• Bulletin of the Korean Chemical Society
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• v.33 no.11
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• pp.3676-3680
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• 2012

An Epstein-Barr virus (EBV)-based plasmid contains the EBV nuclear antigen 1 (EBNA1) gene and EBV replication origin (oriP) sequence. Since EBNA1 (the only EBV-encoded protein) is combined with oriP, it is replicated simultaneously with chromosomal DNA in human, primate, and canine cells and is faithfully segregated at a stable copy number upon cell division. Consequently, it can be used to stably express gene inserts over a prolonged time in target cells. We have previously shown that the polyamidoamine (PAMAM) dendrimer can be surface-modified with L-arginine. Arginine is present at a high frequency in the transactivator of transcription (Tat) sequences of human immunodeficiency virus (HIV). It presents high membrane permeability and permits effective transfer of DNA inside the cells. In this study, we constructed two kinds of recombinant DNA by inserting the luciferase gene and enhanced green fluorescence protein (eGFP) gene as reporter genes into the pCEP4 plasmid vector. We measured dynamic light scattering (DLS) and zeta potential after preparing PAMAM-based cationic polymer/EBV-based plasmid complexes. We performed transfection of HEK 293 cell lines with the polyplexes, and monitored luciferase activity and green fluorescence protein (GFP) expression. Our results show that PAMAM-based cationic polymer/EBV plasmid complexes provide enhanced and sustained gene expression.

• Proceedings of the Korean Society of Sericultural Science Conference
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• 1997.06a
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• pp.23-49
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• 1997

This is a progress report of rice biotechnology including development of gene transformation system, gene cloning and molecular mapping in rice. The scope of the research was focused on the connection between conventional breeding and biotech-researches. Plant transformation via Agrobacterium or particle bombardment was developed to introduce one or several genes to recommended rice cultivars. Two chimeric genes containing a maize ribosome inactivating protein gene (RIP) and a gerbicide resistant gene (bar) were introduced to Nipponbare, a Japonica cultivar, and transmitted to Korean cultivars. The homozygous progenies of herbicide resistant transgenic plant showed good fertility and agronomic characters. To explore the genetic resourses in rice, over 8,000 cDNA clones from immature rice seed have been isolated and sequenced. About 13% of clones were identified as enzymes related to metabolic pathway. Among them, twenty clones have high homology with genes encoding enzymes in the photorespiratory carbon cycle reaction. Up to now about 100 clones were fully sequenced and registered at EMBL and GenBank. For the mapping of quantitative tarits loci (QTL) and eternal recombinant inbred population with 164 F13 lines (MGRI) was developed from a cross between Milyang 23 and Gihobyeo, Korean rice cultivars. After construction of fully saturated RFLP and AFLP map, quantitative traits using MGRI population were analyzed and integrated into the molecular map. Eighty seven loci were determined with 27 QTL characters including yield and yield components on rice chromosomes. Map based cloning was also tried to isolate semi-dwarf (sd-1) gene in rice. A DNA probe, RG 109, the most tightly linked to sd-1 gene was used to screen from bacterial artifical chromosome (BAC) libraries and five over lapping clones presumably containing sd-1 gene were isolated. Rice genetic database including results of biotech reasearch and classical genetics is provided at Korea Rice Genome Server which is accessible with world wide web (www) browser. The server provides rice cDNA sequences and map informations linked with phenotypic images.

• IMMUNE NETWORK
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• v.24 no.1
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• pp.1.1-1.6
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• 2024

IL-18 binding protein (IL-18BP) was originally discovered in 1999 while attempting to identify an IL-18 receptor ligand binding chain (also known as IL-18Rα) by subjecting concentrated human urine to an IL-18 ligand affinity column. The IL-18 ligand chromatography purified molecule was analyzed by protein microsequencing. The result revealed a novel 40 amino acid polypeptide. To isolate the complete open reading frame (ORF), various human and mouse cDNA libraries were screened using cDNA probe derived from the novel IL-18 affinity column bound molecule. The identified entire ORF gene was thought to be an IL-18Rα gene. However, IL-18BP has been proven to be a unique soluble antagonist that shares homology with a variety of viral proteins that are distinct from the IL-18Rα and IL-18Rβ chains. The IL-18BP cDNA was used to generate recombinant IL-18BP (rIL-18BP), which was indispensable for characterizing the role of IL-18BP in vitro and in vivo. Mammalian cell lines were used to produce rIL-18BP due to its glycosylation-dependent activity of IL-18BP (approximately 20 kDa). Various forms of rIL-18BP, intact, C-terminal his-tag, and Fc fusion proteins were produced for in vitro and in vivo experiments. Data showed potent neutralization of IL-18 activity, which seems promising for clinical application in immune diseases involving IL-18. However, it was a long journey from discovery to clinical use although there have been various clinical trials since IL-18BP was discovered in 1999. This review primarily covers the discovery of IL-18BP along with how basic research influences the clinical development of IL-18BP.

