• The Journal of Korean Society of Virology
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• v.28 no.1
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• pp.21-30
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• 1998

Synthetic genes encoding the gag p24 and the part of the envelope protein gp41 of the human immunodeficiency virus (HIV-1) were cloned and overexpressed as fusion proteins in Escherichia coli, using an expression vector carrying T7 promoter and the poly-histidine leader, sequence. The overexpressed p24 fusion protein was purified by centrifugation, Ni-affinity chromatography and CM-sepharose chromatography. The overexpressed gp41 fusion protein was purified by centrifugation, $C_4$ chromatography and DEAE-sepharose chromatography. The purified fusion proteins showed a high level of purity and immunoreactivity in SDS-polyacrylamide gel electrophoresis and western blot analysis. These results suggest that this prokaryotic - expression purification method is suitable for obtaining a large amount of the viral antigen which may be useful for screening of antibodies to HIV-1 in human blood samples.

• Journal of Plant Biology
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• v.38 no.3
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• pp.251-258
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• 1995

The soluble acid invertase ($\beta$-D-fructofuranoside fructohydrolase, EC 3.2.1.26) was isolated and characterized from the hypocotyls of mung bean (Phaseolus radiatus L.). The enzyme was purified to apparent homogeneity by consecutive step using diethylaminoethyl (DEAE)-cellulose anion exchange, Concanavalin (Con) A affinity and Sephacryl S-300 chromatography. The overall purification was about 148-fold with a yield of about 15%. The finally purified enzyme exhibited a specific activity of about 139 $\mu$mol of glucose produced mg-1 protein min-1 at pH 5.0 and appeared to be a single protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and nondenaturing PAGE. The enzyme had the native molecular weight of 70 kD and subunit molecular weight of 70 kD as estimated by Sephadex G-200 chromatography and SDS-PAGE, respectively, suggesting that the enzyme was composed of a monomeric protein. On the other hand, the enzyme appeared to be a glycoprotein containing N-linked high mannose oligosaccharide chain on the basis of its ability to bind to the immobilized C on A. The enzyme had a Km for sucrose of 1.8 mM at pH 5.0 and maximum activity around pH 5.0. The enzyme showed highest enzyme activity with sucrose as substrate, but the activity was slightly measured with raffinose and cellobise. No activity was measured with maltose and lactose. These results indicate the soluble acid invertase is a $\beta$-fructofuranosidase.

• Journal of Plant Biology
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• v.38 no.4
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• pp.349-357
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• 1995

The alkaline invertase ($\beta$-D-fructofuranoside fructohydrolase, EC 3.2.1.26) was isolated and characterized from the hypocotyls of mung bean (Phaseolus radiatus L.). The enzyme was purified by consecutive step using diethylaminoethyl (DEAE)-cellulose anion exchange, 1st Sephadex G-200, DEAE-Sephadex A50 and 2nd Sephadex G-200 chromatography. The overall purification was about 77-fold with a yield of about 6%. The finally purified enzyme exhibited a specific activity of about 48 $\mu$mol of glucose produced mg-1 protein min-1 at pH 7.0 and appeared to be a single protein by nondenaturing polyacrylamide gel electrophoresis (PAGE). The enzyme had the native molecular weight of 450 kD and subunits molecular weight of 63 kD and 38 kD as estimated by Sephadex G-200 chromatography and SDS-PAGE, respectively, suggesting that the enzyme is a heteromultimeric protein composed of two types of subunits. On the other hand, the enzyme appeared to be not a glycoprotein according to the results of Con A chromatography and glycoprotein staining. The enzyme had a Km for sucrose of 19.7 mM at pH 7.0 and maximum activity around pH 7.5. The enzyme was most active with sucrose as substrate, compared to raffinose, cellobiose, maltose and lactose. These results indicate the alkaline invertase is a $\beta$-fructofuranosidase.

• Journal of Microbiology and Biotechnology
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• v.9 no.6
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• pp.881-884
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• 1999

The gene encoding the RNA-dependent RNA polymerase of the hepatitis C virus was cloned and expressed with a C-terminal hexahistidine tag. The protein was purified from Escherichia coli to near homogeneity and characterized in vitro. When the 21 amino acids from the C-terminus of the protein were deleted, an inclusion body was not formed and a better purification yield was achieved. However, the activity of the purified enzyme decreased compared to that of the full length protein. The purified enzyme did exhibit ribonucleotide-incorporation activity on an in vitro transcribed RNA containing the 3' end of the HCV genome. It also possessed ribonucleotide incorporation activity, to a lesser extent, on in vitro transcribed foreign RNA templates when RNA or DNA primers were present. The activity was higher with DNA primers than with RNA primers. Accordingly, this assay system will facilitate the screening of inhibitors for hepatitis C virus replication.

