• Animal cells and systems
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• v.15 no.1
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• pp.29-36
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• 2011

Insect lysozymes are basic, cationic proteins synthesized in fat body and hemocytes in response to bacterial infections and depolymerize the bacterial cell wall. The c-type lysozyme of the insect Spodoptera litura (SLLyz) is a single polypeptide chain of 121 residues with four disulfide bridges and 17 rare codons and is approximately 15 kDa. The full-length SLLyz cDNA is 1039 bp long with a poly(A) tail, and contains an open reading frame of 426 bp long (including the termination codon), flanked by a 54 bp long 5' UTR and a 559 bp long 3' UTR. As a host for the production of high-level recombinant proteins, E. coli is used most commonly because of its low cost and short generation time. However, the soluble expression of heterologous proteins in E. coli is not trivial, especially for disulfide-bonded proteins. In order to prevent inclusion body formation, GST was selected as a fusion partner to enhance the solubility of recombinant protein, and fused to the amplified products encoding mature SLLyz. The expression vector pGEX-4T-1/rSLLyz was then transformed into E. coli BL21(DE3)pLysS for soluble expression of rSLLyz, and the soluble fusion protein was purified successfully. Inhibition zone assay demonstrated that rSLLyz showed antibacterial activity against B. megaterium. These results demonstrate that the GST fusion expression system in E. coli described in this study is efficient and inexpensive in producing a disulfide-bonded rSLLyz in soluble, active form, and suggest that the insect lysozyme is an interesting system for future structural and functional studies.

• The Plant Pathology Journal
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• v.36 no.6
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• pp.579-590
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• 2020

Botrytis cinerea, which causes gray mold disease in more than 200 plant species, is an economically important pathogen that is mainly controlled by synthetic fungicides. Synergistic fungicide mixtures can help reduce fungicide residues in the environment and mitigate the development of fungicide-resistant strains. In this study, we screened microbial culture extracts on Botrytis cinerea to identify an antifungal synergist for tebuconazole. Among the 4,006 microbial extracts screened in this study, the culture extract from Schizophyllum commune displayed the most enhanced activity with a sub-lethal dosage of tebuconazole, and the active ingredient was identified as schizostatin. In combination with 5 ㎍/ml tebuconazole, schizostatin (1 ㎍/ml) showed disease control efficacy against gray mold on tomato leaf similar to that achieved with 20 ㎍/ml tebuconazole treatment alone. Interestingly, schizostatin showed demethylation inhibitor (DMI)-specific synergistic interactions in the crossed-paper strip assay using commercial fungicides. In a checkerboard assay with schizostatin and DMIs, the fractional inhibitory concentration values were 0.0938-0.375. To assess the molecular mechanisms underlying this synergism, the transcription levels of the ergosterol biosynthetic genes were observed in response to DMIs, schizostatin, and their mixtures. Treatment with DMIs increased the erg11 (the target gene of DMI fungicides) expression level 15.4-56.6-fold. However, treatment with a mixture of schizostatin and DMIs evidently reverted erg11 transcription levels to the pre-DMI treatment levels. These results show the potential of schizostatin as a natural antifungal synergist that can reduce the dose of DMIs applied in the field without compromising the disease control efficacy of the fungicides.

• Korean Journal of Microbiology
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• v.36 no.3
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• pp.236-241
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• 2000

A defensin is an inducible antibacterial peptide from a fleshfly and contains 40 residues basic peptide with six cysteines. For the consiruction of recombinant S cerevisiae expressing defensin, the structural gene coding for active defensin was chemically synthesized and fused in fiam to GAP promoter, MFul preprosequence and the GAL7 transcription terminator, generating a recombinant plasnlid pGMD18. S. ce~evisine 2805 Gells were transror~ned to uracil prototroph by the pGMDl8 arid the transformed cells showing antibacterial activity against 111. luteus TAM1056 were selected by growth inhibition zone assay. The optimal culture conditions for the unprovement of the defensin production of a selected tmdonnant were investigated. The optirmzed medium containing 0.4% yeast extract, 2% corn steep liquor, 2.5% glucose and 0.05% $C_2CO_3$, could be determined and the optimum lemperature. and initial pH could be detennnied as $28^{\circ}C$ and pH 3, ~mpectively. The optimized conditioiis revealed the trvofold Increase in the cell growth and the fourfold in the antibaclerial activity. coinpar-ed with tllc Yl'D medium.

