• BMB Reports
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• v.34 no.4
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• pp.334-341
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• 2001

Even though three isotypes of thioredoxins (-f, -m and -h types) have been identified in a variety of plant cells, there are only a few reports on thioredoxin-h that were recently identified. In this study, a cDNA encoding a h-type of thioredoxin was isolated from a cDNA library of Chinese cabbage, and named here CTrx-h. An open reading frame of the gene contained a polypeptide of 133 amino acids with a conserved active center, WCGPC, which appeared in all of the thioredoxin proteins. A deduced amino acid sequence of the CTrx-h showed the highest sequence identity with those of Arabidopsis thioredoxin-h2 (75.2%) and thioredoxin-h5 (46.6%) proteins, but it shared a low sequence homology to other isotypes of plant thioredoxinm and thioredoxin-f. The CTrx-h protein that is expressed in E. coli represented not only an insulin reduction activity, but also electron transferring activity from NADPH to thioredoxin-dependent peroxidase. A genomic Southern blot analysis using the cDNA insert of CTrx-h revealed that the gene consisted of a small multigene family in Chinese cabbage genome. On the contrary to other thioredoxin-h proteins that were widely distributed in most tissues of the plant, the CTrx-h was predominantly expressed in flowers. The expression was very low in other tissues. The data of the Northern blot analysis suggests that the CTrx-h may have other functions in flower development or differentiation, in addition to its defensive role.

• Microbiology and Biotechnology Letters
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• v.32 no.4
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• pp.297-306
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• 2004

Heat shock proteins (HSPs) were induced in cells in the thermal stress, and the HSP90 family is one of the major classes of HSPs. Gene encoding HSPs have been characterized from various mammals and piscine. We have cloned and sequenced the HSP90 cDNA from a brain cDNA library constructed from flounder (Paralichthys oliThe result of sequence analysis shows it to be the HSP90~. The nucleotide sequence of the HSP90$\beta$ was composed of 2791 long, encoding 726 amino acid residues. The flounder hsp90$\beta$ gene showed very high sequence homology with hsp90f3 of European sea bass (96.6%), zebrafish (92.9%), Atlantic salmon (92.0%) and human (89.5%). We also constructed a phylogenetic tree based on HSP90 amino acid sequences from vertebrate species. Gene-specific primers were selected and used in RT-PCR reactions to measure the basal hsp90$\beta$ mRNA. The hsp90f3 gene is constitutively expressed at a fairly high level in all examined tissues (brain, liver, kidney, muscle, and spleen). In order to express protein of flounder hsp90$\beta$ in E. coli, we used the His-tagged pETvector. Then, the expression of flounder HSP90$\beta$ was confirmed by Western blot analysis.

• Journal of Life Science
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• v.15 no.3 s.70
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• pp.317-322
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• 2005

The cycloinulooligosaccharide fructanotransferase (CFTase) gene (cft) from Paenibacillus macerans was subcloned into an E. coli-yeast shuttle vector, pYES2.0, resulting in pYGECFTN. The plasmid pYGECFTN (8.6 kb) was introduced into Saccharomyces cerevisiae SEY2102 cells and then the transformants were selected on the synthetic defined media lacking uracil. The cft gene expression in yeast transformant was demonstrated by the analyses cyclofructan (CF) spots on thin-layer chromatogram. The recombinant CFTase was not secreted into the medium and localized in the periplasmic space. The production of CF was observed after 5 min of the enzymatic reaction with inulin. The optimun pH and temperature for CF production were found to be at pH 8.0 and $45^{\circ}C$, respectively. Enzyme activity was stably maintained up to $55^{\circ}C$. The CF was produced from all inulin sources and was most efficiently produced from dahlia tubers and Jerusalem artichokes.

