• Journal of Microbiology and Biotechnology
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• v.21 no.7
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• pp.679-685
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• 2011

Xanthomonas oryzae pv. oryzae (Xoo) produces a putative effector, XoAvrBs2. We expressed XoAvrBs2 homologously in Xoo with a TAP-tag at the C-terminus to enable quantitative analysis of protein expression and secretion. Addition of rice leaf extracts from both Xoo-sensitive and Xoo-resistant rice cultivars to the Xoo cells induced expression of the XoAvrBs2 gene at the transcriptional and translational levels, and also stimulated a remarkable amount of XoAvrBs2 secretion into the medium. In a T3SS-defective Xoo mutant strain, secretion of the TAPtagged XoAvrBs2 was blocked. Thus, we elucidated the transcriptional and translational expressions of the XoAvrBs2 gene in Xoo was induced in vitro by the interaction with rice and the induced secretion of XoAvrBs2 was T3SSdependent. It is the first report to measure the homologous expression and secretion of XoAvrBs2 in vitro by rice leaf extract. Our system for the quantitative analysis of effector protein expression and secretion could be generally used for the study of host-pathogen interactions.

• International Journal of Industrial Entomology and Biomaterials
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• v.26 no.2
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• pp.119-123
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• 2013

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.

• Journal of Microbiology and Biotechnology
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• v.8 no.1
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• pp.34-41
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• 1998

A method was developed for the controlled expression of an antimicrobial peptide magainin in Escherichia coli. A series of concatemeric magainin genes was constructed with a gene amplification vector, and fused to the 3'end of malE gene encoding the affinity ligand, E. coli maltose-binding protein (MBP). The construct directed the synthesis of the fusion protein with the magainin polypeptide fused to the C-terminus of MBP. The fusion protein was expressed in a tightly regulatable expression system which was under the control of an invertible promoter. The MBP-fused magainin monomer was expressed efficiently. However, the expression level of the MBP-fused magainin in E. coli decreased with the increasing size of multimers possibly because of the transcription and translation inhibition by the multimeric peptides. After purification using an amylose affinity column, the fusion protein was digested by factor Xa at a specific cleavage site between the monomers. The recombinant magainin had an antimicrobial activity identical to that of synthetic magainin. This experiment shows that a biologically active, antimicrobial peptide magainin can be produced by fusing to MBP, along with a promoter inversion vector system.

• Journal of the Korean Society of Tobacco Science
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• v.20 no.1
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• pp.40-49
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• 1998

Protoplasts were isolated from mesophyll of tobacco(Nicotiana glauca) transformed with kanamycin-resistant gene (NPT II gene) and potato hairy root callus containing Ri plasmid of Agrobacterium rhiEogenes, and protoplasm fusion was made between the isolated protoplasts. The transgenic tobacco leaf tissue could grow on the media containing high concentrations of kanamycin, but not on the phytohormone-free media. On the other hand, the potato hairy root calli could be cultured on the phytohormone-free media but not on media containing more than 40 ㎍/ml kanamycin. In these conditions, the viability of both protoplasts were above 90%, These selection markers were used for the selection of protoplasts fused between the two, i.e. protoplast fusion was detected using selection media containing 100㎍/ml kanamycin and with no phytohormone. The mixture of 1.0% cellulase, 0.3% macerozyme, and 0.7M mannitol was best for the maximum protoplast production for tobacco, and that of 2.0% cellulase, 2.0% macerozyme, 1.0% dricelase, and 0.5M mannitol for potato. Both tobacco mesophyll and potato callus protoplasts were fused by using PEG solution on the selectable medium. Cell walls were regenerated after 5 days in this medium, and colonies were alive until 4 weeks after cultural, but died after 6 weeks.

• BMB Reports
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• v.41 no.5
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• pp.400-403
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• 2008

The insect baculovirus expression vector system (BEVS) is useful for producing biologically active recombinant proteins. However, the overexpressions of foreign 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 proteins. Thus, we conclude that bPDI gene fusion is a useful addition to BEVS for the large-scale production of bioactive recombinant proteins.

