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Efficient Production of Recombinant Protein in HSPs (Heat Shock Porteins) Transgenic Silkworm
Sun-Mee Hong,Jae-Man Lee,Takahiro Kusakabe 한국응용곤충학회 2011 한국응용곤충학회 학술대회논문집 Vol.2011 No.05
Many thousands of recombinant proteins have been successfully produced in baculovirus - infected insect cells and larvae. In this study, to improve its value and the yield of recombinant protein production, we constructed transgenic silkworm using Heat shock genes with regard to protein folding. This time, we adapted GAL4/UAS system to express at necessary time point and to carry genes for foreign protein. First, we generated two transgenic cells and silkworm lines that carried the silkworm heat shock proteins, UAS-HOP and UAS-HSC70 and UAS-HSP70 and UAS-HSP40 construct plus 3xP3-DsRED. Subsequently, to drive the GAL4 gene as activatorvector, we engineered Baculoviruses that contain the GAL4 under the P10 promoter linked to the expression cassette of interest foreign genes under the polyhedron promoter. Also, activator vector linked to the GAL4 was designed expressing 6xHis and 6xHis–GST tag. Infection of silkworm larvae with recombinant virus, His-tagged human C3d gene was more efficiently produced transgenic silkworm than that of wild-type, but not His-GST tagged. We show the possibilityin use of HSPs transgenic silkworm system by GAL4/ UAS BmNPV that can generate the efficient production of foreign protein.
조선정,최지현,강주일,임재환,석영식,이재만,Takahiro Kusakabe,홍선미 한국잠사학회 2014 International Journal of Industrial Entomology Vol.29 No.2
Recombinant proteins can be generated quickly and easily in large amounts and at low-costin silkworm larvae by using Bombyx mori nuclear polyhedrosis virus (BmNPV). We searchedfor high-permissive silkworm strains that have high production levels of heterologous proteinsand are thus suitable for use as biofactories. In this study, we performed the analysis usinga BmNPV vector expressing luciferase as a marker, and we confirmed protein expressionby evaluating luciferase activity, determined by western blotting and luciferase ELISA, andconfirmed transcription expression by semi- and quantitative real time PCR. For the selectionof host silkworm strains, we first chose 52 domestic BmNPV sensitive strains and thenidentified 10 high-permissive and 5 low-permissive strains. In addition, to determine whichhybrid of the high-permissive strains would show heterosis, nine strains derived through threewaycrossing were tested for luciferase activity by western blotting, and luciferase ELISA. We found a correlation between luciferase activity and luciferase protein expression, but nottranscription. There was no noticeable difference in protein expression levels between Jam313as the high-permissive control strain and the three-way hybrid strains; however, the three-waycross strains showed lower luciferase activity compared with Jam313. In this study, luciferaseprotein production in the larvae of 52 domestic silkworm strains was elucidated using BmNPV.
홍선미,최지현,조선정,송성규,이재만,Takahiro Kusakabe 한국생물공학회 2015 Biotechnology and Bioprocess Engineering Vol.20 No.3
Korean mistletoe lectins (KMLs) possess many biological activities, including anti-tumor, apoptosis-inducing, anti-metastatic, and anti-angiogenic activities. Recombinant KML-1A (rKML-1A) has been previously expressed in soluble form in Escherichia coli cells. However, the expression of rKML-1B in soluble form remains to be accomplished. In this study, we describe the production, purification, and characterization of recombinant KML-1B by using a baculovirus expression system, which employs silkworm larvae and pupae as hosts. Approximately 495 and 702 μg/mL of a 36-kDa rKML-1B protein were produced without a signal peptide by silkworm larvae and pupae, respectively. Treatment of the recombinant protein with N- or O-deglycosylase led to a decrease in its molecular mass, indicating the N- and/or O-glycosylation of rKML-1B. Purified rKML-1B displayed radical scavenging activities toward 1,1-diphenyl-2-picrylhydrazyl and 2,2'- amino-di-[3-ethylbenzthiazoline sulfonate], with a half maximal inhibitory concentration (IC50) of approximately 95 μg/mL. This activity was discovered to be stable at 65°C. To our knowledge, this is the first report detailing the biological activity of plant-derived, pure rKML-1B.
