• Journal of Microbiology and Biotechnology
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• v.6 no.6
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• pp.451-455
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• 1996

Physiological studies on the expression of Bacillus thuringiensis subsp. tenebrionis (Btt) gene coding for insecticidal protein in recombinant Escherichia coli 537 were carried out to identify optimal culture condition. It was necessary to shift culture temperature from 30 to $42^{\circ}C$ to express the gene. Expression of the Btt toxin gene by recombinant E. coli 537 began within one hour after induction. Complex nitrogen sources increased production of the insecticidal protein. The total insecticidal protein was 0.5 g/I when using yeast extract as a complex nitrogen source. Soybean hydrolysate showed apparently the highest induction efficiency. After induction, the cellular content of the insecticidal protein was 5.4 times higher than it had been before induction. The optimal cultivation strategy was found to grow cells for 7hours at $30^{\circ}C$ and then 5-8 hours at $42^{\circ}C$. The optimal cultivation pH for the production of insecticidal protein was 6.5. The Btt toxin produced by the recombinant E. coli 537 was found to have the same level of potency against Colorado potato beetle as the original toxin.

• Asian-Australasian Journal of Animal Sciences
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• v.12 no.6
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• pp.871-880
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• 1999

The effects of dry roasting (110, 130, $150^{\circ}C$ for 15, 30, 45 min) on potential ruminant protein nutritional values in terms of: a), rumen bypass protein (BCP); b), rumen bypass starch (BST); c), fermented organic matter (FOM); d), true absorbed bypass protein (ABCP); e) microbial protein synthesized in the rumen based on available energy (E_MP); f), microbial protein synthesized in the rumen based on available nitrogen (N_MP); g), true protein supplied to the small intestine (TPSI); h), true absorbed rumen synthesized microbial protein (AMP); i), endogenous protein losses (ENDP); j), true digested protein in the small intestine (DVE); k), degraded protein balance (OEB) of whole lupin seeds (WLS) and faba beans (WFB) were evaluated by the new Dutch DV/OEB protein evaluation system. Dry roasting significantly increased BCP, BST, TPSI, ABCP, DVE (p<0.001) and decreased FOM, E_MP, AMP, N_MP and OEB (p<0.001) with increasing temperatures and times except that when temperature was at $110^{\circ}C$. The values of BCP, BST, TPSI, ABCP and DVE at $150^{\circ}C/45min$ for WLS and WFB were increased 2.2, 3.7; -, 2.0; 1.7, 1.7; 2.3, 3.7 and 1.7, 1.7 times and the values of FOM, E_MP, AMP, N_MP and OEB at $150^{\circ}C/45min$ for WLS and WFB were decreased by 15.3, 25.8; 18.1, 25.8; 18.7, 25.8; 54.6, 41.6 and 82.3% 54.7%, respectively, over the raw WLS and WFB. The results indicated that though dry roasting reduced microbial protein synthesis due to reducing FOM, TPSI didn't decrease but highly increased due to increasing BCP more than enough for compensation of the microbial protein decreasing. Therefore the net absorbable DVE in the small intestine was highly increased. The OEB values were significantly reduced for both WLS and WFB but not to the level of negative. It indicated that microbial protein synthesis might not be impaired due to the sufficient N supplied in the rumen, but the high positive OEB values in the most treatments except of $150^{\circ}C$ for 30 and 45 min of WLS (The OEB values: 54.8 and 26.0 g/kg DM) indicated that there were the large amounts of N loss in the rumen. It was concluded that dry roasting at high temperature was effective in shifting protein degradation from rumen to intestines and it increased the DVE values without reaching the negative OEB values. No optimal treatment was found in WLS due to the too high OEB values in all treatments. But dry roasting at $150^{\circ}C$ for 30 and 45 min might be optimal treatments for WLS due to the very lower OEB values.

