• Journal of Life Science
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• v.17 no.1 s.81
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• pp.132-136
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• 2007

When alginate lyase gene (aly) from Pseudoalteromonas elyakovii was expressed in E. coli, most of the gene product was produced as aggregated insoluble particles known as inclusion bodies. In order to produce a soluble and active form of alginate lyase, E. coli cells fore cotransformed with the plasmids designed to permit coexpression of aly together with molecular chaperones such as DnaK/DnaJ/GrpE or GroEL/ES chaperones. The results revealed that the coexpression of aly together with DnaK/DnaJ/GrpE chaperone had a marked effect on the production of this protein as a soluble and active form, presumably through facilitating correct folding of alginate lyase protein. The optimal concentration of L-arabinose for the induction of DnaK/DnaJ/GrpE chaperone was found to be 0.05 mg/ml. When DnaK/DnaJ/GrpE chaperone was coexpressed, about 34% in the total alginate lyase was produced in the soluble fraction. By addition of 10% cetylpyridinium chloride, a clear zone around the colony coexpressing aly and DnaK/DnaJ/GrpE chaperone was formed, indicating that the alginate in the medium was hydrolyzed by active alginate lyase enzyme.

• Journal of Life Science
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• v.25 no.2
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• pp.130-135
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• 2015

When the alginate lyase gene (aly) from Pseudoalteromonas elyakovii IAM 14594 was expressed in E. coli, most of the gene product expressed was produced as aggregated insoluble particles known as inclusion bodies. In order to produce with an elevated level of a soluble and active form of alginate lyase in E. coli, the hyperthermophilic chaperonins (ApCpnA and ApCpnB) from archaeon Aeropyrum pernix K1 were employed as the coexpression partners. At $25^{\circ}C$ culture temperature, the level of alginate lyase activity was increased from 10.1 unit/g-soluble protein in aly single expression to 83.1 unit/g-soluble protein by coexpressing with ApCpnA and to 100.3 unit/g-soluble protein by coexpressing with ApCpnB. This results indicate that the coexpression of aly with ApCpnA and ApCpnB revealed a marked enhancement, about 8~10 fold, in the production of alginate lyase as a soluble and active form. Based on the results of various examinations on the expression variables, the optimal conditions for the maximal production of alginate lyase were determined as 1.0 mM IPTG for the inducer concentration, $25^{\circ}C$ for the culture temperature after IPTG induction, and ApCpnB for the coexpression partner. The coexpression set in the present report may be useful in the industrial production of functionally or medically important recombinant proteins in E. coli.

• Journal of Microbiology and Biotechnology
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• v.30 no.11
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• pp.1720-1728
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• 2020

We have previously characterized AprESJ4, the major fibrinolytic enzyme from Bacillus subtilis SJ4 (Yao et al., 2019). During that study, we observed a 68 kDa protein with fibrinolytic activity. In this study, we cloned the gene (vprSJ4) encoding the 68 kDa protein, a mature Vpr and minor protease secreted by Bacillus species. vprSJ4 encodes a preproenzyme consisting of 810 amino acids (aa) including signal sequence (28 aa) and prosequence (132 aa). The mature enzyme (650 aa) has a predicted molecular weight of 68,467.35. Unlike Vprs from other B. subtilis strains, VprSJ4 has 4 additional amino acids (DEFA) at the C-terminus. vprSJ4 was overexpressed in Escherichia coli. PreproVprSJ4 was localized in inclusion bodies, and subjected to in vitro renaturation and purification by an affinity column. SDS-PAGE and western blot showed that autoprocessing of preproVprSJ4 occurred and 68 kDa and smaller proteins were produced. The optimum pH and temperature of the recombinant VprSJ4 were pH 7.0 and 40℃, respectively. Kinetic parameters of recombinant VprSJ4 were measured by using an artificial substrate, N-succinyl-ala-ala-pro-phe-p-nitroanilide. Coexpression of vprSJ4 and aprESJ4 using pHY300PLK increased the fibrinolytic activity a further 117% when compared with aprESJ4 single expression using the same vector in B. subtilis WB600.

