| 1. |
Morio A, Lee JM, Fujii T, Mon H, Masuda A, Kakino K, Xu J, Banno Y, Kusakabe T, The biological role of core 1β1-3galactosyltransferase (T-synthase) in mucin-type O-glycosylation in Silkworm, Bombyx mori, Insect Biochemistry and Molecular Biology, 10.1016/j.ibmb.2023.103936, 156, 103936-103936, 2023.05. |
| 2. |
Masuda A, Lee JM, Miyata T, Sato S, Masuda A, Taniguchi M, Fujita R, Ushijima H, Morimoto K, Ebihara T, Hino M, Kakino K, Mon H, Kusakabe T, 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. |
| 3. |
Kato Y, Nishiyama K, Lee JM, Ibuki Y, Imai Y, Noda T, Kamiya N, Kusakabe T, Kanda Y, Nishida M., TRPC3-Nox2 Protein Complex Formation Increases the Risk of SARS-CoV-2 Spike Protein-Induced Cardiomyocyte Dysfunction through ACE2 Upregulation., International Journal of Molecular Sciences, 10.3390/ijms24010102, 24, 1, 2022.12. |
| 4. |
Miyata T, Minamihata K, Kurihara K, Kamizuru Y, Gotanda M, Obayashi M, Kitagawa T, Sato K, Kimura M, Oyama K, Ikeda Y, Tamaki Y, Lee JM, Sakao K, Hamanaka D, Kusakabe T, Tachibana M, Ibrahim HR., Highly efficient protein expression of Plasmodium vivax surface antigen, Pvs25, by silkworm and its biochemical analysis, Protein Expression and Purification, 2022.06. |
| 5. |
Masuda A, LeeJM, Miyata T, Mon H, Sato K, Oyama K, Sakurai Y, Yasuda J, Takahashi D, Ueda T, Kato Y, Nishida M, Karasaki N, Kakino K, Ebihara T, Nagasato T, Hino M, Nakashima A, Suzuki K, Tonooka Y, Tanaka M, Moriyama T, Nakatake H, Fujita R, Kusakabe T., 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, 803647, 2022.01. |
| 6. |
Ohga H, Ito K, Kakino K, Mon H, Kusakabe T, Lee JM, Matsuyama M., Leptin Is an Important Endocrine Player That Directly Activates Gonadotropic Cells in Teleost Fish, Chub Mackerel, Cells, 10.3390/cells10123505, 10, 12, 3505, 2021.12. |
| 7. |
Ebihara T, Masuda A, Takahashi D, Hino M, Mon H, Kakino K, Fujii T, Fujita R, Ueda T, Lee JM, Kusakabe T., 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, 63, 12, 1223-1234, 2021.12. |
| 8. |
Masuda A, Lee JM, Miyata T, Ebihara T, Kakino K, Hino M, Fujita R, Mon H, Kusakabe T., 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, 1-14, 2021.07. |
| 9. |
Takeru Ebihara, Jian Xu, Yoshino Tonooka, Takumi Nagasato, Kohei Kakino, Akitsu Masuda, Kosuke Minamihata, Noriho Kamiya, Hirokazu Nakatake, Yuuka Chieda, Hiroaki Mon, Tsuguru Fujii, Takahiro Kusakabe, Jae Man Lee, Active Human and Murine Tumor Necrosis Factor α Cytokines Produced from Silkworm Baculovirus Expression System, Insects, https://doi.org/10.3390/insects12060517, 12, 6, 517, 2021.06. |
| 10. |
Ryo Nagai, Takeru Ebihara, Kohei Kakino, Akitsu Masuda, Jian Xu, Kosuke Minamihata, Noriho Kamiya, Tatphon Kongkrongtong, Masahiro Kawahara, Hiroaki Mon, Tsuguru Fujii, Takahiro Kusakabe, Jae Man Lee, Production of an active Mus musculus IL-3 using updated silkworm-based baculovirus expression vector system, Journal of Asia-Pacific Entomology, https://doi.org/10.1016/j.aspen.2021.04.011, 2021.04. |
| 11. |
Yuri Kato, Shigeru Yamada, Kazuhiro Nishiyama, Ayano Satsuka, Suyong Re, Daiki Tomokiyo, Jae Man Lee,Tomohiro Tanaka, Akiyuki Nishimura, Kenzo Yonemitsu, Hiroshi Asakura, Yuko Ibuki, Yumiko Imai, Noriho Kamiya, Kenji Mizuguchi, Takahiro Kusakabe, Yasunari Kanda, Motohiro Nishida, Clomipramine suppresses ACE2-mediated SARS-CoV-2 entry, bioRxiv, https://doi.org/10.1101/2021.03.13.435221, 2021.03. |
| 12. |
