Fibroin heavy chain gene replacement with a highly ordered synthetic repeat sequence in Bombyx mori.

The exceptional quality of silkworm silk is attributed to the amino acid sequence of its fibroin heavy chain (Fib-H) protein. The large central domain of Fib-H, which consists of glycine- and alanine-rich crystalline regions interspersed with amorphous motifs of approximately 30 amino acid residues, is considered crucial for fibrilization and determines the properties of the silk fiber. We established a technical platform to modify the Fib-H core region systematically using transcription activator-like effector nuclease-mediated homologous recombination through a somatic and germline gene knockin assay along with PCR-based screening. This efficient knockin system was used to generate a silkworm strain carrying a mutant Fib-H allele, in which the core region was replaced with a highly ordered synthetic repeat sequence of a length comparable with native Fib-H core. Heterozygous knockin mutants produced seemingly normal cocoons, whereas homozygotes did not and exhibited considerable degradation in their posterior silk glands (PSGs). Cross-sectional examination of the PSG lumen and tensile tests conducted on reeled silk threads indicated that the mutant Fib-H, which exhibited reduced stability in the PSG cells and lumen, affected the mechanical properties of the fiber. Thus, sequence manipulation of the Fib-H core domain was identified as a crucial step in successfully creating artificial silk using knockin technology.

Unique Material Properties of Bombyx mori Silk Fiber Incorporated with 3-Azidotyrosine.

Silk fiber produced by the silkworm Bombyx mori is a nature-derived proteinous fiber with excellent mechanical strength and broad biocompatibility. To alter its material properties and make it more suitable for textile, biomedical, and electronics applications, chemical modifications and genetic engineering methods have been extensively studied. Here, we report that the translational incorporation of a synthetic amino acid, 3-azidotyrosine (3-AzTyr), into B. mori silk fiber can improve its material properties. Such an incorporation considerably increased the fiber's mechanical strength and remarkably changed its solubility, whereas its crystalline hierarchical structure was not perturbed, as shown by X-ray analyses. These changes were probably caused by the intra- and/or intermolecular crosslinkings involving the azido group of 3-AzTyr during the degumming process to remove a coating protein. These findings indicate that the incorporation of synthetic amino acids could be an efficient method to improve the properties of silk-based materials.

Development of a filter device for the prevention of aquatic bacterial disease using a single-chain variable fragment (scFv)-conjugated affinity silk.

Infectious disease is one of the most serious problems in the aquaculture industry for ornamental or edible fish. This study attempted to develop a new device for preventing an aquatic bacterial disease, ulcer disease, caused by Aeromonas salmonicida (As), using "affinity silk". Affinity silk is a silk protein-containing fibroin L-chain (FibL) fused to the single-chain variable fragment (scFv). It can be easily processed into different formats such as fibers, gels, sponges, or films. A transgenic silkworm that could express a cDNA construct containing FibL fused to an scFv derived from a monoclonal antibody (MAb) against As was successfully generated. An enzyme-linked immunosorbent assay was used to detect As by employing 96-well plates coated with scFv-conjugated affinity silk. As could be captured efficiently by glass wool coated with affinity silk in the column. Furthermore, the air-lift water filter equipped with the affinity silk-coated wool could considerably reduce the concentration of As in water and was estimated to have sufficient ability to trap a lethal dose of As. These findings show that the "affinity silk filter" is a potential device for the prophylaxis of aquatic animal diseases.

Higher Gene Expression Related to Wound Healing by Fibroblasts on Silk Fibroin Biomaterial than on Collagen.

Silk fibroin (SF), which offers the benefits of biosafety, biocompatibility, and mechanical strength, has potential for use as a good biomedical material, especially in the tissue engineering field. This study investigated the use of SF biomaterials as a wound dressing compared to commercially available collagen materials. After human fibroblasts (WI-38) were cultured on both films and sponges, their cell motilities and gene expressions related to wound repair and tissue reconstruction were evaluated. Compared to the collagen film (Col film), the SF film induced higher cell motility; higher expressions of genes were observed on the SF film. Extracellular matrix production-related genes were up-regulated in WI-38 fibroblasts cultured on the SF sponges. These results suggest that SF-based biomaterials can accelerate wound healing and tissue reconstruction. They can be useful biomaterials for functional wound dressings.

