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JAE MAN LEE Last modified date：2023.11.27

Associate Professor / Agricultural Bioresource Sciences / Department of Bioresource Sciences / Faculty of Agriculture


Papers
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. Mon H, Sato M, Lee JM, Kusakabe T., Construction of gene co-expression networks in cultured silkworm cells and identification of previously uncharacterized lepidopteran-specific genes required for chromosome dynamics., Insect Biochemistry and Molecular Biology, 10.1016/j.ibmb.2022.103875, 151, 103875-103875, 2022.12.
5. Hino M, Tatsuke T, Morio A, Mon H, Lee JM, Masuda A, Kakino K, Tonooka Y, Kusakabe T, Characterization of a Novel Heterochromatin Protein 1 Homolog "HP1c" in the Silkworm, Bombyx mori, Insects, https://doi.org/10.3390/insects13070631, 13, 7, 631, 2022.07.
6. 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.
7. Tanaka M, Fujii T, Mon H, Lee JM, Kakino K, Fukumori H, Ebihara T, Nagasato T, Hino M, Tonooka Y, Moriyama T, Fujita R, Banno Y, Kusakabe T., 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, 2022.04.
8. 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.
9. 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.
10. 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.
11. 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.
12. Fujii T, Kakino K, Fukumori H, Hino M, Lee JM, Kusakabe T, Banno Y. , 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, 2021.11.
13. 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.
14. 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.
15. 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.
16. 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.
17. 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.
18. 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.
19. 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.
20. 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.
21. 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..
22. Fajr A. Aleisa, Kosuke Sakashita, Jae Man Lee, Dina B. AbuSamra, Bader Al Alwan, Shuho Nozue, Muhammad Tehseen, Samir M. Hamdan, X. Satoshi Habuchi, X. Takahiro Kusakabe, Jasmeen S. Merzaban, Functional binding of E-selectin to its ligands is enhanced by structural features beyond its lectin domain, Journal of Biological Chemistry, 10.1074/jbc.RA119.010910, 295, 11, 3719-3733, 2020.03, Selectins are key to mediating interactions involved in cellular adhesion and migration, underlying processes such as immune responses, metastasis, and transplantation. Selectins are composed of a lectin domain, an epidermal growth factor (EGF)-like domain, multiple short consensus repeats (SCRs), a transmembrane domain, and a cytoplasmic tail. It is well-established that the lectin and EGF domains are required to mediate interactions with ligands; however, the contributions of the other domains in mediating these interactions remain obscure. Using various E-selectin constructs produced in a newly developed silkworm-based expression system and several assays performed under both static and physiological flow conditions, including flow cytometry, glycan array analysis, surface plasmon resonance, and cell-rolling assays, we show here that a reduction in the number of SCR domains is correlated with a decline in functional E-selectin binding to hematopoietic cell E- and/or L-selectin ligand (HCELL) and P-selectin glycoprotein ligand-1 (PSGL-1). Moreover, the binding was significantly improved through E-selectin dimerization and by a substitution (A28H) that mimics an extended conformation of the lectin and EGF domains. Analyses of the association and dissociation rates indicated that the SCR domains, conformational extension, and dimerization collectively contribute to the association rate of E-selectin–ligand binding, whereas just the lectin and EGF domains contribute to the dissociation rate. These findings provide the first evidence of the critical role of the association rate in functional E-selectin–ligand interactions, and they highlight that the SCR domains have an important role that goes beyond the structural extension of the lectin and EGF domains..
23. 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, 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..
24. Daigo Tsubokawa, Jae Man Lee, Takeshi Hatta, Fusako Mikami, Haruhiko Maruyama, Takeshi Arakawa, Takahiro Kusakabe, Naotoshi Tsuji, Characterization of the RAGE-binding protein, Strongyloides venestatin, produced by the silkworm-baculovirus expression system, Infection, Genetics and Evolution, 10.1016/j.meegid.2019.103964, 75, 2019.11, The receptor for advanced glycation end products (RAGE) recognizes Ca++-binding proteins, such as members of the S100 protein family released by dead or devitalized tissues, and plays an important role in inflammatory responses. We recently identified the Ca++-binding protein, venestatin, secreted from the rodent parasitic nematode, Strongyloides venezuelensis. We herein characterized recombinant venestatin, which is abundantly produced by the silkworm-baculovirus expression system (silkworm-BES), particularly in its interaction with RAGE. Venestatin from silkworm-BES possessed a binding capacity with Ca++ ions and vaccine immunogenicity against S. venezuelensis larvae in mice, which is similar to venestatin produced by the E. coli expression system (EES). Venestatin from silkworm-BES had a higher affinity for human recombinant RAGE than that from EES, and their affinities were Ca++-dependent. RAGE in the mouse lung co-immunoprecipitated with venestatin from silkworm-BES administered intranasally, indicating that it bound endogenous mouse RAGE. The present results suggest that venestatin from silkworm-BES affects RAGE-mediated pathological processes..
25. Sun Mee Hong, Ji Hyun Choi, Sun Jung Jo, Kwan Sik Min, Dae Jung Kim, Jae Man Lee, Takahiro Kusakabe, Heterologous Production and Glycosylation of Japanese Eel Follitropin Using Silkworm, Biotechnology and Bioprocess Engineering, 10.1007/s12257-019-0045-2, 24, 5, 745-753, 2019.09, Follitropin, an important gonadotropin hormone, participates in vitellogenesis and spermatogenesis. Equine chorionic gonadotropin (eCG) can induce gonadotropin hormone activity in non-equid species and exhibits a long biological half-life. Here, we report the production, using silkworm larval and pupal systems, of biologically active recombinant hybrid-type follitropins based on the coding sequence of the eCG C-terminal peptide (CTP) between the mature β- and α-chains of eel. The three constructs, rJeFSH, rJeFSH·eCG, and rJeFSH·2xeCG were produced and verified to be N- or O-glycosylated and secreted mature peptides. Although rJeFSH·eCG contains more elaborate O-linked carbohydrate chains than rJeFSH, it elicited no significant in vitro oocyte maturation, which may be a result of insufficient terminal sialylation of its N-and O-linked carbohydrate chains. Then, a hybrid of rJeFSH·2xeCG extended with two eCG CTP. Furthermore, the receptor binding assay revealed potency of rJeFSH and rJeFSH·2xeCG to be a few folds greater than that of rJeFSH·eCG. The findings of this study will be useful for the development of more efficient GTHs in teleosts, including eels, when various modifications with two or more extended eCG CTP produced by silkworm are included..
