• Journal of Applied Biological Chemistry
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• v.64 no.3
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• pp.213-216
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• 2021

A gene encoding thermostable cellulase A (TmCelA) was isolated from Thermotoga maritima. The open reading frame of TmCelA gene was 774 bp long which predicted to encode 257 amino acid residues with a molecular weight of 29,732 Da. To examine the biochemical properties, the TmCelA was overexpressed in E. coli BL21, and expressed protein was purified. The optimum temperature of recombinant TmCelA was 90-95 ℃, and the optimum pH of recombinant TmCelA was approximately pH 5.0. Recombinant TmCelA was stable at temperature below 90 ℃.

• Journal of Microbiology and Biotechnology
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• v.24 no.12
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• pp.1719-1727
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• 2014

In the present study, whispovirus immediate early 1 promoter (ie-1) was used to initiate surface expression of the hemagglutinin (HA) protein of Egyptian H5N1 avian influenza virus (AIV) by using the baculovirus expression vector system. The HA gene and whispovirus ie-1 promoter sequence were synthesized as a fused expression cassette (ie1-HA) and successfully cloned into the pFastBac-1 transfer vector. The recombinant vector was transformed into DH10Bac competent cells, and the recombinant bacmid was generated via site-specific transposition. The recombinant bacmid was used for transfection of Spodoptera frugiperda (Sf-9) insect cells to construct the recombinant baculovirus and to induce expression of the HA protein of H5N1 AIV. The recombinant glycoprotein expressed in Sf-9 cells showed hemadsorption activity. Hemagglutination activity was also detected in both extra- and intracellular recombinant HAs. Both the HA and hemadsorption activities were inhibited by reference polyclonal anti-H5 sera. Significant expression of the recombinant protein was observed on the surface of infected insect cells by using immunofluorescence. SDS-PAGE analysis of the expressed protein revealed the presence of a visually distinguishable band of ~63 kDa in size, which was absent in the non-infected cell control. Western blot analysis confirmed that the distinct 63 kDa band corresponded to the recombinant HA glycoprotein of H5N1 AIV. This study reports the successful expression of the HA protein of H5N1 AIV. The expressed protein was displayed on the plasma membrane of infected insect cells under the control of whispovirus ie-1 promoter by using the baculovirus expression vector system.

• Journal of Microbiology and Biotechnology
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• v.17 no.11
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• pp.1898-1903
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• 2007

We attempted to modulate the overall protein expression rate through the addition of a repressor against the araBAD promoter system of Escherichia coli, in which glucose was used as a repressor. Therefore, 0.5% L-arabinose was initially contained as an inducer in culture medium, and either 2% glucose or 2% glycerol was used as a carbon source, and it was found that the expression of recombinant interferon-${\alpha}$ could be observed at the beginning of the batch culture when glycerol was used as a carbon source. However, when glucose was used, the initiation of recombinant interferon-${\alpha}$ expression was delayed compared with that when glycerol was used. Furthermore, when the addition of 0.5% glucose was carried out once or twice after 0.5% L-arabinose induction during DO-stat fed-batch culture, the distributions of soluble and insoluble recombinant interferon-${\alpha}$ were modulated. When glucose was not added after the induction of L-arabinose, all of the expressed recombinant interferon-${\alpha}$ formed an inclusion body during the later half of culturing. However, when glucose was added after induction, the expressed recombinant interferon-${\alpha}$ did not all form an inclusion body, and about half of the total recombinant interferon-${\alpha}$ was expressed in a soluble form. It was deduced that the addition of glucose after the induction of L-arabinose might lower the cAMP level, and thus, CAP (catabolite activator protein) might not be activated. The transcription rate of recombinant interferon-${\alpha}$ in the araBAD promoter system might be delayed by the partial repression. This inhibition of the transcription rate probably resulted in more soluble interferon-${\alpha}$ expression caused by the reduction of the protein synthesis rate.

