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.

Click Decoration of Bombyx mori Silk Fibroin for Cell Adhesion Control.

Silk fibroin produced by the domesticated silkworm, Bombyx mori, has been studied widely as a substrate for tissue engineering applications because of its mechanical robustness and biocompatibility. However, it is often difficult to precisely tune silk fibroin's biological properties due to the lack of easy, reliable, and versatile methodologies for decorating it with functional molecules such as those of drugs, polymers, peptides, and enzymes necessary for specific applications. In this study we applied an azido-functionalized silk fibroin, AzidoSilk, produced by a state-of-the-art biotechnology, genetic code expansion, to produce silk fibroin decorated with cell-repellent polyethylene glycol (PEG) chains for controlling the cell adhesion property of silk fibroin film. Azido groups can act as selective handles for chemical reactions such as a strain-promoted azido-alkyne cycloaddition (SPAAC), known as a click chemistry reaction. We found that azido groups in AzidoSilk film were selectively decorated with PEG chains using SPAAC. The PEG-decorated film demonstrated decreased cell adhesion depending on the lengths of the PEG chains. Azido groups in AzidoSilk can be decomposed by UV irradiation. By partially decomposing azido groups in AzidoSilk film in a spatially controlled manner using photomasks, cells could be spatially arranged on the film. These results indicated that SPAAC could be an easy, reliable, and versatile methodology to produce silk fibroin substrates having adequate biological properties.

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.

Characterization and Scaled-Up Production of Azido-Functionalized Silk Fiber Produced by Transgenic Silkworms with an Expanded Genetic Code.

The creation of functional materials from renewable resources has attracted much interest. We previously reported on the genetic code expansion of the domesticated silkworm Bombyx mori to functionalize silk fiber with synthetic amino acids such as 4-azido-L-phenylalanine (AzPhe). The azido groups act as selective handles for biorthogonal chemical reactions. Here we report the characterization and scaled-up production of azido-functionalized silk fiber for textile, healthcare, and medical applications. To increase the productivity of azido-functionalized silk fiber, the original transgenic line was hybridized with a high silk-producing strain. The F1 hybrid produced circa 1.5 times more silk fibroin than the original transgenic line. The incorporation efficiency of AzPhe into silk fibroin was retained after hybridization. The tensile properties of the azido-functionalized silk fiber were equal to those of normal silk fiber. Scaled-up production of the azido-functionalized silk fiber was demonstrated by rearing circa 1000 transgenic silkworms. Differently-colored fluorescent silk fibers were successfully prepared by click chemistry reactions, demonstrating the utility of the azido-functionalized silk fiber for developing silk-based materials with desired functions.

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.

Effect of supplemented sericin on the development, cell number, cryosurvival and number of lipid droplets in cultured bovine embryos.

Sericin was investigated as an alternative to fetal bovine serum (FBS) for bovine embryo culture. In vitro matured oocytes were developed using 0.05%, 0.1% or 0.15% sericin. The developmental rate, cryosurvival rate and blastulation time of these embryos were compared with those of embryos developed using 5% FBS. The number of lipid droplets was compared among the blastocysts developed using 5% FBS, using 0.05% sericin and in vivo. The rate of cleavage and blastocyst formation was similar among all groups. Blastulation occurred significantly earlier in the embryos developed using 5% FBS than in those developed using sericin at any concentration (P < 0.05). At 72 h after thawing, the cryosurvival rate of the blastocysts developed using 5% FBS and 0.05% sericin were significantly higher compared with those developed using 0.1% and 0.15% sericin (P < 0.05). The blastocysts developed using 0.05% sericin and in vivo produced a significantly fewer number of medium and large lipid droplets than those developed using 5% FBS. These results suggest that the blastocysts developed using 0.05% sericin show characteristics similar to those of the blastocysts developed in vivo and that the use of sericin as an alternative to FBS is feasible.

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.

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.