• International Journal of Industrial Entomology and Biomaterials
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• v.21 no.2
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• pp.151-155
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• 2010

Protein disulfide isomerase (PDI) catalyzes the oxidation of disulfides and the isomerizatiob of incorrect disulfides in new polypeptides during folding in the oxidizing environment of the endoplasmic reticulum (ER). To increase recombinant protein hSCF (human stem cell factor) production, we have developed expression system using the Bombyx mori PDI (bPDI) as a fusion partner. bPDI gene fusion was found to improve the production of recombinant hSCFs. Thus, we conclude that bPDI gene fusion will be very useful for the large-scale production of biologically active recombinant proteins.

• International Journal of Industrial Entomology and Biomaterials
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• v.34 no.1
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• pp.11-15
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• 2017

We constructed the fibroin H-chain expression system to produce enhanced cyan fluorescent proteins (ECFP) in transgenic silkworm cocoon. Fluorescent cocoon could be made by fusing ECFP cDNA to the heavy chain gene and injecting it into a silkworm. The ECFP fusion protein, each with N- and C-terminal sequences of the fibroin H-chain, was designed to be secreted into the lumen of the posterior silk glands. The expression of the ECFP/H-chain fusion gene was regulated by the fibroin H-chain promoter. The use of the 3xP3-driven EGFP cDNA as a marker allowed us to rapidly distinguish transgenic silkworms. The EGFP fluorescence became visible in the ocelli and in the central and peripheral nervous system on the seventh day of embryonic development. A mixture of the donor and helper vector was micro-injected into 1,020 Kumokjam, bivoltin silkworm eggs. We obtained 6 broods. The cocoon was displayed strong blue fluorescence, proving that the fusion protein was present in the cocoon. Accordingly, we suggest that the ECFP fluorescence silk will enable the production of novel biomaterial based on the transgenic silk.

• Journal of Life Science
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• v.19 no.4
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• pp.549-552
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• 2009

Genetic mutations by gene fusion result from chromosomal rearrangement, trans-splicing, and intergenic splicing. Trans-splicing is a phenomenon in which two pre-mRNAs grow together into one. We analyzed the trans-splicing products in embryonic stem cells. By using bioinformatic tools, 70 trans-splicing transcripts were identified. They are classified into 6 types according to fusion pattern: 5'UTR-5'UTR, 5'UTR-3'UTR, 3'UTR-3'UTR, 5'UTR-CDS, 3'UTR-CDS, CDS-CDS. The fusion products are more abundant in CDS regions than in UTR regions, which contain multiple intron numbers. Chromosome analysis showing gene fusion via trans-splicing indicated that chromosomes 17 and 19 were activated. These data are of great use for further studies in relation to fusion genes and human diseases.

• BMB Reports
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• v.31 no.3
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• pp.303-306
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• 1998

Serine proteinase cDNA fragment from protozoan parasite Acanthamoeba culbertsoni was amplified by the reverse transcription-polymerase chain reaction (RTPCR) using degenerate oligonucleotide primers derived from conserved serine proteinase sequences. The amplified DNA fragment was subcloned and sequenced. The sequence analysis and alignment showed significant sequence similarity to other eukaryotic serine proteinases and conservation of the His, Asp, and Ser residues that form the catalytic triad. The cDNA fragment was cloned into the pGEMEX-1 expression vector and expressed in Escherichia coli. A resulting fusion protein of 56 kDa had proteolytic activity. The fusion protein reacted with sera of mice immunized with purified serine proteinase of A. culbertsoni in Western blot. Immune recognition of the fusion protein by mouse antisera suggested that the fusion protein may be valuable as a diagnostic reagent.

• International Journal of Industrial Entomology and Biomaterials
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• v.16 no.2
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• pp.87-92
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• 2008

Bombyx mori nucleopolyhedrovirus (BmNPV) orf47 gene was characterized for the first time. The coding sequence of Bm47 was amplified and subcloned into the prokaryotic expression vector pET-30a(+) in order to produce His-tagged fusion protein in the BL21 (DE3) cells. The His-Bm47 fusion protein was expressed efficiently after induction with IPTG. The purified fusion protein was used to immunize New Zealand white rabbits to prepare polyclonal antibody. As the genome of BmNPV is available in GenBank and the EST database of BmNPV is expanding, identification of novel genes of BmNPV was conceivable by data-mining techniques and bioinformatics tools. Structural bioinformatics approach to analyze the properties of Bm47 encodes protein.