• 한국생물공학회:학술대회논문집
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• 2003.04a
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• pp.571-575
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• 2003

A marine aerobic Gram-negative bacterium, Pseudoalteromonas carrageenovora, has been blown to hydrolyze carrageenans, the sulfated galactans of red algae, and to desulfate oligo kappa-carrageenans. Recently, the gene encoding arylsulfatase (aryl-sulfate sulfohydrolase, E.C.3.1.6.1) of A. carrageenovora was cloned and the nucleotide sequence was reported. Enzymatic hydrolysis of sulfate groups in agaropectin simplifies the process of agarose preparation. In order to overproduce the enzyme, the arylsulfatase gene (astA, 984 bp ORF) from P. carrageenovora genome was amplified by PCR and subcloned into the pET21a vector. When the constructed plasmid pAST-A1 was introduced into E, coli BL21(DE3), the transformant on LB plate containing IPTG showed the hydrolyzing activity for p-nitrophenyl sulfate. Most of arylsulfatase activity was found in the cell lysate, but at $50\;{\sim}\;5000\;{\mu}M$ IPTG concentration the activity was found both in the culture supernatant and the cell lysate. The molecular weight of the recombinant enzyme was estimated to be 34 kDa by SDS-PAGE.

• Journal of Microbiology and Biotechnology
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• v.11 no.1
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• pp.164-166
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• 2001

To examine the characteristics of the recombinant thin aggregative fimbriae of Salmonella, the AgfA subunit gene was amplified from Salmonella enteritidis using a PCR. The maltose binding protein (MBP)-AgfA fusion protein was overproduced in E. coli and purified. The secondary structure of AgfA was then elucidated from the difference CD spectra. An estimation of the secondary structure of AgfA using the self-consistent method revealed a mostly ${\beta}-sheet$ structure.

• Journal of Microbiology and Biotechnology
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• v.27 no.6
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• pp.1106-1111
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• 2017

Escherichia coli was engineered to sense methanol by employing a chimeric two-component system (TCS) strategy. A chimeric MxaY/EnvZ (MxaYZ) TCS was constructed by fusing the Paracoccus denitrificans MxaY with the E. coli EnvZ. Real-time quantitative PCR analysis and GFP-based fluorescence analysis showed maximum transcription of ompC and the fluorescence at 0.01% of methanol, respectively. These results suggested that E. coli was successfully engineered to sense methanol by the introduction of chimeric MxaYZ. By using this strategy, various chimeric TCS-based bacterial biosensors can be constructed and used for the development of biochemical-producing recombinant microorganisms.

• International Journal of Oral Biology
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• v.30 no.1
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• pp.31-37
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• 2005

Treponema maltophilum, a Group IV oral spirochete, is associated with periodontitis and endodontic infections. In this study we analyzed the functional role of the major surface protein of this organism (MspA) in human gingival fibroblasts (HGFs). The full-length gene encoding MspA was cloned and expressed in Escherichia coli by using the expression vector pQE-30. The recombinant protein (rMspA) was purified by affinity chromatography with nickel-nitrilotriacetic acid agarose and possible contamination of E. coli endotoxin in rMspA was removed by using polymyxin B-agarose. rMspA significantly induced the expression of pro inflammatory cytokines like IL-6 and IL-8 and intercellular adhesion molecule (ICAM)-1 in HGFs, when analyzed by reverse transcription-PCR, flow cytometry, and enzyme-linked immunosorbent assay. Our results indicate that MspA of T. maltophilum may play an important role in amplifying the local immune response by upregulating the expression of proinflammatory cytokines and ICAM-1.

• Molecules and Cells
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• v.25 no.2
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• pp.312-316
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• 2008

Flavanone $3{\beta}$-hydroxylases (F3H) are key enzymes in the synthesis of flavonol and anthocyanin. In this study, three F3H cDNAs from Oryza sativa (OsF3H-1 ~3) were cloned by RT-PCR and expressed in E. coli as gluthatione S-transferase (GST) fusion proteins. The purified recombinant OsF3Hs used flavanone, naringenin and eriodictyol as substrates. The reaction products with naringen and eriodictyol were determined by nuclear magnetic resonance spectroscopy to be dihydrokaempferol and taxifolin, respectively. OsF3H-1 had the highest enzymatic activity whereas the overall expression of OsF3H-2 was highest in all tissues except seeds. Flavanone $3{\beta}$-hydroxylase could be a useful target for flavonoid metabolic engineering in rice.