• Journal of Sericultural and Entomological Science
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• v.38 no.1
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• pp.36-41
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• 1996

Recombinant baculoviruses having expanded host range were selected by coinfection of Autographa california NPV and Bombyx mori NPV into Sf-9 and BmN-4 insect cell lines. In order to determine the polyhedra morhplogy of RecS-A6, one of a recombinant baculovirus, polyhedra of RecS-A6 produced in insect cells were observed by phase contrast microscope and scanning electron microscope. The results revealed that the recombinant baculovirus had a various polyhedra morphology which was different from its parental viruses, suggesting that the various morhpology of recombinant baculovirus with an expanded host range was due to the genetic recombination of viral genome. To analyze the genomic recombinantion of the recombinant baculoviruses, genomic DNAs of two parent viruses and RecS-A6 were digested with restriction endonuclease and subjected to agarose gel electrophoresis. Southern blot analysis revealed that RecS-A6 has two polyhedrin gene of AcNPV and BmNPV in a viral genome. Polyhedral protein of recombinant baculovirus was analysed by SDS-PAGE. The result showed that molecular weight of polyhedral protein of RecS-A6 containing two polyhedrin gene of AcNPV and BmNPV was as the 31 kDa band of AcNPV and 30 kDa band of BmNPV.

• Microbiology and Biotechnology Letters
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• v.16 no.4
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• pp.327-333
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• 1988

For optimal production of recombinant human interleukin-2 (IL-2) in E. coli the effect of fermentation conditions on cell growth, IL-2 production, and stability of recombinant cells were investigated. Among the complex nutrients tested in this work, yeast extract, peptone and corn steep liquor were found to be effective for recombinant cell growth. The recombinant cells were maintained stably under repression condition (3$0^{\circ}C$), but the stability of recombinant cells were drastically reduced upon induction of IL-2 expression (42$^{\circ}C$) even under the selection pressure. Addition of antibiotics to the culture medium resulted in the cell growth inhibition without significant improvement in recombinant stability. When the expression of IL-2 gene was induced at different growth phases, highest IL-2 production was achieved by the induction of IL-2 at the middle-exponential growth phase. It was found that the production of IL-2 significantly inhibited the cell growth and the ex-pression of other genes in the plasmid.

• Korean Journal of Veterinary Research
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• v.39 no.4
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• pp.786-796
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• 1999

As an attempt to develop an effective control method against theileriosis, recombinant antigen protein was produced. Thirty-two kDa membrane protein(MP) gene of T sergenti was amplified through RT-PCR from extracted total RNA of T sergenti isolated in Chonbuk, Korea. The amplified 869 bp of Korean T sergenti membrane gene was cloned and the base sequences were analyzed. The amplified gene was cloned into E coli expression vector, pQE32 plasmid vector, and the vector was introduced into E coli strain M15 to produce the recombinant membrane protein. For the induction of T sergenti membrane protein(KTs-MP), the plasmid harboring E coli strain M15 were cultured in the presence of IPTG, and the recombinant protein were purified by $Ni^+$-NTA agarose. Then, to confirm the authenticity of the produced membrane protein, molecular weight of expressed recombinant KTs-MP was analyzed by SDS-PAGE and Western blotting. The molecular weight of expressed recombinant protein was 32 kDa as expected. The recombinant KTs-MP was successfully recognized by anti-His Tag antibody, antisera of T sergenti infected cattle and monoclonal antibody of T sergenti membrane protein. Therefore, we concluded that the authentic 32 kDa membrane protein of T sergenti was produced as immunologically recognizable form.

• The Journal of Korean Society of Virology
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• v.27 no.1
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• pp.29-37
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• 1997