26. 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..
27. 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..
28. 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..
29. 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..
30. Dani Permana, Kosuke Minamihata, Tsuneyuki Tatsuke, Jae M. Lee, Takahiro Kusakabe, Masahiro Goto, Noriho Kamiya, Polymerization of Horseradish Peroxidase by a Laccase-Catalyzed Tyrosine Coupling Reaction, Biotechnology Journal, 10.1002/biot.201800531, 14, 6, 2019.06, The polymerization of proteins can create newly active and large bio-macromolecular assemblies that exhibit unique functionalities depending on the properties of the building block proteins and the protein units in polymers. Herein, the first enzymatic polymerization of horseradish peroxidase (HRP) is reported. Recombinant HRPs fused with a tyrosine-tag (Y-tag) through a flexible linker at the N- and/or C-termini are expressed in silkworm, Bombyx mori. Trametes sp. laccase (TL) is used to activate the tyrosine of Y-tagged HRPs with molecular O2 to form a tyrosyl-free radical, which initiates the tyrosine coupling reaction between the HRP units. A covalent dityrosine linkage is also formed through a HRP-catalyzed self-crosslinking reaction in the presence of H2O2. The addition of H2O2 in the self-polymerization of Y-tagged HRPs results in lower activity of the HRP polymers, whereas TL provides site-selectivity, mild reaction conditions and maintains the activity of the polymeric products. The cocrosslinking of Y-tagged HRPs and HRP-protein G (Y-HRP-pG) units catalyzed by TL shows a higher signal in enzyme-linked immunosorbent assay (ELISA) than the genetically pG-fused HRP, Y-HRP-pG, and its polymers. This new enzymatic polymerization of HRP promises to provide highly active and functionalized polymers for biomedical applications and diagnostics probes..
31. 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..
32. Mai Morishita, Akitsu Masuda, Hiroaki Mon, Man Lee, Takahiro Kusakabe, Kosuke Tashiro, Chisa Yasunaga-Aoki, Kazuhiro Iiyama, Identification of an insecticidal toxin produced by Enterobacter sp. strain 532 isolated from diseased Bombyx mori silkworms, FEMS microbiology letters, 10.1093/femsle/fny295, 366, 2, 2019.01, Although Enterobacter sp. 532 shows pathogenicity in Bombyx mori, the insecticidal mechanisms are unclear. Here, we identified and characterised an insecticidal protein from Enterobacter. The insecticidal protein was purified from the strain and inoculated into B. mori larvae. Intracellular proteins were prepared, purified and separated by preparative native polyacrylamide gel electrophoresis (PAGE); one protein band had insecticidal activity. Sodium dodecyl sulfate-PAGE showed the presence of several bands, indicating that the insecticidal protein formed a complex. Peptide mass fingerprinting of a prominent 255.3-kDa band revealed 64 peptides that matched one protein with 33.0% sequence coverage. This protein was a homologue of the A component of the toxin complex (Tc), and the VRP1 domain was conserved; thus, the gene was named itcA (insecticidal toxin complex A). In the itcA downstream region, B and C component gene homologues were found, and these genes were located on an 86.2-kb contig sequence. Two repA genes and 27 genes related to conjugation transfer of plasmids were located on the contig, suggesting that the contig originated from a mobilisable plasmid. Therefore, these findings suggested that the strain may have acquired the Tc genes by horizontal transfer. This is the first description of Tc produced by the genus Enterobacter..
33. Mai Morishita, Hiroaki Mon, Jae Man Lee, Takahiro Kusakabe, Maximiano Corrêa Cassal, Chisa Yasunaga-Aoki, Kazuhiro Iiyama, Toxin complex is a major virulence determinant of Enterobacter sp. 532 against the silkworm, Bombyx mori, Journal of Insect Biotechnology and Sericology, 10.11416/jibs.88.1_021, 88, 1, 1_021-1_025, 2019.01.
34. 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..
35. 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..
36. 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..
37. Koki Mukai, Ayano Teramoto, Xuchun Qiu, Yohei Shimasaki, Yoko Kato-Unoki, Man Lee, Naohiro Mizoguchi, Mst Ruhina Margia Khanam, Hina Satone, Tsuneyuki Tatsuke, Takahiro Kusakabe, Yuji Oshima, Gene structure and cDNA sequence of 2-Cys peroxiredoxin in the harmful algal bloom species Chattonella marina and its gene transcription under different light intensities, European Journal of Phycology, 10.1080/09670262.2017.1346206, 53, 1, 29-38, 2018.01, We investigated the gene structure and predicted amino acid sequence of the antioxidant enzyme 2-Cys peroxiredoxin (2-Cys Prx) in the raphidophyte Chattonella marina, which is a harmful algal bloom (HAB) species. The open reading frame of 2-Cys Prx was 585 bp long and encoded a protein consisting of 195 amino acids. The putative amino acid sequence contained two cysteine residues located at the 49th and 170th amino acid positions from the N-terminal methionine residue. The sequence also possessed 2-Cys Prx characteristic motifs, F (FFYPLDFTFVCPTEI) and EVCP. The position of the 2-Cys Prx gene relative to several others (ycf59–2-CysPrx–rpl35–rpl20) was the same as that found in the chloroplast genome in the raphidophyte Heterosigma akashiwo. Upstream of the 2-Cys Prx gene, possible TATA and GGA motifs recognized by nuclear-encoded plastid RNA polymerase (NEP), and a possible -10 box and -35 box recognized by plastid-encoded plastid RNA polymerase (PEP) were observed. We measured the transcript levels of 2-Cys Prx in C. marina cells grown under three different light intensities (0, 100, 1000 µmol photons m–2 s–1, 14-h light/8-h dark photoperiod) by quantitative PCR. The 2-Cys Prx transcript level in cells grown under the highest light intensity on day 3 was threefold that on day 0 but two lower light intensities resulted in relatively stable transcription levels. The 2-Cys Prx transcript level was significantly positively related to the H2O2 concentration per cell and the H2O2 scavenging activity per cell. These results suggest that C. marina 2-Cys Prx functions in the chloroplast and its transcription could be regulated by both NEP and PEP. Moreover, the 2-Cys Prx transcript level might increase to remove excessive H2O2 produced under strong light conditions in order to maintain cell proliferation activity..
