• Microbiology and Biotechnology Letters
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• v.30 no.1
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• pp.1-7
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• 2002

Phytase from Escherichia coli WC7 was purified from cell extracts and its molecular mass was estimated to be 45 kDa by SDS-PAGE. Its optimum temperature and pH for phytate hydrolysis was 6$0^{\circ}C$ and pH 5.0, respectively. The enzyme was stable up to 6$0^{\circ}C$ and over broad pH range (pH 2-12). The enzyme had higher affinity for sodium phytate than p-nitrophenylphosphate (pNPP). That is, the apparent Km value for sodium phytate and pNPP were $0.15\pm$0.02 mM and 2.82$\pm$0.05 mM, respectively. The gene encoding the phytase was cloned in E. coli XL1-Blue. Sequence analysis showed an open reading frame of 1241 Up encoding a signal peptide (22 aa) and a mature enzyme (410 aa). WC7 phytase was expressed up to 17.5 U/ml in the transformed E. coli XL1-Blue/pUEP, which was 23-fold higher than the activity from wild strain.

• Molecules and Cells
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• v.25 no.1
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• pp.112-118
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• 2008

Laccases are multicopper-containing oxidases that catalyze the oxidation of many aromatic compounds with concomitant reduction of oxygen to water. Interest in this enzyme has arisen in many fields of industry, including detoxification, wine stabilization, paper processing, and enzymatic conversion of chemical intermediates. In this study, we cloned a laccase gene (GLlac1) from the white-rot fungus Ganoderma lucidum. The cloned gene consists of 4,357 bp, with its coding region interrupted by nine introns, and the upstream region has putative CAAT and TATA boxes as well as several metal responsive elements (MREs). We also cloned a full-length cDNA of GLlac1, which contains an uninterrupted open reading frame (ORF) of 1,560 bp coding for 520 amino acids with a putative 21-residue signal sequence. The DNA and deduced amino acid sequences of GLlac1 were similar but not identical to those of other fungal laccases. GLlac1 was released from the cells when expressed in P. pastoris, and had high laccase activity. In addition, GLlac1 conferred antioxidative protection from protein degradation, and thus may be useful in bio-medical applications.

• The Korean Journal of Food And Nutrition
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• v.15 no.2
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• pp.104-111
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• 2002

The peptidyl prolyl sis-trans isomerase (PPIase, EC 5.2.1.8) from bacillus stearothermophilus was extracted from the cells treated with by lysozyme. PPIase was purified from the cell extracts by heat treatment, ammonium sulfate precipitation, ion exchange chromatography and finally gel filtration, sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE). The molecular weight of the purified PPIase was estimated as 18kDa by SDS-PAGE. The 39 amino acid residues from the N-terminus were determined by the protein sequencer. The enzyme showed the optimum pH at 8.0 and was stable at the range of pH 7.0∼8.0. The enzyme was considerably stable after heat treatment at 60$\^{C}$ for 30minutes, and the enzyme was quite stable up to 65$\^{C}$. The presence of the PPIase in the refolding solution accelerated the isomerization rate of the assay peptide. PPIase gene of Bacillus stearothermophilus was screened from a genomic library by plaque hybridization using the A-l primer as a probe. A PPIase positive plaque contained a 3.0kb insert of the chromosomal DNA. A 3.0kb fragment was subcloned into pUC18, resulting pPI-40. A DNA fragment encoding the N-terminal portion of the PPIase in pPI-40 was amplified by polymerase chain reaction(PCR) method using the A-1 and B-2 primers. The amplified fragment was cloned into the Sma I site of pUC18 and recombinant plasmid was designated as pSN-18. The nucleotide sequence of 167bp fragment was determined. The deduced amino acid sequence of PPIase was completely matched with the determined N-terminal amino acid sequence of PPIase B. stearothermophilus.