Masateru Takahashi, Muhammad Tehseen, Rahul Salunke, Etsuko Takahashi, Sara Mfarrej, Mohamed A Sobhy, Fatimah S Alhamlan, Sharif Hala, Gerardo Ramos-Mandujano, Ahmed A Al-Qahtani, Fadwa S Alofi, Afrah Alsomali, Anwar M Hashem, Asim Khogeer, Naif AM Almontashiri, Jae Man Lee, Hiroaki Mon, Kosuke Sakashita, Mo Li, Takahiro Kusakabe, Arnab Pain, Samir M Hamdan, Quick and Easy Assembly of a One-Step qRT-PCR Kit for COVID-19 Diagnostics Using In-House Enzymes, ACS omega, 10.1021/acsomega.0c05635, 6, 11, 7374-7386, 2021.03. |
| 13. |
Asma S Al-Amoodi, Kosuke Sakashita, Amal J Ali, Ruoyu Zhou, Jae Man Lee, Muhammad Tehseen, Mo Li, Juan Carlos I Belmonte, Takahiro Kusakabe, Jasmeen S Merzaban, Using Eukaryotic Expression Systems to Generate Human α1, 3-Fucosyltransferases That Effectively Create Selectin-Binding Glycans on Stem Cells, Biochemistry, 10.1021/acs.biochem.0c00523, 59, 39, 3757-3771, 2020.09. |
| 14. |
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. |
| 15. |
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, 10.1016/j.aspen.2020.05.001, 23, 3, 694-700, 2020.08. |
| 16. |
Tsuguru Fujii, Kohei Kakino, Miyu Tanaka, Jae Man Lee, Takahiro Kusakabe, Yutaka Banno, A defect in purine nucleotide metabolism in the silkworm, Bombyx mori, causes a translucent larval integument and male infertility, Insect Biochemistry and Molecular Biology, 10.1016/j.ibmb.2020.103458, 2020.08. |
| 17. |
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, 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.. |
| 18. |
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, 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.. |
| 19. |
Yurie Kinoshita, Jian Xu, Akitsu Masuda, Kosuke Minamihata, Noriho Kamiya, Hiroaki Mon, Ryosuke Fujita, Takahiro Kusakabe, Man Lee, Expression and purification of biologically active human granulocyte-macrophage colony stimulating factor (hGM-CSF) using silkworm-baculovirus expression vector system, Protein Expression and Purification, 10.1016/j.pep.2019.03.010, 159, 69-74, 2019.07, Human granulocyte-macrophage colony stimulating factor (hGM-CSF) is a hematopoietic growth factor. It is widely employed as a therapeutic agent targeting neutropenia in cancer patients undergoing chemotherapy and in patients with AIDS or after bone marrow transplantation. In this study, we constructed the recombinant baculoviruses for the expression of recombinant hGM-CSF (rhGM-CSF) with two small affinity tags (His-tag and Strep-tag) at the N or C-terminus. Compared to N-tagged rhGM-CSF, C-tagged rhGM-CSF was highly recovered from silkworm hemolymph. The purified rhGM-CSF proteins migrated as a diffuse band and were confirmed to hold N-glycosylations. A comparable activity was achieved when commercial hGM-CSF was tested as a control. Considering the high price of hGM-CSF in the market, our results and strategies using silkworm-baculovirus system can become a great reference for mass production of the active rhGM-CSF at a lower cost.. |
| 20. |
Takumi Yano, 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, 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.. |
| 21. |
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, 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, 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.. |
| 22. |
Patmawati, Kosuke Minamihata, Tsuneyuki Tatsuke, Man Lee, Takahiro Kusakabe, Noriho Kamiya, Functional horseradish peroxidase−streptavidin chimeric proteins prepared using a silkworm-baculovirus expression system for diagnostic purposes, Journal of Biotechnology, 10.1016/j.jbiotec.2019.03.007, 297, 28-31, 2019.05, Rapid, convenient, sensitive detection methods are of the utmost importance in analytical tools. Enzyme-based signal amplification using horseradish peroxidase (HRP) is commonly implemented in clinical diagnostics kits based on enzyme-linked immunosorbent assay (ELISA), by which the limit of detection is greatly improved. Herein we report the design and preparation of recombinant fusion proteins comprising HRP and streptavidin (Stav), in which HRP was fused to either the N- or C-terminus of Stav ((HRP)
