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( Tae Won Goo ),( Seong Wan Kim ),( Kwang Ho Choi ),( Seong Ryul Kim ),( Seok Woo Kang ),( Seung Won Park ),( Eun Young Yun ) 한국잠사학회 2013 International Journal of Industrial Entomology Vol.26 No.2
The insect baculovirus expression vector system (BEVS) is useful for producing biologically active recombinant proteins. However, the overexpression of heterologous proteins using this system often results in misfolded proteins and the formation of protein aggregates. To overcome this limitation, we developed a versatile baculovirus expression and secretion system using Bombyx mori protein disulfide isomerase (bPDI) as a fusion partner. bPDI gene fusion was found to improve the secretions and antibacterial activities of recombinant nuecin and enbocin proteins. Thus, we conclude that bPDI gene fusion is a useful addition to BEVS for the large-scale production of bioactive recombinant proteins.
Utilization of the Bombyx mori Hypothetical Protein 32 Promoter for Efficient Transgene Expression
( Tae Won Goo ),( Sung Wan Kim ),( Seong Ryul Kim ),( Seung Won Park ),( Seok Woo Kang ),( Kwang Gill Lee ),( O Yu Kwon ),( Eun Young Yun ) 한국잠사학회 2010 International Journal of Industrial Entomology Vol.20 No.2
For stable germline transformation, the promoter of Bombyx mori cytoplasmic actin gene (BmA3) has been used for ubiquitous expression of transgenes. So far, no strong promoter is available for ubiquitous expression in B. mori, excluding BmA3 promoter. To identify more powerful promoter than previously reported BmA3 promoter, we isolated 9 clones that show stronger signal compared to BmA3 by a dot blot hybridization. Among these 9 clones, we focused on one clone which has high amino acid homology (85%) with hypothetical protein 32 gene of Lonomia obliqua. This clone, named bHp32 (B. mori hypothetical protein 32) was ubiquitously expressed in all tissues and developmental stage of fifth instar B. mori larvae. As result of promoter assay using dual luciferase assay system, we found the highest transcription activity region (-1,200/+220) in the 5`-flanking region of bHp32 gene, which has 42-fold more intensive promoter activity than BmA3 promoter. Moreover, the bHp32 promoter was normally regulated in Bm5, Sf9, and S2 cells. Therefore, we suggest that bHp32 promoter may be used more powerful and effectively for transgene expression in various insects containing B. mori as a universal promoter.
Utilization of the Bombyx mori Hypothetical Protein 32 Promoter for Efficient Transgene Expression
Goo, Tae-Won,Kim, Sung-Wan,Kim, Seong-Ryul,Park, Seung-Won,Kang, Seok-Woo,Lee, Kwang-Gill,Kwon, O-Yu,Yun, Eun-Young Korean Society of Sericultural Science 2010 International Journal of Industrial Entomology Vol.20 No.2
For stable germline transformation, the promoter of Bombyx mori cytoplasmic actin gene (BmA3) has been used for ubiquitous expression of transgenes. So far, no strong promoter is available for ubiquitous expression in B. mori, excluding BmA3 promoter. To identify more powerful promoter than previously reported BmA3 promoter, we isolated 9 clones that show stronger signal compared to BmA3 by a dot blot hybridization. Among these 9 clones, we focused on one clone which has high amino acid homology (85%) with hypothetical protein 32 gene of Lonomia obliqua. This clone, named bHp32 (B. mori hypothetical protein 32) was ubiquitously expressed in all tissues and developmental stage of fifth instar B. mori larvae. As result of promoter assay using dual luciferase assay system, we found the highest transcription activity region (-1,200/+220) in the 5'-flanking region of bHp32 gene, which has 42-fold more intensive promoter activity than BmA3 promoter. Moreover, the bHp32 promoter was normally regulated in Bm5, Sf9, and S2 cells. Therefore, we suggest that bHp32 promoter may be used more powerful and effectively for transgene expression in various insects containing B. mori as a universal promoter.
Secretion of the Antibacterial Recombinant Protein Enbocin
Goo, Tae Won,Yun, Eun Young,Kim, Sung Wan,Choi, Kwang Ho,Kang, Seok Woo,Kwon, Kisang,Choi, Joung-Soon,Kwon, O-Yu Walter de Gruyter GmbH 2008 Zeitschrift fur Naturforschung. Section C Vol.63 No.3
<P>The insect baculovirus expression vector system (BEVS) is useful for the production of biologically active recombinant proteins. However, the overexpression of foreign proteins in this system often results in misfolded proteins and the formation of protein aggregates. To overcome this limitation, we have developed a versatile baculovirus expression and secretion system using the Bombyx mori protein disulfide isomerase (bPDI) as a fusion partner. bPDI gene fusion improved the secretion and antibacterial activity of recombinant enbocin proteins. Thus, bPDI gene fusion is a useful addition to the BEVS for the large-scale production of bioactive recombinant proteins</P>
Goo, Tae-Won,Yun, Eun-Young,Kim, Sung-Wan,Park, Kwang-Ho,Hwang, Jae-Sam,Kwon, O-Yu,Kang, Seok-Woo Korean Society of Sericultural Science 2003 International Journal of Industrial Entomology Vol.7 No.2
Protein disulfide isomerase (PDI) found in the endoplasmic reticulum (ER) catalyzes disulfide bond exchange and assists in protein folding of newly synthesized proteins. PDI also functions as a molecular chaperone and has been found to be associated with proteins in the ER. In addition, PDI functions as a subunit of two more complex enzyme systems: the prolyl-4-hydroxylase and the triacylglycerol transfer proteins. A cDNA that encodes protein disulfide isomerase was previously isolated from Bombyx mori (bPDI), in which open reading frame of 494 amino acids contained two PDI-typical thioredoxin active site of WCGHCK and an ER retention signal of the KDEL motif at its C-terminal, and we report its functional characterization here. This putative bPDI cDNA is expressed in insect Sf9 cells as a recombinant proteins using baculovirus expression vector system. The bPDI recombinant proteins are successfully recognized by antirat PDI antibody, and shown to be biologically active in vitro by mediating the oxidative refolding of reduced and scrambled RNase. This suggests that bPDI may play an important role in protein folding mechanism of insects.
A Bombyx mori Transcription Factor, ATFC Binds Directly to the UPRE of Molecular Chaperones
Goo, Tae-Won,Yun, Eun-Young,Kim, Sung-Wan,Park, Kwang-Ho,Hwang, Jae-Sam,Kwon, O-Yu,Kang, Seok-Woo Korean Society of Sericultural Science 2003 International Journal of Industrial Entomology Vol.7 No.2
Cells respond to an accumulation of unfolded proteins in the endoplasmic reticulum (ER) by increasing transcription of genes encoding molecular chaperones and folding enzymes. The information is transmitted from the ER lumen to the nucleus by intracellular signaling pathway, called the unfolded protein response (UPR). In Saccharomyces cerevisiae, such induction is mediated by the cis-acting unfolded response element (UPRE) which has been thought to be recognized by Hac1p transcription factor. We cloned the ATFC gene showing similarity with Hac1p, and then examined to determine whether ATFC gene product specifically binds to UPRE by electrophoretic mobility shift assays. ATFC gene product displayed appreciable binding ${to ^{32}}P-labelled$ UPRE. Therefore, we concluded that ATFC represents a major component of the putative transcription factor responsible for the UPR leading to the induction of ER-localized stress proteins.