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Production of glucocerebrosidase with high-mannose type N-glycan in plants
In Jung Jung,Jeong Chan Moon,Joo Mi Jeon,Wahyu Indra Fanata,Bo Hwa Son,Jae Yong Yoo,Jae Ho Cha,Je Hein Kim,Rikno Harmoko,Ki Seong Ko,Sang Yeol Lee,Kyun Oh Lee 한국당과학회 2009 한국당과학회 학술대회 Vol.2009 No.1
Plant-based expression of recombinant proteins offers significant advantages in terms of the production of pharmaceuticals for human diseases. These include cost-effective production of recombinant proteins due to the ease of scale-up, minimal possibility of product contamination by animal pathogens and the availability of natural storage organs such as tubers, fruits and seeds. Among the many plant-based production systems that have been developed for pharmaceuticals proteins, seeds have the useful advantage of accumulating proteins in a relatively small volume and in stable environment in which they are protected from degradation. In this respect, we overexpressed human glucocerebrosidase (GC) in Arabidopsis seeds driven by cruciferin 3 promoter. In attempt to produce mannose-terminated N-glycan of GC, the construct was introduced into Arabidopsis gnt1 mutant deficient in the activity of N-acetylglucosaminyltransferase I, the first enzyme in the complex glycan biosynthesis. Our results indicate that the human GC is highly expressed in the seed. [Supported by EB-NCRC & BK21 program]
Quantitation and Validation of Atorvastatin using HPLC-UV
Heine, Daniel,Yong, Chul-Soon,Kim, Jung-Sun The Korean Society of Pharmaceutical Sciences and 2007 Journal of Pharmaceutical Investigation Vol.37 No.3
A reversed phase HPLC analysis of atorvastatin (AS) standard solution was performed using diclofenac (DF) as internal standard. Column oven temperature, flow rate and the composition of the mobile phase were varied in order to determine a practical system setup using a C18 column and UV detector. Two C18 columns of different length were compared regarding their influence on the AS peak shape. Based on these preliminary experiments a validation study was performed utilizing a C18 column at $62^{\circ}C$ with a mobile phase consisting of sodium phosphate buffer (0.05 M, pH 4.0), methanol and acetonitrile (40:50:10, v/v/v). The detection limit for AS was $0.1{\mu}g/ml$ and inter- and intra-day calibration curves were linear over a concentration range of $0.2-50{\mu}g/ml$. Accuracy and precision were satisfactory in the AS concentration range of $0.5-50{\mu}g/ml$.
Jung, Wonil,Lee, Sujeong,Yoo, Dongwon,Jeong, Sohee,Miró,, Pere,Kuc, Agnieszka,Heine, Thomas,Cheon, Jinwoo American Chemical Society 2015 JOURNAL OF THE AMERICAN CHEMICAL SOCIETY - Vol.137 No.23
<P>The generation of single-layer 2-dimensional (2D) nanosheets has been challenging, especially in solution-phase, since it requires highly anisotropic growth processes that exclusively promote planar directionality during nanocrystal formation. In this study, we discovered that such selective growth pathways can be achieved by modulating the binding affinities of coordinating capping ligands to the edge facets of 2D layered transition-metal chalcogenides (TMCs). Upon changing the functional groups of the capping ligands from carboxylic acid to alcohol and amine with accordingly modulated binding affinities to the edges, the number of layers of nanosheets is controlled. Single-layer MSe<SUB>2</SUB> (M = Mo, W) TMC nanosheets are obtained with the use of oleic acid, while multilayer nanosheets are formed with relatively strong binding ligands such as oleyl alcohol and oleylamine. With the choice of appropriate capping ligands in the 2D anisotropic growth regime, our solution-based synthetic method can serve a new guideline for obtaining single-layer TMC nanosheets.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jacsat/2015/jacsat.2015.137.issue-23/jacs.5b02772/production/images/medium/ja-2015-02772c_0005.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/ja5b02772'>ACS Electronic Supporting Info</A></P>
The Chirality Conversion Reagent for Amino Acids Based on Salicyl Aldehyde
윤호진,Hein Jung,안윤수,Raju Nandhakumar,김준수,김관묵 대한화학회 2012 Bulletin of the Korean Chemical Society Vol.33 No.5
2-Hydroxy-6-(1-(3-phenylurylphenyl)ethoxy)-benzaldehyde (2) has been synthesized in racemic form from 1,3-Dihydroxybenzene via formylation and reaction with 3-phenyluryl-methylbenzylbromide. The optically pure form of 2 was separated by normal silica column chromatography from the imine diastreomer which was obtained by the reaction of racemic mixture of 2 with optically pure leucinol. The absolute configuration of the separated enantiomer of 2 was decided from the energy calculation of the corresponding imine diastereomers. The activity of 2 as a chirality conversion reagent (CCR) for amino acids was determined by 1H NMR analysis. The efficiency of 2 is not better than the previous CCRs based on binaththol. Compound 2, however, has lower molecular weight compared to other CCRs. This work demonstrates that asymmetric carbon can control the selectivity of amino acids.