To use recombinant viruses with wider host range as viral insecticides, we investigated the characteristics and pathogenicity of host range expanded recombinant viruses in insect cells. We compared host range expanded recombinant viruses, RecS-B6 and RecB-8, constructed by cotransfection of Autographa californica nuclear polyhedrosis virus (AcNPV) and Bombyx mori NPV (BmNPV), to host range expanded AcNPV, Ac-BH, by substitution of the 0.6 Kb fragment of the BmNPV helicase gene. Restriction endonuclease profiles of RecS-B6 and RecB-8 DNAs were different from those of parent viruses. Nucleotide sequence analysis of the 0.6 Kb region in the putative helicase gene of RecS-B6 and RecB-8 showed that their structures were identical to the counterpart region of BmNPV. Comparison of viral replication of these recombinant viruses in Sf-21 and BmN-4 cells showed that Ac-BH, compared to wild type viruses, replicated well in BmN-4 cells but poorly in Sf-21 cells. In contrast, RecS-B6 and RecB-8 replicated relatively well in both cells compared to parent viruses. These results may imply that random genomic recombinant viruses, RecS-B6 and RecB-8, possess better potential as viral pesticides than helicase-mediated recombinant virus, Ac-BH.

• Journal of Periodontal and Implant Science
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• v.38 no.sup2
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• pp.343-354
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• 2008

Purpose: Aggregatibacter actinomycetemcomitans is associated with localized aggressive periodontitis. It produces cytolethal distending toxin (CDT), which induces cell cycle arrest in the G2/M phase. The CDT holotoxin is composed of CdtA, CdtB, and CdtC. CdtB has structural homology to human DNase I and is an active component of the CDT complex acting as a DNase. In particular, the pattern homology seen in the CdtB subunit has been associated with specific DNase I residues involved in enzyme catalysis, DNA binding, and metal ion binding. So, to study the functions and regulation of recombinant CdtB, we made up a quantity of functional recombinant CdtB and tested it in relation to the metal ion effect. Materials and Methods: We constructed the pET28a-cdtB plasmid from A. actinomycetemcomitans Y4 by genomic DNA PCR and expressed it in the BL21 (DE3) Escherichia coli system. We obtained the functional recombinant CdtB by the refolding system using the dialysis method and then analyzed the DNase activity and investigated the metal ion effect from plasmid digestion. Results: The recombinant CdtB subunit was expressed as the inclusion bodies. We were able to obtain functional recombinant CdtB subunit using refolding system. We confirmed that our refolded recombinant CdtB had DNase activity and was influenced by the metal ions $Mg^{2+}$ and $Ca^{2+}$. Conclusion: We suggest that the factors influencing recombinant CdtB may contribute to CDT associated diseases, such as periodontitis, endocarditic, meningitis, and osteomyelitis.

• Journal of Microbiology
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• v.45 no.2
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• pp.179-184
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• 2007

Pasteurella multocida, a Gram-negative facultative anaerobic bacterium, is a causative animal pathogen in porcine atrophic rhinitis and avian fowl cholera. For the development of recombinant subunit vaccine against P. multocida, we cloned and analyzed the gene for outer membrane protein H (ompH) from a native strain of Pasteurella multocida in Korea. The OmpH had significant similarity in both primary and secondary structure with those of other serotypes. The full-length, and three short fragments of ompH were expressed in E. coli and the recombinant OmpH proteins were purified, respectively. The recombinant OmpH proteins were antigenic and detectable with antisera produced by either immunization of commercial vaccine for respiratory disease or formalin-killed cell. Antibodies raised against the full-length OmpH provided strong protection against P. multocida, however, three short fragments of recombinant OmpHs, respectively, showed slightly lower protection in mice challenge. The recombinant OmpH might be a useful vaccine candidate antigen for P. multocida.

• 한국생물공학회:학술대회논문집
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• 2003.10a
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• pp.186-189
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• 2003

Helicobacter pylori is the etiologic agent of human gastritis and peptic ulceration and produces urease as the major protein component on its surface. H. pylori urease is known to serve as a potent immunogen as well as major virulence factor. In order to express the recombinant urease in tobacco plants, a DNA fragment containing the minimal H. pylori urease gene cluster was subcloned into a plant expression vector. The recombinant vector was transformed to tobacco plants. The integration of the recombinant plasmids into tobacco chromosomal genome was verified by genomic PCR. Expression to mRNA was confirmed by Northern blot analysis, and expression to recombinant urease protein was observed by Western blot analysis. These results showed that the recombinant urease can be produced in tobacco plants and will be tested for immune response to use as an edible vaccine.