• Proceedings of the Korea Crystallographic Association Conference
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• 2000.11a
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• pp.7-7
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• 2000

• Molecules and Cells
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• v.25 no.3
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• pp.446-451
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• 2008

We assessed heterologous protein expression in 64 strains obtained from the Escherichia coli Reference (ECOR) collection, a collection representing diverse natural E. coli populations. A plasmid generating a glutathione S-transferase and plant carbonic anhydrase fusion protein (GST-CA) under the control of the tac promoter was introduced into the ECOR strains, and the quantity of the fusion protein was determined by SDS-PAGE. The foreign protein was generated at various levels, from very high (40 strains, high producers) to very low (six strains, low producers). Immunoblotting showed that the high producers expressed approximately 250-500 times more GST-CA protein than the low producers. The results of semi-quantitative RT-PCR showed that the low producers generated mRNA levels comparable to those of the high producers, thereby suggesting that, at least in this case, inefficient translation is a major cause of the low production. We introduced a different plasmid, which expressed a maltose binding protein and plant guanylate kinase fusion protein (MBP-GK) into the six low producers. Interestingly, five of these expressed MBP-GK at very high levels. Thus, we conclude that the production of a particular protein from an expression vector can vary considerably, depending on the host strain. Strains in the ECOR collection could function as useful alternative hosts when a desired level of protein expression is not obtained from commonly used strains, such as E. coli K12 or B derivatives.

• Biomolecules & Therapeutics
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• v.4 no.1
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• pp.103-109
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• 1996

To investigate the effect of the third intracellular loop (i3 loop) peptide of human $\beta$$_2$-adrenergic receptor on receptor agonist binding, we expressed third intracellular loop region of human $\beta$$_2$-adrenergic receptor as glutathione S-transferase fusion protein in E. coli. DNA fragment of the receptor gene which encodes amino acid 221-274 of human $\beta$$_2$-adrenergic receptor was amplified by polymerase chain reaction and subcloned into the bacterial fusion protein expression vector pGEX-CS and expressed as a form of glutathione-S-transferase (GST) fusion protein in E. coli DH5$\alpha$. The receptor fusion protein was identified by SDS-PAGE and Western blot using monoclonal anti-GST antibody. The fusion protein expressed in this study was purified to an apparent homogeneity by glutathione Sepharose CL-4B affinity chromatography. The purified i3 loop fusion proteins at a concentration of 10 $\mu\textrm{g}$/ι caused right shift of the isoproterenol competition curve of [$^3$H]Dihydroalprenolol binding to hamster lung $\beta$$_2$-adrenergic receptor indicating lowered affinity of isoproterenol to $\beta$$_2$-adrenergic receptor possibly due to the uncoupling of receptor and G protein in the presence of the fusion protein. The uncoupling of receptor and G protein suggests that i3 loop region plays a critical role on $\beta$$_2$-adrenergic receptor G protein coupling.

• Animal cells and systems
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• v.12 no.3
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• pp.149-155
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• 2008

We have isolated and characterized Agrius convolvuli cDNA encoding a c-type lysozyme. The cDNA sequence encodes a processed protein of 139 amino acid residues with 19 amino acid residues amino-terminal signal sequence and 120 amino acid residues mature sequence. The amino acid residues responsible for the catalytic activity and the binding of the substrate are conserved. Agrius lysozyme has a high identity to Manduca sexta. Recombinant A. convolvuli lysozyme was expressed in Escherichia coli BL21(DE3) pLysS cells for pGEX 4T-1 expression vector. Their optimal conditions for the fusion protein expression and purification were screened. Lysozyme gene amplified with primers ACLyz BamHI and ACLyz XhoI was ligated into the pGEX 4T-1 vector, which contained the glutathione S-transferase(GST) gene for fusion partner. The fusion protein was induced by IPTG and identified by SDS-PAGE analysis. Molecular weight of the fusion protein was estimated to be about 45 kDa. Recombinant lysozyme, fused to GST, was purified by glutathion-Sepharose 4B affinity chromatography. Western blot analysis of this protein revealed an immunoreactivity with the anti-Agrius lysozyme.