Direct Recovery of the Rare Earth Elements Using a Silk Displaying a Metal-Recognizing Peptide.

Rare earth elements (RE) are indispensable metallic resources in the production of advanced materials; hence, a cost- and energy-effective recovery process is required to meet the rapidly increasing RE demand. Here, we propose an artificial RE recovery approach that uses a functional silk displaying a RE-recognizing peptide. Using the piggyBac system, we constructed a transgenic silkworm in which one or two copies of the gene coding for the RE-recognizing peptide (Lamp1) was fused with that of the fibroin L (FibL) protein. The purified FibL-Lamp1 fusion protein from the transgenic silkworm was able to recognize dysprosium (Dy3+), a RE, under physiological conditions. This method can also be used with silk from which sericin has been removed. Furthermore, the Dy-recovery ability of this silk was significantly improved by crushing the silk. Our simple approach is expected to facilitate the direct recovery of RE from an actual mixed solution of metal ions, such as seawater and industrial wastewater, under mild conditions without additional energy input.

Genetic Code Expansion of the Silkworm Bombyx mori to Functionalize Silk Fiber.

The genetic code in bacteria and animal cells has been expanded to incorporate novel amino acids into proteins. Recent efforts have enabled genetic code expansion in nematodes, flies, and mice, whereas such engineering is rare with industrially useful animals. In the present study, we engineered the silkworm Bombyx mori to synthesize silk fiber functionalized with azidophenylalanine. For this purpose, we developed a bacterial system to screen for B. mori phenylalanyl-tRNA synthetases with altered amino-acid specificity. We created four transgenic B. mori lines expressing the selected synthetase variants in silk glands, and found that two of them supported the efficient in vivo incorporation of azidophenylalanine into silk fiber. The obtained silk was bio-orthogonally reactive with fluorescent molecules. The results showed that genetic code expansion in an industrial animal can be facilitated by prior bacterial selection, to accelerate the development of silk fiber with novel properties.

Development and validation of scFv-conjugated affinity silk protein for specific detection of carcinoembryonic antigen.

The production costs for monoclonal antibodies (MAbs) utilized in medical diagnostic kits are inevitably high because the MAbs are mostly obtained from hybridoma cell culture. Here, we report the development and validation of a novel affinity silk protein produced by transgenic silkworm technology as a possible alternative diagnostic tool for cancers. We generated a transgenic silkworm expressing a cDNA construct containing fibroin L-chain fused to a single-chain variable fragment (scFv) derived from a MAb against carcinoembryonic antigen (CEA). The transgenic cocoons were dissolved in aqueous lithium bromide solution, applied to 96-well plates, and analysed by enzyme-linked immunosorbent assay. The scFv-conjugated affinity silk protein specifically recognized CEA as well as the parental MAb. The binding activity was retained after several months of storage in coated plates or concentrated solution. Thus, the scFv-conjugated affinity silk protein provides a potentially useful alternative to conventional MAbs in medical diagnostic kits.

Fabrication Scheme for Obtaining Transparent, Flexible, and Water-Insoluble Silk Films from Apparently Dissolved Silk-Gland Fibroin of Bombyx mori Silkworm.

Films from silk fibroin protein are one of the most promising biomaterials because of their exquisite balance between mechanical properties and biocompatibility. Numerous schemes have been proposed for processing fibroin film, utilizing liquid silk fibroin (LSF) or regenerated silk fibroin (RSF). The films cast from LSF or RSF in the solution state are water-soluble, and therefore require postproduction treatment inducing ß-sheet formation, to render them insoluble in water. Many kinds of postproduction treatments, using alcohol-water solution, water vapor, or controlled temperature, have been developed. However, the tuning and reproducibility of such treatments are quite sensitive and frequently render the fibroin films less flexible or even brittle because of the formation of an over content of ß-sheet. To overcome this, we developed a novel scheme for fibroin processing using silk-gland fibroin (SGF). The essence of this scheme is to create a softly solidified fibroin-gel state of the silk glands with an imperfect ß-sheet structure, by treating them with an ethanol/water mixture. Such a fibroin gel was found to dissolve in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP). The SGF film cast from the HFIP solution shows a flexible and water-insoluble nature with high reproducibility. In addition to this improvement, the SGF film produced by this method contains a significantly low level of residual HFIP molecules compared to the traditional RSF films prepared from an HFIP solution. The mechanism underlying these advantageous characteristics was investigated from the structural viewpoint, by using techniques such as 13C solid-state NMR, differential scanning calorimetry, and wide-angle X-ray diffraction.

