• Journal of Microbiology and Biotechnology
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• v.20 no.12
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• pp.1756-1763
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• 2010

Pasteurella multocida serogroup D strain, which produces P. multocida toxin (PMT), is a widespread and harmful pathogen of respiratory diseases such as pneumonia and progressive atrophic rhinitis (PAR) in swine. Vaccination has been considered the most desirable and effective approach for controlling the diseases caused by toxigenic P. multocida. To investigate the antigenicity and immunogenicity of partial fragments of recombinant PMT, recombinant proteins of the N-terminal (PMT-A), middle (PMT-B), C-terminal (PMT-C), and middle-C-terminal (PMT2.3) regions of PMT were successfully produced in an Escherichia coli expression system. The molecular masses of PMT-A, PMT-B, PMT-C, and PMT2.3 were ca. 53, 55, 35, and 84 kDa, respectively, purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity column chromatography. All the recombinant proteins except for PMT-A showed immune responses to antisera obtained from a swine showing symptoms of PAR. Moreover, high titers of PMT-specific antibodies were raised from mice immunized with each of the recombinant proteins; however, the immunoreactivities of the antibodies to authentic PMT and heat-inactivated whole bacteria were different, respectively. In the protection study, the highest protection against homologous challenge was shown in the case of PMT2.3; relatively poor protections occurred for the other PMT fragments.

• 한국생물공학회:학술대회논문집
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• 2003.10a
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• pp.391-391
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• 2003

• 한국생물공학회:학술대회논문집
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• 2000.04a
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• pp.117-120
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• 2000

For detecting toxicity of endocrine disruptors (EDs), rapid, sensitive, and simple methods are needed. Therefore, in this study, a new method in which the different toxic effect of EDs can be monitored using 4 different recombinant bacteria was designed and evaluated. It was found that the recombinant bacteria could monitor the toxic effect, not estrogenic effect, due to EDCs through the measurement of bioluminescence and cell growth rate, which were shown to depend upon a form of cellular toxicity, such as DNA damage, protein damage, oxidative damage, and membrane damage. In addition, it was found that the damage done by EDCs can be divided into several groups based upon the toxic mechanisms of the EDCs

• Food Science and Biotechnology
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• v.18 no.2
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• pp.396-401
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• 2009

The nucleotide sequence contains 2 open reading frames encoding a 45-amino-acid protein homologous to a transcriptional repressor protein CopG, and a 203-amino-acid protein homologous to a replication protein RepB. Putative countertranscribed RNA, a double-strand origin, and a single-strand origin were also identified. A shuttle vector, pUCL2.1, for various lactic acid bacteria (LAB) was constructed on the basis of the pCL2.1 replicon, into which an erythromycin-resistance gene as a marker and Escherichia coli ColE1 replication origin were inserted. pUCL2.1 was introduced into E. coli, Lc. lactis, Lactobacillus (Lb.) plantarum, Lb. paraplantarum, and Leuconostoc mesenteroides. The recombinant LAB maintained traits of transformed plasmid in the absence of selection pressure over 40 generations. Therefore, pUCL2.1 could be used as an E. coli/LAB shuttle vector, which is an essential to engineer recombinant LAB strains that are useful for food fermentations.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2000.04a
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• pp.94-99
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• 2000

Bioluminescence is being used as a prevailing reporter of gene expression in microorganisms and mammalian cells. Bacterial bioluminescence draws special attention from environmental biotechnologists since it has many advantageous characteristics, such as no requirement of extra substractes, highly sensitive, and on-line measurability. Using bacterial bioluminescence as a reporter of toxicity has replaced the classical toxicity monitoring technology of using fish or daphnia with a cutting-edge technology. Fusion of bacterial stress promoters, which control the transcription of stress genes corresponding to heat-shock, DNA-, or oxidative-damaging stress, to the bacterial lux operon has resulted in the development of novel toxicity biosensors with a short measurement time, enhanced sensitivity, and ease and convenient usage. Therefore, these recombinant bioluminescent bacteria are expected to induce bacterial bioluminescence when the cells are exposed to stressful conditions, including toxic chemicals. We have used these recombinant bioluminescent bacteria in order to develop toxicity biosensors in a continuous, portable, or in-situ measurement from for air, water, and soil environments. All the data obtained from these toxicity biosensors for these environments were found to be repeatable and reproducible, and the minimum detection level of toxicity was found to be ppb (part per billion) levels for specific chemicals.