4
–Stav or Stav–(HRP)
4
, respectively) using a baculovirus-silkworm expression system. Both (HRP)
4
–Stav and Stav–(HRP)
4
were secreted in the apo form but they were easily converted to the holo form and activated by simple incubation with hemin overnight at 4 °C. The activated (HRP)
4
–Stav and Stav–(HRP)
4
could be combined with a commercial biotinylated anti-OVA IgG antibody to detect ovalbumin (OVA) as the antigen in ELISA. The enzymatic activity of (HRP)
4
–Stav was twofold higher than that of Stav–(HRP)
4
, and the sensitivity of (HRP)
4
-Stav in ELISA was higher than that of a commercial HRP–Stav chemical conjugate. The successful use of (HRP)
4
–Stav chimeric protein as a molecular probe in ELISA shows that the baculovirus-silkworm expression system is promising to produce enzyme–Stav conjugates to substitute for those prepared by chemical methods.. |
| 23. |
Jian Xu, Akihiro Morio, Daisuke Morokuma, Yudai Nagata, Masato Hino, Akitsu Masuda, Zhiqing Li, Hiroaki Mon, Takahiro Kusakabe, 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, 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.. |
| 24. |
Patmawati xxxx, Kosuke Minamihata, Tsuneyuki Tatsuke, Man Lee, Takahiro Kusakabe, Noriho Kamiya, Expression and Activation of Horseradish Peroxidase–Protein A/G Fusion Protein in Silkworm Larvae for Diagnostic Purposes, Biotechnology Journal, 10.1002/biot.201700624, 13, 6, 2018.06, Recombinant protein production can create artificial proteins with desired functions by introducing genetic modifications to the target proteins. Horseradish peroxidase (HRP) has been used extensively as a reporter enzyme in biotechnological applications; however, recombinant production of HRP has not been very successful, hampering the utilization of HRP with genetic modifications. A fusion protein comprising an antibody binding protein and HRP will be an ideal bio-probe for high-quality HRP-based diagnostic systems. A HRP-protein A/G fusion protein (HRP-pAG) is designed and its production in silkworm (Bombyx mori) is evaluated for the first time. HRP-pAG is expressed in a soluble apo form, and is activated successfully by incubating with hemin. The activated HRP-pAG is used directly for ELISA experiments and retains its activity over 20 days at 4 °C. Moreover, HRP-pAG is modified with biotin by the microbial transglutaminase (MTG) reaction. The biotinylated HRP-pAG is conjugated with streptavidin to form a HRP-pAG multimer and the multimeric HRP-pAG produced higher signals in the ELISA system than monomeric HRP-pAG. The successful production of recombinant HRP in silkworm will contribute to creating novel HRP-based bioconjugates as well as further functionalization of HRP by applying enzymatic post-translational modifications.. |
| 25. |
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, 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, 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.. |
| 26. |
Yoshiki Morifuji, Jian Xu, Noriko Karasaki, Kazuhiro Iiyama, Daisuke Morokuma, Masato Hino, Akitsu Masuda, Takumi Yano, Hiroaki Mon, Takahiro Kusakabe, Man Lee, Expression, Purification, and Characterization of Recombinant Human α
1