• Journal of Marine Bioscience and Biotechnology
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• v.1 no.2
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• pp.119-125
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• 2006

To understand the comprehensive mechanisms of biological function for insulin-like growth factor-I (IGF-I) in vertebrates, we have investigated the cDNA sequence of this gene in the korean rockfish (Sebastes schlegeli). The mature form of korean rockfish IGF-I was found to be comprised of 67 amino acid residues, showing about a 7 kDa molecular weight. In this study, we used the polymerase chain reaction (PCR) to obtain a korean rockfish IGF-I (KR IGF-I) cDNA fragment, and methods of rapid amplification of cDNA ends (RACE) to obtain a full length of the KR IGF-I sequence. The KR IGF-I encoded for a predicted amino acid sequence showed identities of 93.6 %, 90.7 %, and 85.4 % in comparison with flounder, chinook salmon, and human IGF-I, respectively. To obtain recombinant biologically active polypeptides, korean rockfish B-C-A-D domains were amplified using the PCR, then the isolated cDNA was expressed in the E. coli BL21(DE3). The recombinant KR IGF-I protein biological function was measured by stimulation of [$^3H$] thymidine incorporation, suggesting the cDNA codes for the korean rockfish proIGF-I.

• Korean Journal of Plant Resources
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• v.17 no.3
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• pp.289-296
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• 2004

A cDNA clone (GenBank accession no. CF924621) homologous to aluminum induced protein gene was isolated and characterized from Codonopsis lanceolata (ClAIP). The ClAIP is 906 nucleotides long and has an open reading frame of 711 bp with a deduced amino acid sequence of 236 residues. The ClAIP shows high homology to A. marina (84%), G. hirsutum(83%), V. radiata (83%), A. thaliana (80%), B. nap us (78%) and T. aestivum (68%). The deduced amino acid sequence of ClAIP also has homology to the N-terminal end of plant Asn synthetase. This region does not contain the active sites of the enzyme and the significance of this conservation is currently not clear. To investigate the expression of ClAIP against several heavy metal stresses, we treated the sliced tap root of C. lanceolata with various heavy metals. The expression of ClAIP was increased by 25 uM $Al_2$(SO$_3$)$_4$ in proportion to incubation time and also increased by 50 uM CdCl$_2$.

• Journal of Plant Biotechnology
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• v.48 no.3
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• pp.148-155
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• 2021

Gibberellins (GAs) are essential phytohormones for plant growth that influence developmental processes and crop yields. Recent functional genomic analyses of model plants have yielded good characterizations of the canonical GA signaling pathways and related genes. Although Panax ginseng has long been considered to have economic and medicinal importance, functional genomic studies of the GA signaling pathways in this crucial perennial herb plant have been rarely conducted. Here, we identified and performed functional analysis of the GA signaling-related genes, including PgGID1s, PgSLY1s, and PgRGAs. We confirmed that the physiological role of GA signaling components in P. ginseng was evolutionarily conserved. In addition, the important functional domains and amino acid residues for protein interactions among active GA, GID1, SCF^SLY1, and RGA were also functionally conserved. Prediction and comparison of crystallographic structural similarities between PgGID1s and AtGID1a supported their function as GA receptors. Moreover, the subcellular localization and GA-dependent promotion of DELLA degradation in P. ginseng was similar to the canonical GA signaling pathways in other plants. Finally, we found that overexpression of PgRGA2 and PgSLY1-1 was sufficient to complement the GA-related phenotypes of atgid1a/c double- and rga quintuple-mutants, respectively. This critical information for these GA signaling genes has the potential to facilitate future genetic engineering and breeding of P. ginseng for increased crop yield and production of useful substances.

• Journal of Microbiology and Biotechnology
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• v.28 no.12
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• pp.2036-2045
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• 2018

