• Journal of Microbiology and Biotechnology
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• 제11권1호
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• pp.164-166
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• 2001

To examine the characteristics of the recombinant thin aggregative fimbriae of Salmonella, the AgfA subunit gene was amplified from Salmonella enteritidis using a PCR. The maltose binding protein (MBP)-AgfA fusion protein was overproduced in E. coli and purified. The secondary structure of AgfA was then elucidated from the difference CD spectra. An estimation of the secondary structure of AgfA using the self-consistent method revealed a mostly ${\beta}-sheet$ structure.

• Biomolecules & Therapeutics
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• 제16권3호
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• pp.184-189
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• 2008

The proto-oncogene protein DEK has been implicated in various human disease including cancer. We have shown that DEK induces caspase-dependent apoptosis in Drosophila by regulating histone acetylation. Reverse transcription-polymerase chain reaction (RT-PCR) method based on annealing control primers was used to screen and identify differentially expressed genes (DEGs) in DEK overexpressed HeLa cells. Among the genes identified, clusterin and fibrillarin have major role in apoptosis pathway regulation. TFIIIC and RPS24 are implicated in HAT mediated transcriptional initiation and cololectal cancer, respectively. To further analyze DEK's role in apoptosis, multiplex PCR was performed. Caspase-3, -7, and -10 and proapoptotic gene bid were newly identified as possible target genes regulated by DEK expression.

• Journal of Microbiology
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• 제40권1호
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• pp.43-50
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• 2002

A cluster of genes encoding the pyruvate dehydrogenase complex (PDC) of Streptomyces seoulensis, a Gram-positive bacterium, was cloned and sequenced. The genes of S. seoulensis consist of four open reading frames. The first gene, lpd, which encodes a lipoamide dehydrogenase, is followed by pdhB encoding a dihydrolipoamide acetyltransferase (E2p), pdhR, a regulatory gene, and pdhA encoding a pyruvate dehydrogenase component (Elp). Elp had an unusual homodimeric subunit, which has been known only in Gram-negative bacteria S. seoulensis E2p contains two lipoyl domains like those of humans and Streptomyces faecalis. The pdhR gene appears to be clustered with the structural genes of S. seoulensis PDC. The PdhR-overexpressed S. seoulensis howed growth retardation and the decrease of Elp, indicating that PdhR regulates the function of PDC by repressing the expression of Elp. A strain of Streptomyces licidans overexpressing S. seoulensis PdhR showed a significant decreasein the level of actinorhodin, implying a regulatory role for Streptomyces PDC in antibiotic biosynthesis.

• BMB Reports
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• 제34권1호
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• pp.39-42
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• 2001

Previous studies indicate that B. subtilis protoporphyrinogen oxidase is poorly inhibited by diphenyl ether herbicides. To better understand the basis of this insensitivity, the enzyme was overexpressed as a soluble protein in E. coli, purified and characterized. The mechanism of oxidation of B. subtilis protoporphyrinogen IX was studied and the enzyme kinetic parameters were determined for protoporpyrinogen IX; $K_m$, and $k_{cat}$ were $6.3\;{\mu}M$ and $0.028\;h-^1$, respectively. The enzyme required flavin adenine dinucleotide as a cofactor and its activity was enhanced by 1 mM n-octylglucopyranoside. The nonenzymatic oxidation rate was dependent on the concentration of protoporphyrinogen IX, suggesting that the reaction involves a pre-equilibrium step followed by a rate-limiting step.

• Toxicological Research
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• 제17권
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• pp.89-96
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• 2001

Recently, considerable attention has been focused on the role of cyclooxygenase-2 (COX-2) in the carcinogenesis as well as in inflammation. Improperly overexpressed COX-2 has been observed in many types of human cancers and transformed cells in culture. Thus, it is conceivable that targeted inhibition of abnormally or improperly up-regulated COX-2 provides one of the most effective and promising strategies for cancer prevention. A ubiquitous eukaryotic transcription factor, NF-kB is considered to be involved in regulation of COX-2 expression. Furthermore, extracellular-regulated protein kinase and p38 mitogen-activated protein (MAP) kinase appear to be key elements of the intracellular signaling cascades involved in NF-kB activation in response to a wide array of external stimuli. Certain chemopreventive phytochemicals suppress activation of NF-kB by blocking one or more of the MAP kinases, which may contribute to their inhibitory effects on COX-2 induction. One of the plausible mechanisms by which chemopreventive phytochemicals inhibit NF-kB activation involves suppression of degradation of the inhibitory unit I kB, which hampers subsequent translocation of p65, the functionally active subunit of NF-kB.