• Journal of Microbiology and Biotechnology
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• v.15 no.2
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• pp.330-336
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• 2005

Lactococcus lactis A164 is a nisin Z-producing strain isolated from kimchi. Its antimicrobial spectrum has been found to be active against most Gram-positive bacteria tested, yet inactive against Gram-negative bacteria [3]. Accordingly, to overcome this drawback, the current study attempted to express human $\beta$-defensin-l (hBD-l), which kills both Gram-positive and Gram-negative bacteria in L. lactis AI64. When the hBD-l cDNA was introduced using a nisin Z-controlled expression cassette, the L. lactis A164 transformants grew very poorly, due to the bactericidal effect of the expressed hBD-l against the transformants. Therefore, a gene fusion system was designed to reduce the toxicity of the expressed heterologous protein against the host cells. As such, the hBD-l gene was fused to the DsbC- Tag of pET -40b(+), then introduced to L. lactis A 164. The transformants expressed an intracellular 35.6-kDa DsbC-hBD-l fusion protein that exhibited slight activity against the host cells, yet not enough to strongly inhibit the cell growth. To obtain the recombinant hBD-l, the DsbC-hBD-l fusion protein was purified by nickel-affinity column chromatography, and the DsbC-Tag removed by cleaving with enterokinase. The cleaved mature hBD-l exhibited strong bactericidal activity against E. coli JM109, indicating that the recombinant L. lactis A 164 produced a biologically active hBD-I. In addition, the recombinant L. lactis A 164 was also found to produce the same level of nisin Z as the wild-type.

• International Journal of Industrial Entomology and Biomaterials
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• v.17 no.1
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• pp.137-141
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• 2008

We previously isolated and cloned a cDNA of abaecin from the Bombus ignitus. In an effort to produce a large amount of soluble abaecin at low cost, we successfully expressed the peptide in Escherichia coli that are highly sensitive to its mature form. For this, we fused the peptide encoding 39 amino acids of mature B. ignitus abaecin to the thioredoxin gene together with a C-terminal 6xHis tag. An enterokinase cleavage site was introduced between the 6xHis tag and mature abaecin to allow final release of the recombinant peptide. A high yield of 9.6 mg soluble fusion protein from 200 ml of bacterial culture was purified by $Ni^{2+}$-charged His-Bind resin affinity column, and 1.4 mg of pure active recombinant abaecin was readily obtained by enterokinase cleavage, followed by affinity chromatograph. The molecular mass of recombinant abaecin peptide was determined by Tricin-SDS-PAGE analysis. The recombinant abaecin exhibited antibacterial activity against Gram-negative bacteria.

• Journal of Marine Bioscience and Biotechnology
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• v.8 no.1
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• pp.1-9
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• 2016

The hydrolysis of biomass to fermentable sugar (saccharification) and to oligosaccharide is an essential process in biotechnology including biorefinery and biofood. Various macroalgae are commercially cultivated in several Asian countries as a useful resource for food and agar production. Agar is a major component of the cell walls of red algae that can be hydrolyzed by agarase. Agarases are classified into ${\alpha}$-agarase (E.C. 3.2.1.158) and ${\beta}$-agarase (E.C. 3.2.1.81) according to the cleavage pattern and grouped in the glycoside hydrolase (GH) family (GH-16, GH-58, GH-86, GH-96, and GH-118) based on the amino acid sequences of the proteins. Agarases have been isolated from various bacteria found in seawater and marine sediments. To increase productivity of the enzyme, a research on recombinant enzymes has been done. The application of recombinant agarase can be possible in the various filed such as energy, food, cosmetics, medical and so on. This paper reviews the source, biochemical characteristics and production system of recombinant agarases for further study.

• Korean Journal of Agricultural Science
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• v.41 no.3
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• pp.245-249
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• 2014

Among dozens of DNA polymerases cloned from thermophilic bacteria, Taq DNA polymerase from Thermus aquaticus has been most frequently used in polymerase chain reaction (PCR) that is being applied to gene cloning, DNA sequencing, gene expression analysis, and detection of infectious and genetic diseases. Since native Taq DNA polymerase is expressed at low level in T. aquaticus, recombinant Escherichia coli system was used to produce Taq DNA polymerase in a large amount. Taq DNA polymerase was expressed as a soluble form under the control of tac promoter in E. coli, and purified by heat treatment and ion exchange chromatographies. The purified Taq DNA polymerase was nearly homogeneous and exhibited a similar DNA amplification activity with a commercial Taq DNA polymerase.

• Korean journal of applied entomology
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• v.55 no.2
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• pp.81-89
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• 2016

Double-stranded RNA (dsRNA) has been applied to control insect pests by its suppressive activity against specific target genes. Integrin is a heterodimer (${\alpha}$ and ${\beta}$) transmembrane protein and plays a critical role in cell-to-cell or cell-to-extracellular matrix interactions in eukaryotes. Suppression of ${\beta}$ subunit integrin gene expression by its specific dsRNA (= dsINT) induces significant mortality against target insects. Furthermore, a recombinant bacterium expressing dsINT is potent to kill target insects. However, it is necessary to develop a formulation technique of the dsRNA-expressing bacteria to apply the bacterial insecticide against field populations. This study formulated the recombinant bacteria by freeze-drying and tested its control efficacy against target insects. The formulation maintained significant insecticidal activity against last instar larvae of Spodoptera exigua. While a commercial Bacillus thuringiensis (Bt) insecticide exhibited only about 60% insecticidal activity against S. exigua last instar, an addition of the dsINT-expressing bacterial formulation significantly enhanced the Bt insecticidal activity. The dsINT-expressing bacterial formulation exhibited relative selectivity to target insects depending on sequence similarity. These results indicate that a freeze-dried form of dsRNA-expressing bacteria keeps its insecticidal activity.