38. 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..
39. 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..
40. 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..
41. 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..
42. Zhiqing Li, Qixin Cui, Jian Xu, Daojun Cheng, Xiaoyan Wang, Bingqian Li, Man Lee, Qingyou Xia, Takahiro Kusakabe, Ping Zhao, SUMOylation regulates the localization and activity of Polo-like kinase 1 during cell cycle in the silkworm, Bombyx mori, Scientific Reports, 10.1038/s41598-017-15884-7, 7, 1, 2017.12, Polo-like kinase 1 (Plk1) is a crucial cell cycle regulator by its specific localization and activity during cell cycle. It has been shown that the phosphorylation and ubiquitylation of Plk1 are required for its own activation and localization. Here, we report that SUMOylation regulates the activity of Plk1 in the lepidopteran insect of Bombyx mori. In the absence of SUMOylation, it causes the lost localization of Plk1 on centrosomes and kinetochores, as well as an uneven distribution in midzone. We further identify that the putative SUMOylation site of Bombyx Plk1 at lysine 466 is required for its localization on centrosomes, and K466 mutation in Plk1 could influence its interaction with Smt3/Ubc9 complex. These findings are also confirmed by Drosophila Polo and human Plk1, which together reveals a conserved role of Plk1 SUMOylation in mammals. Moreover, conjugation of Smt3 to Plk1 SUMOylation mutant promotes its localization on centrosomes and kinetochores, and rescues functional defects of chromosome alignment in cells depleted of endogenous Plk1. Altogether, the present data indicate that the SUMOylation of Plk1 could participate in proper chromosome alignment and segregation during mitosis, and provides a novel layer for the regulation of Plk1 localization and activity throughout cell cycle..
43. Anandrao Ashok Patil, Tsuneyuki Tatsuke, Hiroaki Mon, 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, 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..
44. Ming Ming Ji, 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, 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..
45. Anandrao Ashok Patil, Tsuneyuki Tatsuke, Hiroaki Mon, 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, 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..
46. Hiroaki Mon, Man Lee, Masanao Sato, Takahiro Kusakabe, Identification and functional analysis of outer kinetochore genes in the holocentric insect Bombyx mori, Insect Biochemistry and Molecular Biology, 10.1016/j.ibmb.2017.04.005, 86, 1-8, 2017.07, The kinetochore creates chromosomal attachment sites for microtubules. The kinetochore-microtubule interface plays an important role in ensuring accurate transmission of genetic information to daughter cells. Bombyx mori is known to possess holocentric chromosomes, where spindle microtubules attach along the entire length of the chromosome. Recent evidence suggests that CENP-A and CENP-C, which are essential for centromere structure and function in other species, have lost in holocentric insects, implying that B. mori is able to build its kinetochore regardless of the lack of CENP-A and CENP-C. Here we report the identification of three outer kinetochore genes in the silkworm B. mori by using bioinformatics and RNA interference-based screening. While the homologs of Ndc80 and Mis12 have strong similarity with those of other organisms, the five encoded proteins (BmNuf2, BmSpc24, BmSpc25, BmDsn1 and BmNnf1) are highly diverged from their counterparts in other species. Microscopic studies show that the outer kinetochore protein is distributed along the entire length of the chromosomes, which is a key feature of holocentric chromosomes. We also demonstrate that BmDsn1 forms a heterotrimeric complex with BmMis12 and BmNnf1, which acts as a receptor of the Ndc80 complex. In addition, our study suggests that a small-scale RNAi-based candidate screening is a useful approach to identify genes which may be highly divergent among different species..
47. 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..
48. 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..
49. Kazuhiro Iiyama, Eigo Takahashi, Man Lee, Hiroaki Mon, Mai Morishita, Takahiro Kusakabe, Chisa Yasunaga-Aoki, Alkaline protease contributes to pyocyanin production in Pseudomonas aeruginosa, FEMS Microbiology Letters, 10.1093/femsle/fnx051, 364, 7, 2017.04, The role of the alkaline protease (AprA) in pyocyanin production in Pseudomonas aeruginosa was investigated. AprA was overproduced when a plasmid carrying the aprA gene was introduced to an aprA-deletion mutant strain, EG03; thus, aprA-complemented EG03 was used as an overproducing strain. The complemented strain produced higher pyocyanin than the mutant strain in all commercially available media evaluated. Particularly, pyocyanin production was higher in the complemented than in the parental strain in brain-heart infusion and tryptic soy broths. These results suggested that protein degradation products by AprA were utilized for pyocyanin production. Protein-rich media were used in subsequent validation studies. Similar results were obtained when the basal medium was supplemented with casein or skim milk as the sole organic nitrogen source. However, gelatin failed to induce abundant pyocyanin production in the complemented strain, despite the presence of protein degradation products by AprA as assessed by SDS-PAGE. Thus, gelatin degradation products may not be suitable for pyocyanin synthesis. In conclusion, AprA could contribute to pyocyanin production in the presence of several proteins or peptides..
