| 日野 真人（ひのまさと） | データ更新日：2024.04.09 |
助教 ／
農学研究院
生命機能科学部門
| 1. | Kohei Kakino, Hiroaki Mon, Takeru Ebihara, Masato Hino, Akitsu Masuda, Jae Man Lee, Takahiro Kusakabe, Comprehensive Transcriptome Analysis in the Testis of the Silkworm, Bombyx mori, Insects, 10.3390/insects14080684, 14, 8, 684-684, 2023.08, Spermatogenesis is an important process in reproduction and is conserved across species, but in Bombyx mori, it shows peculiarities, such as the maintenance of spermatogonia by apical cells and fertilization by dimorphic spermatozoa. In this study, we attempted to characterize the genes expressed in the testis of B. mori, focusing on aspects of expression patterns and gene function by transcriptome comparisons between different tissues, internal testis regions, and Drosophila melanogaster. The transcriptome analysis of 12 tissues of B. mori, including those of testis, revealed the widespread gene expression of 20,962 genes and 1705 testis-specific genes. A comparative analysis of the stem region (SR) and differentiated regions (DR) of the testis revealed 4554 and 3980 specific-enriched genes, respectively. In addition, comparisons with D. melanogaster testis transcriptome revealed homologs of 1204 SR and 389 DR specific-enriched genes that were similarly expressed in equivalent regions of Drosophila testis. Moreover, gene ontology (GO) enrichment analysis was performed for SR-specific enriched genes and DR-specific enriched genes, and the GO terms of several biological processes were enriched, confirming previous findings. This study advances our understanding of spermatogenesis in B. mori and provides an important basis for future research, filling a knowledge gap between fly and mammalian studies.. |
| 2. | Akitsu Masuda, Jae Man Lee, Takeshi Miyata, Shintaro Sato, Atsushi Masuda, Masahiro Taniguchi, Ryosuke Fujita, Hiroshi Ushijima, Keisuke Morimoto, Takeru Ebihara, Masato Hino, Kohei Kakino, Hiroaki Mon, Takahiro Kusakabe, High yield production of norovirus GII.4 virus-like particles using silkworm pupae and evaluation of their protective immunogenicity., Vaccine, 10.1016/j.vaccine.2022.12.015, 41, 3, 766-777, 2023.01, Noroviruses (NoVs) are one of the major causes of acute viral gastroenteritis in humans. Virus-like particles (VLPs) without genomes that mimic the capsid structure of viruses are promising vaccine candidates for the prevention of NoVs infection. To produce large amounts of recombinant protein, including VLPs, the silkworm-expression vector system (silkworm-BEVS) is an efficient and powerful tool. In this study, we constructed a recombinant baculovirus that expresses VP1 protein, the major structural protein of NoV GII.4. Expression analysis showed that the baculovirus-infected silkworm pupae expressed NoV VP1 protein more efficiently than silkworm larval fat bodies. We obtained about 4.9 mg of purified NoV VP1 protein from only five silkworm pupae. The purified VP1 protein was confirmed by dynamic light scattering and electron microscopy to form VLPs of approximately 40 nm in diameter. Antisera from mice immunized with the antigen blocked NoV VLPs binding to histo-blood group antigens of pig gastric mucin and also blocked NoV infection in intestinal epithelial cells derived from human induced pluripotent stem (iPS) cells. Our findings demonstrated that NoV VLP eliciting protective antibodies could be obtained in milligram quantities from a few silkworm pupae using the silkworm-BEVS.. |
| 3. | Masato Hino, Tsuneyuki Tatsuke, Akihiro Morio, Hiroaki Mon, Jae Man Lee, Akitsu Masuda, Kohei Kakino, Yoshino Tonooka, Takahiro Kusakabe, Characterization of a Novel Heterochromatin Protein 1 Homolog “HP1c” in the Silkworm, Bombyx mori, Insects, 10.3390/insects13070631, 13, 7, 631-631, 2022.07, Heterochromatin protein 1 plays an important role in chromatin structure and gene expression regulation. Three HP1 genes have been found in Homo sapiens, and five HP1 genes have been reported in Drosophila melanogaster. On the other hand, in Bombyx mori, only two HP1 genes, BmHP1a and BmHP1b, were reported. In this research, we have reported the molecular and functional characterization of a novel Bombyx mori HP1 gene (BmHP1c), which had stronger transcriptional repression activity than BmHP1a. BmHP1a and BmHP1b is reported to form homo- and heterodimers, but in co-immunoprecipitation experiments, no homo- or hetero-dimer formation of BmHP1c with the other silkworm HP1s is detected. The intracellular localization of BmHP1c is not only in the nucleus but also in the cytoplasm like mammalian HP1γ. In contrast to human HP1a and b, all three BmHP1s were localized preferentially in the regions poorly stained with DAPI. Interestingly, the double knockdown of BmHP1a and b, but not BmHP1c with a or b, arrested the cell cycle at the G2/M phase. These results suggest that BmHP1c is not essential for cell progression and plays a different role than BmHP1a and BmHP1b.. |