Aung Thu Hein,Young-Dan Cho,Ye-Hyeon Jo,Dae-Joon Kim,Jung-Suk Han 대한치과보철학회 2018 The Journal of Advanced Prosthodontics Vol.10 No.2
PURPOSE. This study was performed to evaluate the osteogenic potential of 3mol% yttria-stabilized tetragonal zirconia polycrystals (3Y-TZP) and niobium oxide containing Y-TZPs with specific ratios, new (Y,Nb)-TZPs, namely YN4533 and YN4533/Al20 discs. MATERIALS AND METHODS. 3Y-TZP, YN4533 and YN4533/Al20 discs (15 mm diameter and 1 mm thickness) were prepared and their average surface roughness (Ra) and surface topography were analyzed using 3-D confocal laser microscope (CLSM) and scanning electron microscope (SEM). Mouse pre-osteoblast MC3T3-E1 cells were seeded onto all zirconia discs and evaluated with regard to cell attachment and morphology by (CLSM), cell proliferation by PicoGreen assay, and cell differentiation by Reverse-Transcription PCR and Quantitative Real-Time PCR, and alkaline phosphatase (Alp) staining. RESULTS. The cellular morphology of MC3T3-E1 pre-osteoblasts was more stretched on a smooth surface than on a rough surface, regardless of the material. Cellular proliferation was higher on smooth surfaces, but there were no significant differences between 3Y-TZP, YN4533, and YN4533/Al20. Osteoblast differentiation patterns on YN4533 and YN4533/Al20 were similar to or slightly higher than seen in 3Y-TZP. Although there were no significant differences in bone marker gene expression (alkaline phosphatase and osteocalcin), Alp staining indicated better osteoblast differentiation on YN4533 and YN4533/Al20 compared to 3Y-TZP. CONCLUSION. Based on these results, niobium oxide containing Y-TZPs have comparable osteogenic potential to 3Y-TZP and are expected to be suitable alternative ceramics dental implant materials to titanium for aesthetically important areas.
Bo Hwa Son,In Jung Jung,Jeong Chan Moon,Joo Mi Jeon,Wahyu Indra Fanata,Jae Yong Yoo,Jae Ho Cha,Je Hein Kim,Rikno Harmoko,Ki Seong Ko,Sang Yeol Lee,Kyun Oh Lee 한국당과학회 2009 한국당과학회 학술대회 Vol.2009 No.1
N-glycosylation is a major post-translational protein modification, which alters physicochemical properties of the protein, affecting the folding, distribution, stability and thus biological function and efficiency of protein. Plant type complex N-glycans are distinctive from those found in mammalian because they contain β1,2-xylose and core α 1,3-fucose residues attached to the pentasaccharide (Man3GlcNAc2) core structure but no sialic acid residues. The presence of β1,2-xylose and core α1,3-fucose residues on plant type complex N-glycans has long been an irritating limitation in the use of plant-made pharmaceuticals (PMPs) in human therapy, as these N-glycan epitopes are potentially immunogenic in mammals. In this study, to remove the plant specific sugar residues and humanize the N-glycosylation in plant, we isolated mutants of the corresponding plant specific glycosyltransferase genes (α1,3-fucosyltransferase I, α1,3-fucosyltransferase II, β 1,2-xylosyltransferase, β1,3-galactosyltransferase and α1,4-fucosyltransferase). We made double, triple and quadruple mutants by crossing the mutants, and quintuple mutant is on the construction. The triple (fuct1/fuct2/xylt) and quadruple (fuct1/fuct2/xylt/fuct3) mutants do not show significant developmental defects in a normal growth condition and they do not produce the plant specific sugar residues on the N-glycan. The resulting mutant will be transformed by human α1,6-fucosyltransferase and β1,4-galactosyltransferase genes to accomplish further humanized N-glycosylation in plant. [Supported by BK21 program]
Bo Hwa Son,In Jung Jung,Jeong Chan Moon,Joo Mi Jeon,Wahyu Indra Fanata,Jae Yong Yoo,Jae Ho Cha,Je Hein Kim,Rikno Harmoko,Ki Seong Ko,Sang Yeol Lee,Kyun Oh Lee 한국당과학회 2009 한국당과학회 학술대회 Vol.2009 No.1
