• Food Science and Preservation
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• v.15 no.5
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• pp.749-753
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• 2008

To assess the relationship between anti-oxidant and immunomodulatory activities of Diospyros kaki Thunb. fruit, we harvested persimmons on the first Friday of every month from July to October, and conducted a DPPH assay, a T-bet promoter assay, and an ELISA for IL-4 determination. Anti-oxidant activity increased as fruit weight rose. When the fruit was extracted with five different solvents, and the extracts examined for radical scavenging activity, such activity exhibited a pattern similar to that seen when anti-oxidant activity was assessed. T-bet promoter activity decreased on ripening, whereas IL-4 expression increased, as tested in a DNFB-induced animal model. Collectively, the results suggest that inflammation-inhibitory activity, valuable in treatment of some immune diseases, rises as persimmons ripen.

• Journal of Microbiology and Biotechnology
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• v.8 no.2
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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 Animal Science and Technology
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• v.45 no.2
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• pp.169-182
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• 2003

To understand the structure and regulation of bovine ADRP (Adipocyte Differentiation Related Protein) gene, we have isolated the genomic clone of bovine ADRP and determined its sequence. A genomic Southern blot analysis confirmed that ADRP gene is present as a single copy in bovine genome and the ADRP gene spans 12 kb. Bovine ADRP genomic clone, HwADRPg-1, had 8 exons and 7 introns, and all splicing sites conformed to the GT/AG rule with the exon-intron boundaries located exactly. Analysis of the upstream 649 bp of the sequence of HwADRPg-1 showed that it does not contain any canonical TATAA boxes; however Sp1 binding sites and CAAT boxes are found. The promoter contained potential binding sites for AP-1, AP-2 and several putative transcription factor binding sites. The 5'-flanking region of HwADRPg-1 contained muscle specific transcription activator Myo G and C/EBP (CCAAT/ enhancer binding protein) recognizing site. These results suppose that the Myo G transcription activator regulate the transcription of bovine ADRP gene in muscular tissue and its transcriptional activity was triggered by degree of muscular development. Our results provide the necessary analysis for other flanking sequences are needed in addition to the proximal cis elements of this promoter to confer adipocyte differentiation-dependent or growth-dependent transcriptional control.

• Journal of Microbiology and Biotechnology
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• v.8 no.1
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• pp.92-95
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• 1998

Overproduction of Escherichia coli thiol peroxidase in the periplasmic space was achieved by locating the appropriate gene on a downstream region of the strong T7 promoter. E. coli strain BL21 carrying the recombinant plasmid pSK-TPX was induced by IPTG, lysed, and analyzed by SDS-polyacrylamide gel electrophoresis. A large amount of the overexpressed thiol peroxidase was located in the periplasmic space. A homogeneous thiol peroxidase was obtained from E. coli osmotic shock fluid by simple one-step gel permeation chromatography.

• BMB Reports
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• v.35 no.6
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• pp.637-641
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• 2002

Only one natural promoter that interacts with bacteriophage K11 RNA polymerase has so far been identified. To identify more, in the present study restriction fragments of the phage genome were individually assayed for transcription activity in vitro. The K11 genome was digested with two 4-bp-recognizing restriction enzymes, and the fragments cloned in pUC119 were assayed with purified K11 RNA polymerase. Eight K11 promoter-bearing fragments were isolated and sequenced. We report that the nine K11 promoter sequences (including the one previously identified) were highly homologous from -17 to +4, relative to the initiation site at +1. Interestingly, five had -10G and -8A, while the other four had -10A and -8C. The consensus sequences with the natural -10G/-8A and -10A/-8C, and their variants with -10G/-8C and -10A/-8A, showed nearly equal transcription activity, suggesting residues at -10 and -8 do not regulate promoter activity. Using hybridization methods, physical positions of the cloned promoter-bearing sequences were mapped on SalI-and KpnI-restriction maps of the K11 genome. The flanking sequences of six cloned K11 promoters were found to be orthologous with T7 or T3 genomic sequences.

• Journal of Microbiology and Biotechnology
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• v.11 no.1
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• pp.85-91
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• 2001

To construct an efficient Bacillus subtilis expression vector, strong promoters were isolated from the chromosomal DNA libraries of Clostridium acetobutylicum ATCC 4259, Thermoactinomyces sp. E79, and Bacillus thermoglucosidasius KCTC 3400. The $P_{C27}$ promoter cloned from the clostridial chromosmal DNA showed a 5-fold higher promoter strength than the $P_{SP02}$ promoter in the expression of the cat gene, and its sequence was estimated as an upstream region of the predicted hypothetical gene (tet-R family bacterial transcription regulator gene) in C. acetobutylicum. As a promoter element, $P_{C27}$ exhibited putative nucleotide sequences that can bind with bacterial RNAP and the 3'end of the 16S rRNA just upstream of the start codon. In addition, the promoter activity of $P_{C27}$ was distinctively repressed in the presence of glucose. Using $P_{C27}$ as the promoter element, a glucose controllable B. subtilis expression vector was constructed and the lipase gene from Staphylococcus haemolyticus KCTC 8957P was expressed in B. subtilis. When compared with the lipase expression by the T7 promoter induced by IPTG in E. coli, the $P_{C27}$ promoter showed about a 1.5-fold higher expression level in B. subtilis than that without induction.

