• BMB Reports
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• v.33 no.4
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• pp.337-343
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• 2000

The human immunodeficiency virus type 1 (HIV-1), transactivator of transcription (Tat), is one of the viral gene products that is essential for HIV-1 replication. The HIV-l Tat protein regulates transcription from an HIV-1 long terminal repeat (LTR) and affects the gene expression of cellular proteins during infection. In order to develop an expression system to overexpress and simply purify HIV-1 Tat proteins, the HIV-1 Tat coding sequences that contain one or two exons were amplified using PCR and cloned into a pET vector, which contains a consecutive stretch of six histidine residues at the amino-terminus. The reconstituted vectors were overexpressed in the E. coli strain and the soluble recombinant proteins were purified to be homogeneity in a single step by $Ni^{+2}-nitrilotriacetic$ acid Sepharose chromatography under nondenaturing conditions. Recombinant HIV-1 Tat proteins were shown to transactivate the HIV-1 LTR promoter in a dose-dependent manner when introduced into mammalian cells. In addition, treatment of human endothelial cells with purified Tat proteins resulted in a significant increase in the level of vascular cell adhesion molecule-1 (VCAM-1) expression. These results indicate that the recombinant HIV-1 Tat proteins are active in transactivating viral and cellular promoters. The expression and purification system described in this study will facilitate in characterizing the biological functions of the Tat proteins.

• Journal of Microbiology and Biotechnology
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• v.11 no.3
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• pp.482-490
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• 2001

The gene for glycoprotein B (gB2) of HSV-2-strain G was subcloned, sequenced, recombinated into the lacZ-HcNPV, expressed in insect cells, and compared with the homologous gene of other HSV-2 strains. The ORF of the gB2 gene was 2,715 bp. The overall nucleotide sequence homology of te gB2 gene compared ith that of the two previously reported HSV-2 strains appeared to be over 98%. A recombinant virus named Baculo-gB2 protein in insect cells. The recombination was confirmed by a PCR and the expression was demonstrated by radio immunoprecipitation. Insect cells infected with the Baculo-gB2 virus synthesized and processed gB2 with approximately 120 kDa in the cells, and then secreted it into the culture media, where it reacted with a nomoclonal antibody to gB2. The gB2 polypeptide contained two main hydrophobic regions (a signal sequence from 1 to 23 amino acid residues, and a membrane anchor sequence from aa 745 to 798), eight N-glycosylation sites evenly distributed, and was rich in alanine (11.2%). Antibodies to this recombinant protein that were raised in mice recognized the viral gB2 and neutralized the infectivity of the HSV-2 in vitro. There results show that the gB2 protein was successfully porduced in insect cells and could be used to raise a protective neutralizing antibody. Accordingly, this particular recombinant protein may be useful in the development of a subunit vaccine.

• BMB Reports
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• v.38 no.4
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• pp.414-419
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• 2005

The production of recombinant antibodies has been generally recognized as time-consuming and labor-intensive. The aim of our study is to construct mammalian expression vectors containing the cDNA encoding the human constant regions and murine variable regions to massively and cost-effectively produce full-length chimeric antibodies. Unique restriction sites flanking the Ig variable region were designed to allow for the replacement of variable regions generated by PCR. Western blot analysis of the chimeric antibodies revealed that the expressed products were of the predicted size, structure and specificity. The usefulness of the vectors was confirmed by construction of human-mouse chimeric antibody-HCAb which secretes murine antibody against the human colorectal cancer. Selected in medium containing gradually increasing methotrexate (MTX), clones with increased expression of the product gene can be efficiently generated. The secretion of recombinant chimeric antibody-HCAb yielded $30\;pg\;cell^{-1}\;day^{-1}$ at $10^{-6}\;M$ MTX. With this high-level expression from pools, the convenient and rapid production of over 100 milligram amounts per liter of recombinant antibodies may be achieved, which indicates the significant roles of pYR-GCEVH and pYR-GCEVL in the production of chimeric antibodies.