Silk fibroin sponges with cell growth-promoting activity induced by genetically fused basic fibroblast growth factor.

Transgenic silkworm technology has enabled the biological properties of silk fibroin protein to be altered by fusion to recombinant bioactive proteins. However, few studies have reported the fabrication of genetically modified fibroin proteins into three-dimensional spongy structures to serve as scaffolds for tissue engineering. We generated a transgenic silkworm strain that produces fibroin fused to basic fibroblast growth factor (bFGF) and processed the fibroin into a spongy structure using a simple freeze/thaw method. NIH3T3 mouse embryonic fibroblasts grown on bFGF-fused fibroin sponges proliferated and spread out well, showing half the population doubling time of cells cultured on wild-type fibroin sponges. Furthermore, the number of primary rabbit articular chondrocytes growing on bFGF-fused fibroin sponges was around five-times higher than that of the wild-type control at 3-days post cell-seeding. As the physical properties of wild-type and bFGF-fused fibroin sponges were almost identical, it is suggested that bFGF fused to fibroin retained its biological activity, even after the bFGF-fused fibroin was fabricated into the spongy structure. The bFGF-fused fibroin sponge has the potential for widespread application in the field of tissue engineering, and the method of fabricating this structure could be applicable to other recombinant bioactive fibroin proteins.

Development of a FRET-based recombinant tension sensor to visualize cell-material interactions.

A Förster/fluorescence resonance energy transfer (FRET)-based molecular tension sensor was originally reported by the fusion of intracellular molecules, which has contributed to the elucidation of cell mechanotransduction. However, it is still unclear whether recombinant tension sensors can detect forces in the extracellular environment. Here, we developed a recombinant FRET-based tension sensor (rFRET-TS) and immobilized it to a glass surface. Fibroblasts seeded onto the surface likely bound to an RGDS peptide fused to one terminal of the rFRET-TS, and intra-molecular FRET was dominantly observed on the sensor-immobilized surface compared to inter-molecular FRET. Time-lapse FRET imaging showed that the rFRET-TS enabled the real-time visualization of forces between cells and material surfaces that stemmed from focal adhesion formation and actin cytoskeletal reorganization. This sensor is expected to be useful for clarifying cell-scaffold mechanical interactions by its insertion between protein molecules of the scaffold, which will provide clues for the control of cell behavior in/on scaffolds.

Azide-Incorporated Clickable Silk Fibroin Materials with the Ability to Photopattern.

Incorporation of unnatural amino acids (UAAs) bearing bioorthogonal reactive groups into proteins could be a powerful tool for developing novel protein-based biomaterials with innovative and controlled performance. Bombyx mori silk fibroin is one of naturally derived protein materials extensively studied for biomaterials development due to its mechanical strength and biocompatibility. We recently reported the in vivo incorporation of UAAs, 4-substituted analogues of phenylalanine including 4-azidophenylalanine (AzPhe), into silk fibroin by expanding the repertoire of amino acids for protein biosynthesis in silk glands of B. mori using transgenic techniques. We demonstrated that azide groups in AzPhe incorporated into silk fibroin can be selectively modified by bioorthogonal azide-alkyne cycloaddition reactions (click chemistry). However, the incorporation of AzPhe into silk fibroin required a special feeding condition, which led to the limited production of silk fibroin. Here we report more efficient production of an AzPhe-incorporated silk fibroin (termed AzidoSilk) and its modification by click chemistry in varied material forms (thread, film, and porous sponge). Using this methodology, photolithographic micropatterning of fluorescent molecules directly onto silk fibroin film was achieved and should further expand the availability of silk-based biomaterials for cell culture substrates, drug delivery, tissue scaffolds, implantable devices, and so on.

Incorporation of Methionine Analogues Into Bombyx mori Silk Fibroin for Click Modifications.

