (PM2.5 filtration efficiency ~ 98.1%), and a bio-barrier that can sterilize harmful particles and inhibit bacteria proliferation (> 95%). This breathable Janus film (SFNFs/PUNFs-AgNPs/PUNSs, SPAP) with an antibacterial filter shows shape memory stretchability enabled by the thermoplastic PU component, which is mechanically adaptive to human body for wearable protection. This work presents a breathable wearable material for air-filtration and anti-bacteria, promising for applications such as wound dressings, medical masks, protection suits, and multifunctional filters.Graphical abstractAn alternating electrospinning-electrospraying technology was proposed to achieve a silk fibroin-based antibacterial membrane with Janus wettability, as well as good skin affinity and breathability, which serves well as physical and bio-barriers for water resistance, PM2.5 filtration (~98.1%) and bacteria inhibition (efficiency of 95%). This shape memory Janus membrane can adapt mechanically to human body curvatures for functional wearable protections.Supplementary informationThe online version contains supplementary material available at 10.1557/s43578-022-00805-w.

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Impacts of Blended Bombyx mori Silk Fibroin and Recombinant Spider Silk Fibroin Hydrogels on Cell Growth.

Project description:Binary-blended hydrogels fabricated from Bombyx mori silk fibroin (SF) and recombinant spider silk protein eADF4(C16) were developed and investigated concerning gelation and cellular interactions in vitro. With an increasing concentration of eADF4(C16), the gelation time of SF was shortened from typically one week to less than 48 h depending on the blending ratio. The biological tests with primary cells and two cell lines revealed that the cells cannot adhere and preferably formed cell aggregates on eADF4(C16) hydrogels, due to the polyanionic properties of eADF4(C16). Mixing SF in the blends ameliorated the cellular activities, as the proliferation of L929 fibroblasts and SaOS-2 osteoblast-like cells increased with an increase of SF content. The blended SF:eADF4(C16) hydrogels attained the advantages as well as overcame the limitations of each individual material, underlining the utilization of the hydrogels in several biomedical applications.

| S-EPMC8659740 | biostudies-literature

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