50. Hina Satone, Shohei Nonaka, JAE MAN LEE, Yohei Shimasaki, takahiro kusakabe, Shun-ichiro Kawabata, Yuji Oshima, Tetrodotoxin- and tributyltin-binding abilities of recombinant pufferfish saxitoxin and tetrodotoxin binding proteins of Takifugu rubripes, TOXICON, 10.1016/j.toxicon.2016.11.245, 125, 50-52, 2017.01.
51. Kazuhiro Iiyama, Mai Morishita, Man Lee, Hiroaki Mon, Takahiro Kusakabe, Kosuke Tashiro, Taiki Akasaka, Chisa Yasunaga-Aoki, Kazuhisa Miyamoto, A reconsideration of the taxonomic position of two bacterial strains isolated from Flacherie-Diseased silkworms in 1965, Journal of Insect Biotechnology and Sericology, 10.11416/jibs.86.2_035, 86, 2, 35-41, 2017.01, Recent advances in bacterial characterization methodologies have made taxonomic categorization significantly more accurate. Here, we re-evaluated the position of bacterial strains (532 and 652) belonging to the genus Hafnia, isolated from flacherie-diseased silkworms in 1965. Phylogenetic analysis based on the 16S rRNA gene sequences of these strains suggests that they belong to genus Enterobacter. Using multilocus sequence analysis (MLSA), these strains were further classified to MLSA group A, which is a “core” group of Enterobacter containing E. cloacae (the type species of the genus). Although these strains were closely related to E. mori, E. tabaci, and E. asburiae, they also had other MLSA characteristics that distinguished them from these neighboring bacterial species. These data were supported by further biochemical analysis. Thus, it appears that the 532 and 652 strains isolated almost half a century ago belong to genus Enterobacter, and their unique characteristics strongly suggest that they are a novel bacterial species..
52. Md. Nazmul Hasan, Mohammad Jakir Hosen, Prasoon Kumar Thakur, Ruhshan Ahmed Abir, Abdullah Zubaer, Guo Renkai, Mayumi Yoshida, Hiroto Ohta, JAE MAN LEE, takahiro kusakabe, Akinori Hirashima, In vitro screening for inhibitor of cloned Drosophila melanogaster tyramine-β-hydroxylase and docking studies, International Journal of Biological Macromolecules, 93, 889-895, 2016.12.
53. Md Nazmul Hasan, Mohammad Jakir Hosen, Prasoon Kumar Thakur, Ruhshan Ahmed Abir, Abdullah Zubaer, Guo Renkai, Mayumi Yoshida, Hiroto Ohta, Jae Man Lee, Takahiro Kusakabe, Akinori Hirashima, In vitro screening for inhibitor of cloned Drosophila melanogaster tyramine-β-hydroxylase and docking studies, International Journal of Biological Macromolecules, 10.1016/j.ijbiomac.2016.06.026, 93, 889-895, 2016.12, Biogenic amines are common biologically active substances extended within the whole animal kingdom where they play vital roles as signal transducer as well as regulator of cell functions. One of these biogenic amines called octopamine (OA) is synthesized from tyramine (TA) by the catalysis of tyramine-β-hydroxylase (TβH) originated in the insect nervous system. Both TA and OA act as neurotransmitters, neurohormones and neuromodulators in the arthropod nervous system. Herein, the inhibitory activity of 1-arylimidazole-2(3H)-thiones (AITs) was tested on cloned Drosophila tyramine-β-hydroxylase (DmTβH) expressed in Bombyx mori strain. Radiolabelled 3H-TA was used to analyze the activity of AITs exhibited inhibitory effects on DmTβH, whose ID50 values range from 0.02 to 2511 nM where DmTβH was inhibited in a dose-dependent manner at pH 7.6 and 25 °C during a 30 min of incubation. To understand the catalytic role of the TβH, a three dimensional structure of the TβH from Drosophila melanogaster was constructed by homology modeling using the Phyre2 web server with 100% confidence. The modeled three-dimensional structure of TβH was used to perform the docking study with AITs. This may give more insights to precise design of inhibitors for TβH to control insect's population..
54. 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.
55. 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.
56. 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.
57. 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.
58. 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.
59. 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.
60. Erika Taira, 門 宏明, JAE MAN LEE, takahiro kusakabe, Chisa Yasunaga-Aoki, Kazuhiro Iiyama, Virulence of lipopolysaccharide-deficient mutants of Serratia liquefaciens toward the silkworm, Bombyx mori., Journal of Insect Biotechnology and Sericology, 10.11416/jibs.85.1_007, 85, 7-14, 2016.04.
61. Eigo Takahashi, JAE MAN LEE, 門 宏明, Yuuka Chieda, Chisa Yasunaga-Aoki, takahiro kusakabe, Kazuhiro Iiyama, Effect of antibiotics on extracellular protein level in Pseudomonas aeruginosa., Plasmid, 10.1016/j.plasmid.2016.03.001, 84-85, 44-50, 2016.03.
62. 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, 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..
63. Li Zhu, 門 宏明, Jian Xu, takahiro kusakabe, JAE MAN LEE, CRISPR/Cas9-mediated knockout of factors in non-homologous end joining pathway enhances gene targeting in silkworm cells, SCIENTIFIC REPORTS, 10.1038/srep18103, 5, 2015.12.
64. 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.
65. 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.
66. 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.
67. Li Zhu, Tsuneyuki Tatsuke, Jian Xu, Zhiqing Li, 門 宏明, JAE MAN LEE, takahiro kusakabe, Loqs depends on R2D2 to localize in D2 body-like granules and functions in RNAi pathways in silkworm cells, Insect biochemistry and molecular biology, 64, 78-90, 2015.09.
68. 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.
69. 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.
70. 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.