• Journal of Microbiology and Biotechnology
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• v.11 no.4
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• pp.592-597
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• 2001

The pyruvate dehydrogenase complex (PDC) catalyzes the oxidative decarboxylation of pyruvate with the formation of $CO_2$, acetyl-CoA, NADH, and H+. This complex contains multiple copies of three catalytic components including pyruvate dehydrogenase(E1), dihydrolipoamide acetyltransferase(E2), and dihydrolipoamide dehydrogenase (E3). Two regulatory components (E1-kinase and phospho-E1 phosphatase) and functionally less-understood protein (protein X, E3BP) are also involved in the formation of the complex. In this study, cloning and characterization of a gene for human E3BP have been carried out. A cDNA encoding the human E3BP was isolated by database search and cDNA library screening. The primary structure of E3BP has some similar characteristics with that of E2 in the lipoyl domain and the carboxyl-terminal domain, based on the nucleotide sequence and the deduced amino acid sequence. However, the conserved amino acid moiety including the histidine residue for acetyltransferase activity in E2 is not conserved in the case of human E3BP. The human E3BP was expressed and purified in E. coli. The molecular weight of the protein, excluding the mitochondrial target sequence, was about 50 kDa as determined by SDS-PAGE. Cloning of human E3BP and expression of the recombinant E3BP will facilitate the understanding of the role(s) of E3BP in mammalian PDC.

• Korean Journal of Veterinary Research
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• v.49 no.1
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• pp.1-8
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• 2009

The ubiquitin-proteasome mediated protein degradation pathway plays an important role in regulating both cell proliferation and cell death. Proteasome inhibitors are well known to induce apoptosis in various human cancer cell lines. We investigated the effect of combined treatment with proteasome inhibitor and TRAIL, and a possible mechanism of the enhancing apoptosis by the both treatment, on TRAIL-resistant non-small cell lung cancer. A549 cells were exposed to the N-Acetyl-Leu-Leu-Norleu-al (ALLN) as a proteasome inhibitor and then treated with recombinant TRAIL protein. In A549 cells under proteasome inhibition conditions by pretreatment with ALLN, TRAIL treatment significantly decreased cell viability compared to that ALLN and TRAIL alone treatment. Also, the both treatment induced cell damage through DNA fragmentation and p53 expression. In addition, the combined treatment of both markedly increased caspase-8 activation, especially the exposure for 2 h, and Bax expression and induced the dissipation of mitochondrial transmembrane potential in A549 cells. Taken together, these findings showed that proteasome inhibition by ALLN enhanced TRAIL-induced apoptosis via DNA degradation by activated P53 and mitochondrial transmembrane potential loss by caspase-8 activation and bax expression. Therefore, our results suggest that proteasome inhibitor may be used a very effectively chemotherapeutic agent for the tumor treatment, especially TRAIL-resistant tumor cell.

• Journal of Plant Biotechnology
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• v.6 no.1
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• pp.1-7
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• 2004

Forestry sector has witnessed some unprecedented events in the recent past both in terms of galloping biotechnological developments and heated environmental debates over risks associated with release of transgenic trees. Improvements in the in vitro propagation techniques has made it possible to develop tissue culture based plant regeneration protocols just for about any tree species. And with the inclusion of every new species within the realms of tissue culture technology, it becomes a candidate for genetic improvement through recombinant DNA technology, the so called genetic engineering. Poplars and their hybrids serve as the model tree species on which most of the genetic transformation work as been carried out. A lot of work has also gone in genetic transformation of fruit trees and trees of horticultural interests. Trees have been successfully transformed for traits ranging from reduction of length of juvenile phase to alteration of tree architecture to altering wood quality by lignin and cellulose modification. More-over trees have been genetically engineered successfully to combat various types of insect pests and pathogens causing diseases. But all these developments have ignited controversies over the possible benefits and risks associated with transgenic plantations by various environmental agencies and activists. Solutions to most of these concerns can be found out with more intensive prioritized research.