• Korean Journal of Veterinary Research
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• v.44 no.2
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• pp.259-268
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• 2004

Porcine circovirus type 2 (PCV-2) has been associated with various disease in pigs worldwide including postweaning multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS). In this study, monoclonal antibodies (MAbs) against PCV were produced, characterized and applications of MAbs as diagnostic reagents were described. Spleen or lymph node cells from BALB/c mouse immunized respectively with PCV-1, PCV-2 or expressed PCV-2/ORF2 proteins in baculovirus were fused with SP2/0 myeloma cells using polyethylene glycol (PEG) and hybridoma cells producing PCV-1 or PCV-2-specific antibody were screened by an indirect immunofluorescence (IIF) test. A total of fifteen MAbs were produced against PCV. Six MAbs were PCV-1-specific and nine were PCV-2-specific. All PCV-1-specific MAbs reacted with only PCV-1 and all PCV-2-specific MAbs were reactive with only PCV-2 by IIF test. None of the MAbs was reactive with porcine reproductive and respiratory syndrome virus (PRRSV), porcine parvovirus (PPV), porcine rotavirus (PRV), and transmissible gastroenteritis virus (TGEV). Some PCV-2-specific MAbs recognized the PCV-2 infected porcine tissues by IIF or immunohistochemistry (IHC) assay. From this experiment, it was confirmed that MAbs produced in this study were PCV-specific and could be used as reliable diagnostic reagents for PCV-1/PCV-2 detection and differentiation.

• Molecules and Cells
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• v.41 no.5
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• pp.465-475
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• 2018

The advent of massively parallel sequencing, also called next-generation sequencing (NGS), has dramatically influenced cancer genomics by accelerating the identification of novel molecular alterations. Using a whole genome sequencing (WGS) approach, we identified somatic coding and noncoding variants that may contribute to leukemogenesis in 11 adult Korean acute myeloid leukemia (AML) patients, with serial tumor samples (primary and relapse) available for 5 of them; somatic variants were identified in 187 AML-related genes, including both novel (SIN3A, C10orf53, PTPRR, and RERGL) and well-known (NPM1, RUNX1, and CEPBA) AML-related genes. Notably, SIN3A expression shows prognostic value in AML. A newly designed method, referred to as "hot-zone" analysis, detected two putative functional noncoding variants that can alter transcription factor binding affinity near PPP1R10 and SRSF1. Moreover, the functional importance of the SRSF1 noncoding variant was further investigated by luciferase assays, which showed that the variant is critical for the regulation of gene expression leading to leukemogenesis. We expect that further functional investigation of these coding and noncoding variants will contribute to a more in-depth understanding of the underlying molecular mechanisms of AML and the development of targeted anti-cancer drugs.

• Journal of Ginseng Research
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• v.32 no.4
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• pp.300-304
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• 2008

Cysteine proteinases play an essential role in plant growth and development but also in senescence and programmed cell death. They participate in both anabolic and catabolic processes. In addition, they are involved in signalling pathways and in the response to biotic and abiotic stresses. A cDNA clone encoding cysteine proteinase (CP) gene, designated PgCysP1, was isolated from Panax ginseng C. A. Meyer. Reverse transcriptase (RT)-PCR results showed that PgCysP1 expressed at different level in P. ginseng hairy root. Different stresses such as biotic as well as abiotic stresses triggered a significant induction of PgCysP1. The positive responses of PgCysP1 to the various stimuli suggested that PgCysP1 may help to protect the plant against reactive environmental stresses.

• The Plant Pathology Journal
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• v.27 no.4
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• pp.378-384
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• 2011

Tomato yellow leaf curl virus (TYLCV), a member of genus Begomovirus, was isolated in Korea in 2008. We sequenced and analyzed the DNA-A of 51 TYLCV isolates from Korea, and 13 of the TYLCV isolates were selected as type representatives of TYLCV from six Korean provinces. The 13 TYLCV isolates were classified into Korea Group 1 (KG1, nine isolates) and Korea Group 2 (KG2, four isolates) based on the results of phylogenetic analysis and genome size (2774 and 2781 nucleotides, respectively). A recombination detection program 3 (RDP3) revealed two recombinations between the TYLCV Korea isolates and other TYLCV isolates [Thailand (AF206674), Iran (AJ132711), and Israel (X76319)]. TYLCV Jeju isolate was characterized by two recombination events (E1 and E2) caused by the presence of E1 in ORF V1 and C3, which may seem to be the mutations of the high nucleotide transversion and transition rate. Collectively, our results suggest that the occurrence of nucleotide transversions and transitions in TYLCV DNA-A might have induced novel recombination events within the TYLCV Korea isolates.