• The Journal of the Petrological Society of Korea
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• v.23 no.3
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• pp.273-277
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• 2014

This paper presents a brief historical review of various attempts to estimate the age of the Earth, and reappraises the study of Patterson (1956) which revealed for the first time that the age of the Earth is $4550{\pm}70Ma$ by measuring Pb isotope ratios of several meteorites and a marine sediment. The standard model for the planetary formation of early solar system is: formation of solid particles condensed from the cooling of hot nebular gas -> formation of planet-sized bodies by accretion of those solid particles. The Moon is supposed to have formed from the accretion of the relicts produced by the collision of proto-Earth with Mars-sized body. It is not easy to pinpoint the age of the Earth, considering the series of events related to the formation of the Earth. So, I propose that the collision age as that of the Earth, since the present status of the Earth is thought to be the direct product of the collision. According to the previous studies, the collision age can be broadly constrained between the age ($4567.30{\pm}0.16Ma$) of the earliest condensates (CAI, calcium-aluminum rich inclusion) of the nebula gas, i.e., the age of the solar system, and the oldest age ($4,456{\pm}40Ma$) among rocks and minerals of the Earth and the Moon. We need more precise estimation of the collision age, since it is important in estimating time scale for the formation of planet-size body and in revealing thermal evolution of magma oceans of the Earth and the Moon presumably developed right after the collision.

• Conservation Science in Museum
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• v.24
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• pp.17-36
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• 2020

This paper describes the consevation treatment of eleven bigyeokjincheolloe bomb shells that were excavated from the Joseon-period local government office and fortress of Mujang-hyeon (present-day Mujang-myeon) in Gochang in 2018. It also provides information on the production method of the shells revealed through CT scanning, gamma-ray transmission imaging, and metallographic analysis. In preparation for the special exhibition "Bigyeokjincheolloe" at the Jinju National Museum in 2019 (July 16 to August 25), contaminants were removed from the shells and their surface was reinforced during the first phase of conservation treatment. Furthermore, the closures for the shells were identified for the first time. Regarding the production of the shells, the CT scanning and gamma-ray transmission imaging identified many blowholes in the interior of the body and the use of a chaplet on the side of one shell. The side of the body proved to be relatively thinner than the top and bottom. The traces of a hole for pouring molten metal into the center of the bottom indicates that molten metal was indeed emptied into the inverted body. In the metallographic analysis of two of the bodies and one lid, cementite and pearlite structures were identified on the body, indicating that it was made by casting. The presence of the ferrite structure with a partial distribution of the pearlite along with non-metallic inclusion in the lid suggested that the lid was made by forging.

• Korean Journal of Veterinary Research
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• v.22 no.2
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• pp.221-232
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• 1982

