• Current Research on Agriculture and Life Sciences
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• v.17
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• pp.59-63
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• 1999

A cDNA encoding rice chloroplast small HSP, Oshsp21, was introduced into Escherichia coli using the pET expression vector to analyze the possible function of Oshsp21 under heat stress. We compared the viability of E. coli ${\lambda}BL21$ (DE3) cells transformed with recombinant plasmid containing Oshsp21 with the control E. coli cells transformed with pET28a vector under heat stress after IPTG induction. Upon heat treatment at $50^{\circ}C$, those cells that expressed Oshsp21 showed improved viability compared with control cells. When the cell lysates from E. coli transformants were heated at $55^{\circ}C$, the amounts of proteins denatured in the control and pEhsp21-transformed cells were about 60% and 35% of total cell proteins, respectively. These results indicate that rice chloroplast small HSP function as a molecular chaperone in cells.

• Journal of Life Science
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• v.17 no.11
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• pp.1601-1604
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• 2007

The gene for ${\beta}-agarase$ of an Agarivorans sp. JA-1 was expressed in Bacillus subtilis DB104, 168 and ISW1214 strains for mass-production. Among 3 host strains, B. subtilis ISW1214 secreted the highest amount of recombinant ${\beta}-agarase$ with a specific activity of 201 U/mg and 360 mg of protein into culture broth. This was approximately 130-fold higher than the production in E. coli as an expression host. Recombinant enzyme produced neoagarooligosaccharides such as neoagarohexaose, neoagarotetraose, and neoagarobiose from agar. Produced neoagarooligosaccharides showed antibacterial activities against gram-negative E. coli and gram-positive B. subtilis at a concentration of 1.5%. These data suggest that neoagarooligosaccharides could be an useful preservative for food industry.

• Microbiology and Biotechnology Letters
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• v.28 no.1
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• pp.21-25
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• 2000

We have got several clones from Serratia marcescens which stimulated the cells to use maltose as a carbon source in Escherichia. coli TP2139 ( lac, crp). One of the cloned genes, pCKB17, was further analyzed. In order to find whether the increased expression of the gent was under the direction of maltose metabolism, we constructed several recombinant subclones. We have found that the clone, pCKB17AV, codes maltose metabolism stimulation(mms) gene. E. coli transformed with the cloned gene showed increase in the activity of maltose utilzation, The recombinant proteins expressed by multicopy and induction with IPTG, one polypeptide of 29-kDa, was confirmed by SDS-PAGE. The overexpression of maltose-binding proter protein in the presence of mms gene was confirmed by Western blot analysis. Southern hybridization analysis confirmed that the cloned DNA fragment was originated from S. marcescens chromosomal DNA.

• Microbiology and Biotechnology Letters
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• v.40 no.2
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• pp.92-97
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• 2012

Antimicrobial peptides (AMPs) are important components of living organisms acting against Gram-negative and Gram-positive bacterial and fungal pathogens. Cathelicidin human peptides have a variety of biological activities that can be used in clinical applications. AMPs are not produced naturally in large quantities, and chemical synthesis is also economically impractical, especially for long peptides. Therefore, as an alternative, heterologous expression of AMPs by recombinant techniques has been studied as a means to reduce production costs. E. coli is an excellent host for the expression of AMPs, as well as other recombinant proteins, because of the low cost involved and its easy manipulation. However, overexpression of AMPs in E. coli has been shown to cause difficulties resulting from the toxicity of the subsequently produced AMPs. Therefore, fusion expression was theorized to be a solution to this problem. In this study, AMPs were expressed as fused proteins with the glutathione S-transferase (GST) binding protein to protect against the toxicity of AMPs when expressed in E. coli. The LL37, and hybrid gaegurin and LL37 (GGN4(1-16)-LL37(17-32), which we designated as GL32, peptides were expressed as GST-fusion proteins in E. coli and the fusion proteins were then purified by affinity columns. The purified peptides were obtained by removal of GST and were confirmed by western blot analysis. The purified antimicrobial peptides then demonstrated antimicrobial activities against Gram-negative and Gram-positive bacterial strains.

• BMB Reports
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• v.35 no.3
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• pp.297-301
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• 2002

Membrane lipid peroxidation processes yield products that may react with DNA and proteins to cause oxidative modifications. The oxyR gene product regulates the expression of enzymes and proteins that are needed for cellular protection against oxidative stress. Upon exposure to tert-butylhydroperoxide (t-BOOH) and 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH), which induce lipid peroxidation in membranes, the Escherichia coli oxyR overexpression mutant was much more resistant to lipid peroxidation-mediated cellular damage, when compared to the oxyR deletion mutant in regard to growth kinetics, viability, and DNA damage. The deletion of the oxyR gene in E. coli also resulted in increased susceptibility of superoxide dismutase to lipid peroxidation-mediated inactivation. The results indicate that the peroxidation of lipid is probably one of the important intermediary events in free radical-induced cellular damage. Also, the oxyR regulon plays an important protective role in lipid peroxidation-mediated cellular damage.