• Journal of Microbiology and Biotechnology
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• v.14 no.2
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• pp.225-230
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• 2004

A plasminogen kringle domain 1 to 3, rKl-3, was expressed in Escherichia coli under the control of T7 promoter. For the cost-effective production of rKl-3, the induction process was analyzed and optimized. Induction characteristics with lactose were analyzed in terms of induction time and inducer concentration in various culture conditions including batch and high-cell-density fed-batch cultures. In the fed-batch culture, the induction around 6 h after initiation of the DO-stat fed-batch culture resulted in the highest expression level of rKI-3 among the induction points examined. The highest demand of oxygen at this point was crucial for the maximum expression level of rKI-3. As the lactose concentration increased, the expression level also increased, though the expression level showed a plateau above a concentration of 14 mM of lactose. Lactose acted less specifically than IPTG since most of it was hydrolyzed to glucose and galactose. However, using lactose, the cell growth and the maximum expression level of rKl-3 increased by 20% and 24%, respectively, compared with those using IPTG in the fed-batch culture. The lactose seemed to be hydrolyzed by intracellular and extracellular $\beta$-galactosidase liberated by cell lysis at the same time. Residual concentration of glucose was maintained to a a limit of detection by high performance liquid chromatography, and galactose was not consumed by the host strain Escherichia coli BL2l(DE3).

• Journal of Microbiology
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• v.45 no.6
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• pp.593-596
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• 2007

In this study, we describe the development of a simple and efficient method for cell lysis via the insertion of a bacteriophage lambda lysis gene cluster into the pET22b expression vector in the following order; the T7 promoter, a gene for a target protein intended for production, Sam7 and R. This insertion of R and Sam7 into pET22b exerted no detrimental effects on cellular growth or the production of a target protein. The induction of the T7 promoter did not in itself result in the autolysis of cells in culture but the harvested cells were readily broken by freezing and thawing. We compared the efficiency of the cell lysis technique by freezing and thawing to that observed with sonication, and determined that both methods completely disintegrated the cells and released proteins into the solution. With our modification of pET22b, the lysis of cells became quite simple, efficient, and reliable. This strategy may prove useful for a broad variety of applications, particularly in experiments requiring extensive cell breakage, including library screening and culture condition exploration, in addition to protein purification.

• KSBB Journal
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• v.21 no.4
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• pp.236-240
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• 2006

IPTG induction caused a sudden drop of alkali consumption rate during cultivation of a recombinant E. coli with ${\beta}$-galactosidase structural gene under T7 promoter on a plasmid. A series of batch cultivations showed the positive correlation of the decrease of alkali consumption and the level of expression. However, repeated IPTG induction did not cause any variation of alkali consumption rate. Supplementation of medium even at stationary phase enhanced the level of ${\beta}$-galactosidase expression. These results suggests that the drop of alkali consumption rate by IPTG induction represents the rate of expression.

• Applied Biological Chemistry
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• v.40 no.4
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• pp.271-276
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• 1997

We have prepared several chimeric constructs containing the bacteriophage T7 RNA polymerase gene under control of the wound-inducible potato proteinase inhibitor II (pin2) promoter and have transformed Nicotiana tabacum plants with these constructs. Southern blot analyses indicate that either one or two copies of the gene constructs are present in the transgenic plants. Northern blot analyses indicate that mRNA encoding T7 RNA polymerase is expressed in a wound-inducible manner. We purified T7 RNA polymerase and prepared antiserum. This antiserum was used for Western blot analyses to demonstrate that a protein which is cross reactive with T7 RNA polymerase is produced. The molecular mass of this protein is 80 kDa, a size which is consistant with the nicked form of the polymerase as is often seen when expressed in E. coli. RNA polymerase assays were used to indicate that the nicked form of T7 RNA polymerase is active and capable of incorporating labeled nucleotides into transcripts in vitro. Analysis of transgenic plants did indeed show that wound-inducible activation of the T7 RNA polymerase permits the establishment of a genetic system to overexpress genes in plants using T7 RNA polymerase(Received March 20, 1997; accepted May 2, 1997)