• Biomedical Science Letters
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• v.6 no.3
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• pp.165-170
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• 2000

Vacuolar (H$^{+}$)-ATPase (V-ATPase) is an intracellular protein which consists of multiple subunits. It carries out acidification by pumping protons in the cell. This enzyme has also been found in the synaptic vesicles and may play an important role in the neurotransmission. We cloned cDNA fragments encoding the 16 kDa subunit of V-ATPase from the rat brain by RT-PCR and PCR using total RNA or recombinant phage DNA as templates. They contained the full coding sequences (468 bp) and one nucleotide at 3' region turned out to be different (A to C) when compared to the liver counterpart. However, this polymorphic difference did not cause any significant change in the primary structure of the protein because both GCA and GCC code for alanine. Our study would contribute to the understanding of the function of 16 M)a V-ATPase in the brain and of the mechanisms of neurotransmission.

• BMB Reports
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• v.31 no.2
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• pp.111-116
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• 1998

HLA-DR4 is the dominant allele of MHC class II genes in Koreans. In particular, the $DRB1^*0405$ subtype has been reported to be almost exclusively expressed in Far East Asians, and has also been observed to be strongly associated with rheumatoid arthritis in Koreans and the Japanese. Identification of this specific allele has been mainly performed by PCR-based methods, which is often time consuming, costly, and involves tedious procedures such as the isolation of genomic DNA, PCR, and gel electrophoresis. To develop a more convenient tool for screening vast amounts of samples as well as to generate reagents which might also be used in other applications, in this study, antibodies were produced against this specific HLA subtype. By PCR, an allelespecific region covering the ${\beta}1$ domain of $DRB1^*0405$ was amplified and recombinantly expressed in E.coli. Immunization of Lewis rats with the purified protein yielded an allele specific antiserum. Western blot analysis showed the selective detection of the HLA-DR ${\beta}-chain$. Using this antiserum, established cell lines and peripheral blood lymphocytes were analyzed on their HLA haplotype by fluorescence activated flow cytometry. These novel antibodies will provide a powerful tool in the detection and investigation of DR4 alleles.

• Journal of Microbiology and Biotechnology
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• v.11 no.3
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• pp.508-514
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• 2001

A stuructural gene (ycl) encoding novel yeast cell wall hydrolase, YCL, was cloned from alkalophilic Bacillus alcalophilus subsp. YB380 by PCR, and transformed into E. coli JM83. Based on the N-terminal and internal amino acid sequences of the enzyme, primers were designed for PCr. The positive clone that harbors 1.8 kb of the yeast cell wall hydrolase gene was selected by the colony hybridization method with a PCR fragment as a probe. According to the computer analysis, this gene contained a 400-base-paired N-terminal domain of the enzyme. Based on nucletide homology of the cloned gene, a 850 bp fragment was amplified and the C-terminal domain of the enzyme was sequenced. With a combination of the two sequences, a full nucleotide sequence for YCL was obtained. This gene, ycl, consisted of 1,297 nucleotides with 27 nucleotides with 27 amino acids of signal sequence, 83 redundant amino acids of prosequence, and 265 amino acids of the mature protein. This gene was then cloned into the pJH27 shuttle vector and transformed into the Bacillus subtilis DB104 to express the enzyme. It was confirmed that the expressed cell wall hydrolase that was produced by Bacillus subtilis DB104 was the same as that of the donor strain, by Western blot using polyclonal antibody (IgY) prepared from White Leghorn hen. Purified yeast cell wall hydrolase and expressed recombinant protein showed a single band at the same position in the Western blot analysis.

• International Journal of Industrial Entomology and Biomaterials
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• v.23 no.2
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• pp.231-238
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• 2011

Attacin is an insect antibacterial protein that plays an important role in immune response to injury and infection. In this report, we have isolated and characterized of cDNA encoding for the attacin from the immunized larvae of swallowtail butterfly, $Papilio$ $xuthus$. A full length cDNA of $P.$ $xuthus$ attacin was obtained by employing annealing control primer (ACP)-based differential display PCR and 5' RACE. The complete $P.$ $xuthus$ attacin cDNA was comprised of 949 bp encoding a 250 amino acid precursor. It contains a putative 18 amino acid signal peptide sequence, a 42 amino acid propeptide sequence, and a 190 amino acid mature protein with a theoretical molecular mass of 19904.01 and a pI of 9.13. The putative mature protein of $P.$ $xuthus$ attacin showed 48-52% and 24-30% identity in amino acid sequences with that of lepidopteran and dipteran insects, respectively. Semiquantitive RT-PCR results revealed that the transcript of $P.$ $xuthus$ attacin gene was up-regulated at significant levels after injection with bacterial lipopolysaccharide (LPS). We sub-cloned cDNA fragment encoding mature $P.$ $xuthus$ attacin into the expression vector, highly expressed in $E.$ $coli$ BL21 cells, and its antibacterial activity was analyzed. Recombinant $P.$ $xuthus$ attacin evidenced considerably antibacterial activity against Gram-negative bacteria, $E.$ $coli$ ML 35 and $Klebsiella$ $pneumonia$.

