• Bulletin of the Korean Chemical Society
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• v.29 no.9
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• pp.1790-1792
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• 2008

• KSBB Journal
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• v.18 no.4
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• pp.306-311
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• 2003

Solid-phase refolding of immobilized proteins can be an effective way to reuse an immobilized enzyme column. Oriented immobilization methods are known to provide higher activity of the immobilized enzymes. In this study, using recombinant EK (enterokinase) as a model enzyme and a fusion protein, that consisted of recombinant human growth hormone and six His tag that was linked by the peptide of EK-specific recognition sequence, as a model substrate, we evaluated two oriented immobilization methods, i. e., reductive alkylation of N-terminus ${\alpha}$-amine and affinity interaction between poly-histidine tag and Ni-NTA (nickel-nitrilotriacetic acid). The immobilization yield, activity and cleavage of the immobilized enzymes, and the yield of solid-phase refolding were compared. The Ni affinity immobilization and the covalent immobilization yields were about 100％ and 65％, respectively. But the specific activities were the same, about 50％ of that of the soluble enzyme. The cleavage rate by the covalently immobilized EK was higher than the soluble enzyme and the side reaction of cryptic cleavage was significantly decreased. Covalently immobilized EK showed almost 100％ refolding yield but the affinity immobilized EK showed only 70％ yield, which suggested the covalent conjugation provided more rigid ‘reference structure’ for the solid-phase refolding. The monomeric hGH could be easily obtained by capturing the cleaved poly Histidine tag by the Ni affinity column.

• Proceedings of the Korean Society of Sericultural Science Conference
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• 2004.10a
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• pp.25-25
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• 2004

• Journal of the Korean Electrochemical Society
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• v.13 no.1
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• pp.70-74
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• 2010

The immobilization of proteins on silicon surfaces using electrochemical reaction has been studied. Chemical deposition of nitrobenzendiazonium (NiBD) cations is employed to modify silicon surfaces. Electrochemical reduction of nitro-group to primary amine-group have been conducted on the modified surfaces to activate silicon surfaces for the protein immobilization. Attachment of gold nanoparticles was used to prove the reduction. The current method was applied to selective activation of a silicon nanowire and immobilize proteins on the selected nanowire. It has been demonstrated that the use of chemical and electrochemical reaction NiBD is efficient for the selective immobilization of proteins on silicon nanowire surfaces.

• Genomics & Informatics
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• v.4 no.3
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• pp.133-136
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• 2006

The adsorption of proteins on the surface of glass slides is essential for construction of protein chips. Previously, we prepared a nickel-coated plate by the spin-coating method for immobilization of His-tagged proteins. In order to know whether the structural factor is responsible for the immobilization of His-tagged proteins to the nickel-coated glass slide, we executed a series of experiments. First we purified a His-tagged protein after expressing the vector in E. coli BL21 (DE3). Then we obtained the unfolding curve for the His-tagged protein by using guanidine hydrochloride. Fractions unfolded were monitored by internal fluorescence spectroscopy. The ${\Delta}G_{H20}$ for unfolding was $2.27kcal/mol{/pm}0.52$. Then we tested if unfolded His-tagged proteins can be adsorbed to the nickel-coated plate, comparing with $Ni^{2+}-NTA$ (nitrilotriacetic acid) beads. Whereas unfolded His-tagged proteins were adsorbed to $Ni^{2+}-NTA$ beads, they did not bind to the nickel-coated plate. In conclusion, a structural factor is likely to be an important factor for constructing the protein chips, when His-tagged proteins will immobilize to the nickel-coated slides.

• Journal of Physiology & Pathology in Korean Medicine
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• v.19 no.6
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• pp.1585-1593
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• 2005

The genetic effects of restraint stress challenge on HPA axis and the therapeutic effect of Boshimgeonbi-tang on the stress were studied with cDNA microarray analyses, RT-PCR on hypothalamus using an immobilization-stress mice as an animal model. Male CD-1 mice were restrained in a tightly fitted and ventilated vinyl holder for 2hrs once a day, and this challenge was repeated for seven· consecutive days. In the change of body weight it showed that the Boshimgeonbi-tang is effected recovery on weight loss caused by the immobilization-stress. Seven days later, total RNA was extracted from the organs of the mouse, body-labeled with $CyDye^{TM}$ fluorescence dyes and then hybridized to CDNA microarray chip. Scanning and analyzing the array slides were carried out using GenePix4000 series scanner and GenePix $Pro^{TM}$ analyzing program, respectively. The expression profiles of 109 genes out of 6000 genes on the chip were significantly modulated in hypothalamus by the immobilization stress. Energy metabolism-, lipid metabolism-, apoptosis-, stress protein, transcriptional factor, and signal transduction-related genes were transcriptionally activated whereas DNA repair-, protein biosysthesis-, and structure integrity-related genes were down-regulated in hypothalamus. The 58 genes were up-regulated by the mRNA expression folds of 1.5 to 7.9. and the 51 genes were down-regulated by 1.5 - 5.5 fold. The 11 genes among them were selected to confirm the expression profiles by RT-PCR. The mRNA expression levels of Tnfrsf1a (apoptosis), Calm2 (cell cycle), Bag3 (apoptosis), Ogg1 (DNA repair), Aatk (apoptosis), Dffa (apoptosis), Fkbp5 (protein folding) were restored to the normal one by the treatment of Boshimgeonbi-tang.

