• Fisheries and Aquatic Sciences
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• 제9권3호
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• pp.115-117
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• 2006

A micro algal growth-enhancing polysaccharide compound was isolated from the green alga Monostroma nitidum using water extraction, molecular fractionation, a DEAE-cellulose column, and fast protein liquid chromatography using a Superose-12 column. The yield of the compound from the seaweed powder was 8.3$\times$l0$^{-3}$%. At 2 mg/mL concentration, the polysaccharide enhanced Tetraselmis suecica cell growth in f/2 medium by approximately 160%.

• 한국가축번식학회지
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• 제16권1호
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• pp.1-6
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• 1992

These experiments were undertaken as a basic study to understand the role of cumulus cell on in vitro maturation and fertilization process with identifying the cumulus cell-secreted proteins. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and fast protein liquid chromatography(FPLC), the proteins of cumulus cells were identified. The results obtained in these experiments were summarized as follows ; 1. When the proteins secreted from cultured cell for 30 hours were separated by SDS-PAGE, there were a major band (>94,000) and other minor 2 bands with molecular weight ranging 30,000∼67,000 dalton. 2. In addition, the fractionations of these proteins by FPLC were idirectly shown that three bands were hyaluronic acid, chondroitin sulfate, and heparin.

• 미생물학회지
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• 제49권1호
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• pp.95-98
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• 2013

본 연구에는 대장균에서의 과발현 벡터 개발과 FPLC를 사용한 2단계 컬럼 정제과정을 통해 Pseudomonas alkylphenolia의 alkylphenol hydroxylase의 oxygenase 단백질을 다량으로 정제하는 방법을 개발하였다. 재조합 Escherichia coli BL21(DE3)(pJJPMO2)의 50 g의 wet cake로부터 110 mg의 heterodimer이며 화학량론적 철을 갖는 순수한 단백질을 정제하였으며 147nmole/min/mg의 비활성을 보였다.

• 한국수산과학회지
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• 제47권4호
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• pp.370-375
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• 2014

BACE2 is a membrane-bound aspartic protease that is highly homologous with BACE1. While BACE1 processes the amyloid precursor protein (APP) at a key step in generating ${\beta}$-amyloid peptide and presumably causes Alzheimer's disease (AD), BACE2 has not been demonstrated to be involved in APP processing directly, and its physiological functions are unknown. To determine its function and to develop inhibitors from marine sources, we constructed an overexpression vector for producing BACE2. The gene encoding human BACE2 protease was amplified using the polymerase chain reaction and cloned into the pET11a expression vector, resulting in pET11a/BACE2. Recombinant BACE2 protease was overexpressed successfully in E. coli as inclusion bodies, refolded using the rapid-dilution method, and purified via two-step fast protein liquid chromatography using Sephacryl S-300 gel filtration and Resource-Q column chromatography. The BACE2 protease produced was an active form. This study provides an efficient method not only for studying the basic properties of BACE2, but also for developing inhibitors from natural marine sources.

• 한국미생물·생명공학회지
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• 제48권4호
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• pp.480-490
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• 2020

A novel lipase gene, Lip-1420, was isolated from a metagenomic library constructed from reed marsh from Mt. Jumbong in Korea, comprising 112,500 members of recombinant plasmids. The DNA sequence of Lip-1420-subclone (5,513 bp) was found to contain at least 11 ORFs according to the GenBank database. The ORF-3 gene was inserted into the pET21a plasmid containing the C-terminal 6-His tag and transformed into E. coli BL21(DE3) to express the recombinant lipase protein. Lip-1420 was purified using a fast protein liquid chromatography system. The gene was registered in GenBank (MH628529). The values of Km and Vmax were determined as 0.268 mM and 1.821 units, respectively, at 40℃ and pH 8.0, using p-nitrophenyl palmitate as the substrate. This lipase belongs to family IV taxonomically because it has conserved HGGG and GDSAG motifs in the constitutive amino acid sequence. According to the predicted structural model, the binding sites are represented by residues H78, G81, D150, S151, A152, V181, and D236. Finally, Lip-1420 showed triolein selectivity for methanolysis between triolein (18:1) and tristearin (18:0) substrates. Further study of the selective mechanism and structure-function relationship of this new lipase could be useful for more practical applications.

• Bulletin of the Korean Chemical Society
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• 제34권8호
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• pp.2425-2430
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• 2013

A simple, fast and robust analytical method was developed to determine imatinib in human plasma using liquid chromatography-tandem mass spectrometry with electrospray ionization in the positive ion mode. Imatinib and labeled internal standard were extracted from plasma with a simple protein precipitation. The chromatographic separation was performed using an isocratic elution of mobile phase involving 5.0 mM ammonium formate in water-5.0 mM ammonium formate in methanol (30:70, v/v) over 3.0 min on reversed-stationary phase. The detection was performed using a triple-quadrupole tandem mass spectrometer in multiple-reaction monitoring mode. The developed method was validated with lower limit of quantification of 10 ng/mL. The calibration curve was linear over 10-2000 ng/mL ($R^2$ > 0.99). The method validation parameters met the acceptance criteria. The spiked samples and standard solutions were stable under conditions for storage and handling. The reliable method was successfully applied to real sample analyses and thus a pharmacokinetic study in 27 healthy Korean male volunteers.