-Antitrypsin Produced Using Silkworm–Baculovirus Expression System, Molecular Biotechnology, 10.1007/s12033-018-0127-y, 2018.01, Human α
1
-antitrypsin (AAT) is the most abundant serine proteinase inhibitor (serpin) in the human plasma. Commercially available AAT for the medications of deficiency of α
1
-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.. |
| 27. |
Akitsu Masuda, Kosuke Minamihata, Masato Hino, Daisuke Morokuma, Noriko Karasaki, Hiroaki Mon, Noriho Kamiya, Takahiro Kusakabe, 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, 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.. |
| 28. |
Akitsu Masuda, 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, 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.. |
| 29. |
Mitsunori Shiroishi, Yuji Ito, Kenta Shimokawa, Man Lee, Takahiro Kusakabe, Tadashi Ueda, Structure–function analyses of a stereotypic rheumatoid factor unravel the structural basis for germline-encoded antibody autoreactivity, Journal of Biological Chemistry, 10.1074/jbc.M117.814475, 293, 18, 7008-7016, 2018.01, Rheumatoid factors (RFs) are autoantibodies against the fragment-crystallizable (Fc) region of IgG. In individuals with hematological diseases such as cryoglobulinemia and certain B cell lymphoma forms, the RFs derived from specific heavy- and light-chain germline pairs, so-called “stereotypic RFs,” are frequently produced in copious amounts and form immune complexes with IgG in serum. Of note, many structural details of the antigen recognition mechanisms in RFs are unclear. Here we report the crystal structure of the RF YES8c derived from the IGHV1-69/IGKV3-20 germline pair, the most common of the stereotypic RFs, in complex with human IgG1-Fc at 2.8 Å resolution. We observed that YES8c binds to the CH2–CH3 elbow in the canonical antigen-binding manner involving a large antigen–antibody interface. On the basis of this observation, combined with mutational analyses, we propose a recognition mechanism common to IGHV1-69/IGKV3-20 RFs: (1) the interaction of the Leu
432
–His
435
region of Fc enables the highly variable complementarity-determining region (CDR)-H3 to participate in the binding, (2) the hydrophobic tip in the CDR-H2 typical of IGHV1-69 antibodies recognizes the hydrophobic patch on Fc, and (3) the interaction of the highly conserved RF light chain with Fc is important for RF activity. These features may determine the putative epitope common to the IGHV1-69/IGKV3-20 RFs. We also showed that some mutations in the binding site of RF increase the affinity to Fc, which may aggravate hematological diseases. Our findings unravel the structural basis for germline-encoded antibody autoreactivity.. |
| 30. |
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, 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.. |
| 31. |
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, 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.. |
| 32. |
Ming-Ming Ji, JAE MAN LEE, 門 宏明, Jian Xu, Tsuneyuki Tatsuke, takahiro kusakabe, Proteasome inhibitor MG132 impairs autophagic flux through compromising formation of autophagosomes in Bombyx cells, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 10.1016/j.bbrc.2016.09.151, 479, 4, 690-696, 2016.10. |
| 33. |
Jianping Chen, Jian Xu, Masato Hino, Mami Yamashita, Kazuma Hirata, Anandrao Ashok Patil, Tsuneyuki Tatsuke, 門 宏明, Banno Y, 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. |
| 34. |
Masato Hino, Takuji Kawanami, Jian Xu, Daisuke Morokuma, Kazuma Hirata, Mami Yamashita, Noriko Karasaki, Tsuneyuki Tatsuke, 門 宏明, Kazuhiro Iiyama, 神谷 典穂, Banno Y, 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, 313-317, 2016.06. |
