• KSBB Journal
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• v.13 no.6
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• pp.656-661
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• 1998

Hantaan virus belonging to the genus Hantavirus and family Bunyaviridae causes an acute severe illness of human, Haemorrhagic Fever with Renal Syndrome (HFRS). It is a rodent host-borne pathogen and distributed in Asia and Eastern Europe. Hantaviruses have three major antigens, i.e., G1, G2 glycoproteins and nucleocapsid protein (N). Among them, nucleocapsid protein was reported to be the most invaluable antigen as for diagnosis. We have cloned and expressed Hantaan viral nucleocapsid gene in E. coli BL21(DE3). In this study, we have tried to purify the nucleocapsid protein produced by recombinant E. coli, and could attained a purity of >90% by anti-N monoclonal antibody-coupled immunoaffinity chromatography or phenyl sepharose hydrophobic interaction chromatography.

• Environmental Analysis Health and Toxicology
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• v.17 no.3
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• pp.265-272
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• 2002

This study was carried out with the detection for multiresidue of the organophosphorus pesticides such as malathion, parathion. diazinon, and carbamate pesticide such as carbaryl, by enzyme-inhibition method. The acetylcholinesterase (AChE) and cholinesterase (ChE) activities in chicken brain determined by the Ellman's method were 166.6 and 5.8 $\mu$mol/min/g protein, and in chicken plasma were 23.1 and 8.3 $\mu$mol/min/g protein, respectively. The optimum pH of AChE and ChE was 8.2 and 7.8, respectively. The Km of AChE and ChE was 0.034 and 0.045 mM, respectively. I$\_$50/ for AChE and ChE by some organophosphorus was 55.82 and 99.42 mg/L of malathion, 31.16 and 29.13 mg/L of parathion, and 17.89 and 19.62 mg/L of diazinon, respectively. I$\_$50/ for AChE and ChE by carbaryl of carbamate was 0.10 and 0.05 mg/L, respectively. The 0.07 mg/L of drinking water advisory level for carbaryl could be detected with I$\_$50/ of AChE and ChE. Enzyme-Inhibition (EI) method with AChE and ChE was used the multiresidue method to detect the 1 mg/L of the carbamate pesticides.

• BMB Reports
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• v.37 no.6
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• pp.735-740
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• 2004

Hepatitis C virus (HCV) is a causal agent of the chronic liver infection. To understand HCV morphogenesis, we studied the assembly of HCV structural proteins in insect cells. We constructed recombinant baculovirus expression vectors consisting of either HCV core alone, core-E1, or core-E1-E2. These structural proteins were expressed in insect cells and were examined to assemble into particles. Neither core-E1 nor core-E1-E2 was capable of assembling into virus-like particles (VLPs). It was surprising that the core protein alone was assembled into core-like particles. These particles were released into the culture medium as early as 2 days after infection. In our system, HCV structural proteins including envelope proteins did not assemble into VLPs. Instead, the core protein itself has the intrinsic capacity to assemble into amorphous core-like particles. Furthermore, released core particles were associated with HCV RNA, indicating that core proteins were assembled into nucleocapsids. These results suggest that HCV may utilize a unique core release mechanism to evade the hosts defense mechanism, thus contributing to the persistence of HCV infection.

• Journal of Microbiology and Biotechnology
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• v.14 no.1
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• pp.56-61
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• 2004

A plasmid pSDK-l containing the Escherichia coli phosphofructokinase-l gene (pfkA) was constructed, and transferred into extremely acidophilic Acidithiobacillus thiooxidans Tt-7 by conjugation with the aid of plasmid RP4 at a frequency of $10^{-5}$ per recipient. This plasmid was stable in A. thiooxidans. The pfkA gene from E. coli could be expressed in this obligately autotrophic bacterium, but the enzyme activity (21.6 U/g protein) was lower than that in E. coli (K12: 85.9 Dig protein; DF1010 carrying plasmid pSDK-l: 96.6 U/g protein). In the presence of glucose, the Tt-7 transconjugants consumed glucose, leading to a better growth yield.