• BMB Reports
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• v.39 no.6
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• pp.730-736
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• 2006

To evaluated the effect of recombinant adenovirus Ad-ODC-AdoMetDCas which can simultaneously express both antisense ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) on cell cycle distribution in colorectal cancer cell and investigated underlying regulatory responses, human colorectal cancer cells HT-29 were cultured in RPMI 1640 medium and infected with Ad-ODC-AdoMetDCas. Cell cycle progression was detected by flow cytometry analysis. The expression levels of cell cycle regulated proteins were measured by Western blot analysis. The mRNA level of cyclin D1 was measured by RT-PCR. And a luciferase reporter plasmid of cyclin D1 promoter was constructed to observe the effect of Ad-ODC-AdoMetDCas on cyclin D1 promoter activity. The results showed that recombinant adenovirus Ad-ODC-AdoMetDCas significantly induced $G_1$ arrest, decreased levels of cyclin D1 protein and mRNA and suppressed the promoter activity. Ad-ODC-AdoMetDCas also inhibited nuclear translocation of $\beta$-catenin. In conclusion, downregulation of ODC and AdoMetDC mediated by Ad-ODC-AdoMetDCas transfection induces $G_1$ arrest in HT-29 cells and the arrest was associated with suppression of cyclin D1 expression and inhibition of $\beta$-catenin nuclear translocation. As a new anticancer reagent, the recombinant adenovirus Ad-ODC-AdoMetDCas holds promising hope for the therapy of colorectal cancers.

• Horticultural Science & Technology
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• v.17 no.6
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• pp.770-772
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• 1999

This study was conducted to provide a basic system to develop a molecular marker for plant cultivar protection using a recombinant DNA technology. Using Nicotiana tabacum L. plants, the potentiality in the utilization of the developed marker was examined. After homology test with several plant genomes, mouse adenosine deaminase (ADA) gene was selected as DNA source of a molecular marker for cultivar protection. As a result of the digestion of ADA gene with BamHI and Pst I, six DNA fragments were obtained, and 513 bp DNA fragment among them was selected as a possible DNA marker for cultivar protection. Selected 513 bp DNA fragment was efficiently inserted into pBI101 plasmid vector for plant transformation by using phagemid vector pBluescript II SK (+/-) as an intermediate vector. The recombinant pBI101, carrying 513 bp DNA fragment, possible markers for cultivar protection, was transformed into A. tumefaciens LBA4404. Nicotiana tabacum was transformed with A. tumefaciens LBA4404 having the recombinant pBI101 and was confirmed the transfer of 513 bp DNA fragment, a possible molecular marker for cultivar protection.

• Tuberculosis and Respiratory Diseases
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• v.58 no.2
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• pp.142-152
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• 2005

Background : Priming and boosting vaccination strategy has been widely explored for new vaccine development against tuberculosis. As an effort to identify other vaccine candidates, this study was initiated to evaluate protective efficacy of adenylate kinase (AK), nucleoside diphosphate kinase (NdK), and heat shock protein 70 (Hsp70) of Mycobacterium tuberculosis. Method : M. tuberculosis genes encoding AK, NdK, and Hsp70 proteins were amplified by PCR and cloned into E. coli expression vector, pQE30. Recombinant AK, NdK, and Hsp70 was purified through Ni-NTA resin. To evaluate immune responses, we performed enzyme-linked immunosorbent assay (ELISA) for IgG isotype and $IFN-{\gamma}$ after mice were immunized subcutaneously with recombinant proteins delivered in dimethyl dioctadecylammonium bromide (DDA). Immunized- and control groups were challenged by aerosol with M. tuberculosis. The spleens and lungs of mice were removed aseptically and cultured for CFU of M. tuberculosis. Result : Vaccination with recombinant proteins AK, NdK, and Hsp70 delivered in DDA elicited significant level of antibody and $IFN-{\gamma}$ responses to corresponding antigens but no protective immunity comparable to that achieved with Mycobacterium bovis BCG. Conclusion : Recombinant proteins AK, NdK, and Hsp70 do not effectively control growth of M. tuberculosis in mice when immunized with DDA as an adjuvant.

• Korean Journal of Microbiology
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• v.40 no.2
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• pp.81-86
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• 2004

Among the annotated ORFs of Streptomyces coelicolor, SCO7697 was supposed to encode for phytase (myo-inositol hexakisphosphate phosphohydrolase). The DNA fragment containing SCO7697 was cloned by the PCR from the chromosomal DNA of S.coelicolor A3(2)M. The cloned fragment was introduced into E. coli expres-sion vector, pET28a(+), to yield two recombinant plasmids, pET28-SP and pET28-LP, which were designed to encode different length of proteins. When the pET28-SP and pET28-LP were introduced into E. coli BL21, the transformants successfully overexpressed recombinant proteins, but the molecular weights of the expressed pro-teins were appeared bigger than those of expected in SDS-polyacrylamide gel electrophoresis. The shift of cul-tural temperature from 37 to $30^{\circ}C$ made most of expressed protein be solubilized. The expressed protein, however, did not show any phytase activity. When the DNA fragment with its own promoter placed on the E. coli-Streptomyces vector, pWHM3, and introduced into S. lividans, the phytase activity was not detected either. These results suggest that even though the SCO7697 was annotated as a probable phytase with high probability (E value is $6e^{-89}$), the real product doest not have phytase activity.

