• IMMUNE NETWORK
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• v.16 no.5
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• pp.296-304
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• 2016

It has been reported that fatty acid binding proteins (FABPs) do not act only as intracellular mediators of lipid responses but also have extracellular functions. This study aimed to investigate whether extracellular liver type (L)-FABP has a biological activity and to determined serum L-FABP levels in patients with end-stage renal disease (ESRD). We isolated L-FABP complementary deoxyribonucleic acid (cDNA) from the Huh7 human hepatocarcinoma cell line and expressed the recombinant L-FABP protein in Escherichia coli. A549 lung carcinoma and THP-1 monocytic cells were stimulated with the human recombinant L-FABP. Human whole blood cells were also treated with the human recombinant L-FABP or interleukin (IL)-1α. IL-6 levels were measured in cell culture supernatants using IL-6 enzyme-linked immunosorbent assay (ELISA). Human recombinant L-FABP induced IL-6 in a dose-dependent manner in A549, THP-1 cells, and whole blood cells. The blood samples of healthy volunteers and patients with ESRD were taken after an overnight fast. The serum levels of L-FABP in healthy volunteers and ESRD patients were quantified with L-FABP ELISA. The values of L-FABP in patients with ESRD were significantly lower than those in the control group. Our results demonstrated the biological activity of L-FABP in human cells suggesting L-FABP can be a mediator of inflammation.

• 한국생물공학회:학술대회논문집
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• 2000.04a
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• pp.153-156
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• 2000

Xylan, the major hemicellulose component of many plants, occurs naturally in a partially acetylated form and lignin, the most resistant component in plant cell wall degradation, is also attached to ${\beta}-1,4-linked-D-xylose$ backbone through the ester linkage. Esterases are required to release the esterified substituent and acetyl esterases are important in the complete degradation of acetylated polysaccharides, like pectins and xylans. The gene(Axe) encoding acetyl xylan estarase(AXE) was isolated from genomic ${\lambda}$ library from Aspergillus ficuum. Nucleotide sequencing of the Axe gene indicated that the gene was separated with two intervening sequences and the amino acid sequence comparison revealed that it was closely related to that from A. awamori with the 92 % indentity. Heterologous expression of AXE was conducted by using YEp352 and Saccharomyces cerevisae 2805 as a vector and host expression system, respectively. The Axe gene was placed between GAL1 promoter and GAL7 terminator and then this recombinant vector was used to transform S. cerevisiae 2805 strain. Culture filtrate of the transformed yeast was assayed for the presence of AXE activity by spectrophotometry and, comparing with the host strain, four to five times of enzyme activity was detected in culture filtrate of transformed yeast.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.77.1-77
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• 2003

We have isolated a full-length cDNA clone, CaCDPK4 encoding a typical calcium-dependent protein kinase (CDPK) from hot pepper cDNA library. Genomic southern blot analysis showed that it belongs to a multigene family, but represents a single copy gone in hot pepper genome. RNA expression pattern of this gene revealed that it is induced by infiltration of Xanthomonas axonopodis pv. glycines Bra into hot pepper leaves but not by water deficit stress. However, high salt treatment of NaCl (0.4 M) solution to hot pepper plants strongly induced CaCDPK4 gene. In addition, this gene is weakly responsive to the exogenous application of salicylic acid or ethephon. Biochemical study of the GST-CaCDPK4 recominant protein showed that it autophosphorylates in vitro and the presence of EGTA, a calcium chelater, eliminates the kinase activity of the recombinant protein. As a way to identify the in vivo function of CaCDPK4 in plants, VIGS (Virus-Induced Gene Silencing) was employed. Agrobacterium-mediated TRV silencing construct containing the kinase and calmodulin domain of CaCDPK4 resulted in cell death of Nicotiana benthamiana plants. A highly homologous H benthamiana CDPK gene, NbCDPK5, to CaCDPK4 was cloned from N. benthamiana cDNA library. VIGS of NbCDPK5 also resulted in cell death. The molecular characterization of this cell death phenotype is being under investigation.