• Biomedical Science Letters
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• v.2 no.2
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• pp.175-185
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• 1996

The mitogen-activated protein(MAP) kinase signal transduction pathway represents an important mechanism by which mitogen, such as serum and PMA, regulate cell proliferation and differentiation. Target substrates of the MAP kinase are located within several compartments containing plasma membranes and nucleus. We now report that serum addition induces proliferation of the P388 murine leukemia cell, but PMA does not, while both serum and PMA treatment cause translocation of the MAP kinase, mainly p42$^{mapk}$ isoform, from cytosol into the nucleus, which was monitored by immunoblot analysis using polyclonal anti-ERK1 antibodies. We investigated whether the MAP kinase was capable of phosphorylating c-Jun protein and GST-fusion proteins, the P562$^{kk}$N-terminal peptides (1-77 or 1-123 domain) of the T cell tyrosine kinase, using the partially purified MAP kinase by SP-sephadex C-50, phenyl superose and Mono Q column chromatography. We found that the partially purified MAP kinase was able to phosphorylate c-Jun protein and the GST-fusion protein expressed using E.coli DH5$\alpha$ which is transformed with pGEX-3Xb plasmid vector carrying of p562$^{kk}$N-terminal peptide-encoding DNA. These results imply that tyrosine kinase receptor/Ras/Raf/MAP kinase pathway is a major mechanism for mitogen-induced cell proliferation in P388 murine leukemia cell and that the various MAP kinase isoforms may have their own target substrates located in distinct subcellular compartments.

• Bulletin of the Korean Chemical Society
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• v.29 no.2
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• pp.381-388
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• 2008

An X-linked skeletal disorder, SEDT (spondyloepiphyseal dysplasia tarda) is a genetic disease characterized by a disproportionately short trunk and short stature caused by mutations in the SEDL gene. This gene is evolutionarily conserved from yeast to human. The yeast SEDL protein ortholog, Trs20p, has been isolated as a member of a large multi-protein complex called the transport protein particle (TRAPP), which is involved in endoplasmic reticulum (ER)-to-Golgi transport. The interaction between SEDL and partner proteins is important in order to understand the molecular mechanism of SEDL functions. We isolated several SEDL-binding proteins derived from rat cells by an immobilized GST-fusion method. Furthermore, the SEDL-homologue proteins were identified using motif based methods. Common motifs between SEDL-binding proteins and SEDL-homologue proteins were classified into seven types and 78 common motifs were revealed. Sequence similarities were contracted to seven types using phylogenetic trees. In general, types I-III and VI were classified as having the function of acetyl-CoA carboxylase, glycogen phosphorylase, isocitrate dehydrogenase, and enolase, respectively, and type IV was found to be functionally related to the GST protein. Types V and VII were found to contribute to TRAPP vesicle trafficking.

• Korean Journal Plant Pathology
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• v.13 no.4
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• pp.248-254
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• 1997

Expression vector (pGEX-Tu) for the coat protein (CP) gene of turnip mosaic virus Ca strain (TuMV-Ca) was constructed by incorporation of TuMV CP gene into pGEX-KG vector which had ${\beta}$-galactosidase gene and IPTG (isopropylthio-${\beta}$-D-galactoside) induction site. The results of ELISA and western hybridization indicated that optimal condition of the expression were when IPTG and western hybridization indicated that optimal condition of the expression were when IPTG induction was carried out on YTA medium with ampicillin in 2 hours after the E. coli seed inoculation ($A_{595}$=0.1/ml). TuMV CP gene was expressed with GST (Glutathion S-Transferase) gene fusion system, and the size of fusion protein was estimated to be 59kDa, for TuMV CP was 33 kDa and GST was 26 kDa.

• BMB Reports
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• v.30 no.5
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• pp.303-307
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• 1997

Grb2, which is composed of a Src homology 2 (SH2) domain and two Src homology 3 (SH3) domains, is known to serve as an adaptor protein in signaling for Ras activation. Thus, a blocker of the Grb2 interactions with other proteins can be a potential candidate for an anticancer drug. In this study, we have developed a high throughput screening method for SH3 domain binding ligands and blockers. Firstly, we made and purified the glutathione S-transferase (GST)-fusion proteins with the Grb2 SH2 and SH3 domains, and the entire Grb2. This method measures the binding of a biotin-labeled oligopeptide, derived from a Grb2/SH3 binding motif in the hSos, to the GST-fusion proteins, which are precoated as glutathione S-transferase fusion protein on a solid phase. When $1\;{\mu}g$ of each fusion protein was used to coat the wells, both N- and C- terminal SH3 the domains as well as the whole of Grb2 were able to interact with the biotin-conjugated ligand peptide, while the SH2 domain and GST alone showed no binding affinity. Although N- and C- terminal SH3 domains showed an increase of binding to the ligand peptide in proportion to the amount of peptide, the GST fusion protein with Grb2 demonstrated much higher binding affinity. GST-Grb2 coating on the solid phase showed a saturation curve; 66 and 84% of the maximal binding was observed at 100 and 300 ng/$100\;{\mu}l$, respectively. This binding assay system was peptide sequence-specific, showing a dose-dependent inhibition with the unlabeled peptide of SH3 binding motif. Several other peptides, such as SH2 domain binding motifs and PTB domain binding motif, were ineffective to inhibit the binding to the biotin-conjugated ligand peptide. These results suggest that our method may be useful to screen for new anticancer drug candidates which can block the signaling pathways mediated by SH3 domain binding.

