• Journal of Life Science
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• v.19 no.12
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• pp.1690-1697
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• 2009

CD11c and costimulatory molecules such as CD80 and CD86 express mainly in dendritic cells (DCs). In this study, we investigated the biologic effects of recombinant Fms-like tyrosine kinase-3 (Flt-3) ligand on the expression of DC surface markers, including CD11c in leukemia cell lines, such as KG-1, HL-60, NB4, and THP-1 cells. The expression of the Flt-3 receptor was found in NB4 and HL-60 cells, as well as KG-1 cells, but not in THP-1 cells. When KG-1 cells were cultured in a medium containing Flt-3 ligand or granulocyte macrophage-colony stimulating factor (GM-CSF) plus tumor necrosis factor (TNF)-$\alpha$, cell proliferation was inhibited and the expression levels of CD11c, major histocompatibility complex (MHC)-I, and MHC-II were increased in the cells. Flt-3 ligand also increased the expression level of CD11c on HL-60 and NB4 cells, but not on THP-1 cells. In comparison with CD11c expression, the expression level of CD11b on KG-1 cells, but not on NB4 and HL-60 cells, was slightly increased by Flt-3 ligand. Flt-3 ligand induced phosphorylation of extracellular signal-regulated kinase-1/2 (ERK-1/2) and p38-mitogen-activated protein kinase (p38-MAPK) in KG-1 cells, and the up-regulation of CD11c expression by Flt-3 ligand in the cells was abrogated by PD98059, an inhibitor of MEK. The results suggest that Flt-3 ligand up-regulates DC surface markers on $CD34^+$ myelomonocytic KG-1 cells, as well as promyelocytic leukemia cells, and that the differentiation of the leukemia cells into DC-like cells by Flt-3 ligand is mediated by ERK-1/2 activity.

• Journal of Microbiology
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• v.45 no.2
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• pp.158-167
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• 2007

In this study, we have cloned a novel cDNA encoding for a papain-family cysteine protease from the Uni-ZAP XR cDNA library of the polychaete, Periserrula leucophryna. This gene was expressed in Escherichia coli using the T7 promoter system, and the protease was characterized after partial purification. First, the partial DNA fragment (498 bp) was amplified from the total RNA via RT-PCR using degenerated primers derived from the conserved region of cysteine protease. The full-length cDNA of cysteine protease (PLCP) was prepared via the screening of the Uni-ZAP XR cDNA library using the $^{32}P-labeled$ partial DNA fragment. As a result, the PLCP gene was determined to consist of a 2591 bp nucleotide sequence (CDS: 173-1024 bp) which encodes for a 283-amino acid polypeptide, which is itself composed of an 59-residue signal sequence, a 6-residue propeptide, a 218-residue mature protein, and a long 3'-noncoding region encompassing 1564 bp. The predicted molecular weights of the preproprotein and the mature protein were calculated as 31.8 kDa and 25 kDa, respectively. The results of sequence analysis and alignment revealed a significant degree of sequence similarity with other eukaryotic cysteine proteases, including the conserved catalytic triad of the $Cys^{90},\;His^{226},\;and\;Asn^{250}$ residues which characterize the C1 family of papain-like cysteine protease. The nucleotide and amino acid sequences of the novel gene were deposited into the GenBank database under the accession numbers, AY390282 and AAR27011, respectively. The results of Northern blot analysis revealed the 2.5 kb size of the transcript and ubiquitous expression throughout the entirety of the body, head, gut, and skin, which suggested that the PLCP may be grouped within the cathepsin F-like proteases. The region encoding for the mature form of the protease was then subcloned into the pT7-7 expression vector following PCR amplification using the designed primers, including the initiation and termination codons. The recombinant cysteine proteases were generated in a range of 6.3 % to 12.5 % of the total cell proteins in the E. coli BL21(DE3) strain for 8 transformants. The results of SDS-PAGE and Western blot analysis indicated that a cysteine protease of approximately 25 kDa (mature form) was generated. The optimal pH and temperature of the enzyme were determined to be approximately 9.5 and $35^{\circ}C$, respectively, thereby indicating that the cysteine protease is a member of the alkaline protease group. The evaluation of substrate specificity indicated that the purified protease was more active towards Arg-X or Lys-X and did not efficiently cleave the substrates with non-polar amino acids at the P1 site. The PLCP evidenced fibrinolytic activity on the plasminogen-free fibrin plate test.