• Journal of Life Science
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• v.15 no.5 s.72
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• pp.734-738
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• 2005

A gene coding for xylanase from alkali-tolerant Bacillus alcalophilus AX2000 was cloned into Escherichia coli $DH5\alpha$ using pUC19. Among 2,000 transformants, one transformant showed clear zone on the detection agar plate containing oat-spells xylan. Its recombinant plasmid, named pXTY99, was found to carry 7.0 kb insert DNA fragment. When the nucleotide sequence of the cloned xylanase gene (xynT) was determined, xynT gene was found to consist of 1,020 base-pair open reading frame coding for a poly-peptide of 340 amino acids with a deduced molecular weight of 40 kDa. The coding sequence was preceded by a putative ribosome binding site, and the transcription initiation signals. The deduced amino acid sequence of xylanase is similar to those of the xylanases from Bacillus sp. Nl37 and B. stearothermophilus 21 with $61\%$ and $59\%$ identical residues, respectively.

• Journal of Microbiology and Biotechnology
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• v.9 no.4
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• pp.398-403
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• 1999

In our previous studies, we reported that hybridoma B-lymphocytes can be used to determine the virulence of Listeria species in an in vitro cytotoxicity assay. Here, we examined the cytopathic effect, i.e., membrane damage and the nature of cell death induced by Listeria monocytogenes on murine hybridoma B-lymphocytes (Ped-2E9). Membrane damage was assessed by microscopic analyses and by measuring the release of intracellular alkaline phosphatase(AP) and lactate dehydrogenase (LDH). Cell death was determined by DNA fragmentation analyses using agarose gel electrophoresis. Infection by listeriolysin O (LLO)-producing L. monocytogenes strains induced substantial amounts of AP and LDH release from Ped-2E9 hybridoma B-cells, suggesting severe membrane damage in these cells, while an LLO-negative L. monocytogenes mutant strain had no effect. An LLO-producing recombinant L. innocua ($prifA^+hly^+$) strain also induced high AP and LDH release and cytopathic changes in Ped-2E9 cells. Light or scanning electron microscopic examination revealed L. monocytogenes mediated membrane destabilization, pore formation, intense cytoplasmic granulation, bleb formation, and lysis of Ped-2E9 cells. LLO-producing L. monocytogenes and L. innocua ($prifA^{+}hly{^}+$) also induced ladder-like DNA fragmentation in Ped-2E9 cells. Collectively, these results suggest that L. monocytogenes, specifically LLO-producing strains, can induce a severe cytopathic effect leading to apoptosis in hybridoma B-lymphocytes (Ped-2E9).

• BMB Reports
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• v.34 no.3
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• pp.201-205
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• 2001

Thionins are a family of low molecular weight cysteine-rich antimicrobial peptides. We isolated a cDNA encoding thionin gene from a flower bud cDNA library of Chinese cabbage (CFT). The gene contains 611 by nucleotides with 60 bp, and 150 by untranslated regions at its N- and C-terminal, respectively. The deduced amino acid sequence encoded 133 amino acids containing precursor polypeptide. The protein reveals that the precursor has a tripartite structure: a putative signal sequence at the N-terminus, followed by a mature thionin peptide, and a C-terminal acidic domain, which facilitates transport of the mature thionin through membrane. Genomic Southern blot analysis suggests that the CFT gene may be present as a single or two copy gene in the Chinese cabbage genome. Northern blot analysis shows that the gene is specifically expressed in flowers, but not in leaves, stems, or roots. When we analyzed the antifungal activity of the recombinant CFT protein, which was expressed in E. coli using the truncated cDNA region corresponding to the mature protein part, it was not active on fungal growth inhibition.

• Journal of Microbiology and Biotechnology
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• v.7 no.4
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• pp.229-236
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• 1997

A gene, $phbC_{2.4.1}$ encoding poly-3-hydroxybutyric acid (PHB) synthase of Rhodobacter sphaeroides 2.4.1 was cloned by employing heterologous expression in Escherichia coli. R. sphaeroides chromosomal DNA partially digested with MboI was cloned in pUC19 followed by mobilization into E. coli harbouring $phbA,B_{AC}$ in pRK415, which code for ${\beta}$-ketothiolase and acetoacetyl CoA reductase of Alcaligenes eutrophus, respectively. Two E. coli clones carrying R. sphaeroides chromosomal fragment of $phbC_{2.4.1}$ in pUC19 were selected from ca. 10,000 colonies. The PHB-producing colonies had an opaque white appearance due to the intracellular accumulation of PHB. The structure of PHB produced by the recombinant E. coli as well as from R. sphaeroides 2.4.1 was confirmed by [$H^{+}$]-nuclear magnetic resonance (NMR) spectroscopy. Restriction analysis of the two pUC19 clones revealed that one insert DNA fragment is contained as a part of the other cloned fragment. An open reading frame of 601 amino acids of $phbC_{2.4.1}$ with approximate M.W. of 66 kDa was found from nucleotide sequence determination of the 2.8-kb SaiI-PstI restriction endonuclease fragment which had been narrowed down to support PHB synthesis through heterologous expression in the E. coli harbouring $phbA,B_{AC}$. The promoter (s) of the $phbC_{2.4.1}$ were localized within a 340-bp DNA region upstream of the $phbC_{2.4.1}$ start codon according to heterologous expression analysis.