• BMB Reports
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• v.33 no.6
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• pp.483-492
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• 2000

The Herpes simplex virus type 1 (HSV-1) strain F glycoprotein H (gH) gene in the pHLB-4 plasmid was recombinated into a baculovirus expression vector (lacZ-HcNPV) to construct a recombinant virus GH-HcNPV expressing gH. The sequences of gH and its expression were analyzed. The gH gene was located in the 6.41 kb BglII fragment. The open reading frame (ORF) of the gH gene was 2,517 bp and codes 838 amino acid residues. Insect cells infected with this recombinant virus synthesized a high level of the matured and gX-gH fusion protein with approximately 112 kDa. The fusion gH protein was localized on the membrane of the insect cells as seen by using immunofluorescence assay and accumulated in the cultured media by the SDS-PAGE and immunoprecipitation assays. The amino acid sequence presents additional characteristics compatible with the structure of a viral glycoprotein: signal peptide, putative glycosylation sites and a long C-terminal transmembrane sequence. Antibodies raised in mice to this recombinant protein recognized viral gH and neutralized the infectivity of HSV-1 in vitro. These results demonstrate that it is possible to produce a mature protein by gene transfer in eukaryotic cells, and indicate the utility of the HcNPV-insect cell system for producing and characterizing eukaryotic proteins. Furthermore, the neutralizing antibodies would appear to protect mice against HSV; accordingly, this particular recombinant protein may be useful in the development of a subunit vaccine.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.69.1-69
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• 2003

RSH (relA/spoT homolog) has been known to determine the level of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), which are the effector nucleotide of the prokaryotic stringent response and also play a role in antibiotic production and differentiation in Streptomyces species but not a little in eukaryotic organism, especially in plant. Salicylic acid (SA), a critical signal molecule of establishing systemic acquired resistance (SAR), could induce SAR in Pepper (Capcicum annuum) against Phytophthora capsici. And the extent of SAR induction was in proportion to the dosage of SA (or BTH). Suppression subtractive hybridization (SSH), a PCR-based method for cDNA subtraction, was carried out between SA-treated and non-SA-treated pepper leaves to isolate genes which may be responsible for defense signaling against pathogens. Early upregulated gene was selected from reverse northern and kinetics of SSH-genes transcripts in SA-treated pepper leaves upon SA treatment. Full-length cDNA of the gene (PepRSH； Pepper RelA / SpoT homolog) had an open reading frame (ORF) of 2166 bp encoding a protein of 722 amino acids and a significant homology with (p)ppGpp phosphohydrolase or synthetase. Genomic DNA gel blot analysis showed that pepper genome has at least single copy of PepRSH. PepRSH transcripts was very low in untreated pepper leaves but strongly induced by SA and methyljasmonic acid (MeJA), indicating that PepRSH may share common SA and MeJA-mediated signal transduction pathway Functional analysis in E. coli showed PepRSH confers phenotypes associated with (p)ppGpp synthesis through a complementation using active site mutagenesis.

• Korean Journal of Microbiology
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• v.32 no.3
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• pp.186-191
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• 1994

The origin of the leading strand replication (ori) and of lagging strand replication (M-O) of R-plasmid pSBK203 was identified and its base sequence was determined. About 50 bp of ori sequence residues overlapped with the structural gene of rep. Sequence comparison reveals that pSBK-ori shares obvious identities with those of pT181 family and consists of two regions, one is conserved and the other is variable region. Of two palindrome sequence located one after another in upstream region of rep gene, palA' instead of palA which shares sequence homology with diverse family of plasmids such as pOX6, pC194, and pE194 seems to act as a signal for conversion of primarily replicated ssDNA to dsDNA (minus origin (M-O)).