• BMB Reports
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• v.37 no.3
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• pp.282-291
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• 2004

Phytases catalyze the release of phosphate from phytic acid. Phytase-producing microorganisms were selected by culturing the soil extracts on agar plates containing phytic acid. Two hundred colonies that exhibited potential phytase activity were selected for further study. The colony showing the highest phytase activity was identified as Aspergillus niger and designated strain 113. The phytase gene from A. niger 113 (phyI1) was isolated, cloned, and characterized. The nucleotide and deduced amino acid sequence identity between phyI1 and phyA from NRRL3135 were 90% and 98%, respectively. The identity between phyI1 and phyA from SK-57 was 89% and 96%. A synthetic phytase gene, phyI1s, was synthesized by successive PCR and transformed into the yeast expression vector carrying a signal peptide that was designed and synthesized using P. pastoris biased codon. For the phytase expression and secretion, the construct was integrated into the genome of P. pastoris by homologous recombination. Over-expressing strains were selected and fermented. It was discovered that ~4.2 g phytase could be purified from one liter of culture fluid. The activity of the resulting phytase was 9.5 U/mg. Due to the heavy glycosylation, the expressed phytase varied in size (120, 95, 85, and 64 kDa), but could be deglycosylated to a homogeneous 64 kDa species. An enzymatic kinetics analysis showed that the phytase had two pH optima (pH 2.0 and pH 5.0) and an optimum temperature of $60^{\circ}C$.

• Biomolecules & Therapeutics
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• v.23 no.2
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• pp.189-194
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• 2015

P450 1A2 is responsible for the metabolism of clinically important drugs and the metabolic activation of environmental chemicals. Genetic variations of P450 1A2 can influence its ability to perform these functions, and thus, this study aimed to characterize the functional significance of three P450 1A2 allelic variants containing nonsynonymous single nucleotide polymorphisms (P450 $1A2^*8$, R456H; $^*15$, P42R; $^*16$, R377Q). Variants containing these SNPs were constructed and the recombinant enzymes were expressed and purified in Escherichia coli. Only the P42R variant displayed the typical CO-binding spectrum indicating a P450 holoenzyme with an expression level of ~ 170 nmol per liter culture, but no P450 spectra were observed for the two other variants. Western blot analysis revealed that the level of expression for the P42R variant was lower than that of the wild type, however the expression of variants R456H and R377Q was not detected. Enzyme kinetic analyses indicated that the P42R mutation in P450 1A2 resulted in significant changes in catalytic activities. The P42R variant displayed an increased catalytic turnover numbers ($k_{cat}$) in both of methoxyresorufin O-demethylation and phenacetin O-deethylation. In the case of phenacetin O-deethylation analysis, the overall catalytic efficiency ($k_{cat}/K_m$) increased up to 2.5 fold with a slight increase of its $K_m$ value. This study indicated that the substitution P42R in the N-terminal proline-rich region of P450 contributed to the improvement of catalytic activity albeit the reduction of P450 structural stability or the decrease of substrate affinity. Characterization of these polymorphisms should be carefully examined in terms of the metabolism of many clinical drugs and environmental chemicals.

• Journal of Life Science
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• v.17 no.9 s.89
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• pp.1248-1254
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• 2007

