• BMB Reports
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• v.39 no.6
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• pp.717-721
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• 2006

In vitro analyses of type I signal peptidase activities require protein precursors as substrates. Usually, these pre-proteins are expressed in vitro and cleavage of the signal sequence is followed by SDS polyacrylamide gel electrophoresis coupled with autoradiography. Radioactive amino acids have to be incorporated in the expressed protein, since the amount of the in vitro expressed protein is usually very low and processing of the signal peptide cannot be followed by SDS polyacrylamide gel electrophoresis alone. Here we describe a rapid and simple method to express large amounts of a protein precursor in E. coli. We have analyzed the effect of ionophors as well as of azide on the accumulation of expressed protein precursors. Azide blocks the function of SecA and the ionophors dissipate the electrochemical gradient across the cytoplasmic membrane of E. coli. Addition of azide ions resulted in the formation of inclusion bodies, highly enriched with pre-apo-plastocyanine. Plastocyanine is a soluble copper protein, which can be found in the periplasmic space of cyanobacteria as well as in the thylakoid lumen of cyanobacteria and chloroplasts, and the pre-protein contains a cleavable signal sequence at its N-terminus. After purification of cyanobacterial pre-apo-plastocyanine, its signal sequence can be cleaved off by the E. coli signal peptidase, and protein processing was followed on Coomassie stained SDS polyacrylamide gels. We are optimistic that the presented method can be further developed and applied.

• Microbiology and Biotechnology Letters
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• v.20 no.2
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• pp.169-177
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• 1992

An alkaline protease producing microorganism was isolated from soil and identified as Streptomyces griseus HC-1141. The optimum culture condition of Streptomyces griseus HC- 1141 for the production of alkaline protease was as follows; 0.5% casein, 0.05% ammonium chloride, 0.1% ferrous sulfate. 2.0% lactose, pH 8.0 and 84 hrs. The enzyme was purified about 53 folds by ammonium sulfate treatment, DEAE-cellulose ion exchange chromatography and gel filtratioo on Sephadex G-150. The homogeneity of the purified enzyme was verified by polyacrylamide gel electrophoresis. The molecular weight was estimated to be 31,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis. This enzyme consists of glycine and glutamic acid as major amino acids. The N-terminal and C-terminal residues of the alkaline protease were leucine and histidine respectively.

• Applied Biological Chemistry
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• v.29 no.3
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• pp.318-323
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• 1986

We empolyed gel filtraction, polyacrylamide gel electrophoresis, amino acid autoanalyzer, thin layer chromatography for determining protein and lipid composition in walnut. The walnut contained 22.18% of crude protein and 64.23% of crude lipid. Glutamic acid (38.60%) was the major amino acid in soluble protein, followed by arginine and aspartic acid. The sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed 12 band in soluble protein of walnut, and collection rate of main protein fraction purified by Sephadex G-150 was 60.67%. The molecular weight for the main protein was estimated to be 43,000. The lipid fraction obtained by silicic acid column chromatography were mainly composed of about 93.05% neutral lipid, whereas compound lipid was only 7.0% level. Among the neutral lipid by thin layer chromatography, triglyceride was 82.05%, sterol ester and free fatty acid were 3.86% and 4.80%, repectively. The predominant fatty acids of total and neutral lipids were linoleic acid $(64.48{\sim}69.98%)$ and oleic acid $(13.89{\sim}15.36%)$. The major fatty acids of triglyceride separated from neutral lipid were linolenic acid (69.98%).

• Proceedings of the Korean Society of Applied Pharmacology
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• 1993.04a
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• pp.45-45
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• 1993

괄루근으로부터 분리된 다당류에 대하여 continuous gel electrophoresis, SDS-Polyacrylamide gel eletrophoresis, ion exchange column chromatography, Hydroxyapatite column chromatography 및 Gel filtration등의 방법을 이용하여 다음과 같은 결과를 얻었다. 1) 황산암모늄 분별침전법에 의한 렉틴의 정제도는 초추출물의 4.85배이며 DEAE Sephadex A-50 column chromatography법에 의한 정제도는 24.17배로 나타났고, 마지막 정제단계인 Sephacryl S-200 gel filtration에 의한 정제도는 47.34배로 나타났다. 2) 정제된 렉틴의 분자량은 60,000da1ton으로 나타났다. 3) 사람의 혈액형에 따른 응집효과는 90-100％로 특이성은 없었다.