| 35. |
Masato Hino, Daisuke Morokuma, 門 宏明, 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, 85, 21-29, 2016.06. |
| 36. |
Kazuhiro Iiyama, JAE MAN LEE, Tsuneyuki Tatsuke, 門 宏明, takahiro kusakabe, Expression and Characterization of Recombinant Serratia liquefaciens Nucleases Produced with Baculovirus-mediated Silkworm Expression System, MOLECULAR BIOTECHNOLOGY, 10.1007/s12033-016-9937-y, 58, 6, 393-403, 2016.06. |
| 37. |
Ji-Hyun Choi, Dae-Jung Kim, Sun Mee Hong, Sun-Jung Jo, Kwan-Sik Min, Young Chang Sohn, JAE MAN LEE, takahiro kusakabe, Molecular analysis and bioactivity of luteinizing hormone from Japanese eel, Anguilla japonica, produced in silkworm pupae, BIOTECHNOLOGY AND BIOPROCESS ENGINEERING, 10.1007/s12257-016-0042-7, 21, 3, 381-388, 2016.06. |
| 38. |
Jian Xu, Pingbo Zhang, takahiro kusakabe, 門 宏明, Zhiqing Li, Li Zhu, Kazuhiro Iiyama, YUTAKA BANNO, Daisuke Morokuma, JAE MAN LEE, Comparative proteomic analysis of hemolymph proteins from Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-sensitive or -resistant silkworm strains during infections, COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY D-GENOMICS & PROTEOMICS, 10.1016/j.cbd.2015.07.003, 16, 36-47, 2015.12. |
| 39. |
Saki Imai, takahiro kusakabe, Jian Xu, Zhiqing Li, Shintaro Shirai, 門 宏明, Daisuke Morokuma, JAE MAN LEE, Roles of silkworm endoplasmic reticulum chaperones in the secretion of recombinant proteins expressed by baculovirus system, MOLECULAR AND CELLULAR BIOCHEMISTRY, 10.1007/s11010-015-2529-5, 409, 1-2, 255-262, 2015.11. |
| 40. |
Kounosuke Hayashi, JAE MAN LEE, Yusuke Tomozoe, takahiro kusakabe, 神谷 典穂, Heme precursor injection is effective for Arthromyces ramosus peroxidase fusion protein production by a silkworm expression system, JOURNAL OF BIOSCIENCE AND BIOENGINEERING, 10.1016/j.jbiosc.2015.02.013, 120, 4, 384-386, 2015.10. |
| 41. |
Atsushi Masuda, Jian Xu, Takumi Mitsudome, Yudai Nagata, Daisuke Morokuma, 門 宏明, YUTAKA BANNO, takahiro kusakabe, JAE MAN LEE, Mass Production of an Active Peptide-N-Glycosidase F Using Silkworm-Baculovirus Expression System, MOLECULAR BIOTECHNOLOGY, 10.1007/s12033-015-9866-1, 57, 8, 735-745, 2015.08. |
| 42. |
Atsushi Masuda, Jian Xu, Takumi Mitsudome, Daisuke Morokuma, 門 宏明, YUTAKA BANNO, takahiro kusakabe, JAE MAN LEE, Improvement of Endo-β-N-acetylglucosaminidase H production using silkworm–baculovirus protein expression system, Journal of Asia-Pacific Entomology, 18, 2, 175-180, 2015.06. |
| 43. |
Daisuke Morokuma, Jian Xu, 門 宏明, 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, 18, 2, 303-309, 2015.06. |
| 44. |
Daisuke Morokuma, 門 宏明, Banno Y, takahiro kusakabe, JAE MAN LEE, Differential N-Glycan Modifications of Human Alpha 1-Acid Glycoprotein (α1AGP) Produced in Different Silkworm Strains using the Baculovirus Expression System, Journal of Insect Biotechnology and Sericology, 84, 2, 49-53, 2015.05. |
| 45. |
Yasuyuki Hashiguchi, JAE MAN LEE, M. Shiraishi, S. Komatsu, S. Miki, Yohei Shimasaki, N. Mochioka, Yuji Oshima, takahiro kusakabe, Characterization and evolutionary analysis of tributyltin‐binding protein and pufferfish saxitoxin and tetrodotoxin‐binding protein genes in toxic and nontoxic pufferfishes, Journal of evolutionary biology, 28, 5, 1103-1108, 2015.05. |
| 46. |
Jian Xu, takahiro kusakabe, Kimiko Yamamoto, Yoshitaka Suetsugu, 門 宏明, Zhiqing Li, Li Zhu, Kazuhiro Iiyama, YUTAKA BANNO, Kaito Yoshimura, JAE MAN LEE, A novel third chromosomal locus controls susceptibility to Autographa californica multiple nucleopolyhedrovirus in the silkworm, Bombyx mori., Applied Microbiology and Biotechnology, 98, 7, 3049-3058, 2014.04. |