• Journal of Plant Biotechnology
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• v.37 no.1
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• pp.102-109
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• 2010

Fructan has been found to accumulate in various tissues during periods when light levels increased carbon fixation where low temperatures reduced growth rates while photosynthesis continued. In this study, we have cloned 1-sucrose:sucrose fructosyl transferase(1-sst) and 1-fructan: fructan fructosyl transferase (1-fft, a key enzyme for the synthesis of fuctan) from Jerusalem Artichoke (Helianthus tuberosus L.). The recombinant vector with 1-sst and 1-fft has been constructed under the control of 35S promoter of KJGV-B2 vector and transgenic plants obtained by Agrobacterium tumefaciens LBA4404. PCR analysis carried out on the putative transgenic plants for amplification of the coding region of specific gene (1-sst, 1-fft), and HPT genes. Transgenic lines carrying of 1-sst and 1-fft were confirmed for integration into the rice genome using Southern blot hybridization and RT-PCR. The transgenic plants in $T_2$ generation were selected and expression pattern analysis revealed that 1-sst and 1-fft were stable. This analysis confirmed the presence of low-molecular-weight fructan in the seedling of the transgenic rices. Therefore, cold tolerance and carbohydrate metabolism will be possible to develop resistant plants using the transgenic rice.

• Journal of Microbiology and Biotechnology
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• v.25 no.7
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• pp.999-1006
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• 2015

The endo-polygalacturonase gene (endo-pgaA) was cloned from DNA of Aspergillus niger SC323 using the cDNA synthesized by overlapping PCR, and successfully expressed in Saccharomyces cerevisiae EBY100 through fusing the α-factor signal peptide of yeast. The fulllength cDNA consists of 1,113 bp and encodes a protein of 370 amino acids with a calculated molecular mass of 38.8 kDa. After induction by galactose for 48 h, the activity of recombinant endo-PgaA in the culture supernatant can reach up to 1,448.48 U/mg. The recombinant protein was purified to homogeneity by ammonium sulfate precipitation and gel filtration column chromatography and subsequently characterized. The optimal pH and temperature of the purified recombinant enzyme were 5.0 and 50℃, respectively. The Michaelis-Menten constant (K_m) and maximal velocity (V_max) of the enzyme for pectin were 88.54 μmol/ml and 175.44 μmol/mg/min, respectively. The enzyme activity was enhanced by Ca²⁺, Cu²⁺, and Na⁺, and strongly inhibited by Pb²⁺ and Mn²⁺. The pectin hydrolysates were mainly galacturonic acid and other oligo-galacturonates. Therefore, these characteristics suggest that the recombinant endo-PgaA may be of potential use in the food and feed industries.

• Journal of Life Science
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• v.28 no.1
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• pp.99-104
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• 2018

Streptococcus pneumoniae is one of the major pathogens in community-acquired diseases, and it contains several factors that promote its pathogenesis, including pneumolysin (PLY). PLY is a member of the cholesterol-dependent cytolysin family, which attacks cholesterol-containing membranes, thereby forming ring-shaped pores. Thus, it is a major key target for vaccines against pneumococcal disease. We cloned the PLY gene from S. pneumoniae D39 and inserted it into the pQE-30 vector. Recombinant PLY (rPLY) was overexpressed in Escherichia coli M15 and purified by $Ni^{2+}$ affinity chromatography. Similarly, a PLY-EGFP fusion gene was produced by inserting the EGFP gene at the 3' end of the PLY gene in the same vector, and the recombinant protein was purified. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) showed that both recombinant proteins were purified. rPLY exhibited significant hemolytic activity against 1% human red blood cells (RBCs). Complete hemolysis was obtained at 500 ng/ml, and 50% hemolysis was found with a 240 ng/ml concentration. In contrast, rPLY-EGFP did not show hemolytic activity. However, rPLY-EGFP did bind the RBC membrane, indicating that rPLY-EGFP lost hemolytic activity via EGFP fusion, while retaining its membrane-binding ability. These data suggest that PLY's C terminus is important for its hemolytic activity. Therefore, these two recombinant proteins can be extremely useful for investigating the toxin mechanism of PLY and cell damage during pneumonia.