Bombyx mori silk fibroin incorporating three methionine (Met) analogues-homopropargylglycine (Hpg), azidohomoalanine (Aha), and homoallylglycine (Hag)-can be produced simply by adding them to the diet of B. mori larvae. The Met analogues are recognized by methionyl-tRNA synthetase, bound to tRNA(Met), and used for the translation of adenine-uracil-guanine (AUG) codons competitively with Met. In the presence of the standard amount of Met in the diet, incorporation of these analogues remains low. Lowering the amount of Met in the diet drastically improves incorporation efficiencies. Alkyne and azide groups in Hpg and Aha incorporated into silk fibroin can be selectively modified with Cu-catalyzed azide-alkyne cycloaddition reactions (click chemistry). Since Met residues exist only at the N-terminal domain of the fibroin heavy chain and in the fibroin light chain, good access to the reactive sites is expected and domain-selective modifications are possible without perturbing other major domains, including repetitive domains.

High-toughness silk produced by a transgenic silkworm expressing spider (Araneus ventricosus) dragline silk protein.

Spider dragline silk is a natural fiber that has excellent tensile properties; however, it is difficult to produce artificially as a long, strong fiber. Here, the spider (Araneus ventricosus) dragline protein gene was cloned and a transgenic silkworm was generated, that expressed the fusion protein of the fibroin heavy chain and spider dragline protein in cocoon silk. The spider silk protein content ranged from 0.37 to 0.61% w/w (1.4-2.4 mol%) native silkworm fibroin. Using a good silk-producing strain, C515, as the transgenic silkworm can make the raw silk from its cocoons for the first time. The tensile characteristics (toughness) of the raw silk improved by 53% after the introduction of spider dragline silk protein; the improvement depended on the quantity of the expressed spider dragline protein. To demonstrate the commercial feasibility for machine reeling, weaving, and sewing, we used the transgenic spider silk to weave a vest and scarf; this was the first application of spider silk fibers from transgenic silkworms.

Production of Bombyx mori silk fibroin incorporated with unnatural amino acids.

Silk fibroin incorporated with unnatural amino acids was produced by in vivo feeding of p-chloro-, p-bromo-, and p-azido-substituted analogues of L-phenylalanine (Phe) to transgenic silkworms (Bombyx mori) that expressed a mutant of phenylalanyl-tRNA synthetase with expanded substrate recognition capabilities in silk glands. Cutting down the content of Phe in the diet was effective for increasing the incorporation of Phe analogues but simultaneously caused a decrease of fibroin production. The azide groups incorporated in fibroin were active as chemical handles for click chemistry in both the solubilized and the solid (fibrous) states. The azides survived degumming in the boiling alkaline solution that is required for complete removal of the sericin layer, demonstrating that AzPhe-incorporated silk fibroin could be a versatile platform to produce "clickable" silk materials in various forms. This study indicates the huge potential of UAA mutagenesis as a novel methodology to alter the characteristics of B. mori silk.

Cross-linking in the silks of bees, ants and hornets.

Silk production is integral to the construction of nests or cocoons for many Aculeata, stinging Hymenopterans such as ants, bees and wasps. Here we report the sequences of new aculeate silk proteins and compare cross-linking among nine native silks from three bee species (Apis mellifera, Bombus terrestris and Megachile rotundata), three ant species (Myrmecia forficata, Oecophylla smaragdina and Harpegnathos saltator) and three hornets (Vespa analis, Vespa simillima and Vespa mandarinia). The well studied silks of spiders and silkworms are comprised of large proteins that are cross-linked and stabilized predominantly by intra and intermolecular beta sheet structure. In contrast, the aculeate silks are comprised of relatively small proteins that contain central coiled coil domains and comparatively reduced amounts of beta sheet structure. The hornet silks, which have the most beta sheet structure and the greatest amount of amino acid sequence outside the coiled-coil domains, dissolve in concentrated LiBr solution and appear to be stabilized predominantly by beta sheet structure like the classic silks. In contrast, the ant and bee silks, which have less beta sheet and less sequence outside the coiled-coil domains, could not be dissolved in LiBr and appear to be predominantly stabilized by covalent cross-linking. The iso-peptide cross-linker, ε-(γ-glutamyl)-lysine that is produced by transglutaminase enzymes, was demonstrated to be present in all silks by mass spectrometry, but at greater levels in silks of ants and bees. The bee silks and ant cocoons, but not the Oecophylla nest silks, appeared to be further stabilized by tanning reactions.

Production of scFv-conjugated affinity silk film and its application to a novel enzyme-linked immunosorbent assay.