• Journal of Food Hygiene and Safety
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• v.5 no.4
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• pp.219-228
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• 1990

Whey proteins in milk were analyzed by polyacrylamide gel electrophoresis and compared with respect to electrophoregrams, densitograms and concentrations of whey proteins in raw and market milk classified according to 3 kinds of pasteurization by low temperature long time. high temperature short time and ultra-high temperature short time. Relative composition of major whey protein constituents such as bovine serum albumin, ${\alpha}\;-\;lactalbumin\;and\;{\beta}-lactoglobulin$ in raw milk were 3.71:11.44:84.85 and not affected by low temperature long time and high temperature short time pasteurization, even though there were the tendencies of some declining in the actual concentrations. But by ultra-high temperature short time pasteurization compositions of whey protein were changed to 0: 64.75: 35 in which reflected the disapprearance of bovine serum albumin and the extensive decrease of ${\beta}-lactoglobulin$. Storage of low temperature pasteurized milk at $5^{\circ}C$ resulted in a slight decrease of ${\alpha}\;-\;lactalbumin\;a\;{\beta}-lactoglobulin$, but storage at $25^{\circ}C$ did not make any changes until3rd days of storage. Most of whey proteins in high temperature short time pasteurized milk were not affected during storage at $5^{\circ}C\;and\;25^{\circ}C$, but bovine serum albumin and ${\alpha}\;-lactalbumin$ diminished in 2-3 days of storage. Whey proteins of milk treated with ultra-high temeperature were not affected during storage at $5^{\circ}C\;and\;25^{\circ}C$ except a slight decrease of ${\alpha}\;-lactalbumin$ in 2nd day of storage at $5^{\circ}C$.

• Journal of Microbiology and Biotechnology
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• v.16 no.1
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• pp.25-31
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• 2006

Changes of CP4 5-enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) in the glyphosate-tolerant Roundup Ready soybean were examined using purified CP4 EPSPS produced in cloned Escherichia coli as a control. CP4 EPSPS in genetically modified soybean was detected by twodimensional gel electrophoresis (2-DE) and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS) with databases. CP4 EPSPS in soybean products was resolved on 2-DE by first isoelectric focusing (IEF) based on its characteristic pI of 5.1, followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) based on its molecular mass of 47.5 kDa. We quantified various percentages of soybean CP4 EPSPS. The quantitative analysis was performed using a 2D software program on artificial gels with spots varying in Gaussian volumes. These results suggested that 2-DE image analysis could be used for quantitative detection of GM soybean, unlike Western blotting.

• International Journal of Industrial Entomology and Biomaterials
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• v.2 no.1
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• pp.55-59
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• 2001

The vitellin of the red flour beetled Tribolium castaneum Herbst was purified and characterized. The vitellin of T. castaneum was purified by the FPLC techniques, anion exchange chromatography and gel permeation chromatography. In native-polyacrylamide gel electrophoresis, vitellin of T. castaneum was detected as a single band. This native vitellin has molecular weight of 440 kDa. The vitellin of T. castaneum is composed of three polypeptides, designated Vnl (178 kDa), Vn2 (168 kDa) and Vn3 (52 kDa) in SDS-polyacrylamide gel electrophoresis. Three subunits of vitellin were presented in the female adult hemolymph and egg extracts, but not observed in the male. These three polypeptides gradually decreased during embryogenesis. Polyclonal antiserum raised against purified vitellin reacted with the three polypeptides, Vnl, Vn2 and Vn3. Antisera raised against Vn1 and Vn2 cross-reacted with the two large subunits, Vnl and Vn2, respectively. Another subunits Vn3, however, was not cross-reacted with these two antisera. Also, antiserum raised against Vn3 did not cross-react with the Vn1 and Vn2.

