• Journal of the Korean Wood Science and Technology
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• v.33 no.4 s.132
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• pp.45-52
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• 2005

The manganese peroxidase (mnp5) from white-rot fungus Phanerochaete chrysosporium has been heterologously expressed in the methylotrophic yeast Pichia pastoris. The majority of the rMnP5 (recombinant MnP5) produced by P. pastoris exhibited an approximate molecular mass 45 kDa considerably larger than that of the predicting mnp5 due to two glycosylation sites of mnp5. After site direct mutation treatment, the effect of N-linked hyperglycosylation was examined by enzyme activity. Analysis by sodium dodesyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie Brilliant Blue (CBB) staining revealed a major protein band with a molecular mass of 37 kDa. Enzyme activity of M-rMnP5 (mutant recombinant MnP5) was similar to that of rMnP5, indicating that hyperglycosylation did not affect the active site. In this work, active mnp5 was successfully expressed in P. pastoris, suggesting that P. pastoris has potential capability of producing active heme-containing proteins.

• KSBB Journal
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• v.11 no.1
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• pp.8-14
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• 1996

Bacillus subtillis strains with transition state regulator mutations and a spore mutation were developed for the overexpression of apsE and for the enhancement of expression level. Among the many regulator genes, degU and hpr were chosen as a representative positive and negative regulator for the aprE, respectively. Spo II G was used for the construction of asporogeneous strains. All the mutants were constructed from two protease-deleted strain DB104 and the apsE gene was transformed with an integration vector pMK101. DB104(deg$U^h$(32) $his^+$)::pMK101(Cm) and DB104($\Delta$her(Em))::pMKl01(Cm) show 7-fold and about 2-fold increase in aprE expression level, respectively. But the effect of transition state regulator mutation on the aprE expression was diminished when the integrated aprE gene was amplified by the high concentration of chloramphenicol, i. e. 30 $\mu\textrm{g}$/ml. DB104($\Delta$spoIIG(Pm) degUh(32) his+)::pMK101(Cm) and DB104($\Delta$spoIIG(Pm) $\Delta$hpr(Em))::pMK101 double mutant show 10-fold and 3-fold increase in aprE expression level, respectively. The results suggest that sporulation mutation and transition state regulator mutation have independent and additive effect on the aprE expression, and the same gene dosage effect on the transition state regulator mutation was also identified.

• Journal of Life Science
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• v.14 no.1
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• pp.21-25
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• 2004

To identify genes involved in the decolorization of both crystal violet and malachite green, we isolated random mutants generated by transposon insertion in triphenylmethane-decolorizing bacterium, Citrobacter sp. The resulting mutant bank yielded 14 mutants with complete defect in color removal capability of both crystal violet and malachite green. Southern hybridization with a Tn5 fragment as a probe showed a single hybridized band in 5 mutants and these mutants appeared to have insertions at different sites of the chromosome. Tn5-inserted genes were isolated and the DNA sequence flanking Tn5 was determined. From comparison with a sequence database, putative protein products encoded by cmg genes were identified as follows. cmg 2 is MaIC protein in maltose transport system; cmg 6 is transcriptional regulator (LysR-type): cmg 12 is a putative oxidoreductase. The sequences deduced from two cmg genes, cmg 8 and cmg 11, showed no significant similarity to any protein with a known function. Therefore, these results indicate that these two cmg genes encode unidentified proteins responsible for decolorization of both crystal violet and malachite green.

• Applied Chemistry for Engineering
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• v.24 no.6
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• pp.628-632
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• 2013

Arthrospira platensis (A. platensis) is an economically important microalgae because it has carbohydrates, lipids, proteins and a number of phytochemicals. It is also a valuable source used in the production of biodiesel and functional foods. In this study, A. platensis was exposed to electron beam irradation (240 kGy) and induced random mutagenesis for strain improvement. Several mutants were obtained, and the resulting mutant was designated as EB29. The growth rate and chlorophyll content of EB29 was similar to those of wild type. However, the lipid content of EB29 was increased seven-fold compared to that of wild type when comparing the nile red fluorescent intensity. Semi-quantitative analysis of EB29 using the calibration plot of standard lipid, triolein, represented $78.6{\mu}g/mL$, which increased 2 times compared to wild type ($41.4{\mu}g/mL$). When analyzing the fatty acid profile of EB29, polyunsaturated fatty acids (PUFAs), such as gamma-linolenic acid (GLA) in EB29 increased about six-fold. Moreover, fatty acids affecting the quality of biodiesel increased compared to that of wild type. Thus, electron beam could be used for the strain improvement of microalgae in order to accumulate PUFAs and alteration of fatty acid profile for biodiesel.