N-glycosylation is a major post-translational protein modification, which alters physicochemical properties of the protein, affecting the folding, distribution, stability and thus biological function and efficiency of protein. β1,2-xylose and core α1,3-fucose residues on plant type complex N-glycans are potentially immunogenic in mammals. In this study, to remove the plant specific sugar residues and humanize the N-glycosylation in plant, we isolated mutants corresponding plant glycosyltransferase genes (N-acetylglucosaminyltransferaseI, N-acetylglucosaminyltransferaseII, α 1,3-fucosyltransferaseI, α1,3-fucosyltransferaseII, β1,2-xylosyltransferase, β 1,3-galactosyltransferase, α1,4-fucosyltransferase). Double, triple, quadruple and quintuple mutants were made by crossing the mutants and two quadruple mutants (fuct1/fuct2/xylt/gntII), (fuct1/fuct2/xylt/galt) are on the construction. The triple (fuct1/fuct2/xylt) and quadruple (fuct1/fuct2/xylt/fuct3), quintuple (fuct1/fuct2/xylt/fuct3/galt) mutants did not show significant developmental defects in a normal growth condition and they did not produce the plant specific sugar residues on the N-glycan. [Supported by BK21 program]
Jae Yong Yoo,In Jung Jung,Jeong Chan Moon,Joo Mi Jeon,Wahyu Indra Fanata,Bo Hwa Son,Jae Ho Cha,Je Hein Kim,Rikno Harmoko,Ki Seong Ko,Sang Yeol Lee,Kyun Oh Lee 한국당과학회 2009 한국당과학회 학술대회 Vol.2009 No.1
Accumulation of unfolded proteins in the lumen of endoplasmic reticulum (ER) induces ER stress and activates signaling pathway called unfolded protein response (UPR) for relief of ER stress. In yeast and mammalian cells, an ER-located transmembrane receptor protein kinase/ribonuclease called Ire1 transmits the signal to the nucleus culminating in the transcriptional activation of genes encoding an adaptive response. Plants including Arabidopsis are also containing homologous genes. However, it has been unclear whether the homologous genes are involved in similar mechanism in plants. Arabidopsis ire1 mutant did not show different phenotype compared to WT in normal and stress conditions. However, induction of ER chaperone genes including Bip3 were significantly delayed in ire1 compared to WT. To further investigate the role of AtIre1 in plant UPR signaling, we overexpressed AtIre1 under the control of CaMV 35S promoter. The result indicate that AtIre1 is possibly involved in UPR signaling in plants. [Supported by EB-NCRC & BK21 program]
Plant made pharmaceuticals (PMP) as a convenient, safe and economical alternative to the shortfall
Wahyu Indra Fanata,In Jung Jung,Bo Hwa Son,Jae Yong Yoo,Je Hein Kim,Rikno Harmoko,Ki Seong Ko,Sang Yeol Lee,Kyun Oh Lee 한국당과학회 2010 한국당과학회 학술대회 Vol.2010 No.1
Since the completion of the human genome project, development of new therapeutic and diagnostic proteins have been exponentially increased, but widespread use of these molecules has been hampered by production bottlenecks such as low yields, poor and inconsistent product quality and a shortage of production capacity. Especially, we are facing a growing demand for protein diagnostics and therapeutics, but lack the capacity to meet those demands using established facilities. Over the last decade, plants have emerged as a convenient, safe and economical alternative to mainstream expression systems which are based on the large-scale culture of microbes or animal cells, or transgenic animals. The production of plant-made pharmaceuticals and technical proteins is known as Molecular Farming. The objective is to harness the power of agriculture to cultivate and harvest plants or plant cells producing recombinant therapeutics, diagnostics, industrial enzymes and green chemicals. Molecular Farming has the potential to provide virtually unlimited quantities of recombinant antibodies, vaccines, blood substitutes, growth factors, cytokines and enzymes for use as diagnostic and therapeutic tools in health care, the life sciences and the chemical industry. The overall aim of our research is to develop new plant molecular farming systems which are economic, efficient, stable and safe. To accomplish the purpose, we are performing specific research objectives in regards to plant molecular farming.