• Journal of Life Science
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• v.25 no.4
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• pp.385-389
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• 2015

In this study, it is reported that a large polyprotein can be stoichiometrically cleaved by the use of caspase-3-dependent proteolysis. Previously, it has been shown that the proteolytic IETD motif was partially processed when treated with caspase-3, while the DEVD motif was completely cleaved. The cleavage efficiency of the DEVD-based substrate was approximately 2.0 times higher than that of the IETD substrate, in response to caspase-3. Based on this, 3 protein genes of interest were genetically linked to each other by adding two proteolytic cleavage sequences, DEVD and IETD, for caspase-3. Particularly, glutathione-S transferase (GST), maltose binding protein (MBP), and red fluorescent protein (RFP) were chosen as model proteins due to the variation in their size. The expressed polyprotein was purified by immobilized metal ion affinity chromatography (IMAC) via a hexa-histidine tag at the C-terminal end, showing 93 kDa of a chimeric GST:MBP:RFP fusion protein. In response to caspase-3, cleavage products, such as MBP:RFP (68 kDa), MBP (42 kDa), RFP (26 kDa), and GST (25 kDa), were separated from a large precursor GST:MBP:RFP (93 kDa) via SDS-PAGE. The results obtained from this study indicate that a multi-protein can be stoichiometrically produced from a large poly-protein by using proteolytic recognition motifs, such as DEVD and IETD tetra-peptides, for caspase-3.

• Microbiology and Biotechnology Letters
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• v.31 no.4
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• pp.311-321
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• 2003

Bacteria produce lipases, which can catalyze both the hydrolysis and the synthesis of long chain triglycerides. These reactions usually proceed with high regioselectivity and enantioselectivity, and, therefore, lipases have become very important biocatalysts used in organic chemistry. 3D lipase structures were solved from several bacterial lipases. They have an $\alpha/\beta$ hydrolase fold and a catalytic triad consisting of a nucleophilic serine, and an aspartate or glutamate residue that is hydrogen bonded to a histindine. Active sites are covered with $\alpha$-helical lid structure, of which movement is involved in the enzyme's activation at oil/water interface. Four substrate binding pockets were identified for triglycerides: an oxyanion hole and three pockets accommodating the fatty acids bound at positions sn-1, sn-2, and sn-3. These pockets determine the enantiopreference of a lipase. The understanding of structure-function relationships as well as the development of molecular evolution techniques will enable researchers to tailor new lipases for biotechnological applications.

• Journal of the Korea Institute of Information and Communication Engineering
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• v.13 no.7
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• pp.1475-1482
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• 2009

In this paper, a computer simulation is developed for isotachophoretic protein separation in a serpentine micro channel for optimal lab on a chip design using 2D Finite Element Method. This 2D ITP model is composed of 5 components such as hydrochloric acid as Leader, caproic acid as terminator, acetic acid and benzoic acid as two proteins, and histindine as background electrolyte. The computer model is based on mass conservation equation for 5 components, charge conservation equation for electric potential, and electro neutrality condition for pH calculation. For the validation of the 2D spatial ITP model, the results are compared with the Simul5 developed by Bohuslav Gas Group. The simulation results are in a good agreement in a ID planar channel. This proves the precision of our model. The 2Dproteinseparation is conducted in a 2D curved channel for Lab on a chip design and dispersions of proteins are revealed during the electrophoretic process in a curved shape.

• YAKHAK HOEJI
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• v.56 no.6
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• pp.377-381
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• 2012

The histidine-rich $Ca^{2+}$ binding protein (HRC) is a $Ca^{2+}$ binding protein in the sarcoplasmic reticulum (SR). In this study, we examined whether the HRC is involved in the regulation of cardiac contraction and $Ca^{2+}$ signaling using HRC knock-out (KO) mouse ventricular myocytes. In field-stimulated single mouse ventricular myocytes, cell shortenings and $Ca^{2+}$ transients were measured using a video edge detection and a confocal $Ca^{2+}$ imaging, respectively. Compared with the wide-type (WT) myocytes, the magnitudes of cell shortenings were significantly larger in HRC KO cells (P<0.01, WT vs. KO). The rate of contraction and relaxation was significantly accelerated in HRC KO myocytes (P<0.05 and P<0.01, respectively, WT vs. KO). The magnitudes of $Ca^{2+}$ transients were increased by HRC KO (P<0.01, WT vs. KO). In addition, the decay of the $Ca^{2+}$ transient was faster in HRC KO cells than in wild-type cells P<0.01, WT vs. KO). These results suggest that HRC may suppress SR $Ca^{2+}$ releases and decay of $Ca^{2+}$ transients during action potentials, thereby attenuating ventricular contraction and relaxation.

• Journal of Mushroom
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• v.19 no.1
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• pp.33-40
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• 2021

The genome sequence of various Flammulina species has recently been reported, thereby revealing a diverse genetic repertoire. In this study, we identified laccase genes and analyzed their structural characteristics in Flammulina elastica (F. elastica) genome. Through genome analysis and bioinformatics approaches, three laccase genes (Fe-lac1, -lac2, and -lac3) were identified, ranging from 1,548 to 1,602 bp in length. These genes contained a copper ion-binding region with ten histidine residues and one cysteine residue and a disulfide bond-forming region with four cysteine residues. Full-length cDNA sequencing analysis revealed that laccase genes contain 12 to 16 introns and signal peptides between 17 and 22 bp in the N-terminus. Structural characterization of the laccase genes identified in this study should help in better understanding the biomass decomposition of F. elastica.