| 47. |
JAE MAN LEE, Jian Xu, 門 宏明, Takumi Mitsudome, Atsushi Masuda, Kaito Yoshimura, Kazuhiro Iiyama, Yuuka Chieda, takahiro kusakabe, Baculovirus-mediated gene transfer systems in silkworm larvae using constitutive host promoters, JOURNAL OF ASIA-PACIFIC ENTOMOLOGY, 10.1016/j.aspen.2013.10.009, 17, 1, 73-78, 2014.03. |
| 48. |
Yudai Nagata, JAE MAN LEE, 門 宏明, Shigeo Imanishi, Sun Mee Hong,, Shoji Komatsu, Yuji Oshima, takahiro kusakabe, RNAi suppression of β-N-acetylglucosaminidase (BmFDL) for complex-type N-linked glycan synthesis in cultured silkworm cells, Biotechnology letters, 10.1007/s10529-013-1183-9, 35, 7, 1009-1016, 2013.07. |
| 49. |
Tsuneyuki Tatsuke, JAE MAN LEE, takahiro kusakabe, Kazuhiro Iiyama, Hideki Sezutsu, Keiro Uchino, TIGHTLY CONTROLLED TETRACYCLINE-INDUCIBLE TRANSCRIPTION SYSTEM FOR EXPLOSIVE GENE EXPRESSION IN CULTURED SILKWORM CELLS, ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, 10.1002/arch.21083, 82, 4, 173-182, 2013.04. |
| 50. |
Jian Xu, Yudai Nagata, 門 宏明, Zhiqing Li, Li Zhu, Kazuhiro Iiyama, takahiro kusakabe, JAE MAN LEE, Soaking RNAi-mediated modification of Sf9 cells for baculovirus expression system by ectopic expression of Caenorhabditis elegans SID-1, Applied Microbiology and Biotechnology, 2013.03. |
| 51. |
JAE MAN LEE, Yoshito Kojin, Tsuneyuki Tatsuke, 門 宏明, Yoshitaka Miyagawa, takahiro kusakabe, Coexpression of Escherichia coli RNase III in silkworm cells improves the efficiency of RNA interference induced by long hairpin dsRNAs, Insect Science, 10.1111/j.1744-7917.2012.01569.x, 20, 1, 69-77, 2013.02. |
| 52. |
Yasuhiko Soejima, JAE MAN LEE, Yudai Nagata, 門 宏明, Kazuhiro Iiyama, 北野 載, Michiya Matsuyama, takahiro kusakabe, Comparison of signal peptides for efficient protein secretion in the baculovirus-silkworm system, Central European Journal of Biology, 10.2478/s11535-012-0112-6, 8, 1, 1-7, 2013.01. |
| 53. |
Mai Fukushima, Kazuhiro Iiyama, Jun Yamashita, Masutaka Furue, Gaku Tsuji, Shigeo Imanishi, 門 宏明, JAE MAN LEE, takahiro kusakabe, PRODUCTION OF SMALL ANTIBACTERIAL PEPTIDES USING SILKWORM-BACULOVIRUS PROTEIN EXPRESSION SYSTEM, Preparative Biochemistry and Biotechnology, 2013.01. |
| 54. |
Zhiqing Li, Daojun Cheng, 門 宏明, Li Zhu, Jian Xu, Tsuneyuki Tatsuke, JAE MAN LEE, Qingyou Xia, takahiro kusakabe, Cell Cycle-Dependent Recruitment of Polycomb Proteins to the ASNS Promoter Counteracts C/ebp-Mediated Transcriptional Activation in Bombyx mori, PloS one, 10.1371/journal.pone.0052320, 8, 1, e52320, 2013.01. |
| 55. |
Hitoshi Kurio, JAE MAN LEE, takahiro kusakabe, hiroshi iida, Testis-specific Cell Adhesion Molecule, CEACAM6-L, Forms Homophilic Interaction at the Cell Adhesion Site in Vitro, Zoological science, 10.2108/zsj.29.786, 29, 11, 786-793, 2012.11. |
| 56. |
門 宏明, JAE MAN LEE, Mai Fukushima, Yudai Nagata, Mie Fuji, Jian Xu, Oumi Nishi, Kazuhiro Iiyama, takahiro kusakabe, Production and characterization of the celery mismatch endonuclease CEL II using baculovirus/silkworm expression system, Applied microbiology and biotechnology, 2012.11. |
| 57. |
JAE MAN LEE, Naoya Kawakami, 門 宏明, Hitoshi Mitsunobu, Kazuhiro Iiyama, Satoshi Ninaki, Katsumi Maenaka, Enoch Y Park, takahiro kusakabe, Establishment of a Bombyx mori nucleopolyhedrovirus (BmNPV) hyper-sensitive cell line from the silkworm e21 strain, Biotechnology letters, 10.1007/s10529-012-0971-y, 34, 10, 1773-1779, 2012.10. |
| 58. |