| 4. | Miyu Tanaka, Tsuguru Fujii, Hiroaki Mon, Jae Man Lee, Kohei Kakino, Hisayoshi Fukumori, Takeru Ebihara, Takumi Nagasato, Masato Hino, Yoshino Tonooka, Takato Moriyama, Ryosuke Fujita, Yutaka Banno, Takahiro Kusakabe, Silkworm FoxL21 plays important roles as a regulator of ovarian development in both oogenesis and ovariole development, Insect Biochemistry and Molecular Biology, 10.1016/j.ibmb.2022.103737, 143, 103737-103737, 2022.04. |
| 5. | Akitsu Masuda, Jae Man Lee, Takeshi Miyata, Hiroaki Mon, Keita Sato, Kosuke Oyama, Yasuteru Sakurai, Jiro Yasuda, Daisuke Takahashi, Tadashi Ueda, Yuri Kato, Motohiro Nishida, Noriko Karasaki, Kohei Kakino, Takeru Ebihara, Takumi Nagasato, Masato Hino, Ayaka Nakashima, Kengo Suzuki, Yoshino Tonooka, Miyu Tanaka, Takato Moriyama, Hirokazu Nakatake, Ryosuke Fujita, Takahiro Kusakabe, Optimization of SARS-CoV-2 Spike Protein Expression in the Silkworm and Induction of Efficient Protective Immunity by Inoculation With Alum Adjuvants, Frontiers in Immunology, 10.3389/fimmu.2021.803647, 12, 2022.01, The newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing a spread of coronavirus disease 2019 (COVID-19) globally. In order to end the COVID-19 pandemic, an effective vaccine against SARS-CoV-2 must be produced at low cost and disseminated worldwide. The spike (S) protein of coronaviruses plays a pivotal role in the infection to host cells. Therefore, targeting the S protein is one of the most rational approaches in developing vaccines and therapeutic agents. In this study, we optimized the expression of secreted trimerized S protein of SARS-CoV-2 using a silkworm-baculovirus expression vector system and evaluated its immunogenicity in mice. The results showed that the S protein forming the trimeric structure was the most stable when the chicken cartilage matrix protein was used as the trimeric motif and could be purified in large amounts from the serum of silkworm larvae. The purified S protein efficiently induced antigen-specific antibodies in mouse serum without adjuvant, but its ability to induce neutralizing antibodies was low. After examining several adjuvants, the use of Alum adjuvant was the most effective in inducing strong neutralizing antibody induction. We also examined the adjuvant effect of paramylon from |
| 6. | Akitsu Masuda, Jae Man Lee, Takeshi Miyata, Takeru Ebihara, Kohei Kakino, Masato Hino, Ryosuke Fujita, Hiroaki Mon, Takahiro Kusakabe, Stable trimer formation of spike protein from porcine epidemic diarrhea virus improves the efficiency of secretory production in silkworms and induces neutralizing antibodies in mice, Veterinary Research, 10.1186/s13567-021-00971-5, 52, 1, 2021.12, |
| 7. | Takeru Ebihara, Akitsu Masuda, Daisuke Takahashi, Masato Hino, Hiroaki Mon, Kohei Kakino, Tsuguru Fujii, Ryosuke Fujita, Tadashi Ueda, Jae Man Lee, Takahiro Kusakabe, Production of scFv, Fab, and IgG of CR3022 Antibodies Against SARS-CoV-2 Using Silkworm-Baculovirus Expression System, Molecular Biotechnology, 10.1007/s12033-021-00373-0, 2021.07. |
| 8. | Tsuguru Fujii, Kohei Kakino, Hisayoshi Fukumori, Masato Hino, Jae Man Lee, Takahiro Kusakabe, Yutaka Banno, Non-molting dwarf (nm-d) as a mutant of Bombyx mori with a defect in purine synthesis, Insect Biochemistry and Molecular Biology, 10.1016/j.ibmb.2021.103636, 138, 103636-103636, 2021.11. |
| 9. | Ryosuke Fujita, Masato Hino, Takeru Ebihara, Takumi Nagasato, Akitsu Masuda, Jae Man Lee, Tsuguru Fujii, Hiroaki Mon, Kohei Kakino, Ryo Nagai, Miyu Tanaka, Yoshino Tonooka, Takato Moriyama, Takahiro Kusakabe, Efficient production of recombinant SARS-CoV-2 spike protein using the baculovirus-silkworm system, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2020.06.020, 529, 2, 257-262, 2020.08. |
| 10. | Kohei Kakino, Akitsu Masuda, Masato Hino, Takeru Ebihara, Jian Xu, Hiroaki Mon, Ryosuke Fujita, Tsuguru Fujii, Takahiro Kusakabe, Jae Man Lee, Efficient production of recombinant T7 endonuclease I using silkworm-baculovirus expression vector system, Journal of Asia-Pacific Entomology, 23, 3, 694-700, 2020.08. |
| 11. | Masahiko Kobayashi, Jian Xu, Kohei Kakino, Akitsu Masuda, Masato Hino, Naoki Fujimoto, Kosuke Minamihata, Noriho Kamiya, Hiroaki Mon, Hiroshi Iida, Masateru Takahashi, Takahiro Kusakabe, Jae Man Lee, Optimal silkworm larva host for high-level production of Mus musculus IL-4 using a baculovirus expression vector system, Journal of Asia-Pacific Entomology, 10.1016/j.aspen.2019.12.014, 23, 1, 268-273, 2020.04, [URL], Interleukine-4 (IL-4) is a cytokine that plays an important role in the immune system and recognized as a biological medicine. Therefore, there is a demand for the production of IL-4 with high performance. The expression of a recombinant IL-4 protein in the prokaryotic system usually results in the formation of an inclusion body. To date, the solution to obtain those active products without the refolding process remains to be established. In this study, we tried to acquire a biologically active recombinant Mus musculus IL-4 (rMmIL-4) using a silkworm-baculovirus expression vector system (silkworm-BEVS). We constructed two recombinant baculoviruses coding rMmIL-4 with the distinct location of affinity purification tags and succeeded in the expression and purification of rMmIL-4 proteins directly without the refolding process. Both purified proteins displayed comparable biological activity to the commercial proteins produced by the E. coli expression system. Besides, we performed screening of silkworm strains to seek optimal hosts for the mass-production of rMmIL-4. Intriguingly, we found that some silkworm strains showed significantly higher secretion levels of rMmIL-4 in silkworm sera. Our study provides meaningful insights into the industrial-scale production of rMmIL-4 with high productivity for pharmaceutical applications in the future.. |