• Journal of Periodontal and Implant Science
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• v.41 no.6
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• pp.263-272
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• 2011

A number of surface modification techniques using immobilization of biofunctional molecules of Titanium (Ti) for dental implants as well as surface properties of Ti and Ti alloys have been developed. The method using passive surface oxide film on titanium takes advantage of the fact that the surface film on Ti consists mainly of amorphous or low-crystalline and nonstoichiometric $TiO_2$. In another method, the reconstruction of passive films, calcium phosphate naturally forms on Ti and its alloys, which is characteristic of Ti. A third method uses the surface active hydroxyl group. The oxide surface immediately reacts with water molecules and hydroxyl groups are formed. The hydroxyl groups dissociate in aqueous solutions and show acidic and basic properties. Several additional methods are also possible, including surface modification techniques, immobilization of poly(ethylene glycol), and immobilization of biomolecules such as bone morphogenetic protein, peptide, collagen, hydrogel, and gelatin.

• Proceedings of the Korean Society of Sericultural Science Conference
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• 2004.04a
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• pp.23-24
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• 2004

• Journal of Physiology & Pathology in Korean Medicine
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• v.17 no.6
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• pp.1393-1403
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• 2003

The genetic effects of restraint stress challenge on HPA axis and the therapeutic effect of Boshimgeonbi-Tang on the stress were studied with cDNA microarray analyses on hypothalamus using an immobilization-stress mouse as stress model. Male CD-1 mice were restrained in a tightly fitted and ventilated vinyl holder for 2hours once a day, and this challenge was repeated for seven consecutive days. The body weights of the immobilization-stress mice were diminished about 25 percent degree as compared to normal ones. Seven days later, total RNA was extracted from the organs of the mouse, body-labeled with CyDye/sup TM/ fluorescence dyes (Amersham Bioscience Co., NJ), and then hybridized to cDNA microarray chip. Scanning and analyzing the array slides were carried out using GenePix 4000 series scanner and GenePix Pro/sup TM/ analyzing program, respectively. The expression profiles of 109 genes out of 6000 genes on the chip were significantly modulated in hypothalamus by the immobilization stress. Energy metabolism-, lipid metabolism-, apoptosis- and signal transduction-related genes were transcriptionally activated whereas DNA repair-, protein biosynthesis-, and structure integrity-related genes were down-regulated in hypothalamus. The 58 genes were up-regulated by the mRNA expression folds of 1.5 to 7.9. and the 51 genes were down-regulated by 1.5 - 3.5 fold. The 20 genes among them were selected to confirm the expression profiles by RT-PCR. The mRNA expression levels of Tnfrsf1a (apoptosis), Calm2 (cell cycle), Bag3 (apoptosis), Hspe1 (protein folding), Aatk (apoptosis), Dffa (apoptosis), Itgb1 (cell adhesion), Vcam1 (cell adhesion), Fkbp5 (protein folding), BDNF (neuron survival) were restored to the normal one by the treatment of Boshimgeonbi-Tang.

• Journal of Microbiology and Biotechnology
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• v.20 no.2
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• pp.332-339
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• 2010

The lipase from Mucor racemosus NRRL 3631 was partially purified by fractional precipitation using 60% ammonium sulfate, which resulted in a 8.33-fold purification. The partially purified lipase was then immobilized using different immobilization techniques: physical adsorption, ionic binding, and entrapment. Entrapment in a 4% agar proved to be the most suitable technique (82% yield), as the immobilized lipase was more stable at acidic and alkaline pHs than the free enzyme, plus 100% of the original activity was retained owing to the thermal stability of the immobilized enzyme after heat treatment for 60 min at $45^{\circ}C$. The calculated half-lives (472.5, 433.12, and 268.5 min at 50, 55, and $60^{\circ}C$, respectively) and the activation energy (9.85 kcal/mol) for the immobilized enzyme were higher than those for the free enzyme. Under the selected conditions, the immobilized enzyme had a higher $K_m$ (11.11 mM) and lower $V_{max}$ (105.26 U/mg protein) when compared with the free enzyme (8.33 mM and 125.0 U/mg protein, respectively). The operational stability of the biocatalyst was tested for both the hydrolysis of triglycerides and esterification of fatty acids with glycerol. After 4 cycles, the immobilized lipase retained approximately 50% and 80% of its original activity in the hydrolysis and esterification reactions, respectively.