Hideyuki Kajiwara

Identification of Four Major Hornet Silk Genes with a Complex of Alanine-Rich and Serine-Rich Sequences in Vespa simillima xanthoptera Cameron

Hornet silk, a fibrous protein in the cocoon produced by the larva of the vespa, is composed of four major proteins. In this study, we constructed silk-gland cDNA libraries from larvae of the hornet Vespa simillima xanthoptera Cameron and deduced the full amino acid sequences of the four hornet silk proteins, which were named Vssilk 1–4 in increasing order of molecular size. Portions of the amino acid sequences of the four proteins were confirmed by Matrix-assisted laser desorption/ionization-time of flight/mass spectrometry (MALDI-TOF/MS) and N-terminal protein sequencing. The primary sequences of the four Vssilk proteins (1–4) were highly divergent, but the four proteins had some common properties: (i) the amino acid compositions of all four proteins were similar to each other in that the well-defined and characteristic repetitive patterns present in most of the known silk proteins were absent; and (ii) the characteristics of the amino acid sequences of the four proteins were also similar in that Ser-rich structures such as sericin were localized at both ends of the chains and Ala-rich structures such as fibroin were found in the center. These characteristic primary structures might be responsible for the coexisting α-helix and β-sheet conformations that make up the unique secondary structure of hornet silk proteins in the native state. Because heptad repeat sequences of hydrophobic residue are present in the Ala-rich region, we believe that the Ala-rich region of hornet silk predominantly forms a coiled coil with an α-helix conformation.

Protein Profile of Symbiotic Bacteria Mesorhizobium loti MAFF303099 in Mid-growth Phase

Expressed proteins in cultured symbiotic bacteria (Mesorhizobium loti MAFF303099) in the mid-growth phase were proteomically analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and capillary high-performance liquid chromatography coupled with an ion-trap mass spectrometry (MS). The genome sequence data of M. loti were used to identify the analyzed proteins. We identified 114 of the 127 proteins analyzed on 2D-PAGE gel with some microheterogenities which were caused by post-translational modifications.

Detection of specific DNA from crude extracts of rice seed grains using matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

To rapidly detect specific genes, crude extracts prepared from rice seed grains were used as templates for PCR, the PCR products were digested with restriction enzymes or urasil-DNA glycosylase, and then matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF MS) was used to detect amplified DNA. It was possible to amplify small DNA fragments (50-60bp), but not large ones (>200bp), using crude extracts as the PCR template. This method can be completed within 1h, including extractions, and is well suited to automation for high-throughput analyses.

A Hox Gene, Antennapedia, Regulates Expression of Multiple Major Silk Protein Genes in the Silkworm Bombyx mori

Hox genes play a pivotal role in the determination of anteroposterior axis specificity during bilaterian animal development. They do so by acting as a master control and regulating the expression of genes important for development. Recently, however, we showed that Hox genes can also function in terminally differentiated tissue of the lepidopteran Bombyx mori. In this species, Antennapedia (Antp) regulates expression of sericin-1, a major silk protein gene, in the silk gland. Here, we investigated whether Antp can regulate expression of multiple genes in this tissue. By means of proteomic, RT-PCR, and in situ hybridization analyses, we demonstrate that misexpression of Antp in the posterior silk gland induced ectopic expression of major silk protein genes such as sericin-3, fhxh4, and fhxh5. These genes are normally expressed specifically in the middle silk gland as is Antp. Therefore, the evidence strongly suggests that Antp activates these silk protein genes in the middle silk gland. The putative sericin-1 activator complex (middle silk gland-intermolt-specific complex) can bind to the upstream regions of these genes, suggesting that Antp directly activates their expression. We also found that the pattern of gene expression was well conserved between B. mori and the wild species Bombyx mandarina, indicating that the gene regulation mechanism identified here is an evolutionarily conserved mechanism and not an artifact of the domestication of B. mori. We suggest that Hox genes have a role as a master control in terminally differentiated tissues, possibly acting as a primary regulator for a range of physiological processes.

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.