• Journal of Microbiology and Biotechnology
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• 제8권2호
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• pp.141-145
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• 1998

Overproduction of intracellular ${\beta}$-glucosidase was attempted by modifying the promoter region of a ${\beta}$-glucosidase gene cloned from Cellulomonas fimi and expressing it in Bacillus subtilis DB 104. A strong engineered promoter, BJ27UΔ88, was fused to the ${\beta}$-glucosidase gene after removing its native promoter. An effective Shine-Dalgamo sequence (genel0 of phage T7) was inserted between the promoter and the ${\beta}$-glucosidase structural gene. The modified gene was overexpressed in B. subtilis and produced 1121.5 units of ${\beta}$-glucosidase per mg protein which is about $12\%$ of total intracellular protein. Secretion of overproduced intracellular ${\beta}$-glucosidase was attempted by using the signal sequence of the Bacillus endoglucanase gene as well as an in-frame hybrid protein of endoglucanase. The hybrid protein was normally secreted into the culture medium and still retained ${\beta}$-glucosidase activity.

• Journal of Applied Biological Chemistry
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• 제58권2호
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• pp.97-100
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• 2015

The thermostable pectinase gene TmPec isolated from Thermotoga maritima was introduced into the NdeI site of pRSET-B vector and expressed in its intact form in Escherichia coli BL21. The overexpressed intact form of pectinase (TmPecN protein) was partially purified by heat-denaturation procedure. TmPecN showed the highest activity between 85 and $95^{\circ}C$, and at approximately pH 6.5. Enzyme activity was stably maintained at temperatures below $85^{\circ}C$. In the presence of $Ca^{2+}$, pectinase activity of TmPecN increased to 128.4% of normal level. In contrast, $Ba^{2+}$, $Zn^{2+}$, and $Mn^{2+}$ strongly inhibited TmPecN activity. We conclude that the biochemical properties of the intact form of TmPecN are comparable to those of the recombinant protein TmPec reported previously.

• Journal of Microbiology and Biotechnology
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• 제16권7호
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• pp.1144-1148
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• 2006

DNA can oxidatively be deaminated by ROS, which converts DNA base amino groups to keto groups and can trigger abnormal mutations, resulting in mutagenesis in organisms. In this study, a noncanonical purine dNTP pyrophosphatase (AfPPase) from a hyperthermophilic archaeon Archaeoglobus fulgidus, which hydrolyzes aberrant nucleoside triphosphates, was overexpressed in E. coli, purified, and characterized. The purified AfPPase showed remarkably high activity for XTP and dITP, suggesting that the 6-keto group of these nucleotides is critical for the reactivity. Under optimal reaction conditions, the reaction rate for these substrates was about 120 times that with dGTP. Therefore, AfPPase may play a significant role in DNA repair by hydrolysis of noncanonical nucleotides before they are misincorporated into DNA.

• Journal of Microbiology and Biotechnology
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• 제28권5호
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• pp.821-825
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• 2018

A copy number amplification system for yeast artificial chromosomes (YACs) was combined with simultaneous overexpression of genes integrated into a YAC. The chromosome VII (1,105 kb) was successfully split to 887 kb, 44 kb containing the element for copy number amplification, and a 184-kb split-YAC. The 44-kb split-mini YAC was amplified a maximum of 9-fold, and the activity of the reporter enzymes integrated into the split-mini YAC increased about 5-7-fold. These results demonstrate that the mini-YAC containing a targeted chromosome region can be readily amplified, and the specific genes in the mini-YAC could be overexpressed by increasing the copy number.

• Journal of Microbiology and Biotechnology
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• 제12권6호
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• pp.1002-1005
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• 2002

The effects of GroEL/ES chaperone on the production of soluble form of B. macerans cyclodextrin glucanotransferase (CGTase) in recombinant E. coli were investigated. The cgt gene and groEL/ES genes are under the control of T7 promoter and Pzt-1 promoter, respectively. The optimal concentrations of inducers, IPTG and tetracycline, were found to be 1.0 mM and 10 ng/ml, respectively. When tetracycline and IPTG were added at the early exponential phase (2h) and exponential phase (3h) of growth, respectively, about 1.5-fold increase of soluble CGTase activity and 1.6-fold increase of soluble CGTase protein were obtained. An SDS-PAGE analysis revealed that about $37.2\%$ of total CGTase protein was in the soluble fraction when GroEL/ES chaperone was overexpressed.