• BMB Reports
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• v.40 no.1
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• pp.82-87
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• 2007

Our heterologous expression system of the human ferritin H-chain gene (hfH) allowed us to characterize the cellular effects of ferritin in yeasts. The recombinant Saccharomyces cerevisiae (YGH2) evidenced impaired growth as compared to the control, which was correlated with ferritin expression and with the formation of core minerals. Growth was recovered via the administration of iron supplements. The modification of cellular iron metabolism, which involved the increased expression of high-affinity iron transport genes (FET3 and FTR1), was detected via Northern blot analysis. The findings may provide some evidence of cytosolic iron deficiency, as the genes were expressed transcriptionally under iron-deficient conditions. According to our results examining reactive oxygen species (ROS) generation via the fluorescence method, the ROS levels in YGH2 were decreased compared to the control. It suggests that the expression of active H-ferritins reduced the content of free iron in yeast. Therefore, present results may provide new insights into the regulatory network and pathways inherent to iron depletion conditions.

• International Journal of Industrial Entomology and Biomaterials
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• v.7 no.2
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• pp.197-201
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• 2003

The recombinant plasmids harboring a heterologous gene coding mouse endostatin were transfected and expressed stably in Trichoplusia ni Tn 5B1-4 cells and Bombyx mori BmN cells, respectively. Recombinant endostatin expressed in the stably transformed Tn 5B1-4 and BmN cells was secreted into the medium. BmN cells are relatively lower in maximum cell growth and recombinant endostatin production than Tn 5B 1-4 cells. Recombinant endostatin was also purified to homogeneity using a simple one-step ${Ni^2+}$ affinity fractionation method. Purified recombinant endostatin inhibited endothelial cell proliferation in a dose-dependent manner. The concentration at half-maximum inhibition $({ED_50})$ for recombinant endostatin was approximately 0.35 ${\mu}g$/ml.

• Bulletin of the Korean Chemical Society
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• v.33 no.12
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• pp.4041-4046
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• 2012

The recombinant expression of proteins has been the method of choice to meet the demands from proteomics and structural genomics studies. Despite its successful production of many heterologous proteins, Escherichia coli failed to produce many other proteins in their native forms. This may be related to the fact that the stresses resulting from the overproduction interfere with cellular processes. To better understand the physiological change during the overproduction phase, we profiled the metabolites along the time course of the recombinant protein expression. We identified 32 metabolites collected from different time points in the protein production phase. The stress induced by protein production can be characterized by (A) the increased usage of aspartic acid, choline, glycerol, and N-acetyllysine; and (B) the accumulation of adenosine, alanine, oxidized glutathione, glycine, N-acetylputrescine, and uracil. We envision that this work can be used to create a strategy for the production of usable proteins in large quantities.

• Archives of Pharmacal Research
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• v.18 no.3
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• pp.173-178
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• 1995

Ciprofloxacin resistance mechanisms were studied by investigating the inhibitory effect of ciprofloxacin on the gyrase-mediated DNA supercoiling and the intracellular accumulation of ciprofloxacin in clinical isolates of Pseudomonas aeruginosa. A higher amount of ciprofloxacin was required to inhibit the gyrases purified from the ciprofloxacin-resistant strains than that from the sensitive strain. Reconstitution of heterologous gyrase subunits from different strains revealed alterations in the A and/or the B subunits of gyrase in these strains. In addition, the resistant strains accumulated approximately a half amount of ciprofloxacin inside the cells, compared to the sensitive strain. However, when the active efflux was blocked by carbonyl cyanide m-chlorophenyl hydrazone treatment, intracellular concentration of ciprofloxacin was elevated about 4-7 fold in these strains, while the sensitive strain was not significantly affected by this treatment, indicating that the ciprofloxacin-resistant strains developed a drug efflux system. Interestingly, these resistant strains expressed an envelope protein of approximately 51 kD. These studies suggest that alterations in the gyrase as well as the active drug-efflux system conferred dual ciprofloxacin resistance mechanisms to these clinical isolates of P. aeruginosa.

• Archives of Pharmacal Research
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• v.25 no.2
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• pp.208-213
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• 2002

The human polyomavirus JC virus is the etiologic agent of progressive multifocal leukoencephalopathy (PML). As the JC virus early promoter directs cell-specific expression of the viral replication factor large T antigen, transcriptional regulation constitutes a major mechanism of glial tropism in PML. It has been demonstrated that SV4O or JC virus large T antigen interacts with p53 protein and regulates many viral and cellular genes. In this study we founts that p53 represses the JC virus early promoter in both glial and nonglial cells To identify the cis-regulatory elements responsible for p53-mediated repression, deletional and site-directed mutational analyses were performed . Deletion of the enhancer region diminished p53-mediated transcriptional repression. However, point mutations of several transcription factor binding sites in the basal promoter region did not produce any significant changes. In support of this observation, when the enhancer was fused to a heterologous promoter, p53 red reduced the promoter activity about three fold. These results indicate that the enhancer region is important for tole repression of JC virus transcription by p53. Furthermore, coexpression of JC virus T antigen with a p53 protein abolished p53-mediated repression of the JC virus early promoter in non-glial cells, but not in glial cells. This finding suggests that T antigen interacts with p53 and regulates JC virus transcription in a cell-specific manner.

