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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,이재만 한국응용곤충학회 2018 Journal of Asia-Pacific Entomology Vol.21 No.2
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.
Kakino Kohei,Masuda Akitsu,Hino Masato,Ebihara Takeru,Xu Jian,Mon Hiroaki,Fujita Ryosuke,Fujii Tsuguru,Kusakabe Takahiro,Lee Jae Man 한국응용곤충학회 2020 Journal of Asia-Pacific Entomology Vol.23 No.3
Recently, T7 Endonuclease I (T7E1) cleavage assay has been widely employed as an efficient approach for detecting mutations from CRISPR/Cas9 targeted samples. This enzyme is sufficient to detect single- and multiple-base mismatches from various heteroduplex DNA samples. However, T7E1 is quite expensive for researchers to use it only for screening mutations, especially in the condition of a large number of test samples. Regarding the production of this enzyme, to data, only the E. coli system has been reported and the highly overexpressed T7E1 seems toxic to the E. coli host cells. Thus, in this study, we tested whether the silkwormbaculovirus expression vector system (BEVS) is suitable to produce recombinant T7 Endonuclease I (rT7E1). The rT7E1 with N- or C-tags in cultured silkworm cells and silkworm pupae were successfully expressed. Our results demonstrated that the rT7E1-Ntag was highly expressed in silkworm pupae and we obtained rT7E1 proteins in high purity. Moreover, rT7E1 from silkworm-BEVS sufficiently recognized and cleaved the mismatches of designed and CRISPR/Cas9-mediated DNA substrates, which was equivalent to the commercial rT7E1 of the E. coli system. Taken together, our study would greatly support the genome-editing research by providing a cost-effective and active rT7E1 enzyme.
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,이재만 한국응용곤충학회 2019 Journal of Asia-Pacific Entomology Vol.22 No.2
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 Oglycosidase. Thus, our study provides important clues to produce highly active rEndoEF O-glycosidases employing silkworm-BEVS as an alternative.
Morokuma, Daisuke,Hino, Masato,Tsuchioka, Miho,Masuda, Akitsu,Mon, Hiroaki,Fujiyama, Kazuhito,Kajiura, Hiroyuki,Kusakabe, Takahiro,Lee, Jae Man Korean Society of Sericultural Science 2018 International Journal of Industrial Entomology Vol.36 No.1
N-glycosylation is an important posttranslational modification that results in a variety of biological activities, structural stability, and protein-protein interactions. There are still many mysteries in the structure and function of N-glycans, and detailed elucidation is necessary. Baculovirus expression system (BES) is widely used to produce recombinant glycoproteins, but it is not suitable for clinical use due to differences in N-glycan structure between insects and mammals. It is necessary to develop adequate model glycoproteins for analysis to efficiently alter the insect-type N-glycosylation pathway to human type. The previous research shows the recombinant alpha 1-acid glycoprotein (${\alpha}1AGP$) secreted from silkworm cultured cells or larvae is highly glycosylated and expected to be an excellent research candidate for the glycoprotein analysis expressed by BES. Therefore, we improved the ${\alpha}1AGP$ to be a better model for studying glycosylation. The modified ${\alpha}1AGP$ (${\alpha}1AGP{\Delta}$) recombinant protein was successfully expressed and purified by using BES, however, the expression level in silkworm cultured cells and larvae were lower than that of the ${\alpha}1AGP$. Subsequently, we confirmed the detailed profile of N-glycan on the ${\alpha}1AGP{\Delta}$ by LS/MS analysis the N-glycan structure at each glycosylation site. These results indicated that the recombinant ${\alpha}1AGP{\Delta}$ could be usable as a better model glycoprotein of N-glycosylation research in BES.
Nagai Ryo,Ebihara Takeru,Kakino Kohei,Masuda Akitsu,Xu Jian,Minamihata Kosuke,Kamiya Noriho,Kongkrongtong Tatphon,Kawahara Masahiro,Mon Hiroaki,Fujii Tsuguru,Kusakabe Takahiro,Lee Jae Man 한국응용곤충학회 2021 Journal of Asia-Pacific Entomology Vol.24 No.3
Due to the biological significance and therapeutic potential of Interleukin-3 (IL-3) secreted mainly by activated T cells, various protein expression systems have been challenged to produce recombinant IL3 to meet the increasing demands worldwide. Recently, we established an updated silkworm-based baculovirus expression vector system (silkworm-BEVS), which in most cases, produces eukaryotic proteins in biological or enzymatical active forms with considerable amounts. We attempted to reconstruct and express a recombinant mouse IL-3 (rMmIL-3) with C-terminal His8-Strep tags in silkworm-BEVS in the current study. From our results, we gained an active glycosylated rMmIL-3 protein in a substantial amount and quality. As compared with the E. coli expression system, silkworm-BEVS is a better choice regarding the glycosylations attached in rMmIL-3 and upscalable system in case that a commercial amount is required in the future. Collectively, our method shares an excellent model to produce interleukin molecular for approaching pharmaceutical applications.
Masahiko Kobayashi,Jian Xu,Kohei Kakino,Akitsu Masuda,Masato Hino,Naoki Fujimoto,Kosuke Minamihata,Noriho Kamiya,Hiroaki Mon,Hiroshi Iida,Masateru Takahashi,Takahiro Kusakabe,이재만 한국응용곤충학회 2020 Journal of Asia-Pacific Entomology Vol.23 No.1
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.
Takumi Yano,이재만,Jian Xu,Yoshiki Morifuji,Akitsu Masuda,Masato Hino,Daisuke Morokuma,Ryosuke Fujita,Masateru Takahashi,Takahiro Kusakabe,Hiroaki Mon 한국응용곤충학회 2019 Journal of Asia-Pacific Entomology Vol.22 No.2
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 Cterminal 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.
( Daisuke Morokuma ),( Masato Hino ),( Miho Tsuchioka ),( Akitsu Masuda ),( Hiroaki Mon ),( Kazuhito Fujiyama ),( Hiroyuki Kajiura ),( Takahiro Kusakabe ),( Jae Man Lee ) 한국잠사학회 2018 International Journal of Industrial Entomology Vol.36 No.1
N-glycosylation is an important posttranslational modification that results in a variety of biological activities, structural stability, and protein-protein interactions. There are still many mysteries in the structure and function of N-glycans, and detailed elucidation is necessary. Baculovirus expression system (BES) is widely used to produce recombinant glycoproteins, but it is not suitable for clinical use due to differences in N-glycan structure between insects and mammals. It is necessary to develop adequate model glycoproteins for analysis to efficiently alter the insect-type N-glycosylation pathway to human type. The previous research shows the recombinant alpha 1-acid glycoprotein (a1AGP) secreted from silkworm cultured cells or larvae is highly glycosylated and expected to be an excellent research candidate for the glycoprotein analysis expressed by BES. Therefore, we improved the a1AGP to be a better model for studying glycosylation. The modified a1AGP (a1AGPΔ) recombinant protein was successfully expressed and purified by using BES, however, the expression level in silkworm cultured cells and larvae were lower than that of the a1AGP. Subsequently, we confirmed the detailed profile of N-glycan on the a1AGPΔ by LS/MS analysis the N-glycan structure at each glycosylation site. These results indicated that the recombinant a1AGPΔ could be usable as a better model glycoprotein of N-glycosylation research in BES.