• Biotechnology and Bioprocess Engineering:BBE
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• v.9 no.6
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• pp.423-427
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• 2004

A simple purification procedure of bioactive human granulocyte macrophage colony stimulating factor (hGM-CSF) secreted in rice cell suspension culture has previously been described. In this study the protein was purified to apparent homogeneity with an overall yield of $80.1\%$ by ammonium sulfate precipitation and a single chromatographic step involving FPLCanion exchange chromatography. The purified hGM-CSF revealed at least five glycosylated forms ranging from $21.5{\~}29$ kDa, and its biological activity was independent of the glycosylation pattern. This is the first purification report of recombinant hGM-CSF to apparent homogeneity from rice cell suspension cultures.

• Journal of Microbiology and Biotechnology
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• v.26 no.10
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• pp.1808-1816
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• 2016

As a scaffolding subunit of the PIK3C3/VPS34 complex, Beclin 1 recruits a variety of proteins to class III phosphatidylinositol-3-kinase (VPS34), resulting in the formation of a distinct PIK3C3/VPS34 complex with a specific function. Therefore, the investigation of a number of Beclin 1 domains required for the protein-protein interactions will provide important clues to understand the PIK3C3/VPS34 complex, of which Beclin1-VPS34 interaction is the core unit. In the present study, we have designed a bacterial overexpression system for the Beclin 1 domain corresponding to VPS34 binding (Vps34-BD) and set up the denaturing purification protocol due to the massive aggregation of Vps34-BD in Escherichia coli. The expression and purification conditions determined in this study successfully provided soluble and functional Vps34-BD.

• Proceedings of the Zoological Society Korea Conference
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• 1998.10b
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• pp.277.1-277
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• 1998

No Abstract, See Full Text

• Journal of Microbiology and Biotechnology
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• v.8 no.4
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• pp.333-340
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• 1998

Streptomyces griseus trypsin (SGT) is an extracellular proteinase produced by S. griseus. The sprT gene, which encodes premature SGT protein, was cloned into the plasmid pWHM3, a Streptomyces-E. coli shuttle vector. When the recombinant plasmid was introduced into Streptomyces lividans TK24, two proteins with molecular weights of 28 kDa and 42 kDa were detected. The 28-kDa protein was a SGT protein while the larger 42-kDa protein is thought to have been a premature form of the SGT protein. The SGT protein was purified to homogeneity via ammonium sulfate fractionation and many column chromatographies, including CM -sepharose chromatography, Mono-S chromatography, and Superose-12 chromatography, from the culture broth of S. lividans TK24 harboring the sprT gene. The N-terminal amino acid sequence, isoelectric points, and stabilities at various conditions of the SGT proteins purified from the Pronase and transformant were almost identical. The amount of the expressed SGT in S. lividans TK 24 was determined to be 5 times more than that of S. griseus based on the enzymatic activity against artificial substrate.

• Journal of the Korean Magnetic Resonance Society
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• v.14 no.2
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• pp.105-116
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• 2010

SH3YL1, a novel protein containing one Src homology 3 domain at the carboxyl terminus was first detected in mouse anagen skin cDNA. This protein had a significant homology with YHRO 16c/Ysc 84, the yeast Src homology 3 domain-containing protein. The sequence identity was remarkable at the carboxyl and amino-terminal Src homology 3 domain, suggesting that the novel protein is a mouse homolog of the yeast protein and thus was termed as SH3YL1. SH3YL1 is composed of two domains, a DUF500 at N-termini and a SH3 domain at C-termini. In our study we cloned the SH3 domain in bacterial expression system in Escherichia coli using pET32a vector with TEV protease cleavage site and purified as a monomer using affinity chromatography. The N-terminal poly-Histidine tag was cleaved with TEV protease and target protein was used for backbone studies. Our study showed that SH3 domain primarily consists of $\beta$-sheet which is in consistence with previous result performed on the truncated SH3 domain of SH3YL1.

• Proceedings of the KIEE Conference
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• 2008.07a
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• pp.1466-1467
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• 2008

We describe here a novel micro total analysis system for the purification and identification of the affinity-captured proteins. Also we demonstrated the mass analysis of the Carcinoembrionic antigen (CEA) and Alpha femtoprotein which were chosen as the target cancer marker. For MALDI-TOF analyses, the proteins should to be separated from a protein mixture and be concentrated when needed. This procedure usually takes a long time even before protease-digested samples are to be obtained from them. Here, we describe integrated and efficient micro chip for protein purification and digestion for MALDI-TOF analyses. At first, disease protein is purified by passing the micro chamber from a protein mixture or human whole serum and released from the micro affinity beads by thermal heating. Purified protein is then transfer to the hole for trypsin digestion. The final sample is analyzed by MALDI-TOF. All the processes could be finished successfully within one hour, which renders MALDI-TOF analyses of a target protein quite simple.