| 12. | Masato Hino, Noriko Karasaki, Tsuneyuki Tatsuke, Daisuke Morokuma, Akitsu Masuda, Hiroaki Mon, Jae Man Lee, Takahiro Kusakabe, Mild inactivation and inhibition of phenoloxidase in silkworm serum for xeno-free culture of insect cells, Journal of Insect Biotechnology and Sericology, 10.11416/jibs.89.1_009, 89, 1, 9-16, 2020.01, [URL], In insect cell culture, a fetal bovine serum is often used to maintain the cellular physiological conditions, irre-spective of its relatively high cost and unstable supply. Several serum-free media for insects were developed, but it is challenging to adopt some insect cells to these serum-free media. Silkworm serum was used as a re-placement of fetal bovine serum for a long time but not used widely due to the melanization of insect serum catalyzed by phenoloxidase (PO). PO inhibitors reported for insect serum preparation have a significantly nega-tive effect on the long-term culture of healthy insect cells. In this study, we evaluated several kinds of PO inhibi-tors and found the method to prepare silkworm serum, which is suitable for the maintenance of insect cells and the production of the recombinant proteins by the baculovirus expression system. When L-Glutathione (reduced form) or L-Cysteine is used as a PO inhibitor at the time of recovery of silkworm serum, it is possible to sup-press the browning of the silkworm serum. However, in this state of silkworm serum, browning is observed in long-term cell culture use. Therefore, it is preferable for cell culture to add 10% of silkworm serum treated at 50°C for 30 minutes to the medium with 2.7 mM L-Glutathione. Our results suggest that silkworm serum can be an alternative of mammalian serum for cell culture and used for producing the proteins for clinical application without any risk of contamination of zoonotic infectious disease viruses.. |
| 13. | Jian Xu, Jae Man Lee, Tuneyuki Tatsuke, Takeru Ebihara, Akitsu Masuda, Masato Hino, Daisuke Morokuma, Ryosuke Fujita, Hiroaki Mon, Takahiro Kusakabe, Masateru Takahashi, Expression and Purification of Vaccinia Virus DNA Topoisomerase IB Produced in the Silkworm–Baculovirus Expression System, Molecular Biotechnology, 10.1007/s12033-019-00184-4, 61, 8, 622-630, 2019.08, [URL], Type IB DNA topoisomerases are enzymes to change the topological state of DNA molecules and are essential in studying replication, transcription, and recombination of nucleic acids in vitro. DNA topoisomerase IB from Vaccinia virus (vTopIB) is a 32 kDa, type I eukaryotic topoisomerase, which relaxed positively and negatively supercoiled DNAs without Mg2+ and ATP. Although vTopIB has been effectively produced in E. coli expression system, no studies remain available to explore an alternative platform to express recombinant vTopIB (rvTopIB) in a higher eukaryote, where the one can expect post-translational modifications that affect the activity of rvTopIB. Here in this study, rvTopIB with N-terminal tags was constructed and expressed in a silkworm–baculovirus expression vector system (silkworm–BEVS). We developed a simple two consecutive chromatography purification to obtain highly pure rvTopIB. The final yield of rvTopIB obtained from a baculovirus-infected silkworm larva was 83.25 μg. We also evaluated the activity and function of rvTopIB by the DNA relaxation activity assays using a negatively supercoiled pUC19 plasmid DNA as a substrate. With carefully assessing optimized conditions for the reaction buffer, we found that divalent ions, Mg2+, Mn2+, Ca2+, as well as ATP stimulate the DNA relaxation activity by rvTopIB. The functional and active form of rvTopIB, together with the yields of the protein we obtained, suggests that silkworm–BEVS would be a potential alternative platform to produce eukaryotic topoisomerases on an industrial scale.. |
