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Masato Hino,Takuji Kawanami,Jian Xu,Daisuke Morokuma,Kazuma Hirata,Mami Yamashita,Noriko Karasaki,Tuneyuki Tatsuke,Hiroaki Mon,Kazuhiro Iiyama,Noriho Kamiya,Yutaka Banno,Takahiro Kusakabe,이재민 한국응용곤충학회 2016 Journal of Asia-Pacific Entomology Vol.19 No.2
Interleukin 2 (IL-2) is a pharmacologically vital cytokine secreted mainly by activated CD4+ T lymphocyte. Recombinant human IL-2 (rhIL-2) protein has already been globally applied as an immune-therapeutic reagent for various diseases. Therefore, there is great interest in developing an active form of rhIL-2 in very large amounts and with excellent purity for clinical use. In this study, we successfully mass-expressed and purified N- or C-terminal tandem tag-fused rhIL-2 in a baculovirus expression vector system (BEVS) using silkworm larvae as factories. We confirmed that the intrinsic instability of hIL-2 causes the loss and low recovery of N-tagged rhIL-2 and that C-tagged rhIL-2 ismore suitable for mass production. Furthermore, the activity of purified rhIL-2s was further validated by a cell proliferation assay of a human natural killer cell line, and both rhIL-2 proteins produced in the silkworm-BEVS exhibited comparable activity to that of the commercial E.coli-derived rhIL-2. Taken together, our results and strategies could contribute greatly to the mass production of active rhIL-2.
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.
( 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.
Jianping Chen,Jian Xu,Masato Hino,Mami Yamashita,Kazuma Hirata,Anandrao Ashok Patil,Tsuneyuki Tatsuke,Hiroaki Mon,Yutaka Banno,Takahiro Kusakabe,Jae Man Lee 한국응용곤충학회 2016 Journal of Asia-Pacific Entomology Vol.19 No.3
G protein-coupled receptors (GPCRs) are seven transmembrane proteins, which play an essential role in transmitting various extracellular signals into cells. For functional and structural analysis of GPCRs, it is necessary to produce active GPCRs in high quantitieswith outstanding purity. Fortunately, earlier baculovirus expression vector system has been reported as a proven functional GPCR mass-production tool. Therefore, in this study, we selected Bombyx mori allatostatin-C neuropeptide receptor BNGR-A1 as a GPCR reporter protein, which has been already proved successful binding of ligand. We confirmed its expression profile in various silkworm tissues and cell lines and then verified its plasma membrane subcellular localization in cultured silkworm BmN4 cells. In addition, we constructed recombinant baculoviruses for BNGR-A1, its ligand allatostatin-C (BmAST-C) and related eightG proteins (Gs, G12,Gα4, Gq, Gβ2,Gβ3, Gβ5 andGγ) and subsequentlymonitored the extracellular or intracellular expression of BNGR-A1 by co-infection with its ligand and cognate G proteins-expressing viruses. It is interesting to observe that different combinations of G proteins could result in changes or even undetectable of final yields of BNGR-A1, suggesting the essential roles of G proteins involved in the GPCR expression or stabilization. The present study demonstrated that co-infection of recombinant viruses expressing Gα4β3γ trimer enhanced the production of BNGR-A1 on BV fractions. To our knowledge, this is a fine strategy for identifying the specific G protein partner responsible for certain GPCR of interest. These studies would provide a novel idea for improving GPCR expression in the silkworm by baculovirus expression vector system.
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.
Daisuke Morokuma,Jian Xu,Hiroaki Mon,Kazuma Hirata,Masato Hino,Shoko Kuboe,Mami Yamashita,Takahiro Kusakabe,이재만 한국응용곤충학회 2015 Journal of Asia-Pacific Entomology Vol.18 No.2
Glycosylation is an important post-translational modification that confers various biological activities, structural stability, and inter-molecular interactions to proteins. Baculovirus expression vector system (BEVS) is widely used to produce recombinant glycoproteins, which may not be suitable for clinical use due to differences in the N-linked glycan structure between insects and mammals. It is necessary to develop an appropriate model protein-base platform for glycoanalysis to engineer the insect-type N-glycosylation pathway into human type efficiently. In this study,we employed human plasma protein alpha 1-acid glycoprotein (α1AGP). Itwas highly secreted from cultured silkworm cells and larvae when using the BEVS and glycosylated with insect type N-linked glycans. Interestingly, when separated on SDS-PAGE, the purified recombinant α1AGP secreted into silkworm haemolymph generated six distinct products from three alternative translates, suggesting that α1AGP has variations for the recognition or choice of glycosylation sites.
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.
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.
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.