• Journal of Microbiology and Biotechnology
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• v.18 no.8
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• pp.1357-1363
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• 2008

The $P_{entC}$ promoter of the entCERA operon encoding enzymes for enterobactin biosynthesis in Escherichia coli is tightly regulated by the availability of iron in the culture medium. In iron-rich conditions, the $P_{entC}$ promoter activity is strongly repressed by the global transcription regulator Fur (ferric uptake regulator), which complexes with ferrous ions and binds to the Fur box 19-bp inverted repeat. In this study, we have constructed the expression vector pOS2 containing the $P_{entC}$ promoter and characterized its repression, induction, and modulation by quantifying the expression of the lacZ reporter gene encoding $\beta$-galactosidase. $\beta$-Galactosidase activities of E. coli transformants harboring pOS2-lacZ were highly induced in the presence of divalent metal ion chelators such as 2,2'-dipyridyl and EDTA, and were strongly repressed in the presence of excess iron. It was also shown that the basal level $\beta$-galactosidase expression by the $P_{entC}$ promoter was drastically decreased by incorporating the fur gene into the expression vector. Since the newly developed iron chelator-inducible expression system is efficient and cost-effective, it has wide applications in recombinant protein production.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.6
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• pp.850-855
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• 2007

Leptin is the adipocyte-specific product of the obese gene and plays a major role in food intake and energy metabolism. Leptin research was mainly focused on mammalian species, but understanding of leptin and its function in poultry is very poor. In this study, the duck leptin gene was amplified using the reverse transcription-polymerase chain reaction (RT-PCR) from duck liver RNA. The cDNA fragment was inserted into the pET-28a expression vector, and the resulting plasmid was expressed in Escherichia coli BL21 (DE3). Experimental mice were given an intraperitoneal injection of 10 mg/kg leptin dissolved in phosphate buffered saline (PBS), while the control mice were injected with PBS. The effect of leptin on food intake, body weight and fatty deposition in mice was detected. Sequence analysis revealed that duck leptin had a length of 438 nucleotides which encoded a peptide with 146 amino acid residues. The sequence shares highly homology to other animals. The coding sequence of duck leptin was 84 and 86% identical to human and pig leptin nucleotides sequence. Highest identity was with the rat coding sequence (95%). The identity of the amino acid sequence was 84, 82 and 96% respectively compared to that of the human, pig and rat. Results of SDS-PAGE analysis indicated that a fusion protein was specifically expressed in E. coli BL21 (DE3). The purified product was found to be biologically active during tests. Continuous administration of recombinant duck leptin inhibited food intake. Despite the decrease of food intake, leptin significantly induced body weight and fatty deposition. These changes were accompanied by a significant down-secretion of plasma glucose, cholesterol, triglyceride and insulin levels in mice. The observations provide evidence for an inhibitory effect of leptin in the regulation of food intake and for a potential role of duck leptin in the regulation of lipogenesis.

• KSBB Journal
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• v.22 no.5
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• pp.313-317
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• 2007

To enhance the growth of cryopreserved cells of transgenic Nicotiana tabacum, Pluronic F-68 was supplemented in a recovery medium during post-thaw period. As cryoprotective agents, 1 M sucrose, 0.5 M glycerol and 0.5 M dimethyl sulfoxide (DMSO) were added before freezing steps. The post-thaw growth of the cells was improved with Pluronic F-68, ranged from 0.1 to 10 g/L. The interactions of Pluronic F-68 with the cells were confirmed by the changes of hydrophobicity or permeability of the cells. Pluronic F-68 did not show any effect on the activity of $\beta$-glucuronidase (GUS) in all treatments. Therefore, the addition of Pluronic F-68 in a recovery medium was found to be beneficial to enhance the post-thaw growth of cryopreserved transgenic tobacco cells without affecting the production of recombinant protein.