The secretory overexpression of Clostridium thermocellum endoglucanase A gene (celA) was examined in Saccharomyces cerevisiae using Kluyveromyces marxianus exoinulinase (INU1) signal sequence and GAL10 promoter. The two plasmids, pYEG-CT1 with its own signal sequence, and pYInu-CT1 with INU1 signal sequence were introduced to S. cerevisiae SEY2102 and S. cerevisiae 2805 host strains, respectively, and then each transformant was selected on the synthetic defined media lacking uracil. The expression level and secretion efficiency of endoglucanase A was increased by $18{\sim}22%$ and 11%, respectively, by INU1 signal sequence over celA signal sequence. By considering the high level of expression (361 unit/I), plasmid stability (89%), and secretion efficiency (70%), S. cerevisiae 2805 harboring plasmid pYInu-CT1 was selected as the opti-mal host vector system for the production of cellulose-degrading enzyme and recombinant yeast probiotic. The total expression and secretion efficiency of endoglucanase A was 418 unit/l and 73%, respectively, in the batch fermentation of S. cerevisiae 2805/pYlnu-CT1 on galactose medium. The mo-lecular weight of secreted endoglucanase A was found to be greater than 100 kDa, presumably due to the N-linked glycosylation.

• Korean Journal of Microbiology
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• v.28 no.2
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• pp.91-97
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• 1990

Complementary DNA (cDNA) coding for human cytoplasmic superoxide dismutase (SOD1) (superoxide: superoxide oxidoreductase E.C.1.15.1.1) was isolated from human liver cDNA library of $\lambda$gt11 by in situ plaque hybridization. The insery cDNA gas the 5' untranslational region (UTR) and 3'UTR of SOD1 gene. Polymerase Chain Reaction (PCR) method was used fro subcloning of SOD1 structural gene. Using synthetic sense strand primer (24mer) containing a start codon and antisense strand primer (24mer), SOD1 structural gene was selectively amplified. Amplified DNA was directly cloned into the HincII site of pUC19 plasmid. Insery cDNA was subcloned into M13 mp19 and sequenced by dideowy chain termination method with Sequenase. The nucleotide sequence of insert cDNA had an open reading frame (ORF) coding for 153 amino acid residues. The structural gene of cytoplasmic SOD was placed under the control of bacteriophage $\lambda P_{L}$ regulatory sequences, generating a highly efficient expression plasmid. The production of human SOD1 in E. coli cells was about 7% of total cellular proteins and recombinant human SOD1 possessed its own enzymatic acitivity.

• The Plant Pathology Journal
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• v.25 no.4
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• pp.344-351
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• 2009

A biocontrol bacterium Bacillus licheniformis N1 grown in nutrient broth showed no chitinolytic activity, while its genome contains a gene which encodes a chitinase. The gene for chitinase from B. licheniformis N1 was amplified by PCR and the deduced amino acid sequence analysis revealed that the chitinase exhibited over 95% identity with chitinases from other B. licheniformis strains. Escherichia coli cells carrying the recombinant plasmid displayed chitinase activity as revealed by the formation of a clear zone on chitin containing media, indicating that the gene could be expressed in E. coli cells. Chitinase gene expression in B. licheniformis N1 was not detected by RT-PCR analysis. The protein was over-expressed in E. coli BL21 (DE3) as a glutathione S-transferase fusion protein. The protein could also be produced in B. subtilis 168 strain carrying the chitinase gene of N1 strain. The crude protein extract from E. coli BL21 carrying GST fusion protein or culture supernatant of B. subtilis carrying the chitinase gene exhibited enzyme activity by hydrolyzing chitin analogs, 4-methylumbelliferyl-$\beta$-D-N,N'-diacetylchitobioside and 4-methylumbelliferyl-$\beta$-D-N,N',N"-triacetylchitotrioside. These results indicated that even though the chitinase gene is not expressed in the N1 strain, the coding region is functional and encodes an active chitinase enzyme. Furthermore, B. subtilis 168 transformants expressing the chitinase gene exhibited antifungal activity against Fulvia fulva by suppressing spore germination. Our results suggest that the proper engineering of the expression of the indigenous chitinase gene, which will lead to its expression in the biocontrol strain B. licheniformis N1, may further enhance its biocontrol activity.