71. Sun Mee Hong, Ji-Hyun Choi, Sun-Jung Jo, Yohei Shimasaki, Seong Kyu Song, JAE MAN LEE, takahiro kusakabe, Expression of Recombinant Viscum Album Coloratum lectin B-chain in the Silkworm Expression System and Evaluation of Antioxidant Activity, BIOTECHNOLOGY AND BIOPROCESS ENGINEERING, 10.1007/s12257-014-0806-x, 20, 3, 515-522, 2015.06.
72. 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.
73. 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.
74. Takumi Mitsudome, 門 宏明, Jian Xu, Zhiqing Li, JAE MAN LEE, Anandrao Ashok Patil, Atsushi Masuda, Kazuhiro Iiyama, Daisuke Morokuma, takahiro kusakabe, Biochemical characterization of maintenance DNA methyltransferase DNMT-1 from silkworm, Bombyx mori, INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY, 10.1016/j.ibmb.2015.01.008, 58, 55-65, 2015.03.
75. Takumi Mitsudome, 門 宏明, Jian Xu, Zhiqing Li, JAE MAN LEE, Anandrao Ashok Patil, Atsushi Masuda, Kazuhiro Iiyama, Daisuke Morokuma, takahiro kusakabe, Biochemical characterization of maintenance DNA methyltransferase DNMT-1 from silkworm, Bombyx mori, INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY, 10.1016/j.ibmb.2015.01.008, 58, 55-65, 2015.03.
76. Zhiqing Li, 門 宏明, Hitoshi Mitsunobu, Li Zhu, Jian Xu, JAE MAN LEE, takahiro kusakabe, Dynamics of polycomb proteins-mediated histone modifications during UV irradiation-induced DNA damage, INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY, 10.1016/j.ibmb.2014.10.001, 55, 9-18, 2014.12.
77. Ryohei Sugahara, 門 宏明, JAE MAN LEE, Takahiro Shiotsuki, takahiro kusakabe, Differential contribution of the Fanconi anemia-related proteins to repair of several types of DNA damage in cultured silkworm cells, FEBS LETTERS, 10.1016/j.febslet.2014.09.009, 588, 21, 3959-3963, 2014.11.
78. Sun Mee Hong, Ho Sun Sung, Mee Hye Kang, Choong-Gon Kim, Youn-Ho Lee, Dae-Jung Kim, JAE MAN LEE, takahiro kusakabe, Characterization of cryptopygus antarcticus endo-β-1, 4-glucanase from Bombyx mori expression systems, Molecular biotechnology, 56, 10, 878-889, 2014.10.
79. Zhiqing Li, 門 宏明, Jian Xu, Li Zhu, JAE MAN LEE, takahiro kusakabe, A conserved SUMOylation signaling for cell cycle control in a holocentric species Bombyx mori, INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY, 10.1016/j.ibmb.2014.05.008, 51, 71-79, 2014.08.
80. Erika Taira, Kazuhiro Iiyama, 門 宏明, Kazuki Mori, Taiki Akasaka, KOSUKE TASHIRO, Chisa Yasunaga-Aoki, JAE MAN LEE, takahiro kusakabe, Draft genome sequence of entomopathogenic Serratia liquefaciens strain FK01., Genome Announcements, 2, 3, e00609-e00614, 2014.06.
81. Kazuhiro Iiyama, 門 宏明, Kazuki Mori, Takumi Mitsudome, JAE MAN LEE, takahiro kusakabe, Chisa Yasunaga-Aoki, Characterization of KfrA proteins encoded by a plasmid of Paenibacillus popilliae ATCC 14706 T, Meta gene, 4, 29-44, 2014.06.
82. Kazuhiro Iiyama, 門 宏明, Kazuki Mori, Takumi Mitsudome, JAE MAN LEE, takahiro kusakabe, Chisa Yasunaga-Aoki, Characterization of KfrA proteins encoded by a plasmid of Paenibacillus popilliae ATCC 14706 T, Meta gene, 4, 29-44, 2014.06.
83. Kazuhiro Iiyama, Masahiro Otao, Kazuki Mori, 門 宏明, JAE MAN LEE, takahiro kusakabe, KOSUKE TASHIRO, Shin-Ichiro Asano, Chisa Yasunaga-Aoki, Phylogenetic relationship of Paenibacillus species based on putative replication origin regions and analysis of an yheCD-like sequence found in this region, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 10.1080/09168451.2014.905188, 78, 5, 891-897, 2014.05.
84. Takumi Mitsudome, Jian Xu, Yudai Nagata, Atsushi Masuda, Kazuhiro Iiyama, Daisuke Morokuma, Zhiqing Li,, 門 宏明, JAE MAN LEE, takahiro kusakabe, Expression, purification, and characterization of endo-β-N-acetylglucosaminidase H using baculovirus-mediated silkworm protein expression system, Applied Microbiology and Biotechnology, 172, 8, 3978-3988, 2014.04.
85. Ryohei Sugahara, 門 宏明, JAE MAN LEE, takahiro kusakabe, Middle region of FancM interacts with Mhf and Rmi1 in silkworms, a species lacking the Fanconi anaemia (FA) core complex, INSECT MOLECULAR BIOLOGY, 10.1111/imb.12072, 23, 2, 185-198, 2014.04.
86. 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.
87. Jian Xu, Kaito Yoshimura, 門 宏明, Zhiqing Li, Zhu Li, Tatsuke Tsuneyuki, Kazuhiro Iiyama, takahiro kusakabe, JAE MAN LEE, Establishment of Caenorhabditis elegans SID-1-Dependent DNA Delivery System in Cultured Silkworm Cells, MOLECULAR BIOTECHNOLOGY, 10.1007/s12033-013-9694-0, 56, 3, 193-198, 2014.03.
88. 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.
