• Molecular & Cellular Toxicology
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• v.1 no.2
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• pp.87-91
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• 2005

Although 2D-PAGE has been widely used as the primary method for protein separation, difficulties in displaying proteins with an extreme values of isoelectric paint (pI), molecular size and hydrophobicity limit the technique. In addition, time consuming steps involving protein transfer and extraction from the gel-pieces can result in sample loss. Here, we describe a novel protein separation technique with capillary size-exclusion chromatography (CSEC) for rapid protein identification from human plasma. The method includes protein fractionation along with molecular size followed by in-solution tryptic digestion and peptide analysis through reversed phase liquid chromatography (RPLC) coupled to nanoflow electrospray-tandem mass spectrometry (ESI-MS/MS). Tryptic peptides are applied an a $100\;{\mu}m\;i.d.{\times}10mm$ length pre-column and then separated on a $75\;{\mu}m{\times}200mm$ analytical column at -100 nL/min flaw rate. Proteins were identified over the wide ranges of pI (3.7-12.3) when this technique was applied to the analysis of $1-2\;{\mu}L$ of human plasma. This gel-free system provides fast fractionation and may be considered a complementary technique to SDS-PAGE in proteomics.

• KSBB Journal
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• v.14 no.5
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• pp.578-584
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• 1999

The lectin was purified through 0.15 M NaCl extraction, ammonium sulfate precipitation, sepharose 4B affinity chromatography and gel filtration using sephadex G-150 from the leaves of Visum album collected in Mt. Duk Yu. The final gel filtration step resulted in 11.64 folds purification with 0.14% of recovery yield. We also performed biochemical characterization of the purified Visum album lectin. HPLC analysis of lectin purified by gel filtration revealed a singel peak. The analysis of the purified lectin by SDS-PAGE showed a tetramer composed of two identical subunits with molecular weights of 32 and 30 kDa. The lectin was a glycoprotein containing 14.4% carbohydrate, which consist of glucose, fructose, arabinose and xylose, and the amino acids such as phenylalanine, lysine and tyrosine. The purified lectin agglutinated human red blood cell types with similar potency, but when tested against red blood cells from mouse, bovine, rabbit, chicken and porcine, significant difference in potency were observed. Hemaggluting activity was inhibited by D-galactose, D-mannose, D-lactose and D-raffinose, but not by D-glucose, D-glucosamine, D-mannosamine, L-fructose, D-xylose, D-arabinose, D-galacturonic acid, D-fructose, L-rhamnose and N-acetyl-D-galactosamine. The optimal pH and thermal stability of the purified lectin were pH 4.0-7.0 and 20-5$0^{\circ}C$, respectively.

• KSBB Journal
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• v.11 no.3
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• pp.295-302
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• 1996

The measurements of rubisco parameters are important in photosynthetic studies. In this experiment, we used photometric assay method to detect these major parameters, such as activity, carbamylation and amount of rubisco. The main advantages of this method are very simple and as sensitive as conventional methods which usually produce radioactive waste. In this study, with kidney bean (Phaseolus vulgatis L.) leaves grown at normal $CO_2$ (350ppm) and high $CO_2$ (650 ppm), we investigated the effect of $CO_2$ concentration on activation and carbamylation of rubisco by measuring the rubisco activity, carbamylation rate and amount of rubisco using a dual beam (334nm and 405nm) spectrophotometer, and analyzed the polypeptide profiles of rubisco by SDS-PAGE. When $CO_2$ concentration was raised from 350ppm to 650ppm, all parameters of rubisco were decreased : $41.2{\mu}M/m^2/s and 52.2{\mu}M/m^2/s$ to $27.4{\mu}M/m^2/s and 46.1{\mu}M/m^2/s$ for initial and total rubisco activity, respectively ; from 79% to 58.9% for carbamylation rate ; from $1.94 {\mu}M/m^2$ to 1.58{\mu}M/m^2$ for amount of rubisco. These results suggests that the decrease in rubisco activity at high $CO_2$ was caused by carbamylation. The analysis of the preparation by SDS-PAGE showed two major polypeptides at 50 and 14.5 kD which were identified as the large and the small subunits of rubisco. There were no differences in the intensity compared high $CO_2$ to normal $CO_2$ in both 50 kD and 14.5 kD bands. We also found that these inhibitory effects of $CO_2$ were reversible. When high $CO_2$ was switched to normal $CO_2$, the parameters of rubisco changed were almost the same as normal rubisco parameters. These data provide an evidence that activity of rubisco was recovered by $CO_2$ concentration of 350 ppm.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.4
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• pp.334-340
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• 2005