• Microbiology and Biotechnology Letters
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• v.33 no.2
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• pp.84-89
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• 2005

Phosphomannomutase (PMM) is a key enzyme in prokaryotes and eukaryotes, which catalyzes the conversion of ${\alpha}$-D-mannose 6-phosphate to ${\alpha}$-D-mannose 1-phosphate. The latter is the substrate for the synthesis of GDP-mannose, which serves as the mannosyl donor for many metabolic pathways in the cells. We report here on the isolation of a gene from a genomic library of Sphingomonas chungbukensis DJ77, the pmmC gene encoding phosphomannomutase. The gene was cloned into E. coli expression vector, and the sequence was analyzed. The ribosomal binding site GGAAG lays 5 bp upstream of the ORF of 750 bp, which is initiated by ATG codon and terminated by TAG. The predicted sequence of the enzyme consists of 249 amino acids with a molecular mass of 27.4 kDa and showed $86.9\%$ similarity to that of eukaryotic phosphomannomutase after bioinformatical analyses with the conserved domain search of NCBI. The purified gene product revealed the activity of phosphomannomutase. In conclusion, we confirmed that pmmC gene encodes phosphomannomutase actually.

• Korean Journal of Plant Tissue Culture
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• v.24 no.5
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• pp.305-311
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• 1997

Bacillus thuringiensis, a gram-positive soil bacterium, is characterized by its ability to produce crystalline inclusions during sporulation. The crystal proteins exhibit a highly specific insecticidal activity. An insecticidal crystal protein (ICP), Cry II A, is specifically toxic to both lepidopteran and dipteran insects. In this study, tobacco plants transformed by the cry II A gene have been generated. The Cry II A crystal protein was purified from E. coli JM103 harboring cry II A gene by differential solubility. The activated Cry II A was prepared by tryptic digestion. The purified protoxin (70 kDa) and the activated toxin (50 kDa) were analyzed by SDS-PAGE. To generate the transgenic tobacco having cry II A gene, the cry II A gene was subcloned to a plant expression vector, pSRL2, having two CaMV 35S promoters. The recombinant plasmid was transformed into tobacco (N. tabacum var. Petit Havana SR1) by Agrobacterium-mediated leaf disc transformation. Through the regeneration, six putative transgenic tobacco plants were obtained and three transformants were confirmed by Southern blot analysis. It has been found that one plant had single copy of cry II A gene, another had two copies of the gene, and the third had a truncated gene. After the immunochemical confirmation of cry II A expression in plants, the transgenic tobacco plants will be used to study the genetics of future generation with the insecticidal crystal protein gene cry II A.

• Journal of Life Science
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• v.21 no.1
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• pp.96-101
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• 2011

S-Adenosyl-L-methionine synthtase (SAM-s) catalyzes the biosynthesis of SAM from ATP and L-methionine. SAM plays important roles in the primary and secondary metabolism of cells. A metK encoding a SAM-s was searched from Streptomyces natalensis producing natamycin, a predominantly a strong antifungal agent, inhibiting the growth of both yeasts and molds and preventing the formation of aflatoxin in filamentous fungi. To obtain the metK of S. natalensis, PCR using primers designed from the two highly conserved regions for metK genes of Streptomyces strains was carried out, and an intact 1.2-kb metK gene of S. natalensis was cloned by genomic Southern hybridization with PCR product as a probe. To identify the function of the cloned metK gene, it was inserted into pSET152ET for its high expression in the Streptomyces strain, and then introduced into S. lividans TK24 as a host by transconjugation using E. coli ET12567(pUZ8002). The high expression of metK in S. lividans TK24 induced actinorhodin production on R5 solid medium, and its amount in R4 liquid medium was 10-fold higher than that by exconjugant including only pSET152ET.

• Korean Journal of Microbiology
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• v.55 no.2
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• pp.103-111
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• 2019

DNA methylation is involved in diverse processes in bacteria, including maintenance of genome integrity and regulation of gene expression. CcrM, the DNA methyltransferase conserved in Alphaproteobacterial species, carries out $N^6$-adenine or $N^4$-cytosine methyltransferase activities using S-adenosyl methionine as a co-substrate. Celeribacter marinus IMCC12053 from the Alphaproteobacterial group was isolated from a marine environment. Single molecule real-time sequencing method (SMRT) was used to detect the methylation patterns of C. marinus IMCC12053. Gibbs motif sampler program was used to observe the conversion of adenosine of 5'-GANTC-3' to $N^6$-methyladenosine and conversion of $N^4$-cytosine of 5'-GpC-3' to $N^4$-methylcytosine. Exocyclic DNA methyltransferase from the genome of strain IMCC12053 was chosen using phylogenetic analysis and $N^4$-cytosine methyltransferase was cloned. IPTG inducer was used to confirm the methylation activity of DNA methylase, and cloned into a pQE30 vector using dam-/dcm- E. coli as the expression host. The genomic DNA and the plasmid carrying methylase-encoding sequences were extracted and cleaved with restriction enzymes that were sensitive to methylation, to confirm the methylation activity. These methylases protected the restriction enzyme site once IPTG-induced methylases methylated the chromosome and plasmid, harboring the DNA methylase. In this study, cloned exocyclic DNA methylases were investigated for potential use as a novel type of GpC methylase for molecular biology and epigenetics.