• Journal of Microbiology and Biotechnology
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• v.15 no.4
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• pp.880-886
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• 2005

Using carotenoid genes of Erwinia herbicola, metabolic engineering was carried out for lycopene production with the pAC-LYCO4 plasmid, which was composed of a chromosomal DNA fragment of E. herbicola containing the crtE, crtB, and crtI genes under the control of the tetracycline promoter and the ipi gene of Haematococcus pluvialis with the trc promoter. Plasmid pAC-LYCm4 was constructed for efficient expression of the four exogenous genes using a strong RBS sequence and the same tetracycline promoter. The optimized expression construct of pAC-LYCm4 increased Iycopene production three times as compared with pAC-LYCO4. pAC-LYCm5 containing ispA behind the four exogenous genes was constructed. There was no significant difference in Iycopene production and cell growth between pAC-LYCm4 and pAC-LYCm5. FPP synthase encoded by ispA was not rate-limiting for Iycopene production. Each gene of crtE, crtB, crtI, and ipi was overexpressed, using pBAD-crtE, pBAD-crtIB, and pBAD-ipiHPI, in addition to their expression from pAC-LYCm4. However, there was no increase oflycopene production with the additional overexpression of each exogenous gene. The four exogenous genes appeared to be not rate-limiting in cells harboring pAC-LYCm4. When pDdxs, pBAD24 containing dxs, was introduced into cells harboring lycopene synthetic plasmids, lycopene production of pAC-LYCO4, pAC-LYCm4, and pAC-LYCm5 was increased by 4.7-, 2.2-, and 2.2-fold, respectively. Lycopene production of pBAD-DXm4 containing crtE, crtB, crtI, ipi, and dxs was 5.2 mg/g dry cell weight with $0.2\%$ arabinose, which was 8.7-fold higher than that of the initial strain with pAC-LYC04. Therefore, the present study showed that proper regulation of a metabolically engineered pathway is important for Iycopene production.

• Microbiology and Biotechnology Letters
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• v.35 no.1
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• pp.11-16
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• 2007

The arylsulfatase gene (astA, 984 bp ORF) from Pseudoalteromonas carrageenovora genome was amplified by PCR and subcloned into the pHCE-IA vector, in which the hyper consitutive expression (HCE) promoter from the D-amino acid aminotransferase (D-AAT) gene of Geobacillus toevii was employed. The transformant cell, Escherichia coli BL21 (DE3)/pHCE-AST, on LB agar plate containig 4-methylumbelliferyl sulfate, showed an intense fluorescence at 360 nm, indicating that 4-methylumbelliferone was liberated by desulfatate activity. When BL21 (DE3)/pHCE-AST was grown on LB media containing 0.4% glucose or 0.4% glycerol, the arylsulfatase activity was higher at glycerol rather than at glucose. On 2% glycerol medium, the arylsulfatase activity reached 15.0 unit/ml, which was 2.6-fold higher expression level than that with 1% glycerol. The DNA ladder in agarose prepared from agar by this recombinant enzyme revealed similar resolution and migration patterns with a commercial agarose. This results suggests that arylsulfatase overexpressed in E. coli could be applicable to the economic production of electrophoretic-grade agarose.

• Journal of Life Science
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• v.17 no.8 s.88
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• pp.1082-1089
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• 2007

Serological anlysis of recombinant cDNA expression libraries (SEREX) has led to identification of several categories of new antigens recognized by the immune system of cancer patients, which are referred to as the cancer immunome. We analyzed normal testis cDNA expression libraries with serumobtained from non-small lung cancer patient and isolated 40 distinct antigen designated KP-LuT-1 through KP-LuT-40. Among these antigens 20 antigens were previously identified by SEREX analysis of other tumor types, and 20 out of 40 antigens (50%) did not match entries in Cancer Immunome Database and were considered newly identified antigens. Sequencing analysis showed that the anti-gens comprised 26 functional known proteins and 14 noble/uncharacterized gene products. Of these, the hypothetical protein KP-LuT-6 was shown tissue-restricted. RT-PCR showed it to be expressed strongly only in normal testis. In addition to normal tissues-restricted expression, KP-LuT-6 mRNA was detected in lung tumor samples(3/l0), stomach tumor samples(3/l0), and breast tumor samples(l/5), whereas not detected in colon tumor samples(O/I2). These data suggest that KP-LuT-6 is a cancer/testis (CT)-like antigen as a potential target for cancer immunotherapies.