• Korean Journal of Microbiology
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• v.46 no.2
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• pp.122-126
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• 2010

RAD53 functions as an effector kinase of checkpoint pathways in Saccharomyces cerevisiae, which plays a central role to regulate many downstream cellular processes in response to DNA damage. It also involves in transcriptional activation of various genes including RNR genes which encode the key enzyme required for dNTP synthesis. In this study, we identified CYC8 as a suppressor for the hydroxyurea sensitivity of $rad53{\Delta}$ mutation. $Rad53{\Delta}$ mutant transformed with a multi-copy plasmid containing CYC8 showed increased hydroxyurea resistance. In contrast, TUP1 which forms a complex with CYC8 did not function as a suppressor. In the case of mutations, both $cyc8{\Delta}$ and $tup1{\Delta}$ suppressed hydroxyurea sensitivity of $rad53{\Delta}$. Since CYC8 can propagate as a prion in yeast, overexpression of CYC8 induced misfolding of the normal CYC8 proteins, resulting in dominant cyc8-phenotype. Therefore, it is suggested that CYC8 can act as a multi-copy suppressor due to its prion property. It was observed that the levels of RNR transcription were increased in the yeast strains containing either multi-copies of CYC8 gene or $cyc8{\Delta}$ mutation, suggesting that the increased level of RNR will elevate the intracellular pools of dNTPs, which, in turn, suppress the phenotype of $rad53{\Delta}$ mutation.

• Molecules and Cells
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• v.22 no.3
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• pp.343-352
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• 2006

Sialic acid is a sugar typically found at the N-glycan termini of glycoproteins in mammalian cells. Lec3 CHO cell mutants are deficient in epimerase activity, due to a defect in the gene that encodes a bifunctional UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE). Sialic acid modification on the cell surface is partially affected in these cells. We have mutagenized Lec3 CHO cells and isolated six mutants (termed C2m) deficient in the cell surface expression of polysialic acid (PSA). Mutant C2m9 was partially defective in expression of cell-surface PSA and wheat germ agglutinin (WGA) binding, while in the other five mutants, both cell-surface PSA and WGA binding were undetectable. PSA expression was restored by complementation with the gene encoding the CMP-sialic acid transporter (CST), indicating that CST mutations were responsible for the phenotypes of the C2m cells. We characterized the CST mutations in these cells by Northern blotting and RT-PCR. C2m9 and C2m45 carried missense mutations resulting in glycine to glutamate substitutions at amino acids 217 (G217E) and 256 (G256E), respectively. C2m13, C2m39 and C2m31 had nonsense mutations that resulted in decreased CST mRNA stability, and C2m34 carried a putative splice site mutation. PSA and CD15s expression in CST-deficient Lec2 cells were partially rescued by G217E CST, but not by G256E CST, although both proteins were expressed at similar levels, and localized to the Golgi. These results indicate that the novel missense mutations isolated in this study affect CST activity.

• Journal of Life Science
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• v.15 no.4 s.71
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• pp.553-560
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• 2005

The stf (Salmonella typhimurium fimbriae) operon consisting of stfA(CDEFG assumes to encode putative fimbriae. The complete stf operon is existed in S. typhimurium and S. choleraesuis, whereas it is absent in S. typhi. Analyses of the amino acid residues between major subunit StfA of the Stf fimbriae and those of known other fimbriaes suggested that Stf belongs to class I type fimbriae. Through comparison of StfD chaperone with the other fimbrial chaperones, and of C-terminus in subunits of Stf fimbriae, it belongs to FGS (with a short Fl-G1 loop) subfamily. In order to investigate the expression of stf operon, we have constructed a Salmonella strain containing a chromosomal stfA::lacZYA transcriptional fusion, resulting in S. typhimurium $_X8532$. The strain $_X8532$ lacked the expression of \beta-galactosidase$ under normal culture conditions. However, with longer incubation time of the S. typhimurium $_X8532$, we have isolated 21 individual strains exhibiting $Lac^+$ phenotype. $Lac^+$ phenotype was appeared as approximately 0.03 frequency per generation. All isolates expressed lacZ constitutively in the various environmental conditions. Various global regulatory proteins including RpoS, OmpR, and CpxR were not involved in the regulation of the stf operon. A S. typhimurium $_X8661$ mutant lacking stfAC function attenuated 6.7 folds more than that of wild type $_X3761$ in the mouse virulence test, suggesting in the somehow involved in the Salmonella pathogenesis.