• Korean Journal of Microbiology
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• v.35 no.3
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• pp.205-210
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• 1999

16brio cholerne is an important pathogenic organism that causes dimhea in human beings. V ciaoleroe KNIH002 was isolated from patients suffering with dian.heal disease in Korea. From Southern hybridization using the amplified PCR product of 307 bp as a probe. which was obtained from PCR reaction using primer detecting cholera toxin gene, we have found that the c b gene located in 4.5-kb fragmenl double digested with Pstl and BgllI of the chromosome. Therefore, we made mini-libraries of the isolate using PstI and Bgm restriction endonuclease and pBluescript SKU(+) vector. As a result. we cloned 4.5-kb PstI-BglII fragment containing the c a gene encoding a cholera toxin from the constructed mini-libraries of V olzolerae KNlH002 by colony hybridization using the same probes. This recombinant plasmid was named pCTX75. E. coii XL1- Blue harboring pCTX75 showed the cytotoxicity on Chinese Hamster Ovary cells. From the sequencing of he cloned recombinant plasmid, we confinned that it has virulence gene cassette consisting of ace, zot, ctx.4 and cf"~B gene. The ace and zot genes were composed of 291 hp and 1.200 bp with ATG initiation codon and TGA lennination codon, respectively. Nucleotide sequence of the ace gene exhibited 100% identity with that of V cholera E7946 El Tor Ogawa strains. But, nucleolide and amino acid sequence comparison of the zot gene exhibited 99% and 98.8% identity with that of V cholerae 395 Classical Ogawa stram, respectively. Specially. the Ala-100, Ala-272 and Ala-281 sites of Zoi polypeptide presented in V choleme 395 Classical Ogawa strain are replaced by Val in V cholerae KNIH002.

• Journal of Microbiology and Biotechnology
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• v.25 no.5
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• pp.696-703
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• 2015

A gamma-aminobutyric acid (GABA)-producing microorganism was isolated from jeot-gal (anchovy), a Korean fermented seafood. The isolate, A156, produced GABA profusely when incubated in MRS broth with monosodium glutamate (3% (w/v)) at 37℃ for 48 h. A156 was identified as Lactobacillus sakei by 16S rRNA gene sequencing. The GABA conversion yield was 86% as determined by GABase enzyme assay. The gadB gene encoding glutamate decarboxylase (GAD) was cloned by PCR. gadC encoding a glutamate/GABA antiporter was located immediately upstream of gadB. The operon structure of gadCB was confirmed by RT-PCR. gadB was overexpressed in Escherichia coli BL21(DE3) and recombinant GAD was purified. The purified GAD was 54.4 kDa in size by SDS-PAGE. Maximum GAD activity was observed at pH 5.0 and 55℃ and the activity was dependent on pyridoxal 5'-phosphate. The K_m and V_max of GAD were 0.045 mM and 0.011 mM/min, respectively, when glutamate was used as the substrate.

• 한국생물공학회:학술대회논문집
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• 2001.11a
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• pp.652-655
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• 2001

The comparative study of enzymes that catalyze a similar reactions but have different substrate spectrum and/or stereospecificity is a powerful approach to understanding the reaction mechanism between the relative enzymes, and it was also an useful tool to cloning the related enzyme, without the typical cloning from DNA library of genomic pools. For this purpose, we conducted an approach that the comparison at the molecular and protein level of esterases, from various sources including a previously identified (S)-stereospecific esterase of Pseudomonas sp. ES1. As expected, we found an esterase family genes that shared a high similarity at the protein and genetic level in the identical genus Pseudomonad. The striking structural and biochemical identity strongly suggested the family genes to be an identical one. We, hence, aligned the family genes and designated a degenerated primer for PCR-cloning using six Pseudomonas strains as templates. As a result, a recombinant esterase from Pseudomonas fluorescens KCTC 1767 was cloned and high-level expressed with high selectivity to (R,S)-ketoprofen ethyl ester. The enzyme exhibited a high ester-hydrolyzing activity to (S)-ketoprofen but did not hydrolyzed the opposite stereoisomer. Further characteristics were discussed in our presentation.