Lectin is a cell-agglutinating and carbohydrate-binding protein present in many plants. The lectin of Canavalia ensiformis shoot with specific affinity for D-glucose was purified by affinity chromatography using Sephadex G-100, and some of its biochemical characterizations were studied. Lectin was purified 8.87-fold and exhibited final specific activity of 225.74 units/mg protein with a $2.3\%$ yield. SDS-PAGE analysis demonstrated that the purified shoot lectin exists as a tetramer of 102 kD, composed of two subunits with molecular weight of 29 and 22 kD. The purified lectin was observed to agglutinate rabbit blood cell. The optimal temperature for the activity of this lectin was $40^{\circ}C$, and this lectin was relatively stable to heat with the highest activity at $50{\~}60^{\circ}C$. The maximal activity was observed at pH 7.2.

• The Korean Journal of Pesticide Science
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• v.14 no.4
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• pp.446-455
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• 2010

The characteristics of parasporal inclusion body from Bacillus thuringiensis subsp. kurstaki KB099 isolate which is high bioactive to the tobacco cutworm, Spodoptera litura, were examined. Parasporal inclusion of B. thuringiensis subsp. kurstaki KB099 isolate showed only 1 band at 130 kDa compared with B. thuringiensis subsp. kurstaki HD-l isolate producing 2 protein bands at 130 kDa and 60 kDa from by SDS-PAGE analysis without any enzyme treatment. Also, we confirmed that gut extract of sensitive S. litura KB099 isolate had digested only 60 kDa ${\delta}$-endotoxin protein. When the digestive enzyme of sensitive insect responsible for parasporal inclusion from KB099 and HD-l isolate was treated to each of them, protein band 60 KDa of KB099 was maintained up to 12 hours but all bands of HD-l were disappeared within 6 hours. In KB099 isolate, 6 genes (Cry1Aa, Cry1Ab, Cry1Ac, Cry1C, Cry1D and Cry1I) were identified by PCR analysis. Also, $Cry^-$ mutant of KB099 isolate was investigated by phase- contrast microscope, SDS-PAGE and PCR.

• BMB Reports
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• v.35 no.5
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• pp.524-531
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• 2002

Thrombotic thrombocytopenic purpura (TTP) is characterized by widespread platelet thrombi in arterioles and capillaries. Unusually large or multimeric von Willebrand factor, as well as one or ore platelet-agglutinating factors, have been implicated in the pathogenesis of TTP. But, the actual mechanisms of platelet agglutination have not been satisfactorily explained. Recent studies suggested the 37-kDa platelet-agglutinating protein (PAP) p37 to be partially responsible for the formation of platelet thrombi in patients with TTP. We studied mobility in SDS-PAGE, the sequence of N-terminal amino acid residues, DNA and antigenic characteristics of PAP p37, which might be related to the pathogenesis of TTP. PAP p37 was purified from the plasma of a 31-year-old male Korean patient with acute TTP. The findings are as follows: (1) We compared PAP p37 with thrombin through the use of SDS-PAGE, either with or without $\beta$-mercaptoethanol. PAP p37 did not appear to be cleaved between the A- and B-chains of prethrombin 2. However, thrombin did cleave between those of prethrombin 2, but linked with disulfide bridge. (2) N-terminal 21 amino acid sequence of PAP p37 was T-F-G-S-G-E-A-D-X-G-L-R-P-L-F-E-K-K-S-L-E. It appeared to be identical to that of 285-305 amino acid residues of human prothrombin (prethrombin 2). (3) No prothrombin gene DNA mutation was revealed. (4). The antigenicity of PAP p37 was similar to thrombin, which was a result of the competitive binding against the anti-thrombin antibody. With these results, we conclude that PAP p37 has similar characteristics to prethrombin2.

