• IMMUNE NETWORK
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• v.4 no.3
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• pp.184-189
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• 2004

Background: Hu syndrome, a neurological disorder, is characterized by the remote effect of small cell lung cancer on the neural degeneration. The suspicious effectors for this disease are anti-Hu autoantibodies or Hu-related CD8+ T lymphocytes. Interestingly, the same effectors have been suggested to act against tumor growth and this phenomenon may represent natural tumor immunity. For these diagnostic and therapeutic reasons, the demand for antibodies against Hu protein is rapidly growing. Methods: Polyclonal and monoclonal antibodies were generated using recombinant HuR protein. Western blot analyses were performed to check the specificity of generated antibodies using various recombinant proteins and cell lysates. Extracellular stimuli for HuR expression had been searched and HuR-associated proteins were isolated from polysome lysates and then separated in a 2-dimensional gel. Results: Polyclonal and monoclonal antibodies against HuR protein were generated and these antibodies showed HuR specificity. Antibodies were also useful to detect and immunoprecipitate endogenous HuR protein in Jurkat and BJAB. This report also revealed that TNF-${\alpha}$ treatment in BJAB up-regulated HuR expression. Lastly, protein profile in HuR-associated mRNAprotein complexes was mapped by 2-dimensional gel electrophoresis. Conclusion: This study reported that new antibodies against HuR protein were successfully generated. Currently, project to develop a diagnostic kit is in process. Also, this report showed that TNF-${\alpha}$ up-regulated HuR expression in BJAB and protein profile associated with HuR protein was mapped.

• Journal of Microbiology and Biotechnology
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• v.4 no.4
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• pp.250-255
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• 1994

Wild-type and four mutant R100 merR genes were cloned and the proteins overproduced under tac promoter control of pKK223-3. His118Ala, Cys117Ser, Cys126Ser, and wild-type MerR were successfully overproduced although amino-terminal 14 amino acids deletion mutant MerR was not successful. The amount of overproduced wild-type MerR protein as well as other mutant MerR was between 15%-20% of the total protein. The protein was able to be purified up to 95% homogeneity. Specific DNA-protein blotting experiments showed that the 95 bp operator containing DNA fragment could bind to Cys126Ser, His118Ala, and wild- type MerR, but not to Cys117Ser. These results were consistent with the previously reported complementation experiment results that His118Ala, Cys126Ser, and wild-type MerR could repress the mer operon but Cys117Ser could not.

• Journal of Microbiology and Biotechnology
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• v.22 no.11
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• pp.1580-1587
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• 2012

Japanese encephalitis virus (JEV) envelope (E) protein holds great promise for use in the development of a recombinant vaccine. Purified recombinant E (rE) protein may be useful for numerous clinical applications; however, there are limitations in using the Escherichia coli expression system for producing high-quality rE protein. Therefore, in this study, the yeast expression system was used to generate the rE protein. For protein production using the yeast system, the full-length JEV E gene was cloned into Pichia pastoris. SDS-PAGE and immunoblotting analysis demonstrated that the rE protein had a molecular mass of 58 kDa and was glycosylated. The predicted size of the mature unmodified E protein is 53 kDa, suggesting that post-translational modifications resulted in the higher molecular mass. The rE protein was purified to greater than 95% purity using combined ammonium sulfate precipitation and a SP-Sepharose Fast Flow column. This purified rE protein was evaluated for immunogenicity and protective efficacy in mice. The survival rates of mice immunized with the rE protein were significantly increased over that of Hyphantria cunea nuclear polyhedrosis virus E protein (HcE). Our results indicate that the rE protein expressed in the P. pastoris expression system holds great promise for use in the development of a subunit vaccine against JEV.