Sericin accelerates the production of hyaluronan and decreases the incidence of polyspermy fertilization in bovine oocytes during in vitro maturation.

Fetal bovine serum (FBS) has been widely used as a supplement in the maturation medium of bovine oocytes in vitro. However, serum contains many undefined factors and is potentially infectious to humans and animals. As a serum replacement, we evaluated the feasibility of using the silk protein, sericin, derived from the cocoons of silkworm. To examine the rates of oocyte maturation and fertilization, cumulus-oocyte complexes were cultured in TCM-199 supplemented with 0.01%, 0.05%, 0.1% or 0.15% sericin or 5% FBS. The sizes of the perivitelline space that might relate to polyspermy, the expressions of Has2 and CD44 mRNA, the amount of hyaluronan (hyaluronic acid: HA) contained in the oocytes and the rates of blastocyst formation following insemination were then compared between the oocytes cultured with 0.05% sericin and 5% FBS, because the polyspermy rates in oocytes cultured with 0.05% sericin were significantly lower than in those cultured with 5% FBS. After in vitro maturation (IVM), the mean size of the perivitelline space was significantly greater in oocytes cultured with sericin than in those cultured with FBS, although the rates of nuclear maturation, fertilization and blastocyst formation of oocytes under both IVM conditions were not significantly different. The expression of HAS2 and CD44 mRNA and the amount of HA in the denuded oocytes cultured with 0.05% sericin were significantly greater than in those cultured with FBS. These results indicate the feasibility of sericin as an alternative protein supplement for IVM in bovine oocytes.

Drawing-induced changes in morphology and mechanical properties of hornet silk gel films.

Complete amino acid sequences of the four major proteins (Vssilk 1-4) of silk (hornet silk) obtained from yellow hornet ( Vespa simillima , Vespinae, Vespidae) cocoons have been determined. The native structure of the hornet silk (HS), in which Vssilk 1-4 have an alpha-helix domain with coiled-coil alpha-helices and a beta-sheet domain, is restored when hornet silk gel films (HSGFs) are formed by pressing and drying HS hydrogel. Necking occurs when dry HSGFs are drawn; however, wet HSGFs can be uniaxially drawn with a draw ratio (DR) of 2. Drawing helps obtain high-performance films with a maximum tensile strength and tensile modulus of 170 MPa and 5.5 GPa, respectively. Drawing-induced changes in the orientation and conformation of the coiled-coil structure are investigated.

Preparation of gel film from Bombyx mori silk sericin and its characterization as a wound dressing.

Sericin is a highly hydrophilic protein family acting as the glue in Bombyx mori silk. In order to apply sericin as a wound dressing, a novel sericin film named gel film was prepared by a simple process without using any chemical modifications: sericin solution was gelled with ethanol into a sheet shape and then dried. Infrared analysis revealed that the sericin gel film contained water-stable beta-sheet networks formed in the gelation step. This structural feature rendered the gel film morphologically stable against swelling and gave it good handling properties in the wet state. The sericin gel film rapidly absorbed water, equilibrating at a water content of about 80%, and exhibited elastic deformation up to a strain of about 25% in the wet state. A culture of mouse fibroblasts on the gel film indicated that it had low cell adhesion properties and no cytotoxicity. These characteristics of sericin gel film suggest its potential as a wound dressing.

Hornet silk proteins in the cocoons produced by different Vespa species inhabiting Japan.

We compared the components of hornet silk - a fibrous protein occurring in the cocoons produced by hornet larvae - among 6 species of the genus Vespa inhabiting Japan: V. simillima, V. dybowskii, V. crabro, V. mandarinia, V. ducalis, and V. analis. From the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), N-terminal amino acid sequence analysis, and 5'-RACE, it was found that the major component proteins composing hornet silk could be divided into 6 groups. Among these 6 proteins, 5 proteins were common to the hornet silks of all 6 Vespa species. The SDS-PAGE major band corresponding to the remaining protein was observed only in the hornet silks of V. mandarinia and V. ducalis. This correspondence between V. mandarinia and V. ducalis can probably be explained in terms of the phylogenetic relationships of the Vespa species.

Total synthesis of (-)-5,6,11-trideoxytetrodotoxin and its 4-epimer.

[reaction: see text] The first total synthesis of 5,6,11-trideoxytetrodotoxin (1) and its 4-epimer were achieved. The synthesis is characterized by the stereoselective construction of the quaternary amino carbon center at C8a by an asymmetric transferring Strecker synthesis and the highly efficient conversion of cyanohydrin 4 to 1 via intramolecular cyclization reactions.

Phase transition of L-Ser monohydrate crystal studied by (13)C solid-state NMR.

We used gravimetric analysis (GA) and (13)C solid-state nuclear magnetic resonance (NMR) to study solid-phase transition from the transparent single crystal of L-serine (L-Ser) monohydrate to a turbid powder. We found that L-Ser monohydrate loses water molecules and transforms into an anhydrate, thus experimentally demonstrating Frey's assumption. Application of a handmade cross-polarization (CP) NMR probe with a saddle-type coil to the oriented crystal of the L-Ser monohydrate revealed the dehydration mechanism. Furthermore, the chemical shift tensor components of the carboxyl carbon in L-Ser monohydrate were determined. The difference in the tensor component of delta(22) between the monohydrate and anhydrate forms was more than 7 ppm, probably owing to differences in the hydrogen-bonding structure of each form.

Molecular orientation behavior of silk sericin film as revealed by ATR infrared spectroscopy.

This paper reports the structure-dependent molecular orientation behavior of sericin, an adhesive silk protein secreted by silkworm, Bombyx mori. Although application of sericin as a biomaterial is anticipated because of its unique characteristics, sericin's physicochemical properties remain unclear, mainly because of its vulnerability to heat or alkaline treatment during separation from fibroin threads. This study employed intact sericin obtained from fibroin-deficient mutant silkworm to investigate the relationship between molecular orientation and the secondary structure of sericin. Sericin films were artificially stretched after moistening with aqueous ethanol of various concentrations. The resulting molecular orientation was analyzed using polarized infrared spectroscopy. These analyses indicated that formation of aggregated strands among extended sericin chains induced by ethanol treatment is the key to generating molecular orientation. Strong intermolecular hydrogen bonds are inferred to allow aggregated strands' stretching-force transmission, thereby causing molecular orientation.

Preparation of elastic silk sericin hydrogel.

This paper reports a preparation method for silk sericin hydrogel using the Sericin-hope silkworm, whose cocoons consist almost exclusively of sericin. Sericin solution, prepared from Sericin-hope cocoons, contains intact sericin and forms elastic hydrogels with the addition of ethanol. The sericin hydrogel can be prepared without crosslinking by chemicals or irradiation and might be usable as a naturally occurring biomaterial.

Chemical modification of silk sericin in lithium chloride/dimethyl sulfoxide solvent with 4-cyanophenyl isocyanate.

This paper reports chemical modification of silk sericin in LiCl/dimethyl sulfoxide (DMSO) solvent with 4-cyanophenyl isocyanate. Sericin is a highly hydrophilic protein secreted by Bombyx mori, serving as a protein glue in a cocoon. LiCl/DMSO was found to be a good solvent of sericin and useful for homogeneous modification of its abundant hydroxyl groups under nonaqueous condition. Fourier transform infrared (FTIR) analysis of the modified sericins revealed that 4-cyanophenyl groups were incorporated into sericin molecules mainly through urethane linkages. Several characteristics of the modified sericins such as solubility characteristic, hygroscopic property, and thermal stability were investigated. Secondary structure analysis using FTIR spectra suggested that formation of strong intermolecular hydrogen bonds was inhibited by the modification that is probably attributable to the incorporation of bulky 4-cyanophenyl groups. These results demonstrate that chemical modification of sericin using LiCl/DMSO solvent markedly alters its characteristics.