• Preventive Nutrition and Food Science
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• v.3 no.1
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• pp.98-104
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• 1998

As an efffort ot construct LAB (latice acid bacteria), capable of utilizing starch as fermentation substrate without the aid of externally supplied enzymes, plasmid vectors containing the amyL($\alpha$-amylase/pullulansase gene) from Clostridium thermophydrosulfuricum, and glucoamylase cDNA from Asperigillus shirousamii were constructed and introduced itno E. coli and L. lactis. For expression in procaryotes , 1.9kb glucoamylase cDNA encoding the mature form of enzyme was PCR amplified and translationaly fused to a PCR amplified 260 bp fragment containing the promotor and secretion signals of amyl in the same reading frame. The production of $\alpha$-amylase, Apu, and glucoamlase in E. coli and L. lactis was confirmed by enzyme assay and zymography . Enzymeswere detected in both cellpellets and supernatants, indicating theworking of scretion signals in heterologous hosts. The efficiencies of secretion were varibale depending on the gene and host. The highest $\alpha$- amylase acitivity observed was 1.1 units and most activiity was detected from thecell pellets. The degree of gene expression in both hosts and the effect on the growth of hosts were examined.

• Journal of Microbiology and Biotechnology
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• v.5 no.4
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• pp.224-228
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• 1995

Fed-batch fermentations were conducted to produce human lipocortin-I (LC1), a potential anti-inflammatory agent, from recombinant Sacchromyces cerevisiae carrying a galactose-inducible expression system. The cell growth, expression level of LC1, and the plasmid stability were investigated under various LC1 induction modes performed by three different galactose feeding strategies. Galactoe was fed to induce the expression of LCl from the beginning (initial induction) of culture or when the cell concentration reached 120 OD (mid-phase induction) or 300 OD (late induction). Among the three galactose-induction modes tested, the initial induction mode yielded the best result with respect to a final expression level of LC1. Fedbatch fermentation with initial induction mode produced LC1 at a conentration of 220 mg/l, which corresponded to 1.38- and 1.53-fold increases over those produced by mid-phase and late induction modes.

• Journal of Microbiology and Biotechnology
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• v.26 no.8
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• pp.1464-1472
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• 2016

The BLi03719 protein of Bacillus licheniformis DSM13 belongs to the most abundant extracellular proteins under phosphate starvation conditions. In this study, the function of this phosphate starvation inducible protein was determined. An amino-acid sequence analysis of the BLi03719-encoding gene showed a high similarity with genes encoding the barnase of Bacillus amyloliquefaciens FZB42 and binase-like RNase of Bacillus pumilus SARF-032. The comparison of the control strain and a BLi03719-deficient strain revealed a strongly reduced extracellular ribonuclease activity of the mutant. Furthermore, this knockout mutant exhibited delayed growth with yeast RNA as an alternative phosphate and carbon source. These results suggest that BLi03719 is an extracellular ribonuclease expressed in B. licheniformis under phosphate starvation conditions. Finally, a BLi03719 mutant showed an advantageous effect on the overexpression of the heterologous amyE gene under phosphate-limited growth conditions.

• Journal of Microbiology and Biotechnology
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• v.18 no.7
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• pp.1216-1220
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• 2008

Sequence analysis of the metabolically rich genome of Streptomyces peucetius ATCC 27952 revealed a 2,199 bp sesquiterpene alcohol (germacradienol) synthase-encoding gene from the germacradienol synthase/terpene cyclase gene cluster. The gene was named spterp13, and its putative function is as a germacradienol synthase/terpene cyclase. The amino acid sequence of Spterp13 shows 66% identity with SAV2163 (GeoA) from S. avermitilis MA4680 and 65% identity with SCO6073 from S. coelicolor A3(2), which produces germacradienol/geosmin. The full-length recombinant protein was heterologously expressed as a his-tagged fusion protein in Escherichia coli, purified, and shown to catalyze the $Mg^{2+}$-dependent conversion of farnesyl diphosphate to the germacradienol, which was verified by gas chromatography/mass spectrometry.

• Toxicological Research
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• v.12 no.1
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• pp.81-86
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• 1996

Antigenicity of recombinant human interferon $\beta$(LB00013), a newly developed drug for anti-cancer and anti-viral therapeutic use, was investigated in mice and guinea pigs. The following results were obtained: 1. Mice showed no production of antibodies against LB00013 sensitized with aluminum hydroxide gel (Alum) as an adjuvant, when judged by the heterologous passive cutaneous anaphylaxis (PCA) test in rats. Meanwhile, antibodies against ovalbumin (OVA) sensitized with Alum were clearly detected. 2. In guinea pigs, the sensitization of neither LB00013 only nor LB00013 with complete Freund's adjuvant (CFA) produced positive reactions in the homologous active systemic anaphylaxis (ASA) and the PCA tests. Meanwhile, the sensitization of OVA with CFA produced positive reactions in both PCA and ASA. 3. A LB00013-specific reaction was not observed in an indirect hemagglutination(IHA) assay using sera isolated from LB00013 sensitized mice. The present results suggested that LB00013 may have no antigenic potential in mice and guinea pigs.