An endo-${\beta}$-1,4-glucanase gene, cel5L, was cloned using the shot-gun method from Bacillus sp.. The gene, which contained a predicted signal peptide, encoded a protein of 496 amino acid residues, and the molecular mass of the mature Cel5L was estimated to be 51.8 kDa. Cel5L contained a catalytic domain of glycoside hydrolase (GH) family 5 and a carbohydrate-binding module family 3 (CBM_3). Chromatography using HiTrap Q and CHT-II resulted in the isolation of two truncated forms corresponding to 50 (Cel5L-p50) and 35 kDa (Cel5L-p35, CBM_3-deleted form). Both enzymes were optimally active at pH 4.5 and $55^{\circ}C$, but had different half-lives of 4.0 and 22.8 min, respectively, at $70^{\circ}C$. The relative activities of Cel5L-p50 and Cel5L-p35 for barley ${\beta}$-glucan were 377.0 and 246.7%, respectively, compared to those for carboxymethyl-cellulose. The affinity and hydrolysis rate of pNPC by Cel5L-p35 were 1.7 and 3.3 times higher, respectively, than those by Cel5L-p50. Additions of each to a commercial enzyme set increased saccharification of pretreated rice straw powder by 17.5 and 21.0%, respectively. These results suggest CBM_3 is significantly contributing to thermostability, and to affinity and substrate specificity for small substrates, and that these two enzymes could be used as additives to enhance enzymatic saccharification.

• Journal of Applied Biological Chemistry
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• v.62 no.1
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• pp.73-79
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• 2019

A novel esterase gene (est7S) was cloned from an enriched metagenomic library derived from an oil-spill area. The gene encoded a protein of 505 amino acids, and the molecular mass of the Est7S was estimated to be 54,512 Da with no signal peptide. Est7S showed the highest identity of 40% to an esterase from a sludge metagenome compared to the characterized enzymes with their properties, although it showed 99% identity to a carboxylesterase in the genome sequence of Alcanivorax borkumensis SK2. Est7S had catalytic triad residues, Ser183, Glu312, and His420, and the GESAG motif in most family VII lipolytic enzymes. Est7S was purified from the crude extract of clone SM7 using Sephacryl S-200 HR and HiTrap Q column chromatographies. The purified Est7S was optimally active at $50^{\circ}C$ and pH 10.0. Est7S showed a high specific activity of 366.7 U/mg protein. It preferred short length esters, particularly p-nitrophenyl acetate, efficiently hydrolyzed R- and S-enantiomers of methyl-3-hydroxy-2-methylpropionate, and glyceryl tributyrate. These properties of Est7S may provide potential merits in biotechnological applications such as detergent and paper processing under alkaline conditions.

• Molecules and Cells
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• v.44 no.1
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• pp.1-12
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• 2021

The nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) is the master transcriptional regulator in adipogenesis. PPARγ forms a heterodimer with another nuclear receptor, retinoid X receptor (RXR), to form an active transcriptional complex, and their transcriptional activity is tightly regulated by the association with either coactivators or corepressors. In this study, we identified T-cell death-associated gene 51 (TDAG51) as a novel corepressor of PPARγ-mediated transcriptional regulation. We showed that TDAG51 expression is abundantly maintained in the early stage of adipogenic differentiation. Forced expression of TDAG51 inhibited adipocyte differentiation in 3T3-L1 cells. We found that TDAG51 physically interacts with PPARγ in a ligand-independent manner. In deletion mutant analyses, large portions of the TDAG51 domains, including the pleckstrin homology-like, glutamine repeat and proline-glutamine repeat domains but not the proline-histidine repeat domain, are involved in the interaction with the region between residues 140 and 506, including the DNA binding domain, hinge, ligand binding domain and activation function-2 domain, in PPARγ. The heterodimer formation of PPARγ-RXRα was competitively inhibited in a ligand-independent manner by TDAG51 binding to PPARγ. Thus, our data suggest that TDAG51, which could determine adipogenic cell fate, acts as a novel negative regulator of PPARγ by blocking RXRα recruitment to the PPARγ-RXRα heterodimer complex in adipogenesis.

• Journal of Microbiology and Biotechnology
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• v.32 no.4
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• pp.504-513
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• 2022

Chitin deacetylase (CDA) inhibitors were developed as novel antifungal agents because CDA participates in critical fungal physiological and metabolic processes and increases virulence in soil-borne fungal pathogens. However, few CDA inhibitors have been reported. In this study, 150 candidate CDA inhibitors were selected from the commercial Chemdiv compound library through structure-based virtual screening. The top-ranked 25 compounds were further evaluated for biological activity. The compound J075-4187 had an IC50 of 4.24 ± 0.16 µM for AnCDA. Molecular docking calculations predicted that compound J075-4187 binds to the amino acid residues, including active sites (H101, D48). Furthermore, compound J075-4187 inhibited food spoilage fungi and plant pathogenic fungi, with minimum inhibitory concentration (MIC) at 260 ㎍/ml and minimum fungicidal concentration (MFC) at 520 ㎍/ml. Therefore, compound J075-4187 is a good candidate for use in developing antifungal agents for fungi control.