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.11
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• pp.1576-1584
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• 2016

Acetyl xylan esterase (AXE), which hydrolyzes the ester linkages of the naturally acetylated xylan and thus known to have an important role for hemicellulose degradation, was isolated from the anaerobic rumen fungus Neocallimastix frontatlis PMA02, heterologously expressed in Escherichi coli (E.coli) and characterized. The full-length cDNA encoding NfAXE1 was 1,494 bp, of which 978 bp constituted an open reading frame. The estimated molecular weight of NfAXE1 was 36.5 kDa with 326 amino acid residues, and the calculated isoelectric point was 4.54. The secondary protein structure was predicted to consist of nine ${\alpha}$-helixes and 12 ${\beta}$-strands. The enzyme expressed in E.coli had the highest activity at $40^{\circ}C$ and pH 8. The purified recombinant NfAXE1 had a specific activity of 100.1 U/mg when p-nitrophenyl acetate (p-NA) was used as a substrate at $40^{\circ}C$, optimum temperature. The amount of liberated acetic acids were the highest and the lowest when p-NA and acetylated birchwood xylan were used as substrates, respectively. The amount of xylose released from acetylated birchwod xylan was increased by 1.4 fold when NfAXE1 was mixed with xylanase in a reaction cocktail, implying a synergistic effect of NfAXE1 with xylanase on hemicellulose degradation.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.9
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• pp.1286-1295
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• 2008

Leptin is produced by adipocytes and its role in the regulation of lipid metabolism, feed intake, productive and reproductive performance of domestic animal species has been greatly stressed and extensively investigated in recent years. This study was conducted to develop a radioimmunoassay (RIA) for the estimation of plasma bovine leptin and to determine plasma leptin concentration in fattening Japanese Black cattle (Wagyu) and its crossbreds at commercial farms. Relationships of plasma leptin with plasma vitamin A and age of crossbred cattle were also determined. Recombinant bovine leptin (rbleptin) was produced by the E. coli overexpressed leptin as a GST (glutathione S-transferase)-fusion protein. Then antiserum against bovine leptin was obtained by its immunization in rabbits. Using this antiserum, a bovine specific RIA was developed and plasma leptin level was determined in 120 crossbred fattening cattle (WagyuHolstein, 50:50) at commercial farms. The plasma leptin level increased with the age of cattle and its level was greater in the crossbred heifers than in the steers. Plasma vitamin A level was negatively correlated with plasma leptin level in crossbred heifers and steers. This relationship was stronger in heifers than in steers. Plasma leptin was gradually increased with advancing age in fattening Wagyu cattle. In conclusion, development of a bovine specific RIA to estimate plasma leptin will contribute to better understanding of the role of leptin in cattle.

• Biomedical Science Letters
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• v.19 no.4
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• pp.344-347
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• 2013

The S100A8 and S100A9 proteins play important roles in inflammatory diseases. The house dust mite acts as a major allergen that induces allergic diseases. We investigated the effect of the house dust mite on S100A8 and S100A9 protein expression in monocytes. We also examined the effect of CCL2, a powerful monocyte chemoattractant, on the expression of both proteins. Extract of Dermatophagoides pteronissinus (DP), recombinant Der p 1 and Der p 2, or CCL2 had no effect on S100A8 and S100A9 expression in human monocytic THP-1 cells. Monocytes were isolated from healthy donors and treated with DP, Der p 1, and Der p 2. S100A8 expression in monocytes increased after a 24 h stimulation with DP, Der p 1, and Der p 2, and CCL2 also increased S100A8 production. However, S100A9 expression in monocytes was not altered by DP, Der p 1, Der p 2, or CCL2. These results indicate that house dust mite and CCL2 may trigger an inflammatory response by altering S100A8 expression.

• Journal of Microbiology and Biotechnology
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• v.20 no.11
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• pp.1471-1480
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• 2010

An extremely thermostable xylanase gene, xynB, from the hyperthermophilic bacterium Thermotoga maritima MSB8 was successful expressed in Kluyveromyces lactis. The response surface methodology (RSM) was also applied to optimize the medium components for the production of XynB secreted by the recombinant K. lactis. The secretion level (102 mg/l) and enzyme activity (49 U/ml) of XynB in the optimized medium (yeast extract, lactose, and urea; YLU) were much higher than those (56 mg/l, 16 U/ml) in the original medium (yeast extract, lactose, and peptone; YLP). The secretory efficiency of mature XynB was also improved when using the YLU medium. When the mRNA levels of 13 characterized secretion-related genes in the K. lactis cultured in YLP and YLU were detected using a semiquantitative RT-PCR method, the unfolded protein response (UPR)-related genes, including ero1, hac1, and kar2, were found to be up-regulated in the K. lactis cultured in YLU. Therefore, the nutrient ingredients, especially the nitrogen source, were shown to have a significant influence on the XynB secretory efficiency of the host K. lactis.