89. Tsuneyuki Tatsuke, Li Zhu, Zhiqing Li, Hitoshi Mitsunobu, Kaito Yoshimura, 門 宏明, JAE MAN LEE, takahiro kusakabe, Roles of Piwi Proteins in Transcriptional Regulation Mediated by HP1s in Cultured Silkworm Cells, PLOS ONE, 10.1371/journal.pone.0092313, 9, 3, e92313, 2014.03.
90. 門 宏明, JAE MAN LEE, Kazuei Mita, Marian R Goldsmith, takahiro kusakabe, Chromatin-induced spindle assembly plays an important role in metaphase congression of silkworm holocentric chromosomes, Insect biochemistry and molecular biology, 45, 40-50, 2014.02.
91. Jian Xu, 門 宏明, takahiro kusakabe, Zhiqing Li, Zhu Li, Kazuhiro Iiyama, Atsushi Masuda, Takumi Mitsudome, JAE MAN LEE, Establishment of a soaking RNA interference and Bombyx mori nucleopolyhedrovirus (BmNPV)-hypersensitive cell line using Bme21 cell, APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 10.1007/s00253-013-5279-x, 97, 24, 10435-10444, 2013.12.
92. Zhu Li, Tatsuke Tsuneyuki, 門 宏明, Zhiqing Li, Jian Xu, JAE MAN LEE, takahiro kusakabe, Characterization of Tudor-sn-containing granules in the silkworm, Bombyx mori, ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, 10.1016/j.ibmb.2013.04.004, 43, 8, 664-674, 2013.08.
93. 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.
94. 入路　光雄, 北野 載, 清水昭男, LEE JAE MAN, 日下部 宜宏, 山口 明彦, 松山 倫也, Characterization, Localization, and Stage-Dependent Gene Expression of Gonadotropin Receptors in Chub Mackerel (Scomber japonicus) Ovarian Follicles, BIOLOGY OF REPRODUCTION, 10.1095/biolreprod.112.107292, 88, 6, 2013.06.
95. Zhu Li, Y. Masaki, Tatsuke Tsuneyuki, Zhiqing Li, 門 宏明, Jian Xu, JAE MAN LEE, takahiro kusakabe, A MC motif in silkworm Argonaute 1 is indispensible for translation repression, INSECT MOLECULAR BIOLOGY, 10.1111/imb.12023, 22, 3, 320-330, 2013.06.
96. Zhiqing Li, Zhu Li, Jian Xu, 門 宏明, JAE MAN LEE, takahiro kusakabe, AMINO ACID DEPRIVATION-INDUCED EXPRESSION OF ASPARAGINE SYNTHETASE REGULATES THE GROWTH AND SURVIVAL OF CULTURED SILKWORM CELLS, ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, 10.1002/arch.21091, 83, 2, 57-68, 2013.06.
97. 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.
98. Sun Mee Hong, 門 宏明, JAE MAN LEE, takahiro kusakabe, Characterization and recombinant protein expression of ferritin light chain homologue in the silkworm, Bombyx mori, Insect Science, 10.1111/1744-7917.12031, 21, 2, 135-146, 2013.04.
99. 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.
100. Arun M Khurad, Ravindra S Bahekar, Min-Juan Zhang, Ashish D Tiple, JAE MAN LEE, Chuan-Xi Zhang, takahiro kusakabe, Development and characterization of a new Bombyx mori cell line for protein expression, Journal of Asia-Pacific Entomology, 16, 1, 17-22, 2013.03.
101. 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.
102. Kazuhiro Iiyama, Oumi Nishi, 門 宏明, JAE MAN LEE, takahiro kusakabe, Asano Shin-ichiro, Chisa Yasunaga-Aoki, Susumu Shimizu, Phylogenetic analysis of Paenibacillus popilliae and its related taxa based on housekeeping genes., J. Insect Biotech. Seric., , 10.11416/jibs.82.1_001, 82, 1-11, 2013.02.
103. Zhu Li, Zhiqing Li, Tatsuke Tsuneyuki, Daojun Cheng, Jian Xu, Kaito Yoshimura, 門 宏明, Kazuhiro Iiyama, JAE MAN LEE, Qingyou Xia, takahiro kusakabe, Genome-wide identification of Argonaute 1- and Argonaute 2-regulating genes revealed an inhibition of macula-like virus by RNAi pathway in the silkworm, Bombyx mori , J. Insect Biotech. Seric., , 10.11416/jibs.82.1_019, 82, 19-23, 2013.02.
104. 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.
105. 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.
106. 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.
107. 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.
108. 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.
109. 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.
110. 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.
111. 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.
112. 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.
113. Ahmed MH Ali, JAE MAN LEE, Mayumi Yoshida, Kosuke Sakashita, Jumpei Torii, takahiro kusakabe, Akinori Hirashima, Expression and characterization of a recombinant Drosophila tyramine-β-hydroxylase in silkworm infected with recombinant baculovirus, Journal of Asia-Pacific Entomology, http://dx.doi.org/10.1016/j.aspen.2012.06.004, 15, 4, 567-572, 2012.06.
114. 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.
115. 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.
116. 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.
117. Li, Z., Tatsuke, T., Sakashita, K., Zhu, L., Xu, J., Mon, H., Lee, J.M., and Kusakabe, T., Identification and characterization of Polycomb group genes in the silkworm, Bombyx mori., Mol. Biol. Report, , 39, 5575-5588, 2012.01.
118. 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.
119. 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.
120. 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.
121. Iiyama, K., Lee, J.M., Kusakabe, T., Yasunaga-Aoki, C., and Shimizu, S., Improvement in pHERD vectors to express recombinant proteins tagged with hexahistidine at either the NH2- or COOH- terrminal in Pseudomonas aeruginosa., J. Insect Biotech. Seric., , 80, 57-61, 2011.10.
122. Chieda, Y., Iiyama, K., Lee, J.M., Kusakabe, T., Yasunaga-Aoki, C., and Shimizu, S., Virulence of an exotoxin A-deficient strain of Pseudomonas aeruginosa toward the silkworm, Bombyx mori., Microbial. Pathogenesis,, 51, 407-414, 2011.10.