This paper dealt with the pathological and clinical findings on the experimentally induced rodenticide (fluoroacetate, zinc phosphide, thallium sulfate, coumarin) and NaCN poisoning of ruminants (Holstein cattle and/or Korean native goat) for the purpose of the diagnosis in the accidental rodenticide poisoning of cattle. The results observed are summarized as follows: Fluoroacetate poisoning (cattle and goat): in the clinical signs, there were depression, convulsion, dyspnea, groan, grinding of the teeth, vomiting, opisthotonus and post-mortem tympany. In the macroscopical findings, the blood was more or less poor coagulative and dark red, bloody fluid with foam in the trachea, hyperemia and hemorrhage of tracheal mucosa and lung, cloudy swelling and hyperemia of kidney, epicardial hemorrhage(cattle), and hyperemia of abomasum, intestine and brain were observed. In the microscopical findings, there were pulmonary edema and hemorrhage, necrosis of convoluted tubular epithelium and interstitial hemorrhage of kidney, focal coagulative necrosis of myocardium, hemorrhage of pancreas and spleen, dilatation of Virchow-Robin space and hyperemia of brain, and necrosis with desquamation of mucosal epithelia of abomasum and upper small intestine. In the histological lesions of the liver, lobular peripheral hyperemia, centrilobular necrosis and cytoplasmic inclusion bodies of the hetatic cells were observed. The cytoplasmic inclusion body of the hepatic cells was not seen in the affected goat, but hydropic degeneration of the hepatic cells was marked. Zinc phosphide poisoning (cattle and goat): clinically, the affected animals died in recumbent position after ataxia, dyspnea and convulsion. In the macroscopical findings, hyperemia and hemorrhage of lung, cloudy swelling and hyperemia of liver and kidney, hemorrhage of spleen (cattle), and catarrh of abomasum and small intestine were observed. In the microscopical findings, necrosis of the convoluted tubular epithelium and hyperemia of kidney, hemorrhage of spleen, hyperemia of lung, hyperemia or hemorrhage of heart, cloudy. swelling and fatty changes of hepatic cells, dilatation of hepatic central vein, hyperemia of brain, and catarrh of abomasal and small intestinal mucosae were observed. Thallium sulfate poisoning (cattle): in the macroscopical findings dark red color of blood, hyperemia and hemorrhage of lung, bloody fluid with foam in the tracheal mucosa, petechiae of tracheal mucosa, cloudy swelling and hemorrhage of liver, necrotic lesions and hemorrhage of renal cortex and epicardial hemorrhage were observed. In the microscopical findings, severe hemorrhages of the lung, cloudy swelling and necrosis of hepatic cells, hyperemia and hemorrhage of liver, focal coagulative necrosis of mycordium, necrosis of the convoluted tubular epithelium and hyperemia of kidney, hyperemia and hemorrhage of spleen and dilatation of Virchow-Robin apace in brain were observed. Coumarin poisoning (goat): the poisoned animals died in the state of groan and depression. In the macroscopical findings, poor coagulation of blood, hemorrhage of lung, cloudy swelling and severe hemorrhages of liver, cloudy swelling and hemorrhage of kidney, abomasal hemorrhage, catarrh of small intestine, and hyperemia and hemorrhage of the other organs were observed, In the microscopical findings, hyperemia and hemorrhage of lung and kidney, cloudy swelling of the convoluted tubular epithelium of kidney, severe hepatic hyperemia, cloudy swelling and hydropic degeneration of heptatic cell, and hyperemia and hemorrhage of brain and spleen were observed. NaCN poisoning (cattle and goat): clinically, there were convulsion, severe dyspnea, paresis of hind limb, depression and then rigor of four limbs. In the macroscopical findings, bright red color of blood, hyperemia and bright and red tinge of lung cloudy swelling of kidney and liver, and hyperemia of abomasum were observed. In the microscopical findings, cloudy swelling and hydropic degeneration of hepatic cell, hyperemia and edema of lung, necrosis and degeneration of the convoluted tubular epithelium and hemorrhage in kidney, dilatation of Virchow-Robin space of brain and hemorrhage of spleen were observed.

• The Korean Journal of Malacology
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• v.22 no.2
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• pp.157-165
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• 2006

The present study was conducted to confirm that a bivalve Ruditapes philippinarum can be used as a biomarker for the monitoring of the heavy metal pollution in the silt of the marine environment. The clams were collected from the silt of Cheonsu-bay, Buheung-ri, and Tan-island of the West Sea, Korea. To observe the normal structures of the target organs (hepatopancreas and gill), they were dissected out for the immunohistochemical study and the electron microscopy with TEM, SEM, and SEM-EDS. The immunohistochemical study showed that the interdiverticular connective tissues of the hepatopancreas, and the outer epithelium of the gill lamellae was strongly reacted to anti-metallothionein (MT), indicating the presence of MT, a metal-binding protein, involved in metal detoxifying process. According to the examinations under the TEM, the epithelial cells of the hepatopancreas of the clams collected from polluted area (Tan-island) showed certain changes such as swollen rER, swollen nuclear envelope and inclusion bodies in the nulcei. In the SEM-EDS analysis, tissue of the hepatopancreas showed relatively higher concentration of S, Zn, and Cd. These elements are supposed to be concerning with the MT-reaction in the hepatopancreas. Considering that the coastal bivalve R. philippinarum showed immediate subcellular responses to heavy metal pollution in the overall experiments conducted, this species might act as one of efficient biomarkers for the heavy metal contamination in the marine environment.