• BMB Reports
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• v.32 no.5
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• pp.486-491
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• 1999

An engineered cDNA from Phanerochaete chrysosporium encoding both the mature and propeptide-sequence regions of lignin peroxidase H2 (Lip H2) was overexpressed in Escherichia coli BL21 (DE3) to evaluate its catalytic characteristics and potential application as a pollution scavenger. All expressed proteins were aggregated in an inactive inclusion body, which might be due to inherent disulfide bonds. Active enzyme was obtained by refolding with glutathione-mediated oxidation in refolding solution containing $Ca^{2+}$, heme, and urea. Propeptide-sequence region was not processed as evidenced by N-terminal sequence analysis. Recombinant Lip H2 (rLip H2) had the same physical properties of the native protein but differed in the $K_{cat}$. Catalytic efficiency ($k_{cat}/K_m$) of rLip H2 was slightly higher than that of the native enzyme. In order to express an active protein, fusion systems with thioredoxin or Dsb A, which have disulfide isomerase activity, were used. The fused proteins expressed by the Dsb A fusion vector were aggregated, whereas half of the thioredoxin fusion proteins were recovered as a soluble form but still catalytically inactive. These results suggest that Lip H2 may not be expressed as an active enzyme in Escherichia coli although the activity can be recovered by in vitro refolding.

• Journal of Microbiology and Biotechnology
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• v.7 no.3
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• pp.161-166
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• 1997

Nuclei were isolated from the protoplasts of Trichoderma harzianum T95 and treated with colchicine, a polyploid inducer. The nuclei were transferred into the protoplast of multi-auxotrophic Gliocladium virens G88 which cannot grow in minimal medium. The protoplast of G. virens G88 carrying the transferred nuclei were regenerated in a regeneration minimal medium containing $17{\mu}g/ml$ of chloroneb as a haploid inducer. Six intergeneric hybrids between G. virens and T. harzianum were isolated from the regeneration minimal medium. The hybrids could be classified into three types according to morphology, those with an isozyme pattern, those with an protein band and those with an randomly amplified polymorphic DNA(RAPD) pattern produced by random primers and repetitive sequences. The first group was identified to be a haploid recombinant, the second group a heterokaryon, and the third appeared to be petite.

• Journal of Microbiology
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• v.43 no.1
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• pp.34-37
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• 2005

In the present study, the xylA gene encoding a thermostable xylose (glucose) isomerase was cloned from Streptomyces chibaensis J-59. The open reading frame of xylA (1167 bp) encoded a protein of 388 amino acids with a calculated molecular mass of about 43 kDa. The XylA showed high sequence homology (92% identity) with that of S. olivochromogenes. The xylose (glucose) isomerase was expressed in Escherichia coli and purified. The purified recombinant XylA had an apparent molecular mass of 45 kDa, which corresponds to the molecular mass calculated from the deduced amino acid and that of the purified wild-type enzyme. The N-terminal sequences (14 amino acid residues) of the purified protein revealed that the sequences were identical to that deduced from the DNA sequence of the xylA gene. The optimum temperature of the purified enzyme was $85^{\circ}C$ and the enzyme exhibited a high level of heat stability.

• Journal of Plant Biotechnology
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• v.37 no.3
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• pp.262-268
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• 2010

Antibodies are powerful and versatile tools to play a critical role in the diagnosis and treatment of many diseases. Their application has been enhanced significantly with the advanced recombinant DNA and heterologonous expression technologies, allowing to produce immunotherapeutic proteins with improved biofunctional properties. However, with currently available technologies, mammalian cell-based therapeutic antibody production, as an alternative for production in humans and animals, is often not plentiful for passive immunotherapeutics in treatment of many diseases. Recently, plant expression systems for therapeutic antibodies have become well-established. Thus, plants have been considered to provide an attractive alternative production system for therapeutic antibodies, as plants have several advantages such as the lack of human pathogens, and low cost of upstream production and flexible scale-up of highly valuable recombinant glycoproteins. Recent advances in modification of posttranslational processing for human-like glycosylation in transgenic plants will make it possible that plant can become a suitable protein expression system over the animal cellbased current production system. This review will discuss recent advances in plant expression technology and issues for their application to therapeutic antibody production.