• Animal cells and systems
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• v.16 no.6
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• pp.455-461
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• 2012

Amongst the various antimicrobial peptides, lysozyme plays a central role in initiating and maintaining the antibacterial defense response of insect. Here we propose the biosynthesis and refolding of recombinant lysozyme in Escherichia coli expressed in inclusion body form. The Agrius lysozyme gene was amplified using gene specific primers and then ligated into the pGEX-4T-1 vector, which contained the glutathione S-transferase (GST) gene as a fusion partner. A recombinant lysozyme was expressed in E. coli Rosetta cells using a pGEX-4T-1 expression vector, and the fusion protein was induced by ioporpyl-${\beta}$-D-thiogalactopyranoside (IPTG). The recombinant protein produced as an inclusion body was resolubilized in solubilization buffer, and the resultant solution was dialyzed in refolding buffer. After thrombin cleavage, the recombinant lysozyme was purified by ion exchange chromatography and reverse phase chromatography. The recombinant lysozyme was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and immunoreactivity against the anti-Agrius lysozyme was observed by western blot analysis of this protein. The recombinant lysozyme displayed antibacterial activity against Bacillus megaterium and Micrococcus luteus, which was confirmed by the inhibition zone assay.

• BMB Reports
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• v.44 no.3
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• pp.193-198
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• 2011

The chitinase-producing strain SC081 was isolated from Korean traditional soy sauce and identified as Bacillus atrophaeus based on a phylogenetic analysis of the 16S rDNA sequence and a phenotypic analysis. A gene encoding chitinase from B. atrophaeus SC081 was cloned in Escherichia coli and was named SCChi-1 (GQ360078). The SCChi-1 nucleotide sequences were composed of 1788 base pairs and 596 amino acids, which were 92.6, 89.6, 89.3, and 78.9% identical to those of Bacillus subtilis (ABG57262), Bacillus pumilus (ABI15082), Bacillus amyloliquefaciens (ABO15008), and Bacillus licheniformis (ACF40833), respectively. A recombinant SCChi-1 containing a hexahistidine tag at the amino-terminus was constructed, overexpressed, and purified in E. coli to characterize SCChi-1. $H_6SCChi$-1 revealed a hydrolytic band on zymograms containing 0.1% glycol chitin and showed the highest lytic activity on colloidal chitin and acidic chitosan. The optimal temperature and pH for chitinolytic activity were $50^{\circ}C$ and pH 8.0, respectively.

• Korean Journal of Microbiology
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• v.47 no.1
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• pp.7-13
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• 2011

In general, expression of membrane protein in Escherichia coli is very toxic to the host organism, but the mechanism for the toxicity is not clear yet. Expression of human purinergic receptor $P2X_4$ was found to be extremely toxic to the host E. coli. We examined this toxicity by isolation and analysis of less toxic mutant proteins. We could isolate 30 less toxic mutants of $P2X_4$ after hydroxylamine mutagenesis. Western blot showed that all of them produced proteins smaller than the wild type $P2X_4$. DNA sequencing of two largest mutant proteins showed that they were lost its second transmembrane domain. Localization analysis of these mutant proteins showed that they are not in cytoplasmic membrane, but in inclusion bodies. These data showed that inactive truncated $P2X_4$ is not toxic to E. coli and membrane integration and functionality of $P2X_4$ may be needed to show host toxicity.

• Journal of Microbiology and Biotechnology
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• v.10 no.4
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• pp.496-501
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• 2000

Escherichia cole NZN111, which is known as a pfl ldhA double mutant strin, was metabolically engineered to produce succinic acid by overexpressing malic enzyme into the E. coli controlled by a trc promoter. Fermentation studies were carried out in a LB medium by first growing cells aerobically to an $OD_{600}$ of 5. At this point, 0.01 mM IPTG was added to induce the overexpression of malic enzyme and the agitation speed was gradually lowered. When the culture $OD_{600}$ reached 11, a complete anaerobic condition was achieved by flushing with a $CO_3-H_2$ gas mixture. When NZN111(pTrcML) was cultured at $37^{\circ}C$, the final succinic acid concentration of 2.8 g/l could be obtained after 30 h of anaerobic cultivation. The fermentation results were analyzed by the calculation of metabolic fluxes. Metaolic flux analysis showed that about 85% of phosphoenolpyruvate (PEP) was converted to pyruvate, and further converted to malic acid by malic enzyme.

• Plant Biotechnology Reports
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• v.2 no.1
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• pp.41-46
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• 2008

Salinity stress is a major limiting factor in cereal productivity. Many studies report improvements in salt tolerance using model plants, such as Arabidopsis thaliana or standard varieties of rice, e.g., the japonica rice cultivar Nipponbare. However, there are few reports on the enhancement of salt tolerance in local rice cultivars. In this work, we used the indica rice (Oryza sativa) cultivar BR5, which is a local cultivar in Bangladesh. To improve salt tolerance in BR5, we introduced the Escherichia coli catalase gene, katE. We integrated the katE gene into BR5 plants using an Agrobacterium tumefaciens-mediated method. The introduced katE gene was actively expressed in the transgenic BR5 rice plants, and catalase activity in $T_1$ and $T_2$ transgenic rice was approximately 150% higher than in nontransgenic plants. Under NaCl stress conditions, the transgenic rice plants exhibited high tolerance compared with nontransgenic rice plants. $T_2$ transgenic plants survived in a 200 mM NaCl solution for 2 weeks, whereas nontransgenic plants were scorched after 4 days soaking in the same NaCl solution. Our results indicate that the katE gene can confer salt tolerance to BR5 rice plants. Enhancement of salt tolerance in a local rice cultivar, such as BR5, will provide a powerful and useful tool for overcoming food shortage problems.