123. 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.
124. 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.
125. 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., 2011.04.
126. Satone, H., Lee, J.M., Oba, Y., Kusakabe, T., Akahoshi, E., Miki, S., Suzuki, N., Sasayama, Y., Nassef, M., Shimasaki, Y., Kawabata, S., Honjo, T., Oshima, Y., Tributyltin-binding protein type 1, a lipocalin, prevents inhibition of osteoblastic activity by tributyltin in fish scales. , Aquat. Toxicol. , 103, 79-84, 2011.02.
127. Mitsunobu, H., Izumi, M., Iiyama, K., Jikuya, H., Lee, J.M., Mon, H., Kawaguchi, Y., Kusakabe, T. , Molecular Characterization of Core Histones in the Silkworm, Bombyx mori., J. Insect Biotech. Seric., 79, 75-83, 2010.10.
128. Sakashita, K., Tatsuke, T., Masaki, Y., Lee, J., Kawaguchi, Y., Kusakabe, T. , dsRNA Binding Activity of Silkworm Larval Hemolymph is Mediated by Lipophorin Complex , J. Fac. Agr., Kyushu Univ. , 401-406, 2010.04.
129. Tatsuke, T., Sakashita, K., Masaki, Y., Lee, J.M., Kawaguchi, Y., Kusakabe, T. , The telomere-specific non-LTR retrotransposons SART1 and TRAS1 are suppressed by Piwi subfamily proteins in the silkworm, Bombyx mori. , Cell. Mol. Biol. Lett. , 15, 128-133, 2010.04.
130. Hong, S.M., Yamashita, J., Mitsunobu, H., Uchino, K., Kobayashi, I., Tamura, T., Nakajima, H., Miyagawa, H., Lee, J.M., Mon, H., Miyata, T., Kawaguchi, Y., Kusakabe, T., Efficient Soluble Protein Production on Transgenic Silkworms Expressing Cytoplasmic Chaperones using Baculovirus Expression System., Appl. Microbiol. Biotech., 2010.04.
131. Mon, H., Sugahara, R., Hong, S.M., Lee, J.M., Kamachi, Y., Kawaguchi, Y., Kusakabe, T. , Analysis of protein interactions with two-hybrid system in cultured insect cells. , Anal. Biochem. , 392, 180-182., 2009.09.
132. Tatsuke, T., Hong, S.M., Tobata, H., Mon, H., Lee, J.M., Kawaguchi, Y., Kusakabe, T., Construction of piggyBac-based Vectors Using Visible and Drug-resistance Marker for Introducing Foreign Genes into Silkworm Cultured Cells. , J. Fac. Agr., Kyushu Univ. , 54, 397-400, 2009.04.
133. Egami, I., Iiyama, K., Zhang, P., Chieda, Y., Ino, N., Hasegawa, K., Lee, J.M., Kusakabe, T., Yasunaga-Aoki, C. and Shimizu, S.,, Insecticidal bacterium isolated from an ant lion larva from Munakata, Japan., J. Appl. Entomol., 133 2177-2124, 2009.03.
134. Karasaki, N., Mon, M., Takahashi, M., Lee, JM., Koga, K., Kawaguchi, Y., and Kusakabe, T., Establishment of Tetracycline-inducible Gene Expression Systems in the Silkworm, Bombyx mori, Biotechnol. Lett., 31 495-500, 2009.03.
135. Sakashita, K., Tatsuke, T., Lee, J.M. Kawaguchi, Y., and Kusakabe, T., Sequence-nonspecific suppression of gene expression by double-stranded RNA in silkworm cultured cells, J. Insect Biotech. Seric. , 78 33-37, 2009.02.
136. 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.
137. Kawaguchi, Y., Tatsuke, T., Kusakabe, T., Lee, J.M. and Koga, K., Singular compound eye architecture of the varnished eye mutation, ve, in Bombyx mori., J. Insect Biotech. Seric. , 77 53-57 (2008), 2008.02.
138. Iiyama, K., Chieda, Y., Lee, J.M., Kusakabe, T., Yasunaga-Aoki, C. and Shimizu, S., Virulence of phospholipase C mutant of Pseudomonas aeruginosa PA01 against the silkworm, Bombyx mori., J. Insect Biotech. Seric. , 77 115-120 , 2008.02.
139. Kawaguchi, Y., Tatsuke, T., Oike, Y., Kusakabe, T., Lee, J.M. and Koga, K., Fertility and hatching of the vit mutant eggs in Bombyx mori., J. Insect Biotech. Seric. , 77 121-124 , 2008.02.
140. Chieda, Y., Iiyama, K., Lee, J.M., Kusakabe, T., Yasunaga-Aoki, C. and Shimizu, S., Inactivation of pyocyanin synthesis genes has no effect on the virulence of Pseudomonas aeruginosa PAO1 toward the silkworm, Bombyx mori., FEMS Microbiol Lett. , 278(1) 101-107 , 2008.02.
141. Hong, S.M., Kusakabe, T., Lee, J.M., Tatsuke, T., Kawaguchi, Y., Kang, M.W., Kang, S.W., Kim, K.A. and Nho, S.K., Structure and expression analysis of the cecropin-E gene from the silkworm, Bombyx mori., Biosci Biotechnol Biochem. , 72(8), 1992-1998 , 2008.02.
142. 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.
143. Zhang P., Aso Y., Jikuya H., Kusakabe T., Lee JM., Kawaguchi Y., Yamamoto K., Banno Y and Fujii H., Proteomic Profiling of the Silkworm Skeletal Muscle Proteins during Larval-Pupal Metamorphosis., J Proteome Res. , 6, 2295-2303., 2007.07.