| 14. | Takumi Yano, Jae Man Lee, Jian Xu, Yoshiki Morifuji, Akitsu Masuda, Masato Hino, Daisuke Morokuma, Ryosuke Fujita, Masateru Takahashi, Takahiro Kusakabe, Hiroaki Mon, Expression of the thermostable Moloney murine leukemia virus reverse transcriptase by silkworm-baculovirus expression system, Journal of Asia-Pacific Entomology, 10.1016/j.aspen.2019.02.008, 22, 2, 453-457, 2019.06, [URL], Reverse transcriptase from Moloney murine leukemia virus (MMLVRT) is an RNA dependent DNA polymerase, which has been used as a fundamental tool for molecular biology and biotechnology. The secondary structures formed in the RNA templates decrease the accessibility of the reverse transcriptase to the RNA templates; it is important to unfold the RNA secondary structure by increasing the reaction temperature to perform the successful transcription. In this study, we applied silkworm baculovirus expression vector system (silkworm-BEVS) to mass-produce and purify the recombinant MMLVRTs with the N- or C- terminal tandem tags. We confirmed that both of the recombinant MMLVRT enzymes have intact DNA polymerase activity. It is notable that C- terminal tagged MMLVRT outperformed MMLVRT obtained from the E. coli expression system in terms of thermostability and sensitivity to low quantities of RNA template. Taken together, these results demonstrate that silkworm-BEVS is a promising alternative strategy to produce the functional and thermostable reverse transcriptase.. |
| 15. | Akihiro Morio, Jian Xu, Akitsu Masuda, Yurie Kinoshita, Masato Hino, Daisuke Morokuma, Hatsumi M. Goda, Nozomu Okino, Makoto Ito, Hiroaki Mon, Ryosuke Fujita, Takahiro Kusakabe, Jae Man Lee, Expression, purification, and characterization of highly active endo-α-N-acetylgalactosaminidases expressed by silkworm-baculovirus expression system, Journal of Asia-Pacific Entomology, 10.1016/j.aspen.2019.01.009, 22, 2, 404-408, 2019.06, [URL], The O-glycosidase, endo-α-N-acetylgalactosaminidase from Enterococcus faecalis (endoEF) catalyzes the cleavage of core 1 and core 3 type O-linked disaccharides between GalNAc and serine or threonine residues from glycoproteins. The endoEF has broad substrate specificity and thus is extensively utilized for the structural and functional analysis of the O-linked glycans. In this study, we expressed and purified the recombinant endoEF (rEndoEF) by using the silkworm-baculovirus expression vector system (Silkworm-BEVS) and confirmed the deglycosylation activity of rEndoEF targeting reporter glycoproteins, which was equivalent to the commercial O-glycosidase. Thus, our study provides important clues to produce highly active rEndoEF O-glycosidases employing silkworm-BEVS as an alternative.. |
| 16. | Jian Xu, Akihiro Morio, Daisuke Morokuma, Yudai Nagata, Masato Hino, Akitsu Masuda, Zhiqing Li, Hiroaki Mon, Takahiro Kusakabe, Jae Man Lee, A functional polypeptide N-acetylgalactosaminyltransferase (PGANT) initiates O-glycosylation in cultured silkworm BmN4 cells, Applied Microbiology and Biotechnology, 10.1007/s00253-018-9309-6, 102, 20, 8783-8797, 2018.10, [URL], Mucin-type O-glycosylation is initiated by UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts or PGANTs), attaching GalNAc to serine or threonine residue of a protein substrate. In the insect model from Lepidoptera, silkworm (Bombyx mori), however, O-glycosylation pathway is totally unexplored and remains largely unknown. In this study, as the first report regarding protein O-glycosylation analysis in silkworms, we verified the O-glycan profile that a common core 1 Gal (β1-3) GalNAc disaccharide branch without terminally sialylated structure is mainly formed for a baculovirus-produced human proteoglycan 4 (PRG4) protein. Intriguingly, functional screenings in cultured silkworm BmN4 cells for nine Bmpgants reveal that Bmpgant2 is the solo functional BmPGANT for PRG4, implying that Bmpgants may have unique cell/tissue or protein substrate preferences. Furthermore, a recombinant BmPGANT2 protein was successfully purified from silkworm-BEVS and exhibited a high ability to transfer GalNAc for both peptide and protein substrates. Taken together, the present results clarified the functional BmPGANT2 in cultured silkworm cells, providing crucial fundamental insights for future studies dissecting the detailed silkworm O-glycosylation pathways and productions of glycoproteins with O-glycans.. |
| 17. | Akitsu Masuda, Jian Xu, Kosuke Minamihata, Genki Kagawa, Yusei Hamada, Yoshiki Morifuji, Takumi Yano, Masato Hino, Daisuke Morokuma, Noriko Karasaki, Hiroaki Mon, Noriho Kamiya, Takahiro Kusakabe, Jae Man Lee, Production of a biologically active human basic fibroblast growth factor using silkworm-baculovirus expression vector system, Journal of Asia-Pacific Entomology, 10.1016/j.aspen.2018.05.002, 21, 2, 716-720, 2018.06, [URL], As a therapeutic treatment, recombinant human basic fibroblast growth factor (rhbFGF) is usually employed in tissue regeneration, and as an essential component in culture medium for maintaining the induced pluripotent stem (iPS) cell and embryonic stem (ES) cell in an undifferentiated state. Therefore, a large amount of biologically active rhbFGF is required. In this study, silkworm-baculovirus expression vector system (silkworm-BEVS) is employed to achieve a high productivity of recombinant rhbFGF with two small affinity tags (His-tag and STREP-tag) at the N or C-terminus. It is observed that rhbFGF with 30 K signal peptide of silkworm were successfully expressed but are not sufficiently secreted into the culture medium of cultured insect cells. Then we purified the N- or C-tagged intracellular rhbFGF protein and obtained a yield of about 0.7 mg/larva and 1.2 mg/larva, respectively. Although the final yield of the C-tagged rhbFGF is higher than that of the N-tagged, rhbFGF with N-tag demonstrated promising and comparable biological activity, which is evaluated through a mammalian cell proliferation assay. Taken together, these results indicate that silkworm-BEVS could contribute to the mass-production of the biologically active rhbFGF for medical uses.. |