• Journal of Adhesion and Interface
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• v.24 no.4
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• pp.113-119
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• 2023

Since it was reported that the unusual amino acid DOPA in synergy with lysine and histidine residues found in mussel adhesive proteins plays a pivotal role in mussel adhesion in underwater environments, there has been a burgeoning development of various catecholamines-based adhesives for biomedical applications. Among these, catechol-conjugated chitosan, containing catecholamine, featuring multiple catechol groups within its aminerich chitosan backbone, has found versatile utility in fields, such as tissue adhesion, wound dressing, tissue healing, hemostats, drug delivery systems, and tissue engineering scaffolds. Significantly, chitosan-catechol is a mussel-inspired material approved by both US Food and Drug Administration (FDA) and KR Ministry of Food and Drug Safety (MFDS) for its effectiveness in hemostasis. This review focuses on 1) general aspects of catechol-conjugated chitosan, highlighting catechol group integration into chitosan backbones, 2) examination of proposed mechanisms of hemostasis, and 3) exploration of diverse physical forms, including solution, hydrogels, patches, and thin films with practical applications inapplicable to hemostasis.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.13 no.11
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• pp.5350-5355
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• 2012

Human tyrosinase (hTyr) catalyzes the first and rate limiting step in the biosynthesis of a skin color determinant, melanin. Although a number of cosmetic companies have tried to develop hTyr inhibitors for several decades, absence of 3D structure of hTyr make it impossible to design or screen inhibitors by structure-based approach. Therefore, we built a 3D structure by comparative modeling technique based on the crystal structure of tyrosinase from Bacillus megaterium to provide structural information and to search new hit compounds from database. Our model revealed that two copper atoms of active site located deep inside and were coordinated with six strictly conserved histidine residues coming from four-helix-bundle. Substrate binding site had narrow funnel like shape and its entrance was wide and exposed to solvent. In addition, hTyr-tyrosine and hTyr-kojic acid, a well-known inhibitor, complexes were modeled with the guide of solvent accessible surface generated by in-house software. Our model demonstrated that only phenol group or its analogs could fill the binding site near the nuclear copper center, because inside of binding site had narrow shape relatively. In conclusion, the results of this study may provide helpful information for designing and screening new anti-melanogenic agents.

• Journal of Life Science
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• v.17 no.9 s.89
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• pp.1197-1203
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• 2007

A genomic DNA library of the bacterium Paenibacillus sp. DG-22 was constructed and the ${\beta}-xylosi-dase-positive$ clones were identified using the fluorogenic substrate $4-methylumbelliferyl-{\beta}-D-xylopyr-anoside$ $({\beta}MUX)$. A recombinant plasmid was isolated from the clone and 4.3-kb inserted DNA was sequenced. The ${\beta}-xylosidase$ gene (xylA) was comprised of a 2,106 bp open reading frame (ORF) en-coding 701 amino acids with a molecular weight of 78,710 dalton and a pI of 5.0. The deduced amino acid sequence of the xylA gene product had significant similarity with ${\beta}-xylosidases$ classified into family 52 of glycosyl hydrolases. The xylA gene was subcloned into the pQE60 expression vector to fuse with six histidine-tag. The recombinant ${\beta}-xylosidase$ $(XylA-H_6)$ was purified to homogeneity by heat-treatment and immobilized metal affinity chromatography. The pH and temperature optima of the $XylA-H_6$ enzyme were pH 5.5-6.0 and $60^{\circ}C$, respectively.

• Journal of Food Hygiene and Safety
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• v.32 no.3
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• pp.228-233
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• 2017

The aim of the current study was to examine genotoxicological safety of purified bee venom (Apis mellifera L.) The bacterial reverse mutation in Salmonella typhimurium (TA100, TA1535, TA98, and TA1537) and Escherichia coli (WP2 uvrA) were evaluated with purified bee venom at concentrations of 0, 1.5, 5, 15, 50, 150, and $500{\mu}g/plate$. Purified bee venom was negative in Ames test with both in the presence and absence of rat liver microsomal enzyme. According to these results, we concluded that purified bee venom did not cause bacterial reverse mutation. The safety of the purified bee venom at practical doses needs to be further evaluated in in vivo genotoxicity assays.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.14 no.1
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• pp.15-23
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• 1981

Protein compositions of filefish (Navoden modestus) skeletal muscle and their changes in postmortem with reference to freshness kept at $0^{\circ}C$ were investigated. The muscle protein was approximately composed of $31\%\;sarcoplasmic,\;55\%$ myofibrillar, $10\%$residual intracellular, and $4\%$stroma protein. The sarcoplasmic and myofibrillar protein decreased while the residual intracellular protein increased with the decline of freshness during post-mortem lapse. In the analysis of electrophoretograms and its densitograms, the myofibrillar protein resembled to other fishes in protein composition: $70\%$ actin and myosin, $20\%$ regulatory proteins, and $10\%$ unknown proteins. And most of the residual intracellular protein was estimated as myofibrillar protein. Troponin T, troponin C and myosin light chain 2 of the myofibrillar protein constituents were decreased during storage. Amino acid composition of the protein from the at-death muscla was similar to those of other fishes except that tryptophan and sulfur-containing amino acids were scant. Proline and cysteine were remarkably decreased whereas leucine, isoleucine and phenylalanine were slightly increased in the protein from the muscle lapsed of 18 days. In free amino acid composition, alanine, glycine, lysine, and especially taurine were rich in the at-death muscle. The muscle lapsed of 18 days showed an increase of taurine, histidine, valine and methionine, and a decrease of lysine, arginine, aspartic acid, threonine, leucine, and isoleucine.