• Journal of Microbiology
• /
• v.42 no.3
• /
• pp.205-210
• /
• 2004

The sequence coding for carboxymethylcellulase (CMCase, CelC) was isolated from the DNA of Salmonella typhimurium URl. Comparison between the deduced amino acid sequence of CelC (368 amino acid residues, Molecular mass 41 kDa) and that of the previously published CMCase revealed that this enzyme belongs to the cellulase family 8 and D. The protein was overproduced in Escherichia coli using T7 expression system, and its activity was confirmed by CMC-SDS-PAGE. When the overexpressed CelC protein was tested on cellulose-type substrates, the recombinant protein is able to degrade cellulose-type substrates, such as CM-cellulose, xylan, avicel, lichenan, and laminarin. Optimal temperature and pH for enzyme activity were found to be 50$^{\circ}C$ and pH 6.5, respectively.

• Journal of Pharmaceutical Investigation
• /
• v.40 no.spc
• /
• pp.33-43
• /
• 2010

Hyaluronic acid (HA) is a biodegradable, biocompatible, non-toxic, non-immunogenic and non-inflammatory linear polysaccharide, which has been used for various medical applications including arthritis treatment, wound healing, ocular surgery, and tissue augmentation. Because of its mucoadhesive property and safety, HA has received much attention as a tool for drug delivery system development. It has been used as a drug delivery carrier in both nonparenteral and parenteral routes. The nonparenteral application includes the ocular and nasal delivery systems. On the other hand, its use in parenteral systems has been considered important as in the case of sustained release formulation of protein drugs through subcutaneous injection. Particles and hydrogels by various methods using HA and HA derivatives as well as by conjugation with other polymer have been the focus of many studies. Furthermore, the affinity of HA to the CD44 receptor which is overexpressed in various tumor cells makes HA an important means of cancer targeted drug delivery. Current trends and development of HA as a tool for drug delivery will be outlined in this review.

• BMB Reports
• /
• v.29 no.2
• /
• pp.137-141
• /
• 1996

A partial cDNA encoding a Korean radish isoperoxidase was obtained from a cDNA library prepared from 9 day old radish root. In order to obtain Korean radish isoperoxidase cDNA, 5' RACE (rapid amplification cDNA end) PCR was performed and a cDNA (prxK1) encoding a complete structural protein was obtained by RT (reverse transcription)-PCR. Sequence analysis revealed that the length of the cDNA was 945 base pairs, and that of the mRNA transcript was ca. 1.6 kb. The deduced amino acid of the protein were composed of 315 amino acid residues and the protein was 92% homologous to turnip peroxidase, and 46% to 50% homologous to other known peroxidases. The 945 bp cDNA encoding Korean radish isoperoxidase was overexpressed in Escherichia coli up to approximately 9% of total cellular protein. The recombinant fusion protein exhibited 43 kDa on SDS-PAGE analysis and the activity level of the recombinant nonglycosylated protein was two fold higher in IPTG induced cell extracts than that of uninduced ones.

• BMB Reports
• /
• v.29 no.2
• /
• pp.116-121
• /
• 1996

In earlier studies, the genes encoding Escherichia coli thioredoxin and Corynebacterium nephridii thioredoxin C-3 were fused via a common restriction site in the nucleotide sequence coding for the active site of the proteins to generate two chimeric thioredoxins, designated E-C3 (N to C-terminal) and C3-E. The hybrid thioredoxins were overexpressed in E. coli from the cloned chimeric thioredoxin genes by a T7 promoter/polymerase system. To investigate the structure-function relationship of thioredoxin, we purified the E-C3 hybrid thioredoxin through ammonium sulfate fractionation, DEAE-cellulose chromatography, and Sephadex G-50 gel filtration. Its purity was examined on SDS-polyacrylamide gel electrophoresis and the molecular weight of the purified E-C3 hybrid thioredoxin was estimated to be 12,000. On native polyacrylamide gels, the purified E-C3 hybrid thioredoxin shows a much lower mobility than E. coli thioredoxin. E-C3 hybrid thioredoxin exhibits a 40-fold lower catalytic efficiency with E. coli thioredoxin reductase than E. coli thioredoxin. It was shown to catalyze the reduction of insulin disulfide by dithiothreitol. The purified E-C3 hybrid thioredoxin was also characterized in other aspects.

• BMB Reports
• /
• v.34 no.2
• /
• pp.172-175
• /
• 2001

Catechol 1,2-dioxygenase $I_1$ ($CDI_1$) is the first enzyme of the $\beta$-ketoadipate pathway in Acinetobacter lowffii K24. $CDI_1$ has two cysteines (155, 202) and its enzyme activity is inhibited by the cysteine inhibitor, $AgNO_3$. Two mutants, $CDI_1$ C155V and $CDI_1$ C202V, were obtained by site-directed mutagenesis. The two mutants were overexpressed and the mutated amino acid residues (Cys$\rightarrow$Val) were characterized by peptide mapping and amino acid sequencing. Interestingly, $CDI_1$ C155V was inhibited by $AgNO_3$, whereas $CDI_1$ C202V was not inhibited. This suggests that $Cys^{202}$ is the sole inhibition site by $AgNO_3$ and is close to the active site of the enzyme. However, the results of the biochemical assay of mutated $CDI_1s$ suggest that the two cysteines are not directly involved in the activity of the catechol 1,2-dioxygenase of $CDI_1$.