• Journal of Microbiology and Biotechnology
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• v.16 no.12
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• pp.1968-1976
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• 2006

Recombinant E. coli ACV 1003 (recA:: lacZ) was used to measure low concentrations of DNA-damaging chemicals, which produce $\beta$-galactosidase via an SOS regulon system. Very low $\beta$-galactosidase activities of less than 0.01 unit/ml, $\beta$-galactosidase produced through an SOS response corresponding to the 10 ng/ml (ppb) of DNA damaging chemicals in the environment, can be rapidly determined by using an alternative interferometric biosensor with optically flat thin films of porous silicon rather than by the conventional time-consuming Miller's enzyme assay as well as the ELISA method. fu order to enhance the sensitivity in the interferometry, it needs to obtain more uniform distribution and higher biolinking efficiency, whereas interferometric sensing is rapid, cheap, and advantageous in high throughput by using a multiple-well-type chip. In this study, pore size adjusted to 60 nm for the target enzyme $\beta$-galactosidase to be bound on both walls of a Si pore and a calyx crown derivative was apllied as a more efficient biolinker. Furthermore, anti-$\beta$-galactosidase was previously functionalized with the biolinker for the target $\beta$-galactosidase to be specifically bound. When anti-$\beta$-galactosidase was bound to the calyx-crown derivative-linked surface, the effective optical thickness was found to be three times as high as that obtained without using anti-$\beta$-galactosidase. The resolution obtained was very similar to that afforded by the time-consuming ELISA method; however, the reproducibility was still unsatisfactory, below 1 unit $\beta$-galactosidase/ml, owing to the microscopic non-uniform distribution of the pores in the etched silicon surface.

• Applied Biological Chemistry
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• v.45 no.4
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• pp.190-194
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• 2002

Expression of Bacillus subtilis glutamyl-tRNA synthetase (GluRS) in Escherichia coli is lethal for the host, probably because this enzyme misaminoacylates ${tRNA_l}^{Gln}$ with glutamate in vivo. In order to overexpress B. subtilis GluRS, encoded by the gltX gene, in E. coli, this gene was amplified from B. subtilis 168 chromosomal DNA using PCR method and the entire coding region was cloned into a pET11a expression vector so that it was expressed under the control or the T7 Promoter. The resulting recombinant pEBER plasmid was transformed into E. coli Novablue (DE3) bearing the T7 RNA polymerase gene for expression. After IPTG treatment, the overproduced enzyme was purified using ammonium sulfate fractionation, Source Q anion exchange chromatography, Superdex-200 gel filtration, and Mono Q anion exchange chromatography. The purified enzyme yielded 18-fold increase in specific activity over the crude cell extract and its molecular weight was approximately 55 kDa on SDS-PAGE.

• Applied Biological Chemistry
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• v.38 no.1
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• pp.7-12
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• 1995

The purpose of this study was the development of promoter for the lacZ' gene. Two heterologous promoter I and II of lacZ' gene were isolated from chromosomal DNA Bam HI fragment of yeast. The size of the promoter I was estimated to be 2.5 kb and ${\beta}-galactosidase$ activity was 124.6 U/mg protein, and the size of the promoter II was 4.0 kb and its ${\beta}-galactosidase$ activity was 168.8 U/mg protein, respectively. The stability of the recombinant YEp plasmid in the transformant was from 52.7 to 67.4% at minimal medium. YIp plasmid was constructed from YEp plasmid, and expressed both in E. coli and yeast. The promoter I aid II iso-lated from yeast chromosomal DNA can be used for promoter of plasmid YEp and YIp.