• Molecules and Cells
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• v.24 no.1
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• pp.27-36
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• 2007

The hypothetical protein TTC0263 of Thermus thermophilus HB27 is a thermophilic tetratricopeptide repeat (TPR)-containing protein. In the present study, the TPR region (residues 26-230) was resolved at $2.5{\AA}$ with R-factors of $R/R_{free}$ = 23.6%/28.6% $R/R_{free}=23.6%/28.6%$. TTC0263 consists of 11 helices that form five TPR units. Uniquely, it contains one atypical "extended" TPR (eTPR) unit. This comprises extended helical residues near the loop region of TTC0263, such that the helical length of eTPR is longer than that of the canonical TPR sequence. In addition, the hybrid TPR domain of TTC0263 possesses oligomer-forming characteristics. TPR domains are generally involved in forming multi-subunit complexes by interacting with each other or with other subunit proteins. The dynamic structure of TTC0263 described here goes some way to explaining how TPR domains mediate the formation of multi-subunit complexes.

• Journal of Microbiology and Biotechnology
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• v.20 no.3
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• pp.550-557
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• 2010

Escherichia coli (E. coli) heat-labile enterotoxin B subunit (LTB) was regarded as one of the most powerful mucosal immunoadjuvants eliciting strong immunoresponse to coadministered antigens. In the research, the high-level secretory expression of functional LTB was achieved in P. pastoris through high-density fermentation in a 5-1 fermentor. Meanwhile, the protein was expressed in E. coli by the way of inclusion body, although the gene was cloned from E. coli. Some positive yeast and E. coli transformants were obtained respectively by a series of screenings and identifications. Fusion proteins LTB-6$\times$His could be secreted into the supernatant of the medium after the recombinant P. pastoris was induced by 0.5% (v/v) methanol at $30^{\circ}C$, whereas E. coli transformants expressed target protein in inclusion body after being induced by 1 mM IPTG at $37^{\circ}C$. The expression level increased dramatically to 250-300 mg/l supernatant of fermentation in the former and 80-100 mg/l in the latter. The LTB-6$\times$His were purified to 95% purity by affinity chromatography and characterized by SDS-PAGE and Western blot. Adjuvant activity of target protein was analyzed by binding ability with GMI gangliosides. The MW of LTB-6$\times$His expressed in P. pastoris was greater than that in E. coli, which was equal to the expected 11 kDa, possibly resulted from glycosylation by P. pastoris that would enhance the immunogenicity of co-administered antigens. These data demonstrated that P. pastoris producing heterologous LTB has significant advantages in higher expression level and in adjuvant activity compared with the homologous E. coli system.

• Journal of Nutrition and Health
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• v.1 no.2
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• pp.93-105
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• 1968

The variations of both thiamine and riboflavin value in the organs, viz. liver, small intestine, spleen and kidney of the rats were measured for observing some metabolic changes in the animals during fasting and feeding different quality of diets without V-E supplement. The animal used for the experiment was adult female ablino rat from a pure strain, weighing 225-280g. The animals were divided into 6 groups; the control group, the high carbohydrate diet group, the high carbohydrate diet with V-E group, the high protein diet group, the high protein diet with V-E group, and fasting group. The result obtained are summarized as follows; 1. The thiamine contents in the liver were once increased during early stage of starvation compared with the control group, thereafter they were decreased on the 8 days fasting while the contents in the small intestine and spleen were decreased during 1 to 8 days fasting. 2. The riboflavin contents in the liver and kidney were increased during starvation and the content in the small intestine was no marked change compared with control group. 3. The thiamine contents in the liver and small intestine during feeding the high carbohydrate with V-E supplement diet group were lower than that of the diet without V-E group and the content in the spleen was increased by feeding V-E enriched high carbohydrate diet. 4. The thiamine contents in the liver, small intestine and spleen during feeding the V-E supplemented diets were lower than that of the non-supplemented one's. 5. The riboflavin contents in the liver, small intestine, and kidney were increased during feeding the high carbohydrate diet compared to the control group, and they were decreased during feeding the V-E enriched high carbohydrate diet. 6. The riboflavin contents in each organ were increased during feeding the high protein diet compared to the control group, and they were much increased during 20 to 30 days of feeding the V-E supplemented high protein diet. 7. Therefore, as the above results showed, the variation of thiamine value in the each organs were not markedly changed during feeding different quality of the diets. The thiamine and riboflavin contents in the each organ in the V-E enriched high carbohydrate diet group were lower than without V-E supplemented one's The riboflavin contents in each organ were increased during feeding the high protein diet compared with the control group and the centents were increased during 20 to 30 days of the feeding V-E enriched high protein diet.