• BMB Reports
• /
• v.34 no.2
• /
• pp.156-165
• /
• 2001

Leptin is a circulating non-glycosylated protein that is mainly produced in adipocytes. Leptin acts in the brain to regulate food intake and energy expenditure. Previously we reported our success in the isolation of a partial cDNA of the long form of the leptin receptor, OB-Rb, from rat spleen, and showed that leptin might also play a role in peripheral immune organs. In the present study, for the first time, the complete coding region of OB-Rb cDNA was cloned from rat splenocytes, and its nucleotide sequence was determined. The cDNA was then further expressed in E. coli and mammalian cells, thereby confirming the functional integrity of this receptor. Prokaryotically overexpressed OB-R protein was then used as an immunizing antigen in BALE/c mice to produce leptin receptor-specific antibodies. By using them, we confirmed the cell surface expression of OB-Rb in transfected CHO cells. It is our belief that the reagents, as produced in this study, will be of great use in further studies of the biological role of rat leptin.

• Journal of Microbiology and Biotechnology
• /
• v.20 no.4
• /
• pp.821-827
• /
• 2010

Gene delivery that provides targeted delivery of therapeutic genes to the cells of a lesion enhances therapeutic efficacy and reduces toxic side effects. This process is especially important in cancer therapy when it is advantageous to avoid unwanted damage to healthy normal cells. Incorporating cancer-specific ligands that recognize receptors overexpressed on cancer cells can increase selective binding and uptake and, as a result, increase targeted transgene expression. In this study, we investigated whether a peptide capable of homing to hepatocellular carcinoma (HCC) could facilitate targeted gene delivery by cationic liposomes. This homing peptide (HBP) exhibited selective binding to a human hepatocarcinoma cell line, HepG2, at a concentration ranging from 5 to 5,000 nM. When conjugated to a cationic liposome, HBP substantially increased cellular internalization of plasmid DNA to increase the transgene expression in HepG2 cells. In addition, there was no significant enhancement in gene transfer detected for other human cell lines tested, including THLE-3, AD293, and MCF-7 cells. Therefore, we demonstrate that HBP provides targeted gene delivery to HCC by cationic liposomes.

• Journal of Microbiology and Biotechnology
• /
• v.20 no.4
• /
• pp.727-731
• /
• 2010

The gene APE0743 encoding the superoxide dismutase (ApSOD) of a hyperthermophilic archaeon Aeropyrum pernix K1 was cloned and overexpressed as a GST fusion protein at a high level in Escherichia coli. The expressed protein was simply purified by the process of glutathione affinity chromatography and thrombin treatment. The ApSOD was a homodimer of 25 kDa subunits and a cambialistic SOD, which was active with either Fe(II) or Mn(II) as a cofactor. The ApSOD was highly stable against high temperature. This thermostable ApSOD is expected to be applicable as a useful biocatalyst for medicine and bioindustrial processes.

• Environmental Mutagens and Carcinogens
• /
• v.22 no.4
• /
• pp.236-242
• /
• 2002

Using agarose-coupled methotrexate, we have successfully isolated two proteins, which have strong interactions with methotrexate. The two proteins were analyzed by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry and identified as carbamoyl-phosphate synthetase 2 and phosphoribosylglycinamide formyltransferase, respectively. Interestingly, both of these two proteins are essential key enzymes in nucleotide biosynthetic pathways, like dihydrofolate reductase, a well-known methotrexate target. We confirmed the specificity of their interactions between methotrexate and two target proteins by the methods of competition binding assay, which were followed by western blotting using antibody against carbamoyl-phosphate synthetase 2 and phosphoribosylglycinamide formyltransferase, respectively. Moreover, we could observe that carbamoyl-phosphate synthetase 2 is overexpressed in methotrexate-resistant MOLT-3 cells comparing with control MOLT-3 cells. This result indicates that carbamoyl-phosphate synthetase 2 may be a novel target of methotrexate in cancer therapy. We propose that chemical proteomics can be a powerful technique to identify target proteins of a chemical.

• Herbal Formula Science
• /
• v.11 no.2
• /
• pp.95-110
• /
• 2003

Alzheimer's disease(AD) is a geriatric dementia that is widespread in old age. In the near future AD will be the biggest problem in public health service. Although a variety of oriental prescriptions in study Jeonmaedan have been traditionally utilized for the treatment of AD, their pharmacological effects and action mechanisms have not yet fully elucidated. It has been widely believed that A${\beta}$ peptide devided from APP causes apoptotic neurotoxicity in AD brain. However, recent evidence suggests that CTl05(carboxy terminal 105 amino acid peptide fragment of APP) may be an important factor causing neurotoxicity in AD. In addition, AD is one of brain degeneration disease. So we studied on herbal medicine that have a relation of brain degeneration. In Oriental Medicine, Jeonmaedan has been used for disease in relation to brain degeneration. As the result of this study, in Jeonmaedan the apoptosis in the nervous system is inhibited, the repair against the degerneration of SK-N-SH cell lines by CT105 expression is promoted. So Jeonmaedan may be beneficial for the treatment of AD.