• Journal of Microbiology and Biotechnology
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• v.14 no.1
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• pp.35-40
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• 2004

Rotavirus virus-like particle (VLP) composed of VP2, VP6, and VP7 was expressed in the Baculovirus Expression Vector System (BEVS). Sf9 cell, a host of the baculovirus, was cultured from a 0.5-1 spinner flask to the 50-1 bioreactor system. Sf9 cell was maintained at cell density between 3.0E＋05 and 3.0E＋06 cells/ml and grew up to 1.12E＋07 cells/ml in the bioreactor. Growth kinetics was compared under different culture systems and showed similar growth kinetics with 20.1-25.2 h of doubling time. Early exponentially growing cell culture was infected with three recombinant baculoviruses expressing VP2, VP6, and VP7 protein at 1.0, 2.0, and 0.2 moi, respectively. The expression of rotavirus proteins was confirmed by Western blot analysis and its three-layered virus-like structure was observed under an electron microscope. Rotavirus VLP was semipurified and immunized in ICR mice intramuscularly. Rotavirus-specific serum antibody was detected from 2 weeks after the immunization and lasted at least 21 weeks of the post-immunization, indicating its possible use as a vaccine candidate.

• Korean Journal of Animal Reproduction
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• v.21 no.1
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• pp.1-7
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• 1997

Recently the production of transgenic animals to express foreign proteins in mammary glands has been a routine procedure. However, it still takes a considerable time and effort, and is faced with various technical challenges until the protein of interest is successfully made. Thus, a development of an a vitro model for mamm a ary-specific gene expression for recombinant genes was carried out in this study. To this end, bovine $\alpha$$_S1$ casein cDNA was inserted at the multiple cloning site of pMSG vector under the control of MMTV promoter. MCF$_7$ cells were tran sfected with pMSG $\alpha$$_S1$ CN by CaP0$_4$ precipitation. Transfectants were selected in HAT medium and induced with dexamethasone. The cells were analyzed with chicken anti-casein and FITC-labeled rabbit anti-chicken antibodies. The results showed that dexamethasone induced 30-40 fold increase in the MMTV- $\alpha$$_S1$ casein e expression. Therefore MCF$_7$ cells, which have multiple steroid receptors, along with pMSG vector can be used as an in vitro model for the study of mammary-specific gene expression.

• Journal of Microbiology
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• v.44 no.3
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• pp.293-300
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• 2006

The Escherichia coli heat-labile enterotoxin B subunit (HLT-B) is one of the most powerful mucosal immunogens and known mucosal adjuvants. However, the induction of high levels of HLT-B expression in E. coli has proven a difficult proposition. Therefore, in this study, the HLT-B gene was cloned from pathogenic E. coli and expressed as a fusion protein with GST (glutathion S-transferase) in E. coli BL2l (DE3), in an attempt to harvest a large quantity of soluble HLT-B. The culture conditions, including the culture media used, temperature, pH and the presence of lactose as an inducer, were all optimized in order to obtain an increase in the expression of soluble GST-rHLT-B. The biological activity of the purified rHLT-B was assayed in a series of GMI-ELISA experiments. The findings of these trials indicated that the yield of soluble recombinant GST-rHLT-B could be increased by up to 3-fold, as compared with that seen prior to the optimization, and that lactose was a more efficient alternative inducer than IPTG. The production of rHLT-B, at 92 % purity, reached an optimal level of 96 mg/l in a 3.7 L fermentor. The specific GM1 binding ability of the purified rHLT-B was determined to be almost identical to that of standard CTB.