| 18. | Yoshiki Morifuji, Jian Xu, Noriko Karasaki, Kazuhiro Iiyama, Daisuke Morokuma, Masato Hino, Akitsu Masuda, Takumi Yano, Hiroaki Mon, Takahiro Kusakabe, Jae Man Lee, Expression, Purification, and Characterization of Recombinant Human α1-Antitrypsin Produced Using Silkworm–Baculovirus Expression System, Molecular Biotechnology, 10.1007/s12033-018-0127-y, 2018.01, [URL], Human α1-antitrypsin (AAT) is the most abundant serine proteinase inhibitor (serpin) in the human plasma. Commercially available AAT for the medications of deficiency of α1-antitrypsin is mainly purified from human plasma. There is a high demand for a stable and low-cost supply of recombinant AAT (rAAT). In this study, the baculovirus expression vector system using silkworm larvae as host was employed and a large amount of highly active AAT was recovered from the silkworm serum (~ 15 mg/10 ml) with high purity. Both the enzymatic activity and stability of purified rAAT were comparable with those of commercial product. Our results provide an alternative method for mass production of the active rAAT in pharmaceutical use.. |
| 19. | Akitsu Masuda, Kosuke Minamihata, Masato Hino, Daisuke Morokuma, Noriko Karasaki, Hiroaki Mon, Noriho Kamiya, Takahiro Kusakabe, Jae Man Lee, Modular surface display for porcine circovirus type 2 virus-like particles mediated by microbial transglutaminase, Journal of Insect Biotechnology and Sericology, 10.11416/jibs.87.2_053, 87, 2, 53-60, 2018.01, [URL], Virus-like particles (VLPs) are multi-protein complex, which mimic viral particles without viral genomes. Recently various applications of VLPs for medical and pharmaceutical fields are developed. Furthermore, surface modification of VLPs is expected to extend their functional versatility. As an enzymatic strategy for the modification, microbial transglutaminase (MTG) that catalyzes the covalent bond between a glutamine residue and a ly-sine residue or a primary amine is suitable for efficient protein ligation. In the previous study, we demonstrated the efficient production of immunogenic VLPs of porcine circovirus type 2 (PCV2) using silkworm-baculovirus expression vector system (silkworm-BEVS). Herein, we established the display system of exotic molecules to VLPs by enzymatic covalent cross-linking using MTG. For the target modification, Q-tag (YPLQMRG) that is MTG reactive sequence were introduced into the N-terminus, C-terminus, or loop BC of capsid protein of PCV2 and successfully expressed as VLPs in silkworm pupae. Of these, PCV2 VLPs with Q-tag at the loop BC and C-terminus were efficiently conjugated with FITC-cadaverine and K-tagged (MRHKGS) EGFP by MTG. These results showed that flexible surface modification of VLPs mediated by MTG could be used for development of several therapeutic tools such as multivalent vaccines.. |
| 20. | Akitsu Masuda, Jae Man Lee, Takeshi Miyata, Tetsuo Sato, Shizuka Hayashi, Masato Hino, Daisuke Morokuma, Noriko Karasaki, Hiroaki Mon, Takahiro Kusakabe, Purification and characterization of immunogenic recombinant virus-like particles of porcine circovirus type 2 expressed in silkworm pupae, Journal of General Virology, 10.1099/jgv.0.001087, 99, 7, 917-926, 2018.01, [URL], Porcine circovirus type 2 (PCV2) is a primary causative agent of postweaningmultisystemic wasting syndrome (PMWS), which has a significant economic impact on the swine industry. The capsid protein (Cap) encoded by ORF2 of the viral genome has been used effectively as a vaccine against PCV2 infection. The Cap protein can spontaneously assemble into virus-like particles (VLPs) that are safe and highly immunogenic for vaccine applications. Several expression systems, including bacteria, yeast and insect cells, have been utilized to produce PCV2 VLPs. However, in some cases, the recombinant Cap (rCap) proteins produced in bacteria and yeast do not assemble spontaneously. In this study, we expressed rCap protein using a silkworm–baculovirus expression vector system (silkworm–BEVS) for mass production of PCV2 VLPs and established a simple three-step protocol for its purification from pupae: extraction by detergent, ammonium sulfate precipitation and anion exchange column chromatography. Size-exclusion chromatography (SEC) analysis and transmission electron microscope (TEM) observation showed that purified rCap proteins formed VLPs with a similar morphology to that of the original virus. Furthermore, the VLPs produced in silkworms were capable of inducing neutralizing antibodies against PCV2 in mice. Our results demonstrated that the silkworm system is a powerful tool for the production of PCV2 VLPs and will be useful for the development of a reliable and cost-effective PCV2 vaccine.. |