• KSBB Journal
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• v.23 no.3
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• pp.231-238
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• 2008

The second family member of tissue inhibitors of matrix metalloproteinases, TIMP-2, is a 21kDa protein which inhibits matrix metalloproteinases 2 (MMP-2). Expression of mammalian proteins in E. coli often forms inclusion bodies that are made up of mis-folded or insoluble protein aggregates. The requirement for the formation of 6 disulfide bonds in the process of the TIMP-2 folding is likely to be incompatible with the reducing environment of E. coli. However, this incompatibility can be often overcome by introducing a mutagenesis that could lead to enhancement of the protein solubility. In this reason, we have attempted to express the soluble TIMP-2 in E. coli by applying a modified staggered extension process (StEP), one of the in vitro PCR-based recombinant mutagenesis methods, and error-prone PCR. C-terminally located CAT fusion protein with respect to mutated TIMP-2 proteins enables us to differentiate the soluble TIMP-2 from the insoluble in E. coli by virtue of chloramphenicol resistance. According to this scheme, E. coli harboring properly-folded CAT fused to TIMP-2 protein was selected, and some of the resulting colonies exhibited an enhanced, soluble expression of TIMP-2 compared to the wild type, implying (i) the StEP technique is successfully employed to enhance the proper folding thereby increasing the solubility of TIMP-2, and (ii) the CAT dependent screening may be a simple and effective method to differentiate the soluble protein expression in E. coli.

• The Journal of Korean Society of Virology
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• v.27 no.2
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• pp.239-255
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• 1997

Expression of the cDNA of the VP1 gene on the genome RNA B segment of infectious pancreatic necrosis virus (IPNV) DRT strain in E. coli and a recombinant baculovirus were carried out. The VP1 gene in the pMal-pol clone (Lee et al. 1995) was cleaved with XbaI and transferred into baculovirus transfer vector, pBacPAK9 and it was named pBacVP1 clone. The VP1 gene in the pBacVP1 clone was double-digested with SacI and PstI and then inserted just behind T5 phage promoter and the $6{\times}His$ region of the pQE-3D expression vector, and it was called pQEVPl. Again, the $6{\times}$His-tagged VP1 DNA fragment in the pQEVP1 was cleaved with EcoRI and transferred into the VP1 site of the pBacVP1, resulting pBacHis-VP1 recombinant. The pBacHis-VP1 DNA was cotransfected with LacZ-Hyphantria cunea nuclear polyhedrosis virus (LacZ-HcNPV) DNA digested with Bsu361 onto S. frugiperda cells to make a recombinant virus. One VP1-gene inserted recombinant virus was selected by plaque assay. The recombinant virus was named VP1-HcNPV-1. The $6{\times}$His-tagged VP1 protein produced by the pQEVP1 was purified with Ni-NTA resin chromatography and analyzed by SDS-PAGE and Western blot analysis. The molecular weight of the VP1 protein was 94 kDa. The recombinant virus, VP1-HcNPV-1 did not form polyhedral inclusion bodies and expressed VP1 protein with 95 kDa in the infected S. frugiperda cells, which was detected by Western blot. The titer of the VP1-HcNPV-1 in the first infected cells was $2.0{\times}10^5\;pfu/ml$ at 7 days postinfection.

• Journal of The Korean Society of Inherited Metabolic disease
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• v.12 no.1
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• pp.5-13
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• 2012

I-cell disease (mucolipidosis type II; MIM 252500) and pseudo-Hurler polydystrophy (mucolipidosis type III; MIM 252600) are disorders caused by abnormal lysosomal transport in cells. The presence of numerous inclusion bodies in the cytoplasm of fibroblasts, a lack of mucopolysacchariduria, increased lysosomal enzyme activity in serum, and decreased GlcNAc-phosphotransferase activity are hallmark. Here, we attempted to investigate phenotypical and biochemical characteristics of the knockoutmouse of GlcNAc-phosphotransferase ${\alpha}/{\beta}$ subunits; in addition, we also attempted to determine whether the lysosome enriched fraction derived from placenta can be beneficial to phenotype and biochemistry of the knockout mouse.We found that the knockout mouse failed to thrive and had low bone density, as is the case in human. In addition, skin fibroblasts from the animal had the same biochemical characteristics, including increased lysosomal enzyme activity in the culture media, in contrast to the relatively low enzyme activity within the cells. Intravenous injection of the lysosome rich fraction derived from placenta into the tail vein of the animal resulted in a gain of weight, while saline injected animals didn't.In conclusion, our study demonstrated the phenotypical and biochemical similarities of the knockout mouse to a mucolipidosis type II patient and showed the therapeutic potential of the lysosome enriched fraction. We admit that a larger scale animal study will be needed; however, the disease model and the therapeutic potential of the lysosome enriched fraction will highlight the hope for a novel treatment approach to mucopolipidosis type II, for which no therapeutic modality is available.