• Transactions of the Society of Information Storage Systems
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• v.8 no.1
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• pp.1-5
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• 2012

We present a method for preparing a quantum dot fluorescent monolayer film on a glass substrate. Since nanoparticles aggregate easily, it is difficult to prepare a nanoparticle monolayer film. We have used a covalent bond, the peptide bond, to fix quantum dots on the glass substrate. The surface of the quantum dot was functionalized with carboxyl groups, and the glass substrate was also functionalized with amino groups using a silane coupling agent. The carboxyl group can be strongly coupled to the amino group. We were able to successfully prepare a monolayer film of CdSe quantum dots on the glass substrate.

• Journal of Life Science
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• v.10 no.2
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• pp.210-217
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• 2000

A kinetic profile of the catalytic domain of stromelysin-1 (SCD) using the fluorescent peptide substrate has been determined by the stopped-flow technique. The pH profile has a pH optimum of about 5.5 with an extended shoulder above pH 7. Three pKa values, 5.0, 5.7, and 9.8 are found for the free enzyme state and two pH independent Kcat/Km values of 4.1$\times$104 M-1 s-1 and 1.4$\times$104 M-1 s-1 at low and high pH, respectively. The profile is quite different in shape with other MMP family which has been reported, having broad pH optimum with two pKa values. The substrate specificity of SCD towards fluorescent heptapeptide substrates has been also examined by thin layer chromatography. The cleavage sites of the substrates have been identified using reverse-phase HPLC method.SCD cleaves Dns-PLA↓L↓WAR and Dns-PLA↓L↓FAR at two positions. However, the Dns-PLA↓LRAR, Dns-PLE↓LFAR, adn Dns-PLSar↓LFAR are cleaved exclusively at one bond. The double cleavages of Dns-PLALWAR and Dns-PLALFAR by SCD are in marked contrast to the close structurally related matrilysin. A notable feature of SCD catalysis agrees with the structural data that the S1' pocket of SCD is deeper than that of matriysin. The differences observed between SCD and matrilysin may form the basis of understanding the structural relationships and substrate specificities of the MMP family in vivo.

• BMB Reports
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• v.35 no.2
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• pp.206-212
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• 2002

The NS2B-NS3(pro) polyprotein segment from the dengue virus serotype 2 strain 16681 was purified from overexpressing E. coli by metal chelate affinity chromatography and gel filtration. Enzymatic activity of the refolded NS2B-NS3(pro) protease complex was determined in vitro with dansyl-labeled peptide substrates, based upon native dengue virus type 2 cleavage sites. The 12mer substrate peptides and the cleavage products could be separated by reversed-phase HPLC, and were identified by UV and fluorescence detection. All of the peptide substrates (representing the DEN polyprotein junction sequences at the NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 sites) were cleaved by the recombinant protease NS2B-NS3(pro). No cleavage was observed with an enzymatically inactive S135A mutant of the NS3 protein, or with a modified substrate peptide of the NS3/NS4A polyprotein site that contained a K2093A substitution. Enzymatic activity was dependent on the salt concentration. A 50% decrease of activity was observed in the presence of 0.1M sodium chloride. Our results show that the NS3 protease activity of the refolded NS2B-NS3(pro) protein can be assayed in vitro with high specificity by using cleavage-junction derived peptide substrates.

• Microbiology and Biotechnology Letters
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• v.37 no.2
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• pp.105-109
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• 2009

Among signal transduction systems by protein phosphorylation Akt/PKB protein kinase which is one of serine/threonine kinases, is known to regulate the survival and death of the cell and glucose metabolism. Thus, Akt/PKB protein kinase has been used as one of the target proteins to find anti-cancer agents from natural products. In this study, human Akt/PKB protein kinase was expressed in Escherichia coli expression system for the mass production. Human Akt/PKB protein kinase expressed in E. coli formed inclusion body under the general condition. However, most of the expressed protein was solubilized under the culture temperature at $27^{\circ}C$ and 0.01-0.09 mM of IPTG for induction of the protein expression. The expressed protein was purified using $Ni^{2+}$-NTA agarose column and confirmed by using anti-Akt antibody. Subsequently, the purified human Akt/PKB protein kinase was activated by in vitro phosphorylation using cellular extract containing kinases. The activated protein was confirmed to phosphorylate the specific fluorescent peptide specially designed as the artificial substrate for Akt/PKB protein kinase.

• Journal of Life Science
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• v.9 no.2
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• pp.44-48
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• 1999

The ionization of tyrosine residue is known to be involved in the stabilization of transition-state in catalysis of astacin based upon the astacin-transition state analog structure. Two tyrosine residues, Tyr-216 and Tyr-219, are conserved in all MMPs related with astacin family, We replaced Tyr-216 and Tyr-219 into phenylalanine, respectively and the zinc binding properties, kinetic parameters, and pH dependence of each mutant are determined in order to examine the role of tyrosine residue in matrilysin catalysis. Both mutants contain two zinc atoms per mol of enzyme, indicating that either tyrosime does not affect the zinc binding property of the enzyme. Y216F and Y219F mutants are highly active and the kcat/Km values are only decreased 1.1-1.5-fold compared to the wild-type enzyme. The decrease in the activity of the mutants is essentially due to the increase in Km value. The pH dependencies of the kcat/Km values for both mutants are similar to the corresponding dependencies obtained with the wild type enzyme. The pKa values at the alkaline side of both mutants are not changed. These kinetic and pH dependence results indicate that the ionization of active site tyrosine residue of matrilysin is not reflected in the kinetics of peptide hydrolysin as catalyzed by astacin.

• Journal of Life Science
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• v.10 no.2
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• pp.218-227
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• 2000

Bone morphogenetic protein 1 (BMP-1) is part of a complex capable of inducing ectopic bone formation in mammals. Studies on TGF-β1 processing and Drosophila dorsal-ventral patterning have focused attention on BMP-1 as important in mediating the biological activity of this bone inducing complex. Herein, the bacterial expression, refolding, purification, and initial characterization of the BMP-1 proteolytic domain (BPD) are described. A semi-quantitative fluorescence-based thin layer chromatography assay was developed to assist in rapidly screening for optimal renaturation conditions. According to a preliminary screen for optimal conditions for the refolding of BPD , a detectable proteolytic activity against a high turnover substrate for astacin, a homologous protease from crayfish was observed. The conditions identified have allowed the expression of sufficient amounts of BPD for the characterization of the protein. Its proteolytic activity exhibits the same cleavage specificity as astacin against seven substrates that were previously synthesized for studying astacin. Furthermore, this activity is inhibited by the metal chelator 1,10-phenanthroline but not by its analogue 1,7-phenanthroline. The collagenase inhibitor Pro-Leu-Gly hydroxamate was found to inhibit both astacin and BPD activity. The results presented in this paper argue that BMP-1 does in fact possess an intrinsic proteolytic activity.