Bombyx mori (silkworm) silk proteins have been utilized as unique biomaterials for various medical applications. To develop a novel affinity silk material, we generated a transgenic silkworm that spins silk protein containing the fibroin L-chain linked with the single-chain variable fragment (scFv) as a fusion protein. Previously, the scFv-conjugated "affinity" silk powder specifically immunoprecipitated its target protein, Wiskott-Aldrich syndrome protein. To expand the applicability of affinity silk materials, we processed the scFv-conjugated silk protein into a thin film by dissolving it in lithium bromide, then drying it in the wells of 96-well plates. Enzyme-linked immunosorbent assay demonstrated specific detection of Wiskott-Aldrich syndrome protein, both as a recombinant protein and in its native form extracted from mouse macrophages. These findings suggest that this scFv-conjugated silk film serves as the basis for an alternative immunodetection system.

How do chondrocytes aggregate on fibroin substrate.

The effects of substrate material on the spatio-temporal behavior of cells is an important issue. Although cell aggregation has been observed on various fibroin substrates, the mechanisms of this aggregation have yet to be fully clarified. In this study, cell aggregation behavior on fibroin substrates were evaluated, focusing on the distance between each cell and the direction of individual cell migration. Our results showed that on fibroin substrates cells did not attract each other. However cells stayed close to adjacent cells over 24 hours of cultivation.

Quantitative evaluation of fibroblast migration on a silk fibroin surface and TGFBI gene expression.

Cell migration plays important roles in natural processes involving embryonic development, inflammation, wound healing, cancer metastasis and angiogenesis. Cell migration on various biomaterials is also believed to improve the rate of wound healing and implant therapies in the tissue-engineering field. This study measured the distance traversed, or mileage, of mouse fibroblasts on a silk fibroin surface. Fibroblasts on the fibroin surface moved with better progress during 24 h than cells on collagen or fibronectin surfaces. Results obtained by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) revealed that fibroblasts on the fibroin surface expressed transforming growth factor ß-induced protein (TGFBI), which is an extracellular matrix (ECM) protein, stronger than on other surfaces in the early cell-culture stages. These results demonstrate that the fibroin surface shows higher potential to enhance cell migration and the production of ECM than a collagen or fibronectin surface.

Production of scFv-conjugated affinity silk powder by transgenic silkworm technology.

Bombyx mori (silkworm) silk proteins are being utilized as unique biomaterials for medical applications. Chemical modification or post-conjugation of bioactive ligands expand the applicability of silk proteins; however, the processes are elaborate and costly. In this study, we used transgenic silkworm technology to develop single-chain variable fragment (scFv)-conjugated silk fibroin. The cocoons of the transgenic silkworm contain fibroin L-chain linked with scFv as a fusion protein. After dissolving the cocoons in lithium bromide, the silk solution was dialyzed, concentrated, freeze-dried, and crushed into powder. Immunoprecipitation analyses demonstrate that the scFv domain retains its specific binding activity to the target molecule after multiple processing steps. These results strongly suggest the promise of scFv-conjugated silk fibroin as an alternative affinity reagent, which can be manufactured using transgenic silkworm technology at lower cost than traditional affinity carriers.

Identification of hornet silk gene with a characteristic repetitive sequence in Vespa simillima xanthoptera.

Vssilk 5 is a gene encoding a component protein of the silk produced by the larvae of the yellow hornet (Vespa simillima, Vespinae, Vespidae). In this study, we deduced the complete cDNA sequence of Vssilk 5. It was found that 2 silk proteins, Vssilk 5 N and Vssilk 5 C, in the cocoon of the yellow hornet are both encoded by the Vssilk 5 gene. Vssilk 5 N and 5 C are the N- and C-terminal regions, respectively, of the Vssilk 5 pro-protein (Vssilk 5p). The complete amino acid sequences of Vssilk 5 N and Vssilk 5 C were deduced. Although a non-repetitive amino acid sequence and coiled-coil structure are properties common to the major components of silk proteins produced by the larvae of the social superfamilies Apoidea and Vespoidea of the Apocrita, nearly the entire sequence of Vssilk 5 C consisted of a repeated sequence of amino acids, and the calculated coiled-coil probability for this protein was low. Vssilk 5 N is a protein without a repetitive amino acid sequence and has a low coiled-coil probability. Moreover, we found a water soluble protein, Vssilk 5S that is likely segmented from Vssilk 5 C and contains an N-terminal sequence identical to that of Vssilk 5 C.