• Journal of Industrial Technology
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• v.30 no.B
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• pp.61-68
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• 2010

Three chitinase genes (chiA, chiB, and chiC) were cloned into E. coli by PCR amplification from Serratia marcescens KFRI314. The sizes of cloned chitinase genes were 1692 bp, 1500 bp, and 1443 bp which correspond to 563, 499, and 480 amino acids, respectively. Recombinant chitinases were overexpressed using pHCEIA expression vector and purified to homogenity. The molecular weights of chitinases were about 60kDa, 50 kDa, 52 kDa, respectively. Optimum pHs were around pH 5~6 and optimum temperatures were $45{\sim}50^{\circ}C$ while 90% of enzyme activities were stable up to $50^{\circ}C$. The specific activities of ChiA, ChiB, and ChiC were 233.1, 278.8, $111.3{\mu}mol\;(min)^{-1}\;mg^{-1}$ against colloidal chitin. From experiments using TLC and fluorescent substrate analogues, it was demonstrated that ChiA was endo-chitinase while ChiB and ChiC were chitobiosidase.

• Bulletin of the Korean Chemical Society
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• v.32 no.7
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• pp.2405-2409
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• 2011

The psychrophilic bacteria Psychromonas arctica survives at subzero temperatures by having adapted several protective mechanisms against freezing and oxidative stresses. Many reactive oxygen species are likely generated in P. arctica as a result of reduced metabolic turnover rates. A previous study identified the pasod gene for superoxide dismutase from P. arctica using a series of PCR amplifications. Here, upon cloning into a His-tag fused plasmid, the sod gene from P. arctica (pasod) was successfully expressed by IPTG induction. His-tagged PaSOD was subsequently purified by $Ni^{2+}$-NTA affinity chromatography. The purified PaSOD exhibited a higher SOD activity than that of Escherichia coli (EcSOD) at all temperatures. The difference in activity between PaSOD and EcSOD becomes even more significant at 4$^{\circ}C$, indicating that PaSOD plays a functional role in the cold adaptation of P. arctica in the Arctic.

• BMB Reports
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• v.47 no.2
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• pp.92-97
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• 2014

We have examined the effect of bithorax complex genes on the expression of castor gene. During the embryonic stages 12-15, both Ultrabithorax and abdominal-A regulated the castor gene expression negatively, whereas Abdominal-B showed a positive correlation with the castor gene expression according to real-time PCR. To investigate whether ABD-B protein directly interacts with the castor gene, electrophoretic mobility shift assays were performed using the recombinant ABD-B homeodomain and oligonucleotides, which are located within the region 10 kb upstream of the castor gene. The results show that ABD-B protein directly binds to the castor gene specifically. ABD-B binds more strongly to oligonucleotides containing two 5'-TTAT-3' canonical core motifs than the probe containing the 5'-TTAC-3' motif. In addition, the sequences flanking the core motif are also involved in the protein-DNA interaction. The results demonstrate the importance of HD for direct binding to target sequences to regulate the expression level of the target genes.

• Proceedings of the Korean Society for Food Science of Animal Resources Conference
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• 2004.05a
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• pp.353-356
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• 2004

Rotavirus and Norovirus are major causative agents of acute diarrhea and gastroenteritis. In our study, Each viral RNA was isolated from the feces of patients for viral diarrhea in Korea, respectively. And cDNA library were constructed using RT-PCR. Also, cDNAs encoding VP8 derived from Rotavirus and Capsid protein derived from norovirus were subesequently cloned and expressed in Echerichia coli as a fusion antigen. Molecular weight of fusion antigen was approximately 60kDa. Also, substantial overexpression was accomplished. We yielded egg yolk lgY which is potentially useful in controlling of Rotavirus and Norovirus which are one of the most prevalent pathogenic viruses.

• Proceedings of the Korean Biophysical Society Conference
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• 1999.06a
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• pp.55-55
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• 1999

Imperatoxin A (IpTx$_{a}$), a 3.7 kDa peptide from the African scorpion Pandinus imperator, has been known as an agonist of skeletal ryanodine receptor (RyR). In order to study the structure and function of the toxins on RyR, the IpTx$_{a}$ cDNA was PCR-amplified using 3 pairs of primers and the toxin was expressed in E. coli expression system.(omitted)ted)