• Journal of Microbiology and Biotechnology
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• v.14 no.1
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• pp.81-89
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• 2004

Tumor necrosis factor-$\alpha$ (TNF-$\alpha$) and lymphotoxin-$\alpha$ (LT-$\alpha$, TNF-$\beta$) can initiate and perpetuate human diseases such as multiple sclerosis (MS), rheumatoid arthritis (RA), and insulin-dependent diabetes mellitus (IDDM). TNFs can be blocked by the use of soluble TNF receptors. However, since monomeric soluble receptors generally exhibit low affinity or function as agonists, the use of monomeric soluble receptors has been limited in the case of cytokines such as TNF-$\alpha$, TNF-$\alpha$, interleukin (IL)-1, IL-4, IL-6, and IL-13, which have adapted to a multi component receptor system. For these reasons, very high-affinity inhibitors were created for the purpose of a TNFs antagonist to bind the TNFR and trigger cellular signal by using the multistep polymerase chain reaction method. First, recombinant simple TNFR-Ig fusion proteins were constructed from the cDNA sequences encoding the extracellular domain of the human p55 TNFR (CD120a) and the human p75 TNFR (CD120b), which were linked to hinge and constant regions of human $IgG_1$ heavy chain, respectively using complementary primers (CP) encoding the complementary sequences. Then, concatameric TNFR-Ig fusion proteins were constructed using recombinant PCR and a complementary primer base of recombinant simple TNFR-Ig fusion proteins. For high level expression of recombinant fusion proteins, Chinese hamster ovary (CHO) cells were used with a retroviral expression system. The transfected cells produced the simple concatameric TNFR-Ig fusion proteins capable of binding TNF and inactivating it. These soluble versions of simple concantameric TNFR-Ig fusion proteins gave rise to multiple forms such as simple dimers and concatameric homodimers. Simple TNFR-1g fusion proteins were shown to have much more reduced TNF inhibitory activity than concatameric TNFR-Ig fusion proteins. Concatameric TNFR-Ig fusion proteins showed higher affinity than simple TNFR-Ig fusion proteins in a receptor inhibitor binding assay (RIBA). Additionally, concatameric TNFR-Ig fusion proteins were shown to have a progressive effect as a TNF inhibitor compared to the simple TNFR-Ig fusion proteins and conventional TNFR-Fc in cytotoxicity assays, and showed the same results for collagen induced arthritis (CIA) in mice in vivo.

• Journal of Microbiology
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• v.44 no.5
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• pp.515-522
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• 2006

A cDNA library of Ganoderma lucidum has been constructed using a Zap Express cloning vector. A glyceraldehyde-3-phosphate dehydrogenase gene (gpd) was isolated from this library by hybridization of the recombinant phage clones with a gpd-specific gene probe generated by PCR. By comparison of the cDNA and the genomic DNA sequences, it was found that the complete nucleotide sequence encodes a putative polypeptide chain of 338 amino acids interrupted by 6 introns. The predicted amino acid sequence of this gene shows a high degree of sequence similarity to the GPD proteins from yeast and filamentous fungi. The promoter region contains a CT-rich stretch, two CAAT boxes, and a consensus TATA box. The possibility of using the gpd promoter in the construction of new transformation vectors is discussed.

• 한국생물공학회:학술대회논문집
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• 2003.04a
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• pp.467-470
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• 2003

The endoxylanase (642 bp; 213 amino acids) and ${\beta}-xylosidase$ (1,602 bp; 533 amino acids) genes from Bacillus sp. were amplified by PCR and separately inserted downstream of the yeast ADH1 promoters, resulting in the pAEDX-1 and pAEX plasmid. When the yeast transformants, S. cerevisiae SEY2102 harboring pAEDX-1 or pAEX, were grown on YPD medium, the total activities of the enzymes reached about 9.8 unit/mL for endoxylanase and 2.9 unit/mL for ${\beta}-xylosidase$. When the three kinds of xylan from oat spelts, birch wood, and corncob were hydrolyzed by treatment of recombinant endoxylanase and ${\beta}-xylosidase$, it was found that xylose, xylobiose and xylotriose were produced and xylose was the major product after 12 h reaction. In addition, with the higher amount of enzymes, the more amount of xylose was produced.