• Journal of Microbiology and Biotechnology
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• v.17 no.4
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• pp.579-585
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• 2007

Formation of inclusion bodies is usually observed when foreign proteins are overexpressed in E. coli. The formation of inclusion bodies might be prevented by lowering the rate of protein synthesis, and appropriate regulation of the protein expression rate may lead to the soluble expression. In this study, human growth hormone (rhGH) was expressed in a soluble form by slowing down the protein synthesis rate, which was controlled in the transcriptional and translational levels. The transcriptional level was controlled by the regulation of the amount of RNA polymerase specific to the promoter in front of the rhGH gene. For lowering the rate of translation, the T7 transcription terminator-deleted vector was used to synthesize the longer mRNA of the target gene because the longer mRNA is expected to reduce the availability of tree ribosomes. In both methods, the percentage of soluble expression increased when the expression rate slowed down, and more than 93% of rhGH expressed was a soluble form in the T7 transcription terminator-deleted expression system.

• Journal of Microbiology and Biotechnology
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• v.24 no.10
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• pp.1382-1388
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• 2014

Proteus vulgaris K80 lipase was expressed in Escherichia coli BL21 (DE3) cells and immobilized on amine-terminated magnetic microparticles (Mag-MPs). The immobilization yield and activity retention were 84.15% and 7.87%, respectively. A homology model of lipase K80 was constructed using P. mirabilis lipase as the template. Many lysine residues were located on the protein surface, remote from active sites. The biochemical characteristics of immobilized lipase K80 were compared with the soluble free form of lipase K80. The optimum temperature of K80-Mag-MPs was $60^{\circ}C$, which was $20^{\circ}C$ higher than that of the soluble form. K80-Mag-MPs also tended to be more stable than the soluble form at elevated temperatures and a broad range of pH. K80-Mag-MP maintained its stable form at up to $40^{\circ}C$ and in a pH range of 5.0-10.0, whereas soluble K80 maintained its activity up to $35^{\circ}C$ and pH 6.0-10.0. K80-Mag-MPs had broader substrate specificity compared with that of soluble K80. K80-Mag-MPs showed about 80% residual relative activity after five recovery trials. These results indicate the potential benefit of K80-Mag-MPs as a biocatalyst in various industries.

• Korean journal of food and cookery science
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• v.18 no.5
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• pp.487-494
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• 2002

This study was conducted to investigate the thermal stability of water-soluble and fat-soluble vitamins dissolved in water and milk by various heat treatments. Vitamin samples were prepared by dissolving them in water and milk at various concentrations, and were heat treated for 30 min at 65$\^{C}$, 15 sec at 85$\^{C}$, 5 sec at 100$\^{C}$, 121$\^{C}$ at 15 min, the levels of residual vitamin were measured by using HPLC. Milk samples were fortified with vitamins before and after UHT treatment. As heating over 100$\^{C}$, riboflavin in water were destructed more than 92% but fortified in milk showed less than 20% destruction, suggesting that riboflavin was protected by milk components. Also retinol heated ever 100$\^{C}$ was more stable in milk than in water. L-Ascorbic acid and cholecalciferol(D$_3$) showed a similar destruction rate in water and in fortified milk. L-ascorbic acid was easily destructed by UHT treatment. Destruction of thiamin and tocopherol was increased in fortified milk. Among tour capsulated water-soluble vitamins, L-ascorbic acid was much more stable compared with powder form. Nicotinic acid and folic acid either in capsule or powder form showed a slight destruction by heat treatment. The results suggested that the fortification of unstable vitamins such as L-ascorbic acid, thiamin, tocopherol and cholecalciferol(D$_3$) should be made in milk after heat treatment.

• Toxicological Research
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• v.6 no.1
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• pp.99-107
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• 1990

Dopamine beta-hydroxylase (DBH) is a neurotransmitter biosynthetic enzyme which catalyzes the conversion of dopamine to norepinephrine. Although structural studies of the mature form of this enzyme have been extensive, the culmination of these finding had been hightly controversial and contradictory. In this study, biochemical approaches were taken to characterize the structure of mature DBH. Soluble bovine DBH was purified from aderenal medulla. Three bands of 69 kDa, 72 kDa and 75 kDa which were physically separable and similar in structure were observed by SDS-PAGE. Furthermore, gas phase sequence analysis revealed that the 72 kDa band consists of two polypeptides which are present at equimolar concentrations and differed in that one had three extra amino acids at the N-terminus. Taken together, the soluble form of DBH exist in at least four forms, three identified by SDS-PAGE, one of which consists of two polypeptides as identified by N-terminal sequence analysis. The significance of these forms and their possible biosynthetic mechanisms are discussed.

• Bulletin of the Korean Chemical Society
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• v.31 no.9
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• pp.2607-2612
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• 2010

Human tissue-type plasminogen activator (tPA) is a valuable thrombolytic agent used to successfully treat acute myocardial infarction, thromboembolic stroke, peripheral arterial occlusion, and venous thromboembolism. Recombinant tPA is accumulated as an inactive form in inclusion bodies of E. coli and is refolded in vitro, which is accompanied by extensive aggregation. In the present study, a tPA protease domain was expressed in an active soluble form in the cytosol of E. coli Rosetta-gami cells, which allowed disulfide bond formation and supplied the tRNA molecules required for six rarely used codons in E. coli. This strategy increased the amount of soluble protease domain protein and avoided the cumbersome refolding process. The purified protease domain not only degraded tPA substrate peptides but also formed a covalently bound complex with plasminogen activator inhibitor-1, as does full-length tPA. Soluble expression and purification of tPA domains may aid in functional analyses of this multi-domain protein, which has been implicated in many physiological and pathological processes.