| 21. | Anandrao Ashok Patil, Tsuneyuki Tatsuke, Hiroaki Mon, Jae Man Lee, Daisuke Morokuma, Masato Hino, Takahiro Kusakabe, Molecular characterization of mitochondrial Zucchini and its relation to nuage-piRNA pathway components in Bombyx mori ovary-derived BmN4 cells, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2017.09.107, 493, 2, 971-978, 2017.11, [URL], Piwi-interacting RNAs (piRNAs) are a class of small non-coding RNAs that associate with PIWI subfamily proteins, which play an important role in transposon silencing in animal germ cell. The piRNAs biogenesis is divided into two major pathways: primary and secondary, and both pathways are independent of double-stranded RNA-processing enzyme Dicer, which processes the single-stranded RNA transcripts in microRNA (miRNA) and siRNA (small interfering RNA) pathway. Primary piRNAs are processed from long non-coding RNA precursors transcribed from piRNA clusters. Zucchini (Zuc), a mitochondrial phospholipase D (PLD) superfamily protein is conserved among the animals and involved in piRNA biogenesis. Recent studies showed that the Zucchini is an endoribonuclease essential for primary piRNA maturation and production of phased piRNA in secondary piRNA biogenesis of drosophila germ cell. Based on these reports, here we identified and studied the silkworm Zucchini (BmZuc) at subcellular level in ovary-derived BmN4 cell. The silkworm Zuc specifically expressed in germ-related tissues and localized on mitochondria and partially co-localized with perinuclear nuage-piRNA pathway components and nuage marker protein BmVasa. Molecular dissection analyses revealed that the conserved mitochondrial localization sequence, RGV motif, PLDc 2 domain and HKD motif are important for the BmZuc mitochondrial localization. Moreover, the knockdown analyses showed that the piRNA pathway components are independent on BmZuc for their nuage localization, whereas BmZuc depend on piRNA pathway components for the proper localization. Our data provides vital information on mitochondrial BmZuc and its relationship to “nuage” in ovary-derived BmN4 cell.. |
| 22. | Ming Ming Ji, Jae Man Lee, Hiroaki Mon, Kazuhiro Iiyama, Tsuneyuki Tatsuke, Daisuke Morokuma, Masato Hino, Mami Yamashita, Kazuma Hirata, Takahiro Kusakabe, Lipidation of BmAtg8 is required for autophagic degradation of p62 bodies containing ubiquitinated proteins in the silkworm, Bombyx mori, Insect Biochemistry and Molecular Biology, 10.1016/j.ibmb.2017.08.006, 89, 86-96, 2017.10, [URL], p62/Sequestosome-1 (p62/SQSTM1, hereafter referred to as p62) is a major adaptor that allows ubiquitinated proteins to be degraded by autophagy, and Atg8 homologs are required for p62-mediated autophagic degradation, but their relationship is still not understood in Lepidopteran insects. Here it is clearly demonstrated that the silkworm homolog of mammalian p62, Bombyx mori p62 (Bmp62), forms p62 bodies depending on its Phox and Bem1p (PB1) and ubiquitin-associated (UBA) domains. These two domains are associated with Bmp62 binding to ubiquitinated proteins to form the p62 bodies, and the UBA domain is essential for the binding, but Bmp62 still self-associates without the PB1 or UBA domain. The p62 bodies in Bombyx cells are enclosed by BmAtg9-containing membranes and degraded via autophagy. It is revealed that the interaction between the Bmp62 AIM motif and BmAtg8 is critical for the autophagic degradation of the p62 bodies. Intriguingly, we further demonstrate that lipidation of BmAtg8 is required for the Bmp62-mediated complete degradation of p62 bodies by autophagy. Our results should be useful in future studies of the autophagic mechanism in Lepidopteran insects.. |
| 23. | Anandrao Ashok Patil, Tsuneyuki Tatsuke, Hiroaki Mon, Jae Man Lee, Daisuke Morokuma, Masato Hino, Takahiro Kusakabe, Characterization of Armitage and Yb containing granules and their relationship to nuage in ovary-derived cultured silkworm cell, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2017.06.008, 490, 2, 134-140, 2017.08, [URL], PIWI-interacting RNAs (piRNAs) are a class of endogenous small non-coding RNAs, which are mostly 24–32 nucleotides in length and interact specifically with PIWI subfamily of argonaute proteins. Despite the significant research progress in germ line piRNA pathway, its role in somatic cell is not well known. In Drosophila ovarian somatic cell, maturation of primary piRNA and its loading onto Piwi occurs at perinuclear Yb body. The Armitage (Armi) and Yb proteins are the major components of Yb body and specially expressed in ovarian somatic cell. Based on the reports, here we studied the BmArmi and BmYb in Bombyx mori ovary-derived BmN4 cells expressing BmVasa. In this study, we show that BmArmi and BmYb co-localized with BmVasa at nuage. The helicase domains of BmArmi and BmYb are important for nuage localization. Moreover, RNAi of piRNA components reveal that BmArmi depend on BmAgo3 for nuage localization, and BmArmi and BmYb form cytoplasmic granules independently in the absence of BmVasa. Our results provide evidence that the BmArmi and BmYb coexist with BmVasa and play an important role in perinuclear nuage granules formation in ovary-derived BmN4 cell.. |