• Asian-Australasian Journal of Animal Sciences
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• v.19 no.11
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• pp.1665-1670
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• 2006

The objective of this research was to provide the characterization and method for producing anti-E. coli O157:H7 antibodies in egg-laying hens and to determine if the antibody can restrain the proliferation of E. coli O157:H7 in-vitro. Selected antigenic fractions (whole cell, outer membrane protein and lipopolysaccharide (LPS)) from E. coli O157:H7 were injected to hens in order to produce anti-E. coli O157:H7 antibodies. The immune response and the egg yolk antibodies of laying hens against the whole cell, outer membrane protein and LPS antigens were monitored by ELISA. The level of antibodies against whole cell antigen monitored through ELISA sharply increased after the initial immunization, and it was found to be maximum on day 49 however, the level was maintained up to day 70. Antibodies (5 mg/ml) directed against the whole cell inhibited E. coli proliferation 10-13 times more than outer membrane protein or LPS. The antibody response against the whole cell antigens appeared to have higher activity in restraining the proliferation of E. coli O157:H7 than antibody against outer membrane protein or LPS. Results reflected that increasing the IgY's in the egg yolk could prevent greater economic losses due to human and animal health from pathogenic bacteria i.e. E. coli O157:H7.

• Journal of Microbiology and Biotechnology
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• v.14 no.3
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• pp.466-469
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• 2004

The recombinant bioluminescent Escherichia coli strains, DPD2511 and TV 1061 containing the katG and grpE promoters, respectively, from Vibrio fischeri fused to luxCDABE, were used to detect the adaptive and repair responses to oxidative damage caused by hydrogen peroxide $(H_2O_2)$, and protein damage due to phenol. The response ratio, represented as the bioluminescence induced in subsequent inductions of DPD2511 and TV1061 with the mother cells previously induced by each chemical, i.e., $H_2O_2$ and phenol during the previous induction stage, decreased suddenly compared with the ratio of the control culture of each strain, meaning there is a possible adaptive response to stress caused by chemicals. Protein damage due to phenol was completely repaired by the second culturing after the initial induction, as was oxidative damage caused by $H_2O_2$ which was also rapidly repaired, as detected by the recovery of bioluminescence level. This result suggests that E. coli promptly adapt and repair oxidative and protein damage by $H_2O_2$ and phenol completely.

• Archives of Pharmacal Research
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• v.21 no.3
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• pp.291-297
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• 1998

Cop protein has been overexpressed in Escherichia coli using a T7 RNA polymerase system. Purification to apparent homogeneity was achieved by the sequential chromatography on ion exchange, affinity chromatography, and reverse phase high performance liquid chromatography system. The molecular weight of the purified Cop was estimated as 6.1 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). But the molecular mass of the native state Cop was shown to be 19 kDa by an analytical high performance size exclusion chromatography, suggesting a trimer-like structure in 50 mM Tris-HCI buffer (pH 7.5) containing 100 mM NaCl. Cop protein Was calculated to contain $39.1% {\alpha}-helix, 16.8% {\beta}-sheet$, 17.4% turn, and 26.8% random structure. The DNA binding property of Cop protein expressed in E. coli Was preserved during the expression and purification process. The isoelectric point of Cop was determined to be 9.0. The results of amino acid composition analysis and N-terminal amino acid sequencing of Cop showed that it has the same amino acid composition and N-terminal amino acid sequence as those deduced from its DNA sequence analysis, except for the partial removal of N-terminal methionine residue by methionyl-aminopeptidase in E. coli.

• Journal of Microbiology and Biotechnology
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• v.16 no.9
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• pp.1422-1428
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• 2006

An ABC transporter apparatus of the Gram-negative bacterial type I secretion pathway can be used as a secretory protein expression system in Escherichia coli. Four types of coexpression systems for the Pseudomonas fluorescens lipase gene, tliA, and its cognate ABC transporter gene cluster, tliDEF, were constructed. When the relative expression levels were changed by adding different concentrations of IPTG, the secretion (16.9 U/ml of culture) of TliA in E. coli [pTliDEFA-223+pACYC184] was significantly higher than E. coli [pKK223-3+pTliDEFA-184] secreting the lowest level of TliA (5.2 U/ml of culture). Maximal accumulation of the lipase secreted occurred in the mid-exponential phase, implying that the efficient protein secretion via an ABC transporter was restricted only to actively growing cells. Finally, the secretion level of TliA in E. coli [pTliDEFA-223+pACYC184] was increased to 26.4 U/ml by inducing gene expression at the culture initiation time. These results indicate that a significant increase in the ABC transporter-dependent protein secretion can be achieved by simply controlling the relative expression levels between the ABC transporter and its passenger protein, even in the recombinant E. coli cells.