• Asian Pacific Journal of Cancer Prevention
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• 제15권20호
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• pp.8947-8950
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• 2014

Immunological functions of heat shock proteins (HSPs) have long been recognized. In this study we aimed to efficiently purify HSP70 from renal cell carcinoma and test it as a tumor antigen for pulsing dendritic cells in vitro. HSP70 was purified from renal cell carcinoma specimens by serial column chromatography on Con A-sepharose, PD-10, ADP-agarose and DEAE-cellulose, and finally subjected to fast protein liquid chromatography (FPLC). Dendritic cells derived from the adherent fraction of peripheral blood mononuclear cells were cultured in the presence of IL-4 and GM-CSF and exposed to tumor HSP70. After 24 hours, dendritic cells were phenotypically characterized by flow cytometry. T cells obtained from the non-adherent fraction of peripheral blood mononuclear cells were then co-cultured with HSP70-pulsed dendritic cells and after 3 days T cell cytotoxicity towards primary cultured renal cell carcinoma cells was examined by Cell Counting Kit-8 assay. Dendritic cells pulsed in vitro with tumor-derived HSP70 expressed higher levels of CD83, CD80, CD86 and HLA-DR maturation markers than those pulsed with tumor cell lysate and comparable to that of dendritic cells pulsed with tumor cell lysate plus TNF-${\alpha}$. Concomitantly, cytotoxic T-lymphocytes induced by HSP70-pulsed dendritic cells presented the highest cytotoxic activity. There were no significant differences when using homologous or autologous HSP70 as the tumor antigen. HSP70 can be efficiently purified by chromatography and induces in vitro dendritic cell maturation in the absence of TNF-${\alpha}$. Conspecific HSP70 may effectively be used as a tumor antigen to pulse dendritic cells in vitro.

• Asian-Australasian Journal of Animal Sciences
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• 제24권1호
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• pp.82-87
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• 2011

A study was conducted to determine the relationships between triacylglycerol (TAG) of plasma, very low density lipoprotein (VLDL) and fat deposition in two different breeds of chickens. The VLDL apolipoproteins of both breeds were also characterised. The breeds used were crossbred village chicken (AK) (Sasso crossed) and commercial broiler (CB) (Avian). They were housed in six pens with 30 female and 30 male birds of each breed per pen. Three male and three female birds from each pen were slaughtered and the blood was collected. The VLDL was isolated and sub-fractionated using Fast Protein Liquid Chromatography (FPLC). VLDL TAG of CB was significantly lower than AK. The particle size was negatively correlated with VLDL TAG and positively correlated with abdominal fat. Sub-fraction 2 contained more apo E that will enhance the lipolysis process of the VLDL TAG than subfraction 1. CB had a higher proportion of sub-fraction 2 than AK. The results showed that the proportion of sub-fraction 2 was negatively correlated with VLDL TAG concentration and positively correlated with abdominal fat.

• Journal of Microbiology and Biotechnology
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• 제20권4호
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• pp.693-699
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• 2010

An antiproliferative ribonuclease with a new N-terminal sequence was purified from fruiting bodies of the edible wild mushroom Russula delica in this study. This novel ribonuclease was unadsorbed on DEAE-cellulose, but absorbed on SP-Sepharose and Q-Sepharose. It had a molecular mass of 14 kDa, as judged by fast protein liquid chromatography on Superdex 75 and SDS-polyacrylamide gel electrophoresis. Its optimal pH and optimal temperature were pH 5 and $60^{\circ}C$, respectively. The ranking of its activity toward various polyhomoribonucleotides was poly C> poly G>poly A>poly U. It could inhibit proliferation of HepG2 and MCF-7 cancer cells with an $IC_50$ value of $8.6\;{\mu}M$ and $7.2\;{\mu}M$, respectively. It was devoid of antifungal and HIV-1 reverse transcriptase inhibitory activity.

• Mass Spectrometry Letters
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• 제6권4호
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• pp.116-119
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• 2015

Globotriaosylsphingosine (lyso-Gb3) is considered as one of the biological marker for Fabry disease. To date, a reliable biomarker that reflects disease severity and progression has not been discovered to guide the management of Fabry disease. A new method included a simple protein precipitation with acetonitrile in 100 μL of plasma following analyte separation on an Phenomenex Kintex- C18 column using a gradient elution (0.1% formic acid in 5-90% acetonitrile). Total run time was within 12 min including sample preparation and MS/MS analysis. The limit of detection and limit of quantitation were 1 ng/mL and 2 ng/mL, respectively. The calibration curve was linear over the concentration range of 2.0-200.0 ng/mL (r² = 0.9999). Inter-day accuracy and precision at 7 level were 93.4-100.7% with RSD of 0.55-5.97%. Absolute recovery was 97.6-98.6%. The method was applied to human and mice plasma, proved the suitability for quantification of lyso-Gb3 for screening, diagnosis and therapeutic monitoring of Fabry disease patients.