• Reproductive and Developmental Biology
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• v.31 no.3
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• pp.161-167
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• 2007

In this study, we constructed and tested retrovirus vectors designed to express the human thrombopoietin gene under the control of the tetracycline-inducible promoters. To increase the hTPO gene expression at him-on state, WPRE sequence was also introduced into retrovirus vector at downstream region of either the hTPO gene or the sequence encoding reverse tetracycline-controlled transactivator (rtTA). Primary culture cells (PFF, porcine fetal fibroblast; CEF, chicken embryonic fibroblast) infected with the recombinant retrovirus were cultured in the medium supplemented with or without doxycycline for 48hr, and induction efficiency was measured by comparing the hTPO gene expression level using RT-PCR, western blot and ELISA. Higher hPTO expression and tighter expression control were observed from the vector in which the WPRE sequence was placed at downstream of the hTPO (in CEF) or rtTA(in PFF) gene. This resulting tetracycline inducible vector system may be helpful in solving serious physiological disturbance problems which have been a major obstacle in successful production of transgenic animals.

• Korean Journal of Microbiology
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• v.43 no.1
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• pp.19-22
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• 2007

Acetohydroxyacid synthase (E.C.2.2.1.6., AHAS) is the enzyme that catalyses the first step in the synthesis of the branched-chain amino acids valine, leucine and isoleucine. The AHAS gene (TIGR access code HI2585) from Heamophilus influenzae was cloned into the bacterial expression vector pET-28a and expressed in the Escherichia coli strain BL21(DE3). The expressed enzyme was purified by $Ni^{2+}-charged$ HiTrap chelating HP column. The purified enzyme appears as a single band on SDS-PAGE with a molecular mass of about 63.9 kDa. The enzyme exhibits absolute dependence on the three cofactors FAD, $MgCl_{2}$ and thiamine diphosphate for activity. Specific activity of purified enzyme has 3.22 unit/mg and optimum activity in the pH 7.5 at $37^{\circ}C$. This enzyme activity has an effect on the buffer. When comparing the enzyme activity against the organic solvent, it followed in type and the difference it is but even from the aqueous solution where the organic solvent is included with the fact that the enzyme activity is maintained.

• Journal of Sericultural and Entomological Science
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• v.52 no.1
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• pp.45-51
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• 2014

A cecropin-A gene was isolated from the immunized larvae of the Japanese oak silkworm, Antheraea yamamai and designed Ay-CecA. The complete Ay-CecA cDNA consists of 419 nucleotides with 195 bp open reading frame encoding a 64 amino acid precursor that contains a putative 22-residue signal peptide, a 4-residue propetide and a 37-residue mature peptide with a theoretical mass of 4046.81. The deduced amino acid sequence of the peptide evidenced a significant degree of identity (62 ~ 78% identity) with other lepidopteran cecropins. Like many insect cecropin, Ay-CecA also harbored a glycine residue for C-terminal amidation at the C-end, which suggests potential amidation. To understand this peptide better, we successfully expressed bioactive recombinant Ay-CecA in Escherichia coli that are highly sensitive to the mature peptide. For this, we fused mature Ay-CecA gene with insoluble protein ketosteroid isomerase (KSI) gene to avoid the cell death during induction. The fusion KSI-CecA protein was expressed as inclusion body. The expressed fusion protein was purified by Ni-NTA immobilized metal affinity chromatography (IMAC), and cleaved by cyanogen bromide (CNBr) to release recombinant Ay-CecA. The purified recombinant Ay-CecA showed considerably antibacterial activity against Gram-negative bacteria, E. cori ML 35, Klebsiella pneumonia and Pseudomonas aeruginosa. Our results proved that this peptide with a potent antibacterial activity may play a role in the immune response of Japanese oak silkworm.