• Mass Spectrometry Letters
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• v.6 no.2
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• pp.31-37
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• 2015

Phosphorylation upon protein is well known to a key regulator that implicates in modulating many cellular processes like growth, migration, and differentiation. Up to date, grafting of multidimensional separation techniques onto advanced mass spectrometry (MS) has emerged as a promising tool for figuring out the biological functions of phosphorylation in a cell. However, advanced MS-based phosphoproteomics is still challenging, due to its intrinsic issues, i.e., low stoichiometry, less susceptibility in positive ion mode, and low abundance in biological sample. To overcome these bottlenecks, diverse techniques (e.g., SCX, HILIC, ERLIC, IMAC, TiO₂, etc.) are continuously developed for on-/off-line enrichment of phosphorylated protein (or peptide) from biological samples, thereby helping qualitative/quantitative determination of phosphorylated protein and its phosphorylated sites. In this review, we introduce to the overall views of enrichment tools that are universally used to selectively isolate targeted phosphorylated protein (or peptide) from ordinary ones before MS-based phospoproteomic analysis.

• Journal of Nutrition and Health
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• v.19 no.5
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• pp.323-332
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• 1986

This study was performed to see the effects of lead poisoning and dietary protein levels(6, 15 and 40 % casein diets) on growth, protein and lipid metabolisms in growing rats. It was also investigated whether the high protein intake would alleviate lead toxicity by decreasing Pb absorption and/or increasing Pb excretion. The results obtained were summarized as follows ; 1) Weight gain, F.E.R liver weight, weight and length of bone in Pb-administered groups were lower than in Pb-free groups. However, these values in the 40% casein diet group with Pb were increased to the level in 15% casein diet group without Pb. 2) Hematocrt and hemoglobin content in blood were lower in Pb -adminstered groups than in Pb free groups. Especially, these levels were lower in 6% casein diet group with Pb than in any other group. 3) Plasma protein level in th e 40% casein casein diet group was the highest of all groups and those of Pb-administered groups tended to be lower than those of Pb-free groups. Plasma lipid and cholesterol levels were increased with decreasing dietary protein level, and these levels were higher in the animals exposed to Pb than in free groups.4) Total liver protein, lipid and cholesterol contents were increased with increasing dietary protein level, and these contents were lower in Pb-administered groups than in Pb free groups. 5) Fecal Pb excretion was not different between 6 and 40% casein diet groups. However, urinary Pb excretion was higher in the 40% casein diet group than in the 6% casein diet group. Above results suggest that, in exposing to the Pb pollution, sufficient protien intake must be recommended. High protein intake seemed to alleviate lead toxicity by increasing urinary Pb excretion.

• Molecules and Cells
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• v.21 no.1
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• pp.21-28
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• 2006

The interferon-induced, double-stranded RNA (dsRNA)-dependent protein kinase PKR plays a key role in interferon-mediated host defense against viral infection, and is implicated in cellular transformation and apoptosis. We have isolated a cDNA of simian PKR encoding a product with 83% amino acid identity to the human homolog and showed that PKR expression is significantly attenuated in the Vero E6 African green monkey kidney cells devoid of type I interferon genes. A variant form of PKR lacking the exon 12 in the kinase domain is produced in these cells, presumably from an alternatively spliced transcript. Unlike wild type PKR, the variant protein named PKR-${\Delta}E12$ is incapable of auto-phosphorylation and phosphorylation of eIF2-${\alpha}$, indicating that the kinase sub-domains III and IV embedded in exon 12 are indispensable for catalytic function. PKR-${\Delta}E12$ had no dominant negative effect but was weakly phosphorylated in trans by wild type PKR.