• Korean Journal of Microbiology
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• v.33 no.2
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• pp.97-104
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• 1997

Herpes simpex virus type 2 (HSV-2) infects the genital and oral mucosae of human and other animals. HSV-2 infection is a widespread health problem causing various clinical syndromes including oral, genital, and ocular lesions, viral encephalitis, and recurrent diseases. Hybridorna cell lines secreting a monoclonal antibody (mAb) against the HSV-2 were produced by fusing spleen cells of HSV-2-immunized mice with Sp2/0-AgI4 myeloma cells. One hybridoma cell line was established and its monoclonal C-2, IgM, recognized the antigens of 134, 86, and 43 kDa in western blot analysis. In SDS-P AGE analysis of HSV -2 antigens, 25 bands were separated between 3D kDa and 159 kDa. In indirect immunofluorescent assay, mAbs exhibited binding to the virus antigen expressed on Vero cell infected with HSV-2.

• Biotechnology and Bioprocess Engineering:BBE
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• v.6 no.2
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• pp.100-106
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• 2001

This investigation was performed to study the influence of benomyl on photosynthetic pigments and enzymes in soybean leaves. Chlorophyll and pheophytin levels were reduced by benomyl 45 days after greening. These results indicate that chlorophyll a and b, and pheophytin must be controlled by benomyl. SDS-PAGE analysis showed that 50 and 14.5 kD polypeptides represented as large and small subunits of rubisco. In the both of these subunits, the band intensity of the control was significantly higher than that after benomyl treatment, indicating that these two subunits are affected by benomyl. Benomyl strongly inhibited both the activity and content of rubisco as its concentration was gradually increased. However, it remains unclear whether this reduction of rubisco level was due to a reduced level of rubisco activase. Two major polypeptides of 46 and 42 kD were identified as rubisco activase subunits by SDS-PAGE. The intensity of these two bands was shown to be higher in the control than after benomyl treatment. These results indicate that the rubisco decrease resulting from increased benomyl concentrations was caused by rubisco activase. A significant decrease in both the activity and content of rubisco activase by benomyl was also observed. There results suggest that the decrease in rubisco level caused by benomyl is accompanied by a decrease in both the activity and content of rubisco activase.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.43 no.4
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• pp.307-313
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• 2010

Stanniocalcin 1 (STC1) is a glycoprotein hormone that is important in the maintenance of calcium and phosphate homeostasis in both fish and mammals. STC1 and its paralog STC2 are expressed in multiple tissues in fishes, although the physiological roles of piscine STCs are still unclear compared with those of mammals. In this study, we cloned olive flounder STC1 (ofSTC1) and ofSTC2 cDNAs into pET28a vector and used E. coli Rosetta (DE3) as the host strain for protein expression. Expression experiments were carried out using isopropyl-$\beta$-D-thiogalactoside (IPTG) and nickel affinity chromatography. We could identify the recombinant proteins as single 29.5 kDa (ofSTC1) and 33.2 kDa (ofSTC2) bands in the insoluble fraction on sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE). These results indicate that ofSTC1 and ofSTC2 were expressed as insoluble proteins in E. coli. Furthermore, the injection of ofSTC1 protein into juvenile tilapia resulted in a decrease of the serum calcium level. These results suggest that the purified fish STC1 and STC2 proteins may be used to elucidate the physiological role of STCs in fishes.

• Microbiology and Biotechnology Letters
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• v.31 no.1
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• pp.75-82
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• 2003

The cellular responses of RDX-degrading bacterium, Pseudomonas sp. HK-6 to explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) were examined. Strain HK-6 grown at different RDX concentrations was found to demonstrate the survival rate in proportional to the rate of the stress shock proteins produced in this bacterium. Analysis of total cellular fatty acid acids showed that lipids 10:0 iso and 14:1 $\omega$5c/$\omega$5t increased approx three times in strain HK-6 grown on RDX media than TSA media. SDS-PAGE and Western blot using anti-DnaK and GroEL revealed that several stress shock proteins including 70 kDa DnaK and 60 kDa CroEL were newly synthesized in strain HK-6 exposed to different RDX concentrations in exponentially growing cultures. 2-D PAGE of soluble protein fractions from the culture of HK-6 exposed to RDX demonstrated that approximately 300 spots were observed on the silver stained gel ranging from pH 3 to pH 10. As a result, 10 spots were significantly induced and expressed in response to RDX. Scanning electron microscopy fur the cells treated with 0.135 mM RDX for 12 hrs showed the presence of perforations and irregular rod shapes with wrinkled surfaces.