144. Kawaguchi Y., Yoshida Y., Kusakabe T., Lee JM., and Koga K., Characteristic of Se, the white-sided egg mutation in Bombyx mori., J. Insect Biotech. Seric., 76, 71-78., 2007.06.
145. Iiyama K., ChiedaY., Lee J.M., Kusakabe T., Yasunaga-Aoki C. and Shimizu S. , Effect of inactivation of the superoxide dismutase genes on the virulence of Pseudomonas aeruginosa PAO1 to silkworm, Bombyx mori. , Appl. Environ. Microbiol. , 73(5):1569-75, 2007.03.
146. Sugahara R., Mon H., Yamashita J., Mitsunobu H., Lee J.M., Kawaguchi Y., Koga K. and Kusakabe T. , Heterotrimeric Complex of Replication Protein A, a Single-Stranded DNABinding Protein, from the Silkworm, Bombyx mori. , J. Insect Biotech. Seric. , In press, 2007.03.
147. Yamashita J., Miyagawa Y., Sugahara R., Mon H., Mitsunobu H., Lee J.M., Kawaguchi Y.and Kusakabe T. , Molecular Cloning of Silkworm Cdc37 and its Interaction with Hsp90 Chaperon. , J. Insect Biotech. Seric. , In press, 2007.03.
148. 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.
149. Iiyama,K., Chieda,Y., Lee, JM., Kusakabe, T., Yasunaga-Aoki, C., Lee, J., Kusakabe, T., Shimizu, S., Effect of inactivation of the superoxide dismutase genes on the virulence of Pseudomonas aeruginosa PAO1 to silkworm, Bombyx mori., Appl. Environ. Microbiol., 73, 1569-1575., 2007.03.
150. Takahashi M., Lee J.M., Mon H., Yoshida H., Kawaguchi Y., Maekawa H., Koga K., and Kusakabe T., Radiation resistance and its inheritance in the silkworm, Bombyx mori., J. Fac. Agr., Kyushu Univ., 2006.01.
151. Kawaguchi Y., Kusakabe T., Lee J.M., Nakajima Y., and Koga K., Micropylar structure of chorion of the female sterile mutation, bd, in, Bombyx mori., J. Insect Biotech. Seric., 75, 9-14., 2006.01.
152. Takahashi M., Lee J.M., Mon H., Kawaguchi Y., Koga K., and Kusakabe T., Cell Cycle Arrest induced by Radiation in Cultured Silkworm Cells., J. Insect Biotech. Seric., 75, 23-30, 2006.01.
153. Mitsunobu H., Sakashita K., Mon H., Lee J.M., Kawaguchi Y., Koga K., and Kusakabe T., Construction of gateway-based destination vectors for detecting subcellular localization of proteins in the silkworm, Bombyx mori., J. Insect Biotech. Seric., 75,141-145., 2006.01.
154. Tsukioka H., Takahashi M., Mon H., Okano K., Mita K., Shimada T., Lee J.M., Kawaguchi Y., Koga K., and Kusakabe T., Role of the Silkworm Argonaute2 Homologue Gene in Double-Strand Break Repair of Extrachromosomal DNA., Nucleic Acids Res., 34. 1092-1101., 2006.01.
155. Maeda, T., Kusakabe, T., Lee, J.M., Miyagawa, Y., Kawaguchi, Y. and Koga, K., Efficient nonviral gene transfer mediated by polyethylenimine in an insect cell line., J. Insect Biotech. Seric., 74(1) 21-26, 2005.01.
156. Maeda, T., Lee, J.M., Miyagawa, Y., Koga, K., Kawaguchi, Y. and Kusakabe, T., Cloning and characterization of a ribonuclease L inhibitor from the silkworm, Bombyx mori., DNA Sequence, 10.1080/10425170400028871, 16, 1, 21-27, 16(1) 21-27, 2005.01.
157. Chieda, Y., Iiyama, K., Yasunaga-Aoki, C., Lee, J.M., Kusakabe, T. and Shimizu, S., Pathogenicity of gacA mutant of Pseudomonas aeruginosa PA01 in the silkworm, Bombyx mori., FEMS Microbiol. Lett., 10.1016/j.femsle.2005.01.032, 244, 1, 181-186, 244(1) 181-186, 2005.01.
158. Miyagawa, Y., Lee, J.M., Maeda, T., Koga, K., Kawaguchi, Y. and Kusakabe, T., Differential expression of a Bombyx mori AHA1 homologue during spermatogenesis., Insect Mol. Biol., 10.1111/j.1365-2583.2004.00553.x, 14, 3, 245-253, in press, 2005.01.
159. 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.
160. Mon, H., Kusakabe, T., Lee, J.M., Kawaguchi, Y. and Koga, K., In vivo DNA double-strand breaks enhance gene targeting in cultured silkworm cells., Comp. Biochem. Phys. Part B, 10.1016/j.cbpc.2004.06.013, 139, 1, 99-106, 139 99-106, 2004.01.
161. Lee, J.M., Kusakabe, T., Kawaguchi, Y., Takahashi, M., Mon, H., Nho, S.K. and Koga, K., Molecular cloning and characterization of the translationally controlled tumor protein (TCTP) gene in Bombyx mori., Comp. Biochem. Phys. Part B, 10.1016/j.cbpc.2004.06.004, 139, 1, 35-43, 139 35-43, 2004.01.
162. Miyagawa, Y., Kusakabe, T., Lee, J.M., Maeda, T., Kawaguchi, Y. and Koga, K., Isolation and characterization of differently expressed cDNAs in a meiotic recombination strain of Bombyx mori., J. Insect Biotech. Seric., 73(3) 117-127, 2004.01.
163. Lee, J.M., Kusakabe, T., Kawaguchi, Y., Aoki, C., Nho, S.K., Nakajima, Y. and Koga, K., Molecular characterization of Heat Shock Cognate 70-4 promoter from the silkworm, Bombyx mori, J. Insect Biotech. Seric., 72 (1) 33-39, 2003.01.


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