• Journal of Microbiology and Biotechnology
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• v.22 no.2
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• pp.234-238
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• 2012

Recombinant Escherichia coli displaying organophosphorus hydrolase (OPH) was used to overcome the diffusion barrier limitation of organophosphorus pesticides. A new anchor system derived from the N-terminal domain of ice-nucleation protein from Pseudomonas syringae InaV (InaV-N) was used to display OPH onto the surface. The designed sequence was cloned in the vector pET-28a(+) and then was expressed in E. coli. Tracing of the expression location of the recombinant protein using SDS-PAGE showed the presentation of OPH by InaV-N on the outer membrane, and the ability of recombinant E. coli to utilize diazinon as the sole source of energy, without growth inhibition, indicated its significant activity. The location of OPH was detected by comparing the activity of the outer membrane fraction with the inner membrane and cytoplasm fractions. Studies revealed that recombinant E. coli can degrade 50% of 2 mM chlorpyrifos in 2 min. It can be concluded that InaV-N can be used efficiently to display foreign functional protein, and these results highlight the high potential of an engineered bacterium to be used in bioremediation of pesticide-contaminated sources in the environment.

• Molecules and Cells
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• v.42 no.12
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• pp.840-849
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• 2019

The spatiotemporal mitotic processes are controlled qualitatively by phosphorylation and qualitatively by ubiquitination. Although the SKP1-CUL1-F-box protein (SCF) complex and the anaphase-promoting complex/cyclosome (APC/C) mainly mediate ubiquitin-dependent proteolysis of mitotic regulators, the E3 ligase for a large portion of mitotic proteins has yet to be identified. Here, we report c-Cbl as an E3 ligase that degrades DDA3, a protein involved in spindle dynamics. Depletion of c-Cbl led to increased DDA3 protein levels, resulting in increased recruitment of Kif2a to the mitotic spindle, a concomitant reduction in spindle formation, and chromosome alignment defects. Furthermore, c-Cbl depletion induced centrosome over-duplication and centriole amplification. Therefore, we concluded that c-Cbl controls spindle dynamics and centriole duplication through its E3 ligase activity against DDA3.

• Journal of the Society of Cosmetic Scientists of Korea
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• v.30 no.4 s.48
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• pp.479-483
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• 2004

An attempt was made to develop a proteinchip for screening of MITF (microphthalmia transcription factor) inhibitor. Binding of MITF to E-box causes transcription of several pigmenting genes including tyrosinase gene. We investigated binding of MITF and its DNA binding site (E-box) using a protein-DNA chip with various detection methods including flurorescence (Cyt3). SPR (surface plasmon resonance) and SPRi (surface plasmon resonance imaging). A fusion protein (MITF-Maltose Binding Protein) was attached on the glass plate by chemical modification. An inhibitory synthetic DNA oligomer, artificially designed based on the E-box sequence, inhibited the binding of MITF and E-box. These results showed the potentials of flurorescence-based MITF protein chip as a microarray for high throughput screening (HTS) system of depigmenting agents.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.15 no.3
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• pp.219-225
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• 1982

Four heat conduction models were examined for defatted soy-protein curds in order to get the 'intrinsic' thermal conductivity of soy-protein. As the result of examination, the 'intrinsic', thermal conductivities of soy-protein, frozen and unfrozen states, were determined on the basis of series model to be 0.488 W/m.K and 0.300 W/m.K, respectively. By using the 'intrinsic' thermal conductivity values of soybean protein and the series model, the effective thermal conductivity of soybean curds, with and without fat, at frozen and unfrozen states, was predicted satisfactorily, The temperature dependency of the effective thermal conductivity of soybean curd was mostly observed to correlate with the thermal conductivity of water and ice.