| 24. | Mami Yamashita, Jian Xu, Daisuke Morokuma, Kazuma Hirata, Masato Hino, Hiroaki Mon, Masateru Takahashi, Samir M. Hamdan, Kosuke Sakashita, Kazuhiro Iiyama, Yutaka Banno, Takahiro Kusakabe, Jae Man Lee, Characterization of Recombinant Thermococcus kodakaraensis (KOD) DNA Polymerases Produced Using Silkworm-Baculovirus Expression Vector System, Molecular Biotechnology, 10.1007/s12033-017-0008-9, 59, 6, 221-233, 2017.06, [URL], The KOD DNA polymerase from Thermococcus kodakarensis (Tkod-Pol) has been preferred for PCR due to its rapid elongation rate, extreme thermostability and outstanding fidelity. Here in this study, we utilized silkworm-baculovirus expression vector system (silkworm-BEVS) to express the recombinant Tkod-Pol (rKOD) with N-terminal (rKOD-N) or C-terminal (rKOD-C) tandem fusion tags. By using BEVS, we produced functional rKODs with satisfactory yields, about 1.1 mg/larva for rKOD-N and 0.25 mg/larva for rKOD-C, respectively. Interestingly, we found that rKOD-C shows higher thermostability at 95 °C than that of rKOD-N, while that rKOD-N is significantly unstable after exposing to long period of heat-shock. We also assessed the polymerase activity as well as the fidelity of purified rKODs under various conditions. Compared with commercially available rKOD, which is expressed in E. coli expression system, rKOD-C exhibited almost the same PCR performance as the commercial rKOD did, while rKOD-N did lower performance. Taken together, our results suggested that silkworm-BEVS can be used to express and purify efficient rKOD in a commercial way.. |
| 25. | Daisuke Morokuma, Jian Xu, Masato Hino, Hiroaki Mon, Jasmeen S. Merzaban, Masateru Takahashi, Takahiro Kusakabe, Jae Man Lee, Expression and Characterization of Human β-1, 4-Galactosyltransferase 1 (β4GalT1) Using Silkworm–Baculovirus Expression System, Molecular Biotechnology, 10.1007/s12033-017-0003-1, 59, 4-5, 151-158, 2017.05, [URL], Baculovirus expression vector system (BEVS) is widely known as a mass-production tool to produce functional recombinant glycoproteins except that it may not be always suitable for medical practice due to the differences in the structure of N-linked glycans between insects and mammalian. Currently, various approaches have been reported to alter N-linked glycan structures of glycoproteins derived from insects into terminally sialylated complex-type N-glycans. In the light of those studies, we also proposed in vitro maturation of N-glycan with mass-produced and purified glycosyltransferases by silkworm–BEVS. β-1,4-Galactosyltransferase 1 (β4GalT1) is known as one of type II transmembrane enzymes that transfer galactose in a β-1, 4 linkage to accepter sugars, and a key enzyme for further sialylation of N-glycans. In this study, we developed a large-scale production of recombinant human β4GalT1 (rhβ4GalT1) with N- or C-terminal tags in silkworm–BEVS. We demonstrated that rhβ4GalT1 is N-glycosylated and without mucin-type glycosylation. Interestingly, we found that purified rhβ4GalT1 from silkworm serum presented higher galactosyltransferase activity than that expressed from cultured mammalian cells. We also validated the UDP-galactose transferase activity of produced rhβ4GalT1 proteins by using protein subtracts from silkworm silk gland. Taken together, rhβ4GalT1 from silkworms can become a valuable tool for producing high-quality recombinant glycoproteins with mammalian-like N-glycans.. |
| 26. | Jianping Chen, Jian Xu, Masato Hino, Mami Yamashita, Kazuma Hirata, Anandrao Ashok Patil, Tsuneyuki Tatsuke, Hiroaki Mon, Yutaka Banno, Takahiro Kusakabe, Jae Man Lee, Co-expression of silkworm allatostatin-C receptor BNGR-A1 with its cognate G protein subunits enhances the GPCR display on the budding baculovirus, Journal of Asia-Pacific Entomology, 10.1016/j.aspen.2016.07.007, 19, 3, 753-760, 2016.09, [URL], G protein-coupled receptors (GPCRs) are seven transmembrane proteins, which play an essential role in transmitting various extracellular signals into cells. For functional and structural analysis of GPCRs, it is necessary to produce active GPCRs in high quantities with outstanding purity. Fortunately, earlier baculovirus expression vector system has been reported as a proven functional GPCR mass-production tool. Therefore, in this study, we selected Bombyx mori allatostatin-C neuropeptide receptor BNGR-A1 as a GPCR reporter protein, which has been already proved successful binding of ligand. We confirmed its expression profile in various silkworm tissues and cell lines and then verified its plasma membrane subcellular localization in cultured silkworm BmN4 cells. In addition, we constructed recombinant baculoviruses for BNGR-A1, its ligand allatostatin-C (BmAST-C) and related eight G proteins (Gs, G12, Gα4, Gq, Gβ2, Gβ3, Gβ5 and Gγ) and subsequently monitored the extracellular or intracellular expression of BNGR-A1 by co-infection with its ligand and cognate G proteins-expressing viruses. It is interesting to observe that different combinations of G proteins could result in changes or even undetectable of final yields of BNGR-A1, suggesting the essential roles of G proteins involved in the GPCR expression or stabilization. The present study demonstrated that co-infection of recombinant viruses expressing Gα4β3γ trimer enhanced the production of BNGR-A1 on BV fractions. To our knowledge, this is a fine strategy for identifying the specific G protein partner responsible for certain GPCR of interest. These studies would provide a novel idea for improving GPCR expression in the silkworm by baculovirus expression vector system.. |
| 27. | Masato Hino, Takuji Kawanami, Jian Xu, Daisuke Morokuma, Kazuma Hirata, Mami Yamashita, Noriko Karasaki, Tuneyuki Tatsuke, Hiroaki Mon, Kazuhiro Iiyama, Noriho Kamiya, Yutaka Banno, Takahiro Kusakabe, Jae Man Lee, High-level expression and purification of biologically active human IL-2 using silkworm-baculovirus expression vector system, Journal of Asia-Pacific Entomology, 10.1016/j.aspen.2016.03.014, 19, 2, 313-317, 2016.06, [URL], Interleukin 2 (IL-2) is a pharmacologically vital cytokine secreted mainly by activated CD4+ T lymphocyte. Recombinant human IL-2 (rhIL-2) protein has already been globally applied as an immune-therapeutic reagent for various diseases. Therefore, there is great interest in developing an active form of rhIL-2 in very large amounts and with excellent purity for clinical use. In this study, we successfully mass-expressed and purified N- or C-terminal tandem tag-fused rhIL-2 in a baculovirus expression vector system (BEVS) using silkworm larvae as factories. We confirmed that the intrinsic instability of hIL-2 causes the loss and low recovery of N-tagged rhIL-2 and that C-tagged rhIL-2 is more suitable for mass production. Furthermore, the activity of purified rhIL-2s was further validated by a cell proliferation assay of a human natural killer cell line, and both rhIL-2 proteins produced in the silkworm-BEVS exhibited comparable activity to that of the commercial E.coli-derived rhIL-2. Taken together, our results and strategies could contribute greatly to the mass production of active rhIL-2.. |
| 28. | Masato Hino, Daisuke Morokuma, Hiroaki Mon, Jae Man Lee, Takahiro Kusakabe, Characterization of the roles of DNA polymerases, clamp, and clamp loaders during s-phase progression and cell cycle regulation in the silkworm, bombyx mori, Journal of Insect Biotechnology and Sericology, 10.11416/jibs.85.2_021, 85, 2, 21-29, 2016.01, [URL], DNA replication is one of key event in cell-cycle progression, yet due to their importance and lethality, the chronological phenotypes of DNA synthesis machineries after the depletion of corresponding genes have proved difficult to study. In the present study, mRNAs for three DNA polymerases, a clamp, and three clamp loaders were gradually depleted from cultured silkworm cells by soaking RNAi. Interestingly, the depletion of these DNA synthesis factors had different effects on the cell growth rate and arrest of cell-cycle progression during time-lapse observation. The depletion of DNA polymerases immediately arrested the cell-cycle progression at the S phase, while that of PCNA, a DNA clamp, required more time to slow cell growth and finally induced apoptosis. Surprisingly, silkworm cells continued to undergo several rounds of cell division when the components of clamp loaders were knocked down.. |
| 29. | Daisuke Morokuma, Jian Xu, Hiroaki Mon, Kazuma Hirata, Masato Hino, Shoko Kuboe, Mami Yamashita, Takahiro Kusakabe, Jae Man Lee, Human alpha 1-acid glycoprotein as a model protein for glycoanalysis in baculovirus expression vector system, Journal of Asia-Pacific Entomology, 10.1016/j.aspen.2015.03.006, 18, 2, 303-309, 2015.06, [URL], Glycosylation is an important post-translational modification that confers various biological activities, structural stability, and inter-molecular interactions to proteins. Baculovirus expression vector system (BEVS) is widely used to produce recombinant glycoproteins, which may not be suitable for clinical use due to differences in the N-linked glycan structure between insects and mammals. It is necessary to develop an appropriate model protein-base platform for glycoanalysis to engineer the insect-type N-glycosylation pathway into human type efficiently. In this study, we employed human plasma protein alpha 1-acid glycoprotein (α1AGP). It was highly secreted from cultured silkworm cells and larvae when using the BEVS and glycosylated with insect type N-linked glycans. Interestingly, when separated on SDS-PAGE, the purified recombinant α1AGP secreted into silkworm haemolymph generated six distinct products from three alternative translates, suggesting that α1AGP has variations for the recognition or choice of glycosylation sites.. |
本データベースの内容を無断転載することを禁止します。