• IMMUNE NETWORK
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• v.13 no.4
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• pp.148-156
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• 2013

The $PrP^C$ is expressed in many types of immune cells including monocytes and macrophages, however, its function in immune regulation remains to be elucidated. In the present study, we examined a role for $PrP^C$ in regulation of monocyte function. Specifically, the effect of a soluble form of $PrP^C$ was studied in human monocytes. A recombinant fusion protein of soluble human $PrP^C$ fused with the Fc portion of human IgG1 (designated as soluble $PrP^C$-Fc) bound to the cell surface of monocytes, induced differentiation to macrophage-like cells, and enhanced adherence and phagocytic activity. In addition, soluble $PrP^C$-Fc stimulated monocytes to produce pro-inflammatory cytokines such as $TNF-{\alpha}$, $IL-1{\beta}$, and IL-6. Both ERK and $NF-{\kappa}B$ signaling pathways were activated in soluble $PrP^C$-treated monocytes, and inhibitors of either pathway abrogated monocyte adherence and cytokine production. Taken together, we conclude that soluble $PrP^C$-Fc enhanced adherence, phagocytosis, and cytokine production of monocytes via activation of the ERK and $NF-{\kappa}B$ signaling pathways.

• Korean Journal of Pharmacognosy
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• v.28 no.4
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• pp.264-270
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• 1997

This Study was carried out develop antitumor effect of the n-butanol soluble of fraction of Perilla frutescens on human skin melanoma cells. The antitumor activity of various fractions obtained form n-butanol soluble fraction of Perilla frutescens was evaluated in human skin melanoma cells. The antitumor activity of the n-butanol soluble fraction in human skin melanoma cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, neutral red (NR) assay and sulforhordamine B protein (SRB) assay of colorimetic assay methods. The light microscopic study was carried out to observe morphological changes of cultured human skin melanoma cells. These results were obtained follows; The fractions 5 and 6 of the n-butanol soluble fraction of P frutescens were shown significant antitumor activities. The number of human skin melanoma cells were decreased and tend to form cell cluster by treatment with actions 5 and 7 of the n-butanol soluble fraction of P. frutescens. The fraction 6 of the the n-butanol soluble fraction showed the highest antitumor activity on P. frutescens. These results suggest that the fraction 6 of the n-butanol soluble fraction of P. frutescens may be a valuable choice for the studies on the treatment of human skin tumors.

• Preventive Nutrition and Food Science
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• v.11 no.1
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• pp.73-77
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• 2006

Ground pork was irradiated with an electron beam (e-beam) at a dose of 0, 1.5, 3, 5 and 10 kGy and the changes in various functional and other associated properties of salt-soluble proteins extracted from the pork were evaluated. Irradiation did not affect turbidity and the disulfide content of pork salt-soluble protein, but the content of sulfhydryls and the hydrophobocity of salt-soluble protein increased. This indicates that protein degradation occurred when the pork was e-beam irradiated and that the sulfhydryls and hydrophobic moieties buried inside the proteins were exposed to the outside environment. However, these degraded protein molecules did not form large protein aggregates through disulfide bridges. The emulsifying capacity of the pork increased with irradiation, which could be the result from increased hydrophobicity of pork salt-soluble protein. Water holding capacity of pork was not affected bye-beam irradiation.

• KSBB Journal
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• v.14 no.4
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• pp.429-433
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• 1999

$\beta$-Glucosidaes from Cellvibrio gilvus(CG) was successfully overproduced in soluble form in E. coli with the coexpression of GroEL/ES/. Without the GroEL/ES protein, the $\beta$-glucosidase overexpressed in E. coli constituted a huge amount(80%) of total cellular protein, but was localized in the insoluble fraction, and little activity was detected in the soluble fraction. Coexpression of the E. coli GroEL/ES had a drastic impact on the proper folding of the $\beta$-glucosidase; 20% of the overexpressed enzyme was recovered in the soluble fraction in active form. Similar effects of GroEL/ES were also observed on the overexpressed $\beta$-glucosidase from Agrobacterium tumefaciens(AT). And pET28(a)-RGRAR, partially deleted mutant lacking 5-amino acid residues at carboxy teminus also could be folded into an active form when expressed with the molecular chaperonin GroEL/ES, and its activity was higher than that of the without GroEL/ES system, In addition, the synergistic effect of GroEL/ES and the low induction temperature were important factors for solubilization of the inclusion body from overproduced $\beta$-glucosidases.

• Biotechnology and Bioprocess Engineering:BBE
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• v.11 no.5
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• pp.414-419
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• 2006

When the alginate lyase gene (aly) from Pseudoalteromonas elyakovii was expressed in E. coli, most of the gene product was organized as aggregated insoluble particles known as inclusion bodies. To examine the effects of chaperones on soluble and nonaggregated form of alginate lyase in E. coli, we constructed plasm ids designed to permit the coexpression of aly and the DnaK/DnaJ/GrpE or GroEL/ES chaperones. The results indicate that coexpression of aly with the DnaK/DnaJ/GrpE chaperone together had a marked effect on the yield alginate lyase as a soluble and active form of the enzyme. It is speculated this result occurs through facilitation of the correct folding of the protein. The optimal concentration of L-arabinose required for the induction of the DnaK/DnaJ/GrpE chaperone was found to be 0.05mg/mL. An analysis of the protein bands on SDS-PAGE gel indicated that at least 37% of total alginate lyase was produced in the soluble fraction when the DnaK/DnaJ/GrpE chaperone was coexpressed.