JAE MAN LEE, Yoshito Kojin, Tsuneyuki Tatsuke, 門 宏明, Yoshitaka Miyagawa, takahiro kusakabe, RNA Interference Induction by Long Hairpin dsRNAs Expressed from Chromosomal DNA of Bombyx mori Cells, Journal of the Faculty of Agriculture, Kyushu University, 57, 2, 441-445, 2012.09.  |
| 59. |
Zhu, L., Tatsuke, T., Li, Z., Mon, H., Xu, Z., Lee, J.M., and Kusakabe T.,, Molecular cloning of BmTUDOR-SN and analysis of its role in RNAi pathway in the silkworm, Bombyx mori (Lepidoptera: Bombycidae). , Appl. Entomol. Zool., 2012.06. |
| 60. |
Lee, J.M., Mon, H., Banno, Y., Iiyama, K., Kusakabe, T., Bombyx mori strains useful for efficient recombinant protein production using a baculovirus vector., J. Biotech. Biomater., 2012.06. |
| 61. |
Sugahara, R., Mon, H., Lee, J.M., and Kusakabe, T.,, Monoubiquitination-Dependent Chromatin Loading of FancD2 in Silkworms, a Species Lacking the FA Core Complex., Gene, 2012.05. |
| 62. |
Nagata, Y., Sakashita, K., Imanishi, S., Lee, J.M., and Kuskabe T. , Expression of glycosylated mucin-like domain using baculovirus expression system in silkworm, Bombyx mori., J. Fac. Agr. Kyushu Univ., , 57, 83-86, 2012.04. |
| 63. |
Fujii, M., Takahashi, M., Mon, H., Tatsuke, T., Lee, J.M, and Kuskabe T. , Molecular cloning of the silkworm p53R2 homolog gene. , J. Fac. Agr. Kyushu Univ., , 57, 79-82, 2012.04. |
| 64. |
Mitsunobu, H., Izumi, H., Mon, H., Tatsuke, T., Lee, J.M., Kusakabe, T., Molecular Characterization of heterochromatin proteins 1a and 1b from the Silkworm, Bombyx mori., Insect Mol. Biol. , 21, 9-20, 2012.01. |
| 65. |
Mon, H., Kobayashi, I., Ohkubo, S. Tomita, S., Lee, J.M., Sezutsu, H., Tamura, T., Kusakabe, T., Effective RNA interference in cultured silkworm cells mediated by overexpression of Caenorhabditis elegans SID-1., RNA Biol., , 9, 40-46, 2012.01. |
| 66. |
Li, Z., Cheng, D., Mon, H., Tatsuke, T., Zhu, L., Xu, J., Lee, J.M., Xia, Q., and Kusakabe, T., Genome-wide identification of Polycomb target genes reveals a functional association of Pho with Scm in Bombyx mori., PLoS ONE, , 7, e34330, 2012.01. |
| 67. |
Mon, H., Lee, J.M., Kawaguchi, Y., Kusakabe, T., Double-strand breaks repair by gene conversion in silkworm holocentric chromosomes, Mol. Genet. Genomics, 2011.09. |
| 68. |
Mon, H., Izumi, M., Mitsunobu, H., Tatsuke, T., Iiyama, K., Jikuya, H., Lee, J.M., Kusakabe, T., Post-translational modifications of the N-terminal tail of histone H3 in holocentric chromosomes of bombyx mori. Insect Biochem. , Mol. Genet. Genomics, 2011.09. |
| 69. |
Lee JM, Takahashi M, Mon H, Mitsunobu H, Koga K, Kawaguchi Y, Nakajima Y, Kusakabe T., Construction of gene expression systems in insect cell lines using promoters from the silkworm, Bombyx mori., J Biotechnol, 133(1) 9-17 , 2008.02. |
| 70. |
Kawakami, N., Lee, J.M., Mon, H., Kubo, Y., Banno, Y., Kawaguchi, Y., Maenaka, K., Park, E.Y., Koga, K. and Kusakabe, T., Efficient protein expression in Bombyx mori larvae of the strain d17 highly sensitive to B. mori nucleopolyhedrovirus., Mol Biotechnol. , 40(2), 180-185 , 2008.02. |
| 71. |
Lee J.M., Mon H., Takahashi M., Kawakami N. Yoshida Y., Banno Y., Koga K., Kawaguchi Y. and Kusakabe T. , Screening of high-permissive silkworm strains for efficient recombinant protein production in Autographa californica nuclear polyhedrosis virus (AcNPV). , J. Insect Biotech. Seric. , In press, 2007.03. |
| 72. |
Lee J.M., Takahashi M., Mon H., Koga K., Kawaguchi Y., and Kusakabe T., Efficient gene transfer into silkworm larval tissues by a combination of sonoporation and lipofection., Cell Biol. Int., 10.1016/j.cellbi.2005.07.007, 29, 11, 976-979, 29, 976-979., 2005.01. |