• Korean Journal of Environmental Agriculture
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• v.28 no.4
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• pp.435-441
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• 2009

In the present study, we analyzed the defensin protein deduced from Korean radish (Raphanus sativus L.) seeds.To express the genes in E. coli, we constructed a recombinant expression vector with a defensin gene, named rKRs-AFP gene isolated from Korean radish seeds. Over expressed rKRs-AFP proteins was separated by SDS-PAGE to determine the purity, and protein concentration was determined by the Bradford method. Antifungal activity was assessed by disk assay method against the tested fungi. As a result, when 500 mL of cell culture were disrupted by sonicator, 32.5 mg total proteins were obtained. The purified protein showed a single band on SDS-PAGE with estimated molecular weight about 6 KDa, consistent with the molecular mass calculated from the deduced amino acid sequence. The purified rKRs-AFP protein showed remarkable antifungal activities against several fungi including Aspergillus niger, Botrytis cinerea causing the gray mold disease, and Candida albicans. In field tests using the purified rKRs-AFP protein, the protein showed the reducing activity of disease spot and the mitigating effect of spreading of disease like agrichemicals. The immuno-assay of rKRs-AFP protein showed that the purified protein entirely accumulated at B. cinerea cytoplasm through the hyphal septa shown by fluorescence imaging. There was no fluorescence inside the cell, when the hypha was incubated without the protein. These all results indicate that the recombinant rKRs-AFP proteins can be utilized as a potential antifungal drug to control harmful plant fungal pathogens.

• Proceedings of the Korean Biophysical Society Conference
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• 2003.06a
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• pp.45-45
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• 2003

We isolated a novel ankyrin-repeat containing protein, rSIAP (rSlo Interacting Ankyrin-repeat Protein), as an interacting protein to the cytosolic domain of the alpha-subunit of rat large-conductance Ca$\^$2+/-activated K$\^$+/ channel (rSlo) by yeast two-hybrid screening. Affinity pull-down assay showed the direct and specific interaction between rSIAP and rSlo domain. The channel-binding proteins can be classified into several categories according to their functional effects on the channel proteins, i.e. signaling adaptors, scaffolding net, molecular tuners, molecular chaperones, etc. To obtain initial clues on its functional roles, we investigated the cellular localization of rSIAP using immunofluorescent staining. The results showed the possible co-localization of rSlo and rSIAP protein near the plasma membrane, when co-expressed in CHO cells. We then investigated the functional effects of rSIAP on the rSlo channel using electrophysiological means. The co-expression of rSIAP accelerated the activation of rSlo channel. These effects were initiated at the micromolar [Ca$\^$2+/]$\_$i/ and gradually increased as [Ca$\^$2+/]$\_$i/ raised. Interestingly, rSIAP decreased the inactivation kinetics of rSlo channel at micromolar [Ca$\^$2+/]$\_$i/, while the rate was accelerated at sub-micromolar [Ca$\^$2+/]$\_$i/. These results suggest that rSIAP may modulate the activity of native BK$\_$Ca/ channel by altering its gating kinetics depending on [Ca$\^$2+/]$\_$i/. To localize critical regions involved in protein-protein interaction between rSlo and rSIAP, a series of sub-domain constructs were generated. We are currently investigating sub-domain interaction using both of yeast two-hybrid method and in vitro binding assay.

• Biomolecules & Therapeutics
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• v.27 no.6
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• pp.514-521
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• 2019

G protein-coupled receptors (GPCRs) are membrane receptors whose agonist-induced dynamic conformational changes trigger heterotrimeric G protein activation, followed by GRK-mediated phosphorylation and arrestin-mediated desensitization. Cytosolic regions of GPCRs have been studied extensively because they are direct contact sites with G proteins, GRKs, and arrestins. Among various cytosolic regions, the role of helix 8 is least understood, although a few studies have suggested that it is involved in G protein activation, receptor localization, and/or internalization. In the present study, we investigated the role of helix 8 in dopamine receptor signaling focusing on dopamine D1 receptor (D1R) and dopamine D2 receptor (D2R). D1R couples exclusively to Gs, whereas D2R couples exclusively to Gi. Bioinformatic analysis implied that the sequences of helix 8 may affect GPCR-G protein coupling selectivity; therefore, we evaluated if swapping helix 8 between D1R and D2R changed G protein selectivity. Our results suggest that helix 8 is not involved in D1R-Gs or D2R-Gi coupling selectivity. Instead, we observed that D1R with D2R helix 8 or D1R with an increased number of hydrophobic residues in helix 8 relative to wild-type showed diminished ${\beta}$-arrestin-mediated desensitization, resulting in increased Gs signaling.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.3
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• pp.248-253
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• 2005