• Journal of Life Science
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• v.9 no.3
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• pp.253-261
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• 1999

Leptin gene, an obesity gene, has been known to involve in the regulation of food intake and body weight. It is also thought to be related to the glucose metabolism, insulin secretion and type II diabetes mellitus. Recently, the production of recombinant leptin protein has been attempted for the application in the treatment of obesity and the correction of hereditary obesity and type II diabetes. In the present study, leptin cDNA was cloned from mouse fat cells by RT-PCR and prokaryotic expression of leptin was attempted in order ot prepare a leptin-specific antigen. Immunization of a rabbit with the leptin-specific antigen into a rabbit resulted in the generation of leptin-specific antiserum that could be useful in the detection of leption expressed in various tissues. The sequence of leptin cDNA prepared in the present study wa identical to the previously reported one. Transformation of E. coli(DH5a) cells with the leptin cDNA-inserted translation vector, pGEX-4T-3-leptin followed by treatment with IPTG (0.1mM) resulted in the expression of a large amount of GST-leptin fusion protein with a molecular weight of 44 KDa as an inclusion body. Denaturation of the insoluble fusion protein by 8M urea, 6M guanidium-HCI or 0.1% 2-mercaptoethanol followed by a slow oxidation could not solubilize the inclusion body. The cell extract was subjected to SDS-PAGE and GST-leptin protein electroeluted from the gel was then injected into a rabbit subcutaneously for the immunization. Anti-GST-leptin rabbit antiserum which had a cross reactivity to the GST-leptin protein was generated. Leptin protein expressed in mouse brain and fat tissues was detected by Western blot immunodetection system using the antiserum generated in the present study.

• Korean Journal of Veterinary Service
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• v.44 no.4
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• pp.185-194
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• 2021

Polyglutamine extension in the coding sequence of mutant huntingtin causes neuronal degeneration associated with the formation of insoluble polyglutamine aggregates in Huntington's disease (HD). Mutant huntingtin can form aggregates within the nucleus and processes of neurons possibly due to misfolding of the proteins. To better understand the mechanism by which an elongated polyglutamine causes aggregates, we have developed an in vitro binding assay system of polyglutamine tract from truncated huntingtin. We made GST-HD exon1 fusion proteins which have expanded polyglutamine epitopes (e.g., 17, 23, 32, 46, 60, 78, 81, and 94 CAG repeats). In the present emergence of new study adjusted nanotechnology on protein chip such as surface plasmon resonance strategy which used to determine the substance which protein binds in drug discovery platform is worth to understand better neurodegenerative diseases (i.e., Alzheimer disease, Parkinson disease and Huntington disease) and its pathogenesis along with development of therapeutic measures. Hence, we used strengths of surface plasmon resonance (SPR) technology which is enabled to examine binding specificity and explore targeted molecular epitope using its electron charged wave pattern in HD pathogenesis utilize conjugated mutant epitope of HD protein and its interaction whether wild type GST-HD interacts with mutant GST-HD with maximum binding affinity at pH 6.85. We found that the maximum binding affinity of GST-HD17 with GST-HD81 was higher than the binding affinities of GST-HD17 with other mutant GST-HD constructs. Furthermore, our finding illustrated that the mutant form of GST-HD60 showed a stronger binding to GST-HD23 or GST-HD17 than GST-HD60 or GST-HD81. These results indicate that the binding affinity of mutant huntingtin does not correlate with the length of polyglutamine. It suggests that the aggregation of an expanded polyglutamine might have easily occurred in the presence of wild type form of huntingtin.