• Microbiology and Biotechnology Letters
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• v.23 no.6
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• pp.695-701
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• 1995

An alkaline lipase producing bacteria was isolated from soil and identified as Serratia liquefaciens AL-11. from the results of analysis of its morphological, biochemical and physiological properties. This strain showed the highest productivity of alkaline lipase when grown at pH 9.0 and 30$\circ$C for 42 hours in the medium of 1% peptone, 0.5% tryptone, 0.9% yeast extract, 1% starch, 1% tween 80, 0.05% CaCl$_{2}$ and 0.05% NaCl. The enzyme was purified by ammonium sulfate treatment, Sephadex G-100 gel filtration and DEAE-Sephadex A-50 column chromatography. The specific activity of the purified enzyme was 27 unit/mg protein and the yield of enzyme activity was 61.3%. The homogeneity of the purified enzyme was verified by polyacrylamide gel disc electrophoresis. Molecular weight of the purified enzyme was estimated about 53,000 by sodium dodecyl sulfate- polyacrylamide gel electrophoresis. This enzyme is composed of 17 amino acids of which glycine, proline and glutamic acid were three miajor acids.

• Korean Journal of Microbiology
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• v.27 no.1
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• pp.35-42
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• 1989

The mode of transglycosylation reaction observed during the action of low-molecular-weigh $\beta$-D-glucosidase ($\beta$-D-glucoside glucohydrolase, EC3.2.1.21) purified from Trichoderma koningii ATCC 26113 was investigated using $^{1}H$-NMR spectroscopy. The enzyme was purified by the series of procedures including ammonium sulfate precipitation, and fractionations by column chromatographies on Bio-Gel P-150, DEAE-Sephadex A-50, and SP-Sephadex C-50. The final purification was performed by the band eluation after preparative polyacrylamide gel electrophoresis. The enzyme showed its molecular size of 78,000 through the analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and its isoelectric point of 5.80 through the analysis of analytical isoelectric focusing. The H-1 proton resonances were analyzed. After the reaction of the enzyme with cellobiose, the reaction products were separated by high performance liquid chromatography using refractive index detector. H-1 resonances of the products were consisted with those of gentiobiose [$\beta$-D-glucopyranosyl--(1,6)-D-glucopyranose], and cellotriose [$\beta$-D glucopyranosyl-(1,4)-$\beta$-D-glucopyranosyl]-(1,4)-D-glucopyranose] with minor resonances of sophorose [$\beta$-D-glucopyranosyl-(1,2)-D-glucopyranose], respectively.

• Journal of the Korean Society of Food Science and Nutrition
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• v.24 no.4
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• pp.570-577
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• 1995

Aspergillus sp. SB-2704 was selected for its strong polygalacturonase activity among various strain of mold found in soil. It was found that production of polygalacturonase reached to maximum when the wheat bran medium containing 1% polypepton, 1% glucose, and 0.2% FeSO4 were cultured for 3 days at 35$^{\circ}C$. Polygalacturonase was purified 20.90 fold from Aspergillus SB-2704. The purification procedures include ammonium sulfate treatment, gel filtration on Sephdex G-150 and DEAE-cellulose ion exchange chromatography. Yield of the enzyme purification was 4.34%. Purified enzyme was confirmed as a single band by the polyacrylamide gel electrophoresis. When the purified enzyme was applied to SDS-polyacrylamide gel electrophoresis, the molecular weight was estimated to be 36,000. The optimum pH for the enzyme activity was 5.5 and optimum temperature was 5$0^{\circ}C$. The enzyme is stable in acidic condition. The activity of purified enzyme was inhibited by Pb2+, Hg2+ and Ba2+, whereas activated by Cu2+, Mn2+, Mg2+ and Fe2+. The activity of polygalacturonase was inhibited by the treament wit maleic anhydride, iodine, and EDTA. The result indicate the possible involvement of histidine and metal ion at active site.