• Korean Journal of Microbiology
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• v.32 no.2
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• pp.102-108
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• 1994

Autonomously replicating sequence Binding Factor 1(ABF1) is a DNA-binding protein that specifically recognizes the $RTCRYN_5ACG$ at many sites in the yeast genome including the promoter element, mating-type silencer and ARS. To express the intact full-length ABF1 gene in E. coli, the ABF1 gene has been cloned into pMAL-c2 and His-61, Leu-353 and Leu-360 were substituted with other amino acid. ABF1 fusion proteins of wild type ABF1 and H61A, L353R and L360R nutants were purified by amylose resin affinity chromatography. Fusion protein of MBP and ABF1 was digested by Factor Xa and Characterized by gel retardation assay and complementation test. As aresult, we suggested that other DNA binding motif except atypical inc-finger motif is in the middle region of ABF1.

• The Korean Journal of Physiology and Pharmacology
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• v.27 no.4
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• pp.417-426
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• 2023

The TRPM4 gene encodes a Ca²⁺-activated monovalent cation channel called transient receptor potential melastatin 4 (TRPM4) that is expressed in various tissues. Dysregulation or abnormal expression of TRPM4 has been linked to a range of diseases. We introduced the hemagglutinin (HA) tag into the extracellular S6 loop of TRPM4, resulting in an HA-tagged version called TRPM4-HA. This TRPM4-HA was developed to investigate the purification, localization, and function of TRPM4 in different physiological and pathological conditions. TRPM4-HA was successfully expressed in the intact cell membrane and exhibited similar electrophysiological properties, such as the current-voltage relationship, rapid desensitization, and current size, compared to the wild-type TRPM4. The presence of the TRPM4 inhibitor 9-phenanthrol did not affect these properties. Furthermore, a wound-healing assay showed that TRPM4-HA induced cell proliferation and migration, similar to the native TRPM4. Co-expression of protein tyrosine phosphatase, non-receptor type 6 (PTPN6 or SHP1) with TRPM4-HA led to the translocation of TRPM4-HA to the cytosol. To investigate the interaction between PTPN6 and tyrosine residues of TRPM4 in enhancing channel activity, we generated four mutants in which tyrosine (Y) residues were substituted with phenylalanine (F) at the N-terminus of TRPM4. The YF mutants displayed properties and functions similar to TRPM4-HA, except for the Y256F mutant, which showed resistance to 9-phenanthrol, suggesting that Y256 may be involved in the binding site for 9-phenanthrol. Overall, the creation of HA-tagged TRPM4 provides researchers with a valuable tool to study the role of TRPM4 in different conditions and its potential interactions with other proteins, such as PTPN6.

• Journal of Life Science
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• v.30 no.3
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• pp.285-290
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• 2020

Foot-and-mouth disease virus (FMDV), a member of the genus Aphthovirus in the Picornaviridae family, affects wild and domesticated ruminants and pigs. FMDV causes various clinical symptoms, including severe inflammation in infected tissue. Genome RNA of FMDV shows a positive single-strand chain approximately 8.3 kb long and encodes a single long open reading frame (ORF). The ORF is translated into structural and non-structural proteins by viral proteases. The FMDV 2C protein is one of the non-structural proteins encoded by FMDV and plays a critical role in FMD pathogenesis, including inflammation, apoptosis, and viral replication. In this study, we examined whether FMDV 2C induces intracellular expression of pro-inflammatory cytokine tumor necrosis factor alpha (TNFα). FMDV 2C expression in pig IBRS-2 cells increased mRNA and protein expression of TNFα at the transcriptional level via activation of TNFα promoter. Treatment with 4-phenylbutyric acid, an endoplasmic reticulum (ER) stress reducer, decreased TNFα expression induced by FMDV 2C. Activating transcription factor 4 (ATF4), a transcription factor mediating ER stress response, induced transactivation of TNFα promoter and expression of mRNA and protein of TNFα. However, the dominant negative mutant of ATF4 did not induce FMDV 2C-mediated TNFα expression. The results indicate that FMDV 2C protein increases clinical inflammation via ATF4-mediated TNFα expression and is associated with ER stress induction.