AfsR2, originally identified from Streptomyces lividans, is a global regulatory protein which stimulates antibiotic biosynthesis. Through its stable chromosomal integration, the high level of gene expression of afsR2 significantly induced antibiotic production as well as the sporulation of S. lividans, implying the presence of yet-uncharacterized AfsR2-target proteins. To identify and evaluate the putative AfsR2-target proteins involved in antibiotic regulation, the proteomics-driven approach was applied to the wild-type S. lividans and the afsR2-integrated actinorhodin overproducing strain. The 20 gel-electrophoresis gave approximately 340 protein spots showing different protein expression patterns between these two S. lividans strains. Further MALDI-TOF analysis revealed several AfsR2-target proteins, including glyceraldehyde-3-phosphate dehydrogenase, putative phosphate transport system regulator, guanosine penta phosphate synthetase/polyribonucleotide nucleotidyltransferase, and superoxide dismutase, which suggests that the AfsR2 should be a pleiotropic regulatory protein which controls differential expressions of various kinds of genes in Streptomyces species.

• Journal of Life Science
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• v.20 no.8
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• pp.1181-1185
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• 2010

This study was carried out to evaluate the vaccination effects of recombinant fusion protein rVP19+28 against WSSV in shrimp, Litopenaeus vannamei. The VP19+28 gene fused with VP19 and VP28 genes was inserted into pET-28a(+) expression vector and cloned in E. coli BL21 (DE3) to produce fused gene product recombinant VP19+VP28 as a single protein. For the vaccination, the shrimps were fed with pellets coated with purified recombinant protein, rVP19+28, for 2 weeks. Then, constant amounts of WSSV at $1{\times}10^2$ diluted stocks were injected to the muscle of the shrimp for the in vivo challenge tests. Non-vaccinated shrimps showed a cumulative mortality of 100% at 11 days post-challenge. The shrimps vaccinated with the inactivated E. coli BL21 as a host cell control showed cumulative mortality of 100% at 17 days post-challenge. The shrimps vaccinated with rVP19, rVP28 and rVP19+28 showed mortalities of 66.7%, 41.7% and 41.7% at 21 days post-challenge, respectively. These results indicated that the rVP28 and rVP19+28 had relatively high vaccination effects against WSSV infection. However, this study suggests that the fusion protein rVP19+28 was more effective for the protection of shrimp against WSSV than rVP28, even though the cumulative mortalities were the same 21 days post-challenge.

• BMB Reports
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• v.43 no.12
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• pp.795-800
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• 2010

Huntingtin-interacting protein 1-related (HIP1r) is known to function in clathrin-mediated endocytosis and regulation of the actin cytoskeleton, which occurs continuously in non-dividing cells. This study reports a new function for HIP1r in mitosis. Green fluorescent protein-fused HIP1r localizes to the mitotic spindles. Depletion of HIP1r by RNA interference induces misalignment of chromosomes and prolonged mitosis, which is associated with decreased proliferation of HIP1r-deficeint cells. Chromosome misalignment leads to missegregation and ultimately production of multinucleated cells. Depletion of HIP1r causes persistent activation of the spindle checkpoint in misaligned chromosomes. These findings suggest that HIP1r plays an important role in regulating the attachment of spindle microtubules to chromosomes during mitosis, an event that is required for accurate congression and segregation of chromosomes. This finding may provide new insights that improve the understanding of various human diseases involving HIP1r as well as its fusion genes.

• The Korean Journal of Physiology and Pharmacology
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• v.15 no.2
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• pp.95-100
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• 2011

DREAM (downstream regulatory element antagonistic modulator) is a calcium-binding protein that regulates dynorphin expression, promotes potassium channel surface expression, and enhances presenilin processing in an expression level-dependent manner. However, no molecular mechanism has yet explained how protein levels of DREAM are regulated. Here we identified group I mGluR (mGluR1/5) as a positive regulator of DREAM protein expression. Overexpression of mGluR1/5 increased the cellular level of DREAM. Up-regulation of DREAM resulted in increased DREAM protein in both the nucleus and cytoplasm, where the protein acts as a transcriptional repressor and a modulator of its interacting proteins, respectively. DHPG (3,5-dihydroxyphenylglycine), a group I mGluR agonist, also up-regulated DREAM expression in cortical neurons. These results suggest that group I mGluR is the first identified receptor that may regulate DREAM activity in neurons.