Heterologous Production and Glycosylation of Japanese Eel Follitropin Using Silkworm
홍선미,최지현,조선정,민관식,김대정,이재만,Takahiro Kusakabe 한국생물공학회 2019 Biotechnology and Bioprocess Engineering Vol.24 No.5
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 Nand 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.
최지현,김대중,홍선미,조선정,민관식,손영창,이재만,Takahiro Kusakabe 한국생물공학회 2016 Biotechnology and Bioprocess Engineering Vol.21 No.3
Luteinizing hormone (LH), a gonadotropin hormone (GTH) of the pituitary glycoprotein family, is important in oocyte maturation, ovulation, and spermiation. In this study, we generated a Japanese eel LH (JeLH) with and without the equine chorionic gonadotropin (eCG) carboxyl-terminal peptides under the control of the polyhedron in the silkworm pupae BES to determine their expression levels, glycosylation profile, and in vitro bioactivity. The target proteins were highly expressed in the pupae hemolymph. Recombinant JeLH·eCG and JeLH were N- or O-glycosylated, as shown by periodic-acid Schiff staining, deglycosidase enzyme treatment, and lectin blot analyses, and showed no significant difference in the in vitro bioactivity. Both single-chain hormones and salmon pituitary extract induced maturation of Japanese eel oocytes in vitro. Recombinant LHs produced in silkworm pupae might be suitable candidates for in vivo experiments, because they can be produced in sufficient amount and can undergo N-glycosylation.
Atsushi Masuda,Jian Xu,TakumiMitsudome,Daisuke Morokuma,Hiroaki Mon,Yutaka Banno,Takahiro Kusakabe,이재만 한국응용곤충학회 2015 Journal of Asia-Pacific Entomology Vol.18 No.2
Endo-β-N-acetylglucosaminidase H (Endo H) catalyzes cleavage between the GlcNAc residues of the chitobiose core of N-linked glycans, leaving one GlcNAc residues attached to asparagine. Endo H cleaves high mannose and hybrid, but not complex, N-linked oligosaccharides on glycoproteins. Because of its unique specificity, Endo H is widely used for the structural and functional analyses of glycoproteins. In our previous study, the recombinant Endo H was produced as a secreted protein using silkworm–baculovirus expression system, but the yield was low (30 μg Endo H/10 ml larval hemolymph) compared to that of Escherichia coli. In this study, we purified active recombinant Endo H as an intracellular protein from fat body of silkworm infected with the recombinant baculovirus expressing Endo H without the exogenous signal peptide. Remarkably, the yield (9.3mgfrom20 silkwormlarvae)was about 310-fold higher than that secreted into larval hemolymph as reported previously. In addition,we screened the silkwormstrains maintained in Kyushu University and identified n17 as a high-level expression strain for Endo H.
Development and characterization of a new Bombyx mori cell line for protein expression
Arun M. Khurad,Ravindra S. Bahekar,Min-Juan Zhang,Ashish D. Tiple,Jae Man Lee,Chuan Xi Zhang,Takahiro Kusakabe 한국응용곤충학회 2013 Journal of Asia-Pacific Entomology Vol.16 No.1
A Bombyx mori continuous cell line, designated DZNU-Bm-17, was established from larval ovaries. The cells were initially grown in MGM-448 insect cell culture medium supplemented with 10% fetal bovine serum and 3% heat inactivated B. mori hemolymph at 25±1 °C and later adapted gradually to TNM-FH medium. Partially adhered refractive cells were the predominant cell type in the culture. The cells took about 1055 days to complete 100 passages in TNM-FH medium. The population doubling time of the cell line was about 30–34 h at 25±1 °C. The cell population was largely diploid, but a few triploids and tetraploids were also observed. DNA profiles using simple sequence repeat loci established the differences between the DZNU-Bm-1, Bm-5, DZNU-Bm-12, DZNU-Bm-17, and BmN cell lines. The cell line was susceptible to budded virus of B. mori nucleopolyhedrovirus (BmNPV), and 85–92% of the cells harbored BmNPV with an average of 15 occlusion bodies/infected cell. The cells expressed the luciferase and green fluorescent proteins using the BmNPV bacmid vector.Wesuggest the usefulness of the DZNU-Bm-17 cell line for BmNPV-based baculoviral expression studies.
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.