Hidetoshi Teramoto

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 β-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.

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.

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.

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.

Expansion of the Amino Acid Repertoire in Protein Biosynthesis in Silkworm Cells

Introduction of unnatural amino acids (UAAs) into proteins has become a powerful tool for biological studies and creating proteins with novel functions. To date, in vivo UAA incorporation into proteins has been achieved with various organisms, including unicellular organisms such as bacteria 5] and yeast, 7] cultured (single) cells of mammals 9] and of an insect (Drosophila melanogaster), and even a multicellular organism (Caenorhabditis elegans). Here we report the extension of UAA incorporation methodology to cultured cells of the domesticated silkworm, Bombyx mori. Incorporation of UAAs into proteins holds enormous potential for increasing the utility of protein-based materials. Silk proteins produced by B. mori are under extensive study as textile, biomedical, electronic, and photonics materials, due to their excellent textures, mechanical strengths, moldabilities, and biocompatibilities. The addition of UAAs to proteins would be a promising approach to creating compounds beyond those achievable with the 20 standard natural amino acids, thereby expanding the scope of silk applications. The introduction of novel functionalities through the UAAs might result not only in new structural and physicochemical characteristics of silk but also in chemical handles for chemoselective modifications. The future goal of our study is to genetically modify B. mori larvae so that they can incorporate UAAs into protein biosynthesis and to produce UAA-incorporated silk proteins for their biomedical and industrial applications. However, UAA incorporation into proteins synthesized in multicellular animals such as B. mori is a challenging task. Greiss and Chin recently succeeded in site-specifically incorporating UAAs into proteins in a multicellular animal, C. elegans. Because the site-specific incorporation strategy can specify the positions of UAAs in target proteins, perturbation to the living systems of host animals could be minimized. 19] At the same time, this strategy requires multiple gene manipulations, including introduction of distinct codons for UAAs in target proteins. In contrast to the site-specific method, residue-specific incorporation of UAAs can be achieved in relatively simple ways. 20] In fact, residue-specific incorporation requires only the engineering of aminoacyl-tRNA synthetases (aaRSs), 21–23] which catalyze the charging of specific tRNAs with their cognate amino acids with strict substrate specificity to ensure the accuracy of the genetic code during protein synthesis. E. coli phenylalanyl-tRNA synthetase (PheRS), for example, was engineered by simple point mutations at its amino acid binding site to exhibit broader substrate recognition capacity. Through overexpression of such mutants in E. coli, proteins containing a wide variety of phenylalanine (Phe) analogues have been obtained. Because all Phe residues in all proteins are likely to be replaced with their analogues by this strategy, the living systems of host animals might be subject to some adverse effects. Organ-specific expression of engineered aaRSs in host animals might decrease such negative effects on the living systems. Phe is considered an appropriate target for achieving residue-specific UAA incorporation into silk because Phe is an essential amino acid for B. mori, making it possible to control the uptake of Phe by feeding, and because Phe is incorporated in the major protein component of B. mori silk in a periodic manner. We have therefore cloned two genes encoding two subunits (a and b) of B. mori PheRS (BmPheRS) and have succeeded in generating its mutant by the same strategy as employed in E. coli. This mutant BmPheRS has an Ala450-to-Gly mutation at the amino acid binding site in the a subunit and was found to catalyze aminoacylation of B. mori tRNA with p-Cland p-Br-substituted Phe analogues in vitro. Here, to verify the function of this BmPheRS mutant in protein synthesis in vivo, incorporation of Phe analogues into proteins was investigated with a cell line of B. mori : BmN. Figure 1 illustrates the assay procedure employed in this study. BmN cells were transfected with expression plasmids separately encoding the two subunits (a and b) of BmPheRS, in which the a subunit was either the wild type or the A450G mutant (see Figure S1 in the Supporting Information). As a reporter protein, an expression plasmid encoding enhanced green fluorescent protein (EGFP) was also introduced. We chose EGFP because it can be expressed at a high level in BmN cells and can be easily handled for purification and analyses. The transfected cells were cultured in Grace’s insect medium for nine days in the presence or the absence of p-Clor p-Br-Phe (1 mm ; pictures of the cells during culture are shown in Figure S2). The synthesized EGFP obtained in the supernatants of the cell lysates was affinity-purified by using Strep and His6 tags fused at its N and C termini, respectively (see Figure S3). Because EGFP was purified with two different affinity tags fused at its N and C termini, only the full-length EGFP was obtained, without any detectable contaminants in SDS-PAGE (Figure S4). The purified EGFP was subjected to [a] Dr. H. Teramoto, Dr. K. Kojima, Dr. J. Ishibashi Genetically Modified Organism Research Center National Institute of Agrobiological Sciences 1-2 Ohwashi, Tsukuba, Ibaraki 305-8634 (Japan) E-mail : teramoto@affrc.go.jp [b] Dr. H. Kajiwara Agrogenomics Research Center